Baicalin improves podocyte injury in rats with diabetic nephropathy by inhibiting PI3K/Akt/mTOR signaling pathway
- Yi Ou
,Wenjuan Zhang,Shaopeng Chen andHaihua Deng
Abstract
Objective
To investigate the effect of baicalin on diabetic nephropathy (DN) rats and podocytes and its mechanism.
Methods
The rat models with DN were established by high-fat and high-sugar diet and intraperitoneal injection of streptozotocin. The fasting blood glucose (FBG) and weight of rats in each group were measured at 0, 1, 2, 3, and 4 weeks. Their biochemical indicators, expression of inflammatory, and antioxidant factors were measured using an automatic biochemical analyzer together with ELISA. Hematoxylin–eosin staining and periodic acid-schiff staining were used to observe the morphological changes in the kidneys of rats in each group. Finally, the expressions of related molecules and PI3K/Akt/mTOR signaling pathway proteins in renal tissues and podocytes were examined by qRT-PCR and Western blot.
Results
Compared with the DN group, the FBG and weight, serum creatinine, blood urea nitrogen, total cholesterol, triacylglycerol, microalbumin, and albumin/creatinine ratio were all significantly decreased in the Baicalin treatment groups in a concentration-dependent manner. The levels of inflammatory factors in kidney tissue and podocytes were decreased. In addition, the activities of lactate dehydrogenase and malondialdehyde in tissue were decreased, while the superoxide dismutase was increased. The pathological sections showed that glomerular atrophy and glomerular basement membrane thickening caused by hyperglycemia were improved in the Baicalin treatment groups. Meanwhile, baicalin inhibited the downregulation of Nephrin and Podocin expressions and upregulation of Desmin expression caused by DN, and inhibited the expressions of p-PI3K, p-Akt, and p-mTOR proteins.
Conclusion
Baicalin slows down podocyte injury caused by DN by inhibiting the activity of PI3K/Akt/mTOR signaling pathway.
1 Introduction
Diabetic nephropathy (DN) is a disease in which diabetes mellitus (DM) induces vascular lesions, thus promoting glomerulosclerosis [1]. DN is characterized by diffuse thickening of the glomerular basement membrane (GBM), proliferation and dilatation of the mesangial matrix, and other morphological changes, inducing renal dysfunction. DN will develop into end stage renal disease if not treated in time [2]. According to the latest statistics from the International Diabetes Federation, there are currently about 425 million DM patients worldwide and the number is expected to reach 629 million by 2045, of which 30–40% of DM patients will suffer from DN [3,4]. It has been shown that the persistent hyperglycemic state in the peripheral blood of DM patients changes hemodynamics and increases oxidative stress in the body, thus causing severe injury to local renal cells such as glomerular capillary endothelial cells and podocytes [5]. Among them, podocytes, as terminally differentiated cells, are the important functional cells in the renal glomerulus. Because of the terminal differentiation, podocytes cannot regenerate once subjected to destruction [6]. Hyperglycemia destroys the GBM, resulting in podocyte hypertrophy and detachment. So podocyte content in urine was proposed to be used as a marker to determine the early stage of DN, which is more accurate than urinary albumin [7]. Some studies have pointed out that avoiding podocyte injury is the key to prevent the occurrence of DN [8].
Baicalin (5,6,7-trihydroxyflavone) is a flavonoid extracted from scutellaria baicalensis georgi [9]. It has a wide range of biological functions, including antibacterial, antiviral, and anti-tumor functions [10,11]. Cheng showed that baicalin reduced lipopolysaccharide-induced liver inflammation in chicken by suppressing TLR4-mediated NF-κB signaling pathway [12]. Meanwhile, baicalin was found to improve renal function in DN patients by inhibiting excessive inflammation and oxidative stress, and thereby effectively delaying the development of DN [13]. Baicalin is also used in clinical applications. Haixia [14] found that baicalin could reduce proteinuria in patients with early DN. Yang et al. [15] also found that baicalin can improve the renal function of patients with DN and delay the progression of DN through various pathways such as anti-inflammation and antioxidation. However, at present, the effect of baicalin on podocytes in DM-induced DN tissues and its mechanism has not been reported. Therefore, this study investigated DN rats and podocytes to provide a certain theoretical and experimental basis for subsequent research on the treatment of DM-induced DN.
2 Materials and methods
2.1 Establishment and grouping of DN rat models
A total of 30 Sprague Dawley (SD) male rats aged 8 weeks and weighing 180−200 g were selected. After 1 week of adaptive culture, the rats were randomly divided into five groups (6 rats/group). Subsequently, the DN rat models were established by intraperitoneal injection of 35 mg/kg streptozotocin (STZ) after 4 weeks of high-fat and high-sugar diet. After 72 h, the rats with fasting blood glucose (FBG) higher than 13.9 mmol/L and random blood glucose higher than 16.7 mmol/L were considered as diabetic rats. After successful modeling, the diabetic rats were divided into 4 test groups: DN group, baicalin low-concentration group (Baicalin-L), baicalin medium-concentration group (Baicalin-M), and baicalin high-concentration group (Baicalin-H). In these test groups, same amount of saline, 50 mg/kg baicalin, 100 mg/kg baicalin, and 200 mg/kg baicalin were administered, respectively, once daily for 4 weeks by gavage [16]. Meanwhile, the rats with a normal diet were intraperitoneally injected with the same amount of sodium citrate buffer for 4 weeks as the control group. After 4 weeks, 24 h urine and peripheral blood were collected, followed by anesthesia and cervical dislocation to sacrifice the rats and then their renal tissues were collected.
Ethical approval: This trial was approved by the ethics committee of Shenzhen Fuyong People’s Hospital.
2.2 Cell culture and grouping
SD male rats aged 8 weeks were injected intraperitoneally with 10% chloral hydrate, and the rats were anesthetized and then sacrificed by cervical dislocation. Their kidneys were collected under aseptic conditions, soaked and rinsed with pre-cooled sterile PBS solution. After the renal capsule was removed, the kidneys were cut into small tissue masses (1 mm3) using ophthalmic scissors and then collected in centrifuge tubes to digest at 150 rpm with 0.1% of collagenase type IV at 37°C. RPMI-1640 complete culture medium was added 25 min later to terminate digestion. Subsequently, the cell suspension passed through the 100, 150, and 200 mesh sieves in turn. Next the cell suspension was collected into centrifuge tube, centrifuged at 1,000 rpm at 4°C for 5 min, resuspended with complete medium, and inoculated in culture flasks to culture in an incubator (Thermo, USA) with 5% of CO2 at 37°C. Finally,in vitro podocytes of the rats were obtained. The podocytes as the Test groups were cultured with RPMI-1640 complete medium containing 30 mmol/L of glucose and then treated with 10% of serum from the rats in each test group, named as high glucose (HG) group, Baicalin-L group, Baicalin-M group, and Baicalin-H group. Meanwhile, the other part of the podocytes cultured in RPMI-1640 medium were treated with 10% of serum from the rats in the control group, named as the Control group.
2.3 Measurement of FBG and weight
FBG was measured in the control, DN, Baicalin-L, Baicalin-M, and Baicalin-H groups at 0, 1, 2, 3, and 4 weeks, respectively. Then 5 μL of blood collected from the caudal vein was dropped onto glucose test strips and FBG was measured by a blood glucose meter (Accu-Check Active, Roche). Their weights were also measured and recorded.
2.4 Detection of urine biochemical indicators
The rats in each group were fed in the promethion cages (Sable Systems International, USA), and 24 h urine was collected 4 weeks later. After the total amount of urine was determined, 3 mL of urine was centrifuged at 3,000 rpm for 10 min at 4°C, and the supernatant was taken. The 24 h microalbumin (mALB) in urine was detected using an automatic biochemical analyzer (Olympus, Japan) to calculate the urinary albumin/creatinine ratio (ACR).
2.5 Detection of blood biochemical indicators and inflammatory factors
The peripheral blood was collected from the orbital venous plexus of the rats and centrifuged at 3,000 rpm for 20 min. Then, the serum from the upper layer was collected and placed into new centrifuge tubes. The serum creatinine (SCr), blood urea nitrogen (BUN), total cholesterol (TC), and triacylglycerol (TG) in the serum of the rats were measured using the automatic biochemical analyzer.
In the meanwhile, the serum or podocyte supernatant of rats from each group was detected by the expressions of TNF-α, IL-1β, and IL-6 using ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), which were operated in strict accordance with the experimental instructions.
2.6 Detection of oxidative damage index
Fifty milligram of kidney tissues from rats in each group were fully ground and centrifuged at 9,000 rpm for 10 min at 4°C. The supernatant was isolated to detect the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) in kidney tissues of rats from each group using LDH, SOD, and MDA assay kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions.
2.7 Hematoxylin–eosin (HE) staining and periodic acid-schiff (PAS) staining
The collected renal tissues were rinsed with PBS buffer. After the renal capsule was removed, the right renal tissues were fixed with 10% of neutral formaldehyde and embedded in paraffin for sectioning. The sections were stained with hematoxylin at room temperature for 5 min, followed by differentiation with hydrochloric acid alcohol. The sections appearing as blue color was washed using 1% (volume/volume%) of ammonia and then were washed in running tap water before being stained with eosin for 30 s. Finally, the sections were dehydrated with alcohol, cleared in xylene, and covered with slips. The pathological changes in the kidneys were observed under a biomicroscope.
The prepared renal tissue sections were deparaffinized at 65°C, washed with distilled water, and oxidized in 1% of periodic acid solution for 5–10 min. The sections were rinsed with distilled water again and stained with Schiff reagent for 10 min. Subsequently, the tissue sections were rinsed with sodium bisulfite solution for 3 times (2 min/time) and incubated with hematoxylin solution for 3 min. Finally, the sections were dehydrated with alcohol, treated with xylene, and covered with slips. The pathological changes in the kidneys were observed under the biomicroscope. Semiquantitative measurement of the changes was performed using Image J software according to the previous study [17].
2.8 MTT assay
After treatment, podocytes in the logarithmic growth phase were seeded in 96-well plates at a density of 500 cells/well and cultured for 24, 48, and 72 h, respectively. Twenty microliter of 5 mg/mL of MTT solution was added to each group of cells and continued culturing in the incubator for another 4 h. The culture supernatant in the wells was pipetted and discarded. Afterwards, 150 μL of DMSO was added and shaken for 15 min. The absorbance of each well was measured at a wavelength of 490 nm by using the enzyme mark instrument.
2.9 Flow cytometry (FCM)
The cells from each group were digested into a centrifuge tube by utilizing trypsin. After that, they were rinsed twice with pre-cooled sterile PBS and then adjusted the cell concentration to 5 × 105 cells/mL. Two hundred microliter of cell suspension was extracted in order to add 10 μL of Annexin V-FITC followed by 10 μL of PI solution at the concentration of 20 mg/L. Finally, it was incubated in the dark at room temperature for 10 min. After adding 500 μL of PBS, apoptosis was detected by FCM.
2.10 qRT-PCR
Total RNA was extracted from renal tissues or cells using Trizol (Invitrogen) and the RNA concentration was measured by spectrophotometer. The total RNA was reverse transcribed into cDNA using MLV reverse transcriptase. qPCR was performed in the detection system of SYBR Green Master Mix (Promega, USA). GAPDH was used as an internal reference and the results were calculated using the 2−△△Ct method. The primers used are shown inTable 1.
Primer sequences
| RNA | Sequences(5′ to 3′) |
|---|---|
| Nephrin | F: 5′-GCATAGCCAGAGGTGGAAATCC |
| R: 5′-GAACGGTCATCACCAGCACACT | |
| Podocin | F: 5′-GTGGAAGCTGAGGCACAAAGAC |
| R: 5′-CAGCGACTGAAGAGTGTGCAAG | |
| Desmin | F: 5′-GCGGCTAAGAACATCTCTGAGG |
| R: 5′-ATCTCGCAGGTGTAGGACTGGA | |
| GAPDH | F: 5′-CATCACTGCCACCCAGAAGACTG |
| R: 5′-ATGCCAGTGAGCTTCCCGTTCAG |
2.11 Western blot
RIPA lysis buffer was added to 50 mg of renal tissues and homogenized and then centrifuged at 12,000 rpm for 30 min at 4°C; the supernatant was taken as total protein. Meanwhile, RIPA lysis buffer was added to the podocytes and then centrifuged at 12,000 rpm for 30 min at 4°C; the supernatant was taken as total protein. The protein concentrations were determined using the BCA protein quantification kit (Thermo Fisher). In immunoblotting, 20 µg of proteins were separated using 10% of SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Following the blocking step with 5% of skim milk for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies: p-PI3K (Abcam UK), PI3K (Abcam UK), p-Akt (Abcam UK), Akt (Abcam UK), p-mTOR (Abcam UK), and mTOR (Abcam UK). Subsequently, they were incubated with the corresponding secondary antibodies for 1 h at room temperature. GAPDH was used as an internal reference when scanning immunoblot bands at gray scale. The gray values of the bands were analyzed using Image LabTM Software.
2.12 Statistical analysis
SPSS 24.0 was used for one-way analysis of variance (ANOVA) and independent samplet-test. The results were expressed as mean values ± standard deviation, andp < 0.05 indicated significant differences.
3 Results
3.1 Effects of baicalin on FBG and weight in DN rats
The blood glucose of rats in each group was measured by the blood glucose meter. The results showed that the level of FBG in the control group was in the normal range, while that in the DN group and Baicalin treatment groups were always at high levels. In the DN group, FBG level was higher than 16.7 mmol/L all the time. In the Baicalin treatment groups, FBG level showed a downward trend with the increase in baicalin treatment time. At the 4th week, compared with the DN group, the FBG of rats was significantly decreased in the Baicalin treatment groups (Figure 1a,p < 0.05), and in a concentration-dependent manner. The FBG in the Baicalin-H group was closest to that in the control group. At the 0th week, the weight of rats in the test groups was significantly higher than that in the control group. Compared with the DN group, the weights in the Baicalin treatment groups were gradually decreased (Figure 1b). These results indicated that baicalin has an inhibitory effect on blood glucose and weight in DN rats.
Effects of baicalin on FBG and weight in DN rats. The DN rats were administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage. The FBG (a) and weight (b) of the rats in each group are measured weekly.#p < 0.05 vs control group; *p < 0.05 and **p < 0.01 vs DN group.
3.2 Effects of baicalin on serum and urine biochemical indicators in DN rats
The changes in serum and urine biochemical indicator in DN rats before and after baicalin treatment were observed. The results of the automatic biochemical analyzer revealed that compared with the control group, the levels of SCr, BUN, TC, and TG in the serum in the DN group were significantly increased (Figure 2a–d,p < 0.05), while the 24 h mALB and ACR in the urine were significantly increased (Figure 2e and f,p < 0.05). Compared with the DN group, the levels of SCr, BUN, TC, and TG in the Baicalin treatment groups were significantly decreased (Figure 2a–d,p < 0.05) in a concentration-dependent manner; the higher the baicalin concentration, the lower the levels of SCr, BUN, TC, and TG. Meanwhile, compared with the DN group, the 24 h mALB and ACR in urine in the Baicalin treatment groups were significantly decreased (Figure 2e–f,p < 0.05), also in a concentration-dependent manner.
Effects of baicalin on serum and urine biochemical indicators in DN rats. The DN rats are administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage. (a) SCr levels in the serum of rats in each group; (b) BUN levels in the serum of rats in each group; (c) TC levels in the serum of rats in each group; (d) TG levels in the serum of rats in each group; (e) mALB levels in the urine of rats in each group; (f) ACR levels in the urine of rats in each group.##p < 0.01 vs control group; *p < 0.05 and **p < 0.01 vs DN group.
3.3 Effects of baicalin on serum inflammatory factors and renal oxidative stress factors in DN rats
The ELISA results showed (Figure 3a) that the expressions of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum of rats from the DN group were significantly increased compared with that from the control group. Fortunately, baicalin could inhibit the expressions of TNF-α, IL-1β, and IL-6 in the serum of DN rats. Furthermore, the lower the concentration of baicalin, the lower the expressions of TNF-α, IL-1β, and IL-6.
Effects of baicalin on serum inflammatory factors and renal oxidative stress factors in DN rats. The DN rats are administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage. (a) the expression of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum of rats in each group; (b) the activities of LDH, SOD, and MDA in the renal tissue of rats in each group;##p < 0.01 vs Control group; *p < 0.05, **p < 0.01, and ***p < 0.001 vs DN group.
Oxidative stress is closely related to DN incidence [18].Figure 3b indicated that compared with the control group, the activities of LDH and MDA in the renal tissue from the DN group were significantly increased, while the activity of SOD was significantly decreased. Adding to that, compared with the DN group, the activities of LDH and MDA from different concentrations of baicalin group were markedly decreased, while the activities of SOD were significantly increased, and all of which were baicalin concentration-dependent.
3.4 Effects of baicalin on renal pathomorphology in DN Rats
The pathological morphological changes in the renal tissues after baicalin treatment in DN rats were examined using histopathology. The results of HE staining showed no obvious pathological changes in the renal tissues in the control group; the glomerular structure was intact, the renal tubules were arranged neatly and uniform in size, and there was no inflammatory cell infiltration. Compared with the control group, the renal tissues in the DN group were significantly damaged; the glomeruli were atrophic, the glomerular mesangial matrix was increased, the renal tubules showed vacuolar degeneration, and a large number of inflammatory cell infiltration were observed. After baicalin treatment, renal pathological injury was improved in different degrees, and the higher the baicalin concentration, the more significant the improvement (Figure 4a). The results of PAS staining showed no significant pathological changes in the renal tissues in the control group; the basement membrane was intact, and the glomerular vascular loops were thin and clear. Compared with the control group, the basement membrane in the DN group was significantly thickened. After baicalin treatment, the thickening of GBM was improved, and the higher the baicalin concentration, the more significant the improvement (Figure 4b). These results confirmed that baicalin improved renal tissue injury in DN rats in a concentration-dependent manner.
Effects of baicalin on renal pathological morphology in DN rats. The DN rats are administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage, and their renal tissues are collected. (a) HE staining images of renal tissues of rats in each group; (b) PAS staining images of renal tissues of rats in each group.##p < 0.01 vs Control group; **p < 0.01 vs DN group.
3.5 Effects of baicalin on the expressions of podocyte-related molecules and PI3K/Akt/mTOR signaling pathway in the renal tissues of rats
To investigate the molecular mechanism by which baicalin improved renal tissue injury in DN rats, the expressions of podocyte-related molecules (Nephrin, Podocin, and Desmin) and PI3K/Akt/mTOR signaling pathway proteins were detected. Compared with the control group, the mRNA expressions of Nephrin and Podocin were significantly decreased, while that of Desmin significantly increased in the renal tissues in the DN group (Figure 5a,p < 0.05). Compared with the DN group, the expressions of Nephrin and Podocin were significantly increased, while that of Desmin significantly decreased in the renal tissues in the baicalin treatment groups (Figure 5a,p < 0.05) in a concentration-dependent manner. In terms of protein, compared with the control group, the expressions of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR were significantly increased in the renal tissues in the DN group (Figure 5b,p < 0.05). Compared with the DN group, the expressions of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR in the baicalin treatment groups were significantly decreased (Figure 5b,p < 0.05) in a concentration-dependent manner.
Effects of baicalin on the expressions of podocyte-related molecules and PI3K/Akt/mTOR signaling pathway in renal tissues of rats. The DN rats are administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage, and their renal tissues are collected. (a) qRT-PCR to detect the mRNA expressions of Nephrin, Podocin, and Desmin in the renal tissues of rats in each group; (b) Western blot to detect the expressions of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR proteins in the renal tissues of rats in each group.##p < 0.01 vs Control group; *p < 0.05 and **p < 0.01 vs DN group.
The results further confirmed that baicalin ameliorates renal injury in DN rats by inhibiting PI3K/Akt/mTOR. Besides, after the PI3K/Akt signaling pathway was activated via 740Y-P, the results showed that (Figure 6a and b) the expressions of Nephrin and Podocin in the renal tissue of rats from the DN group were inhibited, but the protein expression of Desmin, like p-PI3K, p-Akt, and p-mTOR, was promoted. Expressions of these proteins in rat kidney tissue were reversed upon baicalin treatment. In short, the results confirmed that baicalin could ameliorate renal injury by inhibiting the activation of PI3K/Akt/mTOR signaling pathway in DN rats.
Baicalin ameliorates renal injury by inhibiting the activation of PI3K/Akt/mTOR signaling pathway in DN rats. 30 SD rats are randomly divided into 5 groups (6 rats in each group) which are control group, DN group, 740Y-P group (3.5 mg/kg PI3K activator), Baicalin group (200 mg/kg baicalin), Baicalin + 740Y-P group (200 mg/kg baicalin + 3.5 mg/kg 740Y-PA), with 4 weeks of gavage. (a) qRT-PCR to detect the mRNA expressions of Nephrin, Podocin, and Desmin in the kidney tissues of rats in each group; (b) Western blot to detect the protein expressions of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR in the kidney tissues of rats in each group. *p < 0.05 vs DN group;&p < 0.05 vs Baicalin group; and#p < 0.05 vs DN + 740Y-P group.
3.6 Effect of baicalin on podocyte injury induced by HG
In order to further confirm the results ofin vitro trails, primary culture of rat podocytes was carried out and podocyte models of HG were induced using 30 mmol/L of glucose and then the podocytes were treated with 10% of serum from the rats in each test group. The results ofFigure 7a–c show that compared with the control group, the viability of podocytes in the HG group was significantly decreased with an increase in the rate of apoptosis. However, the expressions of inflammatory factors such as TNF-α, IL-1β, and IL-6 were significantly upregulated. Baicalin, on the one hand, could promote the proliferation of podocytes. On the other hand, it could inhibit apoptosis and the expression of TNF-α, IL-1β, and IL-6.
Effect of baicalin on podocyte injury induced by HG. Primary culture of rat podocytes was carried out and podocyte models of HG were induced using 30 mmol/L of glucose and then the podocytes were treated with 10% of serum from the rats in each test group. (a) MTT assay for podocyte viability; (b) flow assay for podocyte apoptosis; (c) ELISA for the expression of inflammatory factors TNF-α, IL-1β, and IL-6.##p < 0.01 vs control group; *p < 0.05, **p < 0.01, and ***p < 0.001 vs HG group.
3.7 Effect of baicalin on PI3K/Akt/mTOR signaling pathway after podocyte injury induced by HG
Compared with the Control group, the mRNA expressions of Nephrin and Podocin were significantly decreased, while that of Desmin significantly increased in the HG group (Figure 8a,p < 0.05). Compared with the HG group, the mRNA expressions of Nephrin and Podocin were significantly upregulated, while that of Desmin was significantly downregulated in the Baicalin treatment groups (Figure 8a,p < 0.05) in a concentration-dependent manner. In terms of protein, compared with the control group, the expressions of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR were significantly increased in the HG group (Figure 8b,p < 0.05). Compared with the HG group, the expressions of p-PI3K/PI3K, p-Akt/AKT, and p-mTOR/mTOR proteins were significantly decreased in the Baicalin treatment groups (Figure 8b,p < 0.05) in a concentration-dependent manner. These results of trailsin vitro andin vivo were similar.
In vitro trails to detect effects of baicalin on rat podocytes. Primary culture of rat podocytes is carried out and the podocyte models of HG are induced using 30 mmol/L of glucose and then the podocytes are treated with 10% of serum from the rats in each test group. (a) qRT-PCR to detect the mRNA levels of Nephrin, Podocin, and Desmin in podocytes of each group; (b) Western blot to detect the expressions of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR proteins in podocytes of each group.##p < 0.01 vs Control group; *p < 0.05 and **p < 0.01 vs DN group.
4 Discussion
Current treatments for DN patients include diet control, exercise enhancement, weight control, and drug therapy. The first three are known as non-drug therapies, in which weight control can effectively control diabetes and slow down the development of DN [19]. In addition, drug therapy is to control blood glucose in DN patients. Many studies have shown that blood glucose controlled within the normal range is the basis for the treatment of DN [20], while poor blood glucose control induces DN development as well as other serious microvascular complications [21]. In addition, the control of urinary protein is also the key to the treatment of DN [22]. In this study, diabetic rat models with DN were established by high-fat and high-sugar diet and STZ injection. In the test groups, DN rats had significantly increased FBG and weight, significant proteinuria (upregulation of 24 h mALB and urinary ACR), increased SCr, BUN, TC, and TG in serum, and significant changes in podocyte-related molecules (downregulation of Nephrin and Podocin expressions and upregulation of Desmin expression). Many studies have pointed out that baicalin can reduce diabetic kidney injury. Zhang et al. [23] showed that baicalin significantly inhibited DM-induced fibrosis of proximal renal tubular epithelial cells. Zheng et al. [24] demonstrated that baicalin–lysin conjugate ameliorated renal fibrosis in STZ-induced DN rats. Li et al. revealed that baicalin could prevent the apoptosis of podocytes due to HG, thereby delaying the development of DN [25]. In this study, during the treatment of DN rats with baicalin, FBG and body weight of rats treated with different concentrations of baicalin showed a downward trend, and longer treatment time and higher concentration cause more significant decrease in FBG and weight in rats. Meanwhile, baicalin significantly inhibited the levels of SCr, BUN, TC, and TG in the blood and urinary mALB and ACR. Furthermore, by observing the pathological sections of the kidneys, it can be seen that baicalin improved GBM thickening and glomerular atrophy caused by hyperglycemia. Baicalin could also significantly upregulate the expressions of Nephrin and Podocin, but downregulate the expression of Desmin. Some studies have pointed out that upregulation of Nephrin and Podocin and downregulation of Desmin are beneficial to protect the morphology, structure, and function of podocytes, and to inhibit the epithelial-mesenchymal transition of the podocytes [26]. Therefore, the result indicated that baicalin maintained the homeostasis of podocyte-related functional proteins to protect the normal functions of podocytes. All these studies confirmed that baicalin could improve renal injury in DN rats.
An increasing number of studies have shown that hyperglycemia induced oxidative stress or inflammatory factors, leading to podocyte detachment from GBM and apoptosis, which is the major factor in the early development of DN [18]. In this study, we found that the expressions of inflammatory factors, namely, TNF-α, IL-1β, and IL-6 were all upregulated in the serum of rats in the DN group. Furthermore, the activities of LDH and MDA were increased, but the activity of SOD was decreased in the renal tissue. At the same time, HG-induced podocyte viability was reduced, but apoptotic ability was enhanced, along with upregulation of TNF-α, IL-1β, and IL-6 expressions. Baicalin, on the other hand, could reverse these changes. These results confirmed that baicalin could ameliorate the damage of renal podocytes in DN rats. DM will cause hemodynamic changes, leading to the abnormal activation of PI3K/Akt/mTOR pathway. This abnormal activation induces autophagy of podocytes, thus inhibiting the adhesive ability of podocytes and resulting in detachment of podocytes from GBM [27]. Many studies have explored new therapeutic ideas for DN based on the effect mention above. Wu et al. [28] found that Huangkui capsule alleviated glomerular pathological changes in the early stage of DN by inhibiting the activity of Akt/mTOR signaling pathway. Huang et al. [29] revealed that Notoginsenoside R1 could inhibit autophagy and apoptosis of podocytes to reduce glucose-induced podocyte injury through inhibiting the activity of the PI3K/Akt/mTOR signaling pathway. Wu et al. [30] also found that curcumin inhibited the autophagic transformation of podocytes through suppressing the activity of this signaling pathway, thus reducing the damage caused by DN to the body. Therefore, it is speculated that baicalin also has a benign effect on the development of DN by regulating this signaling pathway. From our results of Western blot, baicalin significantly inhibited the expression of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR in bothin vivo andin vitro experiments. So, we believed the possibility that baicalin protected podocytes by downregulating the activity of the PI3K/Akt/mTOR signaling pathway.
5 Conclusion
In summary, baicalin can not only reduce the increase in FBG, body weight, and biochemical indicator in serum and urine due to DM, but also slow down the damage of podocytes caused by hyperglycemia by inhibiting the activity of PI3K/Akt/mTOR signaling pathway, thereby delaying the progression of DM-induced DN. The results of this trial provide effective data for the clinical application of baicalin in the treatment of DN.
Funding information: Not applicable.
Author contributions: Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work: All authors. Drafting the work or revising it critically for important intellectual content: YO. Final approval of the version to be published: All authors. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: All authors.
Conflict of interest: The authors have no conflicts of interest to declare.
Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
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© 2021 Yi Ouet al., published by De Gruyter
This work is licensed under the Creative Commons Attribution 4.0 International License.
Abstract
Objective
To investigate the effect of baicalin on diabetic nephropathy (DN) rats and podocytes and its mechanism.
Methods
The rat models with DN were established by high-fat and high-sugar diet and intraperitoneal injection of streptozotocin. The fasting blood glucose (FBG) and weight of rats in each group were measured at 0, 1, 2, 3, and 4 weeks. Their biochemical indicators, expression of inflammatory, and antioxidant factors were measured using an automatic biochemical analyzer together with ELISA. Hematoxylin–eosin staining and periodic acid-schiff staining were used to observe the morphological changes in the kidneys of rats in each group. Finally, the expressions of related molecules and PI3K/Akt/mTOR signaling pathway proteins in renal tissues and podocytes were examined by qRT-PCR and Western blot.
Results
Compared with the DN group, the FBG and weight, serum creatinine, blood urea nitrogen, total cholesterol, triacylglycerol, microalbumin, and albumin/creatinine ratio were all significantly decreased in the Baicalin treatment groups in a concentration-dependent manner. The levels of inflammatory factors in kidney tissue and podocytes were decreased. In addition, the activities of lactate dehydrogenase and malondialdehyde in tissue were decreased, while the superoxide dismutase was increased. The pathological sections showed that glomerular atrophy and glomerular basement membrane thickening caused by hyperglycemia were improved in the Baicalin treatment groups. Meanwhile, baicalin inhibited the downregulation of Nephrin and Podocin expressions and upregulation of Desmin expression caused by DN, and inhibited the expressions of p-PI3K, p-Akt, and p-mTOR proteins.
Conclusion
Baicalin slows down podocyte injury caused by DN by inhibiting the activity of PI3K/Akt/mTOR signaling pathway.
1 Introduction
Diabetic nephropathy (DN) is a disease in which diabetes mellitus (DM) induces vascular lesions, thus promoting glomerulosclerosis [1]. DN is characterized by diffuse thickening of the glomerular basement membrane (GBM), proliferation and dilatation of the mesangial matrix, and other morphological changes, inducing renal dysfunction. DN will develop into end stage renal disease if not treated in time [2]. According to the latest statistics from the International Diabetes Federation, there are currently about 425 million DM patients worldwide and the number is expected to reach 629 million by 2045, of which 30–40% of DM patients will suffer from DN [3,4]. It has been shown that the persistent hyperglycemic state in the peripheral blood of DM patients changes hemodynamics and increases oxidative stress in the body, thus causing severe injury to local renal cells such as glomerular capillary endothelial cells and podocytes [5]. Among them, podocytes, as terminally differentiated cells, are the important functional cells in the renal glomerulus. Because of the terminal differentiation, podocytes cannot regenerate once subjected to destruction [6]. Hyperglycemia destroys the GBM, resulting in podocyte hypertrophy and detachment. So podocyte content in urine was proposed to be used as a marker to determine the early stage of DN, which is more accurate than urinary albumin [7]. Some studies have pointed out that avoiding podocyte injury is the key to prevent the occurrence of DN [8].
Baicalin (5,6,7-trihydroxyflavone) is a flavonoid extracted from scutellaria baicalensis georgi [9]. It has a wide range of biological functions, including antibacterial, antiviral, and anti-tumor functions [10,11]. Cheng showed that baicalin reduced lipopolysaccharide-induced liver inflammation in chicken by suppressing TLR4-mediated NF-κB signaling pathway [12]. Meanwhile, baicalin was found to improve renal function in DN patients by inhibiting excessive inflammation and oxidative stress, and thereby effectively delaying the development of DN [13]. Baicalin is also used in clinical applications. Haixia [14] found that baicalin could reduce proteinuria in patients with early DN. Yang et al. [15] also found that baicalin can improve the renal function of patients with DN and delay the progression of DN through various pathways such as anti-inflammation and antioxidation. However, at present, the effect of baicalin on podocytes in DM-induced DN tissues and its mechanism has not been reported. Therefore, this study investigated DN rats and podocytes to provide a certain theoretical and experimental basis for subsequent research on the treatment of DM-induced DN.
2 Materials and methods
2.1 Establishment and grouping of DN rat models
A total of 30 Sprague Dawley (SD) male rats aged 8 weeks and weighing 180−200 g were selected. After 1 week of adaptive culture, the rats were randomly divided into five groups (6 rats/group). Subsequently, the DN rat models were established by intraperitoneal injection of 35 mg/kg streptozotocin (STZ) after 4 weeks of high-fat and high-sugar diet. After 72 h, the rats with fasting blood glucose (FBG) higher than 13.9 mmol/L and random blood glucose higher than 16.7 mmol/L were considered as diabetic rats. After successful modeling, the diabetic rats were divided into 4 test groups: DN group, baicalin low-concentration group (Baicalin-L), baicalin medium-concentration group (Baicalin-M), and baicalin high-concentration group (Baicalin-H). In these test groups, same amount of saline, 50 mg/kg baicalin, 100 mg/kg baicalin, and 200 mg/kg baicalin were administered, respectively, once daily for 4 weeks by gavage [16]. Meanwhile, the rats with a normal diet were intraperitoneally injected with the same amount of sodium citrate buffer for 4 weeks as the control group. After 4 weeks, 24 h urine and peripheral blood were collected, followed by anesthesia and cervical dislocation to sacrifice the rats and then their renal tissues were collected.
Ethical approval: This trial was approved by the ethics committee of Shenzhen Fuyong People’s Hospital.
2.2 Cell culture and grouping
SD male rats aged 8 weeks were injected intraperitoneally with 10% chloral hydrate, and the rats were anesthetized and then sacrificed by cervical dislocation. Their kidneys were collected under aseptic conditions, soaked and rinsed with pre-cooled sterile PBS solution. After the renal capsule was removed, the kidneys were cut into small tissue masses (1 mm3) using ophthalmic scissors and then collected in centrifuge tubes to digest at 150 rpm with 0.1% of collagenase type IV at 37°C. RPMI-1640 complete culture medium was added 25 min later to terminate digestion. Subsequently, the cell suspension passed through the 100, 150, and 200 mesh sieves in turn. Next the cell suspension was collected into centrifuge tube, centrifuged at 1,000 rpm at 4°C for 5 min, resuspended with complete medium, and inoculated in culture flasks to culture in an incubator (Thermo, USA) with 5% of CO2 at 37°C. Finally,in vitro podocytes of the rats were obtained. The podocytes as the Test groups were cultured with RPMI-1640 complete medium containing 30 mmol/L of glucose and then treated with 10% of serum from the rats in each test group, named as high glucose (HG) group, Baicalin-L group, Baicalin-M group, and Baicalin-H group. Meanwhile, the other part of the podocytes cultured in RPMI-1640 medium were treated with 10% of serum from the rats in the control group, named as the Control group.
2.3 Measurement of FBG and weight
FBG was measured in the control, DN, Baicalin-L, Baicalin-M, and Baicalin-H groups at 0, 1, 2, 3, and 4 weeks, respectively. Then 5 μL of blood collected from the caudal vein was dropped onto glucose test strips and FBG was measured by a blood glucose meter (Accu-Check Active, Roche). Their weights were also measured and recorded.
2.4 Detection of urine biochemical indicators
The rats in each group were fed in the promethion cages (Sable Systems International, USA), and 24 h urine was collected 4 weeks later. After the total amount of urine was determined, 3 mL of urine was centrifuged at 3,000 rpm for 10 min at 4°C, and the supernatant was taken. The 24 h microalbumin (mALB) in urine was detected using an automatic biochemical analyzer (Olympus, Japan) to calculate the urinary albumin/creatinine ratio (ACR).
2.5 Detection of blood biochemical indicators and inflammatory factors
The peripheral blood was collected from the orbital venous plexus of the rats and centrifuged at 3,000 rpm for 20 min. Then, the serum from the upper layer was collected and placed into new centrifuge tubes. The serum creatinine (SCr), blood urea nitrogen (BUN), total cholesterol (TC), and triacylglycerol (TG) in the serum of the rats were measured using the automatic biochemical analyzer.
In the meanwhile, the serum or podocyte supernatant of rats from each group was detected by the expressions of TNF-α, IL-1β, and IL-6 using ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), which were operated in strict accordance with the experimental instructions.
2.6 Detection of oxidative damage index
Fifty milligram of kidney tissues from rats in each group were fully ground and centrifuged at 9,000 rpm for 10 min at 4°C. The supernatant was isolated to detect the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) in kidney tissues of rats from each group using LDH, SOD, and MDA assay kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions.
2.7 Hematoxylin–eosin (HE) staining and periodic acid-schiff (PAS) staining
The collected renal tissues were rinsed with PBS buffer. After the renal capsule was removed, the right renal tissues were fixed with 10% of neutral formaldehyde and embedded in paraffin for sectioning. The sections were stained with hematoxylin at room temperature for 5 min, followed by differentiation with hydrochloric acid alcohol. The sections appearing as blue color was washed using 1% (volume/volume%) of ammonia and then were washed in running tap water before being stained with eosin for 30 s. Finally, the sections were dehydrated with alcohol, cleared in xylene, and covered with slips. The pathological changes in the kidneys were observed under a biomicroscope.
The prepared renal tissue sections were deparaffinized at 65°C, washed with distilled water, and oxidized in 1% of periodic acid solution for 5–10 min. The sections were rinsed with distilled water again and stained with Schiff reagent for 10 min. Subsequently, the tissue sections were rinsed with sodium bisulfite solution for 3 times (2 min/time) and incubated with hematoxylin solution for 3 min. Finally, the sections were dehydrated with alcohol, treated with xylene, and covered with slips. The pathological changes in the kidneys were observed under the biomicroscope. Semiquantitative measurement of the changes was performed using Image J software according to the previous study [17].
2.8 MTT assay
After treatment, podocytes in the logarithmic growth phase were seeded in 96-well plates at a density of 500 cells/well and cultured for 24, 48, and 72 h, respectively. Twenty microliter of 5 mg/mL of MTT solution was added to each group of cells and continued culturing in the incubator for another 4 h. The culture supernatant in the wells was pipetted and discarded. Afterwards, 150 μL of DMSO was added and shaken for 15 min. The absorbance of each well was measured at a wavelength of 490 nm by using the enzyme mark instrument.
2.9 Flow cytometry (FCM)
The cells from each group were digested into a centrifuge tube by utilizing trypsin. After that, they were rinsed twice with pre-cooled sterile PBS and then adjusted the cell concentration to 5 × 105 cells/mL. Two hundred microliter of cell suspension was extracted in order to add 10 μL of Annexin V-FITC followed by 10 μL of PI solution at the concentration of 20 mg/L. Finally, it was incubated in the dark at room temperature for 10 min. After adding 500 μL of PBS, apoptosis was detected by FCM.
2.10 qRT-PCR
Total RNA was extracted from renal tissues or cells using Trizol (Invitrogen) and the RNA concentration was measured by spectrophotometer. The total RNA was reverse transcribed into cDNA using MLV reverse transcriptase. qPCR was performed in the detection system of SYBR Green Master Mix (Promega, USA). GAPDH was used as an internal reference and the results were calculated using the 2−△△Ct method. The primers used are shown inTable 1.
Primer sequences
| RNA | Sequences(5′ to 3′) |
|---|---|
| Nephrin | F: 5′-GCATAGCCAGAGGTGGAAATCC |
| R: 5′-GAACGGTCATCACCAGCACACT | |
| Podocin | F: 5′-GTGGAAGCTGAGGCACAAAGAC |
| R: 5′-CAGCGACTGAAGAGTGTGCAAG | |
| Desmin | F: 5′-GCGGCTAAGAACATCTCTGAGG |
| R: 5′-ATCTCGCAGGTGTAGGACTGGA | |
| GAPDH | F: 5′-CATCACTGCCACCCAGAAGACTG |
| R: 5′-ATGCCAGTGAGCTTCCCGTTCAG |
2.11 Western blot
RIPA lysis buffer was added to 50 mg of renal tissues and homogenized and then centrifuged at 12,000 rpm for 30 min at 4°C; the supernatant was taken as total protein. Meanwhile, RIPA lysis buffer was added to the podocytes and then centrifuged at 12,000 rpm for 30 min at 4°C; the supernatant was taken as total protein. The protein concentrations were determined using the BCA protein quantification kit (Thermo Fisher). In immunoblotting, 20 µg of proteins were separated using 10% of SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Following the blocking step with 5% of skim milk for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies: p-PI3K (Abcam UK), PI3K (Abcam UK), p-Akt (Abcam UK), Akt (Abcam UK), p-mTOR (Abcam UK), and mTOR (Abcam UK). Subsequently, they were incubated with the corresponding secondary antibodies for 1 h at room temperature. GAPDH was used as an internal reference when scanning immunoblot bands at gray scale. The gray values of the bands were analyzed using Image LabTM Software.
2.12 Statistical analysis
SPSS 24.0 was used for one-way analysis of variance (ANOVA) and independent samplet-test. The results were expressed as mean values ± standard deviation, andp < 0.05 indicated significant differences.
3 Results
3.1 Effects of baicalin on FBG and weight in DN rats
The blood glucose of rats in each group was measured by the blood glucose meter. The results showed that the level of FBG in the control group was in the normal range, while that in the DN group and Baicalin treatment groups were always at high levels. In the DN group, FBG level was higher than 16.7 mmol/L all the time. In the Baicalin treatment groups, FBG level showed a downward trend with the increase in baicalin treatment time. At the 4th week, compared with the DN group, the FBG of rats was significantly decreased in the Baicalin treatment groups (Figure 1a,p < 0.05), and in a concentration-dependent manner. The FBG in the Baicalin-H group was closest to that in the control group. At the 0th week, the weight of rats in the test groups was significantly higher than that in the control group. Compared with the DN group, the weights in the Baicalin treatment groups were gradually decreased (Figure 1b). These results indicated that baicalin has an inhibitory effect on blood glucose and weight in DN rats.
Effects of baicalin on FBG and weight in DN rats. The DN rats were administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage. The FBG (a) and weight (b) of the rats in each group are measured weekly.#p < 0.05 vs control group; *p < 0.05 and **p < 0.01 vs DN group.
3.2 Effects of baicalin on serum and urine biochemical indicators in DN rats
The changes in serum and urine biochemical indicator in DN rats before and after baicalin treatment were observed. The results of the automatic biochemical analyzer revealed that compared with the control group, the levels of SCr, BUN, TC, and TG in the serum in the DN group were significantly increased (Figure 2a–d,p < 0.05), while the 24 h mALB and ACR in the urine were significantly increased (Figure 2e and f,p < 0.05). Compared with the DN group, the levels of SCr, BUN, TC, and TG in the Baicalin treatment groups were significantly decreased (Figure 2a–d,p < 0.05) in a concentration-dependent manner; the higher the baicalin concentration, the lower the levels of SCr, BUN, TC, and TG. Meanwhile, compared with the DN group, the 24 h mALB and ACR in urine in the Baicalin treatment groups were significantly decreased (Figure 2e–f,p < 0.05), also in a concentration-dependent manner.
Effects of baicalin on serum and urine biochemical indicators in DN rats. The DN rats are administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage. (a) SCr levels in the serum of rats in each group; (b) BUN levels in the serum of rats in each group; (c) TC levels in the serum of rats in each group; (d) TG levels in the serum of rats in each group; (e) mALB levels in the urine of rats in each group; (f) ACR levels in the urine of rats in each group.##p < 0.01 vs control group; *p < 0.05 and **p < 0.01 vs DN group.
3.3 Effects of baicalin on serum inflammatory factors and renal oxidative stress factors in DN rats
The ELISA results showed (Figure 3a) that the expressions of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum of rats from the DN group were significantly increased compared with that from the control group. Fortunately, baicalin could inhibit the expressions of TNF-α, IL-1β, and IL-6 in the serum of DN rats. Furthermore, the lower the concentration of baicalin, the lower the expressions of TNF-α, IL-1β, and IL-6.
Effects of baicalin on serum inflammatory factors and renal oxidative stress factors in DN rats. The DN rats are administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage. (a) the expression of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum of rats in each group; (b) the activities of LDH, SOD, and MDA in the renal tissue of rats in each group;##p < 0.01 vs Control group; *p < 0.05, **p < 0.01, and ***p < 0.001 vs DN group.
Oxidative stress is closely related to DN incidence [18].Figure 3b indicated that compared with the control group, the activities of LDH and MDA in the renal tissue from the DN group were significantly increased, while the activity of SOD was significantly decreased. Adding to that, compared with the DN group, the activities of LDH and MDA from different concentrations of baicalin group were markedly decreased, while the activities of SOD were significantly increased, and all of which were baicalin concentration-dependent.
3.4 Effects of baicalin on renal pathomorphology in DN Rats
The pathological morphological changes in the renal tissues after baicalin treatment in DN rats were examined using histopathology. The results of HE staining showed no obvious pathological changes in the renal tissues in the control group; the glomerular structure was intact, the renal tubules were arranged neatly and uniform in size, and there was no inflammatory cell infiltration. Compared with the control group, the renal tissues in the DN group were significantly damaged; the glomeruli were atrophic, the glomerular mesangial matrix was increased, the renal tubules showed vacuolar degeneration, and a large number of inflammatory cell infiltration were observed. After baicalin treatment, renal pathological injury was improved in different degrees, and the higher the baicalin concentration, the more significant the improvement (Figure 4a). The results of PAS staining showed no significant pathological changes in the renal tissues in the control group; the basement membrane was intact, and the glomerular vascular loops were thin and clear. Compared with the control group, the basement membrane in the DN group was significantly thickened. After baicalin treatment, the thickening of GBM was improved, and the higher the baicalin concentration, the more significant the improvement (Figure 4b). These results confirmed that baicalin improved renal tissue injury in DN rats in a concentration-dependent manner.
Effects of baicalin on renal pathological morphology in DN rats. The DN rats are administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage, and their renal tissues are collected. (a) HE staining images of renal tissues of rats in each group; (b) PAS staining images of renal tissues of rats in each group.##p < 0.01 vs Control group; **p < 0.01 vs DN group.
3.5 Effects of baicalin on the expressions of podocyte-related molecules and PI3K/Akt/mTOR signaling pathway in the renal tissues of rats
To investigate the molecular mechanism by which baicalin improved renal tissue injury in DN rats, the expressions of podocyte-related molecules (Nephrin, Podocin, and Desmin) and PI3K/Akt/mTOR signaling pathway proteins were detected. Compared with the control group, the mRNA expressions of Nephrin and Podocin were significantly decreased, while that of Desmin significantly increased in the renal tissues in the DN group (Figure 5a,p < 0.05). Compared with the DN group, the expressions of Nephrin and Podocin were significantly increased, while that of Desmin significantly decreased in the renal tissues in the baicalin treatment groups (Figure 5a,p < 0.05) in a concentration-dependent manner. In terms of protein, compared with the control group, the expressions of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR were significantly increased in the renal tissues in the DN group (Figure 5b,p < 0.05). Compared with the DN group, the expressions of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR in the baicalin treatment groups were significantly decreased (Figure 5b,p < 0.05) in a concentration-dependent manner.
Effects of baicalin on the expressions of podocyte-related molecules and PI3K/Akt/mTOR signaling pathway in renal tissues of rats. The DN rats are administered with 50 mg/kg (Baicalin-L), 100 mg/kg (Baicalin-M), and 200 mg/kg (Baicalin-H) baicalin for 4 weeks by gavage, and their renal tissues are collected. (a) qRT-PCR to detect the mRNA expressions of Nephrin, Podocin, and Desmin in the renal tissues of rats in each group; (b) Western blot to detect the expressions of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR proteins in the renal tissues of rats in each group.##p < 0.01 vs Control group; *p < 0.05 and **p < 0.01 vs DN group.
The results further confirmed that baicalin ameliorates renal injury in DN rats by inhibiting PI3K/Akt/mTOR. Besides, after the PI3K/Akt signaling pathway was activated via 740Y-P, the results showed that (Figure 6a and b) the expressions of Nephrin and Podocin in the renal tissue of rats from the DN group were inhibited, but the protein expression of Desmin, like p-PI3K, p-Akt, and p-mTOR, was promoted. Expressions of these proteins in rat kidney tissue were reversed upon baicalin treatment. In short, the results confirmed that baicalin could ameliorate renal injury by inhibiting the activation of PI3K/Akt/mTOR signaling pathway in DN rats.
Baicalin ameliorates renal injury by inhibiting the activation of PI3K/Akt/mTOR signaling pathway in DN rats. 30 SD rats are randomly divided into 5 groups (6 rats in each group) which are control group, DN group, 740Y-P group (3.5 mg/kg PI3K activator), Baicalin group (200 mg/kg baicalin), Baicalin + 740Y-P group (200 mg/kg baicalin + 3.5 mg/kg 740Y-PA), with 4 weeks of gavage. (a) qRT-PCR to detect the mRNA expressions of Nephrin, Podocin, and Desmin in the kidney tissues of rats in each group; (b) Western blot to detect the protein expressions of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR in the kidney tissues of rats in each group. *p < 0.05 vs DN group;&p < 0.05 vs Baicalin group; and#p < 0.05 vs DN + 740Y-P group.
3.6 Effect of baicalin on podocyte injury induced by HG
In order to further confirm the results ofin vitro trails, primary culture of rat podocytes was carried out and podocyte models of HG were induced using 30 mmol/L of glucose and then the podocytes were treated with 10% of serum from the rats in each test group. The results ofFigure 7a–c show that compared with the control group, the viability of podocytes in the HG group was significantly decreased with an increase in the rate of apoptosis. However, the expressions of inflammatory factors such as TNF-α, IL-1β, and IL-6 were significantly upregulated. Baicalin, on the one hand, could promote the proliferation of podocytes. On the other hand, it could inhibit apoptosis and the expression of TNF-α, IL-1β, and IL-6.
Effect of baicalin on podocyte injury induced by HG. Primary culture of rat podocytes was carried out and podocyte models of HG were induced using 30 mmol/L of glucose and then the podocytes were treated with 10% of serum from the rats in each test group. (a) MTT assay for podocyte viability; (b) flow assay for podocyte apoptosis; (c) ELISA for the expression of inflammatory factors TNF-α, IL-1β, and IL-6.##p < 0.01 vs control group; *p < 0.05, **p < 0.01, and ***p < 0.001 vs HG group.
3.7 Effect of baicalin on PI3K/Akt/mTOR signaling pathway after podocyte injury induced by HG
Compared with the Control group, the mRNA expressions of Nephrin and Podocin were significantly decreased, while that of Desmin significantly increased in the HG group (Figure 8a,p < 0.05). Compared with the HG group, the mRNA expressions of Nephrin and Podocin were significantly upregulated, while that of Desmin was significantly downregulated in the Baicalin treatment groups (Figure 8a,p < 0.05) in a concentration-dependent manner. In terms of protein, compared with the control group, the expressions of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR were significantly increased in the HG group (Figure 8b,p < 0.05). Compared with the HG group, the expressions of p-PI3K/PI3K, p-Akt/AKT, and p-mTOR/mTOR proteins were significantly decreased in the Baicalin treatment groups (Figure 8b,p < 0.05) in a concentration-dependent manner. These results of trailsin vitro andin vivo were similar.
In vitro trails to detect effects of baicalin on rat podocytes. Primary culture of rat podocytes is carried out and the podocyte models of HG are induced using 30 mmol/L of glucose and then the podocytes are treated with 10% of serum from the rats in each test group. (a) qRT-PCR to detect the mRNA levels of Nephrin, Podocin, and Desmin in podocytes of each group; (b) Western blot to detect the expressions of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR proteins in podocytes of each group.##p < 0.01 vs Control group; *p < 0.05 and **p < 0.01 vs DN group.
4 Discussion
Current treatments for DN patients include diet control, exercise enhancement, weight control, and drug therapy. The first three are known as non-drug therapies, in which weight control can effectively control diabetes and slow down the development of DN [19]. In addition, drug therapy is to control blood glucose in DN patients. Many studies have shown that blood glucose controlled within the normal range is the basis for the treatment of DN [20], while poor blood glucose control induces DN development as well as other serious microvascular complications [21]. In addition, the control of urinary protein is also the key to the treatment of DN [22]. In this study, diabetic rat models with DN were established by high-fat and high-sugar diet and STZ injection. In the test groups, DN rats had significantly increased FBG and weight, significant proteinuria (upregulation of 24 h mALB and urinary ACR), increased SCr, BUN, TC, and TG in serum, and significant changes in podocyte-related molecules (downregulation of Nephrin and Podocin expressions and upregulation of Desmin expression). Many studies have pointed out that baicalin can reduce diabetic kidney injury. Zhang et al. [23] showed that baicalin significantly inhibited DM-induced fibrosis of proximal renal tubular epithelial cells. Zheng et al. [24] demonstrated that baicalin–lysin conjugate ameliorated renal fibrosis in STZ-induced DN rats. Li et al. revealed that baicalin could prevent the apoptosis of podocytes due to HG, thereby delaying the development of DN [25]. In this study, during the treatment of DN rats with baicalin, FBG and body weight of rats treated with different concentrations of baicalin showed a downward trend, and longer treatment time and higher concentration cause more significant decrease in FBG and weight in rats. Meanwhile, baicalin significantly inhibited the levels of SCr, BUN, TC, and TG in the blood and urinary mALB and ACR. Furthermore, by observing the pathological sections of the kidneys, it can be seen that baicalin improved GBM thickening and glomerular atrophy caused by hyperglycemia. Baicalin could also significantly upregulate the expressions of Nephrin and Podocin, but downregulate the expression of Desmin. Some studies have pointed out that upregulation of Nephrin and Podocin and downregulation of Desmin are beneficial to protect the morphology, structure, and function of podocytes, and to inhibit the epithelial-mesenchymal transition of the podocytes [26]. Therefore, the result indicated that baicalin maintained the homeostasis of podocyte-related functional proteins to protect the normal functions of podocytes. All these studies confirmed that baicalin could improve renal injury in DN rats.
An increasing number of studies have shown that hyperglycemia induced oxidative stress or inflammatory factors, leading to podocyte detachment from GBM and apoptosis, which is the major factor in the early development of DN [18]. In this study, we found that the expressions of inflammatory factors, namely, TNF-α, IL-1β, and IL-6 were all upregulated in the serum of rats in the DN group. Furthermore, the activities of LDH and MDA were increased, but the activity of SOD was decreased in the renal tissue. At the same time, HG-induced podocyte viability was reduced, but apoptotic ability was enhanced, along with upregulation of TNF-α, IL-1β, and IL-6 expressions. Baicalin, on the other hand, could reverse these changes. These results confirmed that baicalin could ameliorate the damage of renal podocytes in DN rats. DM will cause hemodynamic changes, leading to the abnormal activation of PI3K/Akt/mTOR pathway. This abnormal activation induces autophagy of podocytes, thus inhibiting the adhesive ability of podocytes and resulting in detachment of podocytes from GBM [27]. Many studies have explored new therapeutic ideas for DN based on the effect mention above. Wu et al. [28] found that Huangkui capsule alleviated glomerular pathological changes in the early stage of DN by inhibiting the activity of Akt/mTOR signaling pathway. Huang et al. [29] revealed that Notoginsenoside R1 could inhibit autophagy and apoptosis of podocytes to reduce glucose-induced podocyte injury through inhibiting the activity of the PI3K/Akt/mTOR signaling pathway. Wu et al. [30] also found that curcumin inhibited the autophagic transformation of podocytes through suppressing the activity of this signaling pathway, thus reducing the damage caused by DN to the body. Therefore, it is speculated that baicalin also has a benign effect on the development of DN by regulating this signaling pathway. From our results of Western blot, baicalin significantly inhibited the expression of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR in bothin vivo andin vitro experiments. So, we believed the possibility that baicalin protected podocytes by downregulating the activity of the PI3K/Akt/mTOR signaling pathway.
5 Conclusion
In summary, baicalin can not only reduce the increase in FBG, body weight, and biochemical indicator in serum and urine due to DM, but also slow down the damage of podocytes caused by hyperglycemia by inhibiting the activity of PI3K/Akt/mTOR signaling pathway, thereby delaying the progression of DM-induced DN. The results of this trial provide effective data for the clinical application of baicalin in the treatment of DN.
Funding information: Not applicable.
Author contributions: Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work: All authors. Drafting the work or revising it critically for important intellectual content: YO. Final approval of the version to be published: All authors. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: All authors.
Conflict of interest: The authors have no conflicts of interest to declare.
Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
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- Disparity in clinical characteristics between 2019 novel coronavirus pneumonia and leptospirosis
- Use of microspheres in embolization for unruptured renal angiomyolipomas
- COVID-19 cases with delayed absorption of lung lesion
- A triple combination of treatments on moderate COVID-19
- Social networks and eating disorders during the Covid-19 pandemic
- Letter
- COVID-19, WHO guidelines, pedagogy, and respite
- Inflammatory factors in alveolar lavage fluid from severe COVID-19 pneumonia: PCT and IL-6 in epithelial lining fluid
- COVID-19: Lessons from Norway tragedy must be considered in vaccine rollout planning in least developed/developing countries
- What is the role of plasma cell in the lamina propria of terminal ileum in Good’s syndrome patient?
- Case Report
- Rivaroxaban triggered multifocal intratumoral hemorrhage of the cabozantinib-treated diffuse brain metastases: A case report and review of literature
- CTU findings of duplex kidney in kidney: A rare duplicated renal malformation
- Synchronous primary malignancy of colon cancer and mantle cell lymphoma: A case report
- Sonazoid-enhanced ultrasonography and pathologic characters of CD68 positive cell in primary hepatic perivascular epithelioid cell tumors: A case report and literature review
- Persistent SARS-CoV-2-positive over 4 months in a COVID-19 patient with CHB
- Pulmonary parenchymal involvement caused byTropheryma whipplei
- Mediastinal mixed germ cell tumor: A case report and literature review
- Ovarian female adnexal tumor of probable Wolffian origin – Case report
- Rare paratesticular aggressive angiomyxoma mimicking an epididymal tumor in an 82-year-old man: Case report
- Perimenopausal giant hydatidiform mole complicated with preeclampsia and hyperthyroidism: A case report and literature review
- Primary orbital ganglioneuroblastoma: A case report
- Primary aortic intimal sarcoma masquerading as intramural hematoma
- Sustained false-positive results for hepatitis A virus immunoglobulin M: A case report and literature review
- Peritoneal loose body presenting as a hepatic mass: A case report and review of the literature
- Chondroblastoma of mandibular condyle: Case report and literature review
- Trauma-induced complete pacemaker lead fracture 8 months prior to hospitalization: A case report
- Primary intradural extramedullary extraosseous Ewing’s sarcoma/peripheral primitive neuroectodermal tumor (PIEES/PNET) of the thoracolumbar spine: A case report and literature review
- Computer-assisted preoperative planning of reduction of and osteosynthesis of scapular fracture: A case report
- High quality of 58-month life in lung cancer patient with brain metastases sequentially treated with gefitinib and osimertinib
- Rapid response of locally advanced oral squamous cell carcinoma to apatinib: A case report
- Retrieval of intrarenal coiled and ruptured guidewire by retrograde intrarenal surgery: A case report and literature review
- Usage of intermingled skin allografts and autografts in a senior patient with major burn injury
- Retraction
- Retraction on “Dihydromyricetin attenuates inflammation through TLR4/NF-kappa B pathway”
- Special Issue Computational Intelligence Methodologies Meets Recurrent Cancers - Part I
- An artificial immune system with bootstrap sampling for the diagnosis of recurrent endometrial cancers
- Breast cancer recurrence prediction with ensemble methods and cost-sensitive learning
Articles in the same Issue
- Research Articles
- Identification of ZG16B as a prognostic biomarker in breast cancer
- Behçet’s disease with latentMycobacterium tuberculosis infection
- Erratum
- Erratum to “Suffering from Cerebral Small Vessel Disease with and without Metabolic Syndrome”
- Research Articles
- GPR37 promotes the malignancy of lung adenocarcinoma via TGF-β/Smad pathway
- Expression and role of ABIN1 in sepsis:In vitro andin vivo studies
- Additional baricitinib loading dose improves clinical outcome in COVID-19
- The co-treatment of rosuvastatin with dapagliflozin synergistically inhibited apoptosis via activating the PI3K/AKt/mTOR signaling pathway in myocardial ischemia/reperfusion injury rats
- SLC12A8 plays a key role in bladder cancer progression and EMT
- LncRNA ATXN8OS enhances tamoxifen resistance in breast cancer
- Case Report
- Serratia marcescens as a cause of unfavorable outcome in the twin pregnancy
- Spleno-adrenal fusion mimicking an adrenal metastasis of a renal cell carcinoma: A case report and embryological background
- Research Articles
- TRIM25 contributes to the malignancy of acute myeloid leukemia and is negatively regulated by microRNA-137
- CircRNA circ_0004370 promotes cell proliferation, migration, and invasion and inhibits cell apoptosis of esophageal cancer via miR-1301-3p/COL1A1 axis
- LncRNA XIST regulates atherosclerosis progression in ox-LDL-induced HUVECs
- Potential role of IFN-γ and IL-5 in sepsis prediction of preterm neonates
- Rapid Communication
- COVID-19 vaccine: Call for employees in international transportation industries and international travelers as the first priority in global distribution
- Case Report
- Rare squamous cell carcinoma of the kidney with concurrent xanthogranulomatous pyelonephritis: A case report and review of the literature
- An infertile female delivered a baby after removal of primary renal carcinoid tumor
- Research Articles
- Hypertension, BMI, and cardiovascular and cerebrovascular diseases
- Case Report
- Coexistence of bilateral macular edema and pale optic disc in the patient with Cohen syndrome
- Research Articles
- Correlation between kinematic sagittal parameters of the cervical lordosis or head posture and disc degeneration in patients with posterior neck pain
- Review Articles
- Hepatoid adenocarcinoma of the lung: An analysis of the Surveillance, Epidemiology, and End Results (SEER) database
- Research Articles
- Thermography in the diagnosis of carpal tunnel syndrome
- Pemetrexed-based first-line chemotherapy had particularly prominent objective response rate for advanced NSCLC: A network meta-analysis
- Comparison of single and double autologous stem cell transplantation in multiple myeloma patients
- The influence of smoking in minimally invasive spinal fusion surgery
- Impact of body mass index on left atrial dimension in HOCM patients
- Expression and clinical significance of CMTM1 in hepatocellular carcinoma
- miR-142-5p promotes cervical cancer progression by targeting LMX1A through Wnt/β-catenin pathway
- Comparison of multiple flatfoot indicators in 5–8-year-old children
- Early MRI imaging and follow-up study in cerebral amyloid angiopathy
- Intestinal fatty acid-binding protein as a biomarker for the diagnosis of strangulated intestinal obstruction: A meta-analysis
- miR-128-3p inhibits apoptosis and inflammation in LPS-induced sepsis by targeting TGFBR2
- Dynamic perfusion CT – A promising tool to diagnose pancreatic ductal adenocarcinoma
- Biomechanical evaluation of self-cinching stitch techniques in rotator cuff repair: The single-loop and double-loop knot stitches
- Review Articles
- The ambiguous role of mannose-binding lectin (MBL) in human immunity
- Case Report
- Membranous nephropathy with pulmonary cryptococcosis with improved 1-year follow-up results: A case report
- Fertility problems in males carrying an inversion of chromosome 10
- Acute myeloid leukemia with leukemic pleural effusion and high levels of pleural adenosine deaminase: A case report and review of literature
- Metastatic renal Ewing’s sarcoma in adult woman: Case report and review of the literature
- Burkitt-like lymphoma with 11q aberration in a patient with AIDS and a patient without AIDS: Two cases reports and literature review
- Skull hemophilia pseudotumor: A case report
- Judicious use of low-dosage corticosteroids for non-severe COVID-19: A case report
- Adult-onset citrullinaemia type II with liver cirrhosis: A rare cause of hyperammonaemia
- Clinicopathologic features of Good’s syndrome: Two cases and literature review
- Fatal immune-related hepatitis with intrahepatic cholestasis and pneumonia associated with camrelizumab: A case report and literature review
- Research Articles
- Effects of hydroxyethyl starch and gelatin on the risk of acute kidney injury following orthotopic liver transplantation: A multicenter retrospective comparative clinical study
- Significance of nucleic acid positive anal swab in COVID-19 patients
- circAPLP2 promotes colorectal cancer progression by upregulating HELLS by targeting miR-335-5p
- Ratios between circulating myeloid cells and lymphocytes are associated with mortality in severe COVID-19 patients
- Risk factors of left atrial appendage thrombus in patients with non-valvular atrial fibrillation
- Clinical features of hypertensive patients with COVID-19 compared with a normotensive group: Single-center experience in China
- Surgical myocardial revascularization outcomes in Kawasaki disease: systematic review and meta-analysis
- Decreased chromobox homologue 7 expression is associated with epithelial–mesenchymal transition and poor prognosis in cervical cancer
- FGF16 regulated by miR-520b enhances the cell proliferation of lung cancer
- Platelet-rich fibrin: Basics of biological actions and protocol modifications
- Accurate diagnosis of prostate cancer using logistic regression
- miR-377 inhibition enhances the survival of trophoblast cells via upregulation of FNDC5 in gestational diabetes mellitus
- Prognostic significance of TRIM28 expression in patients with breast carcinoma
- Integrative bioinformatics analysis of KPNA2 in six major human cancers
- Exosomal-mediated transfer of OIP5-AS1 enhanced cell chemoresistance to trastuzumab in breast cancer via up-regulating HMGB3 by sponging miR-381-3p
- A four-lncRNA signature for predicting prognosis of recurrence patients with gastric cancer
- Knockdown of circ_0003204 alleviates oxidative low-density lipoprotein-induced human umbilical vein endothelial cells injury: Circulating RNAs could explain atherosclerosis disease progression
- Propofol postpones colorectal cancer development through circ_0026344/miR-645/Akt/mTOR signal pathway
- Knockdown of lncRNA TapSAKI alleviates LPS-induced injury in HK-2 cells through the miR-205/IRF3 pathway
- COVID-19 severity in relation to sociodemographics and vitamin D use
- Clinical analysis of 11 cases of nocardiosis
- Cis-regulatory elements in conserved non-coding sequences of nuclear receptor genes indicate for crosstalk between endocrine systems
- Four long noncoding RNAs act as biomarkers in lung adenocarcinoma
- Real-world evidence of cytomegalovirus reactivation in non-Hodgkin lymphomas treated with bendamustine-containing regimens
- Relation between IL-8 level and obstructive sleep apnea syndrome
- circAGFG1 sponges miR-28-5p to promote non-small-cell lung cancer progression through modulating HIF-1α level
- Nomogram prediction model for renal anaemia in IgA nephropathy patients
- Effect of antibiotic use on the efficacy of nivolumab in the treatment of advanced/metastatic non-small cell lung cancer: A meta-analysis
- NDRG2 inhibition facilitates angiogenesis of hepatocellular carcinoma
- A nomogram for predicting metabolic steatohepatitis: The combination of NAMPT, RALGDS, GADD45B, FOSL2, RTP3, and RASD1
- Clinical and prognostic features of MMP-2 and VEGF in AEG patients
- The value of miR-510 in the prognosis and development of colon cancer
- Functional implications of PABPC1 in the development of ovarian cancer
- Prognostic value of preoperative inflammation-based predictors in patients with bladder carcinoma after radical cystectomy
- Sublingual immunotherapy increases Treg/Th17 ratio in allergic rhinitis
- Prediction of improvement after anterior cruciate ligament reconstruction
- Effluent Osteopontin levels reflect the peritoneal solute transport rate
- circ_0038467 promotes PM2.5-induced bronchial epithelial cell dysfunction
- Significance of miR-141 and miR-340 in cervical squamous cell carcinoma
- Association between hair cortisol concentration and metabolic syndrome
- Microvessel density as a prognostic indicator of prostate cancer: A systematic review and meta-analysis
- Characteristics of BCR–ABL gene variants in patients of chronic myeloid leukemia
- Knee alterations in rheumatoid arthritis: Comparison of US and MRI
- Long non-coding RNA TUG1 aggravates cerebral ischemia and reperfusion injury by sponging miR-493-3p/miR-410-3p
- lncRNA MALAT1 regulated ATAD2 to facilitate retinoblastoma progression via miR-655-3p
- Development and validation of a nomogram for predicting severity in patients with hemorrhagic fever with renal syndrome: A retrospective study
- Analysis of COVID-19 outbreak origin in China in 2019 using differentiation method for unusual epidemiological events
- Laparoscopic versus open major liver resection for hepatocellular carcinoma: A case-matched analysis of short- and long-term outcomes
- Travelers’ vaccines and their adverse events in Nara, Japan
- Association between Tfh and PGA in children with Henoch–Schönlein purpura
- Can exchange transfusion be replaced by double-LED phototherapy?
- circ_0005962 functions as an oncogene to aggravate NSCLC progression
- Circular RNA VANGL1 knockdown suppressed viability, promoted apoptosis, and increased doxorubicin sensitivity through targeting miR-145-5p to regulate SOX4 in bladder cancer cells
- Serum intact fibroblast growth factor 23 in healthy paediatric population
- Algorithm of rational approach to reconstruction in Fournier’s disease
- A meta-analysis of exosome in the treatment of spinal cord injury
- Src-1 and SP2 promote the proliferation and epithelial–mesenchymal transition of nasopharyngeal carcinoma
- Dexmedetomidine may decrease the bupivacaine toxicity to heart
- Hypoxia stimulates the migration and invasion of osteosarcoma via up-regulating the NUSAP1 expression
- Long noncoding RNA XIST knockdown relieves the injury of microglia cells after spinal cord injury by sponging miR-219-5p
- External fixation via the anterior inferior iliac spine for proximal femoral fractures in young patients
- miR-128-3p reduced acute lung injury induced by sepsis via targeting PEL12
- HAGLR promotes neuron differentiation through the miR-130a-3p-MeCP2 axis
- Phosphoglycerate mutase 2 is elevated in serum of patients with heart failure and correlates with the disease severity and patient’s prognosis
- Cell population data in identifying active tuberculosis and community-acquired pneumonia
- Prognostic value of microRNA-4521 in non-small cell lung cancer and its regulatory effect on tumor progression
- Mean platelet volume and red blood cell distribution width is associated with prognosis in premature neonates with sepsis
- 3D-printed porous scaffold promotes osteogenic differentiation of hADMSCs
- Association of gene polymorphisms with women urinary incontinence
- Influence of COVID-19 pandemic on stress levels of urologic patients
- miR-496 inhibits proliferation via LYN and AKT pathway in gastric cancer
- miR-519d downregulates LEP expression to inhibit preeclampsia development
- Comparison of single- and triple-port VATS for lung cancer: A meta-analysis
- Fluorescent light energy modulates healing in skin grafted mouse model
- Silencing CDK6-AS1 inhibits LPS-induced inflammatory damage in HK-2 cells
- Predictive effect of DCE-MRI and DWI in brain metastases from NSCLC
- Severe postoperative hyperbilirubinemia in congenital heart disease
- Baicalin improves podocyte injury in rats with diabetic nephropathy by inhibiting PI3K/Akt/mTOR signaling pathway
- Clinical factors predicting ureteral stent failure in patients with external ureteral compression
- Novel H2S donor proglumide-ADT-OH protects HUVECs from ox-LDL-induced injury through NF-κB and JAK/SATA pathway
- Triple-Endobutton and clavicular hook: A propensity score matching analysis
- Long noncoding RNA MIAT inhibits the progression of diabetic nephropathy and the activation of NF-κB pathway in high glucose-treated renal tubular epithelial cells by the miR-182-5p/GPRC5A axis
- Serum exosomal miR-122-5p, GAS, and PGR in the non-invasive diagnosis of CAG
- miR-513b-5p inhibits the proliferation and promotes apoptosis of retinoblastoma cells by targeting TRIB1
- Fer exacerbates renal fibrosis and can be targeted by miR-29c-3p
- The diagnostic and prognostic value of miR-92a in gastric cancer: A systematic review and meta-analysis
- Prognostic value of α2δ1 in hypopharyngeal carcinoma: A retrospective study
- No significant benefit of moderate-dose vitamin C on severe COVID-19 cases
- circ_0000467 promotes the proliferation, metastasis, and angiogenesis in colorectal cancer cells through regulating KLF12 expression by sponging miR-4766-5p
- Downregulation of RAB7 and Caveolin-1 increases MMP-2 activity in renal tubular epithelial cells under hypoxic conditions
- Educational program for orthopedic surgeons’ influences for osteoporosis
- Expression and function analysis of CRABP2 and FABP5, and their ratio in esophageal squamous cell carcinoma
- GJA1 promotes hepatocellular carcinoma progression by mediating TGF-β-induced activation and the epithelial–mesenchymal transition of hepatic stellate cells
- lncRNA-ZFAS1 promotes the progression of endometrial carcinoma by targeting miR-34b to regulate VEGFA expression
- Anticoagulation is the answer in treating noncritical COVID-19 patients
- Effect of late-onset hemorrhagic cystitis on PFS after haplo-PBSCT
- Comparison of Dako HercepTest and Ventana PATHWAY anti-HER2 (4B5) tests and their correlation with silverin situ hybridization in lung adenocarcinoma
- VSTM1 regulates monocyte/macrophage function via the NF-κB signaling pathway
- Comparison of vaginal birth outcomes in midwifery-led versus physician-led setting: A propensity score-matched analysis
- Treatment of osteoporosis with teriparatide: The Slovenian experience
- New targets of morphine postconditioning protection of the myocardium in ischemia/reperfusion injury: Involvement of HSP90/Akt and C5a/NF-κB
- Superenhancer–transcription factor regulatory network in malignant tumors
- β-Cell function is associated with osteosarcopenia in middle-aged and older nonobese patients with type 2 diabetes: A cross-sectional study
- Clinical features of atypical tuberculosis mimicking bacterial pneumonia
- Proteoglycan-depleted regions of annular injury promote nerve ingrowth in a rabbit disc degeneration model
- Effect of electromagnetic field on abortion: A systematic review and meta-analysis
- miR-150-5p affects AS plaque with ASMC proliferation and migration by STAT1
- MALAT1 promotes malignant pleural mesothelioma by sponging miR-141-3p
- Effects of remifentanil and propofol on distant organ lung injury in an ischemia–reperfusion model
- miR-654-5p promotes gastric cancer progression via the GPRIN1/NF-κB pathway
- Identification of LIG1 and LIG3 as prognostic biomarkers in breast cancer
- MitoQ inhibits hepatic stellate cell activation and liver fibrosis by enhancing PINK1/parkin-mediated mitophagy
- Dissecting role of founder mutation p.V727M in GNE in Indian HIBM cohort
- circATP2A2 promotes osteosarcoma progression by upregulating MYH9
- Prognostic role of oxytocin receptor in colon adenocarcinoma
- Review Articles
- The function of non-coding RNAs in idiopathic pulmonary fibrosis
- Efficacy and safety of therapeutic plasma exchange in stiff person syndrome
- Role of cesarean section in the development of neonatal gut microbiota: A systematic review
- Small cell lung cancer transformation during antitumor therapies: A systematic review
- Research progress of gut microbiota and frailty syndrome
- Recommendations for outpatient activity in COVID-19 pandemic
- Rapid Communication
- Disparity in clinical characteristics between 2019 novel coronavirus pneumonia and leptospirosis
- Use of microspheres in embolization for unruptured renal angiomyolipomas
- COVID-19 cases with delayed absorption of lung lesion
- A triple combination of treatments on moderate COVID-19
- Social networks and eating disorders during the Covid-19 pandemic
- Letter
- COVID-19, WHO guidelines, pedagogy, and respite
- Inflammatory factors in alveolar lavage fluid from severe COVID-19 pneumonia: PCT and IL-6 in epithelial lining fluid
- COVID-19: Lessons from Norway tragedy must be considered in vaccine rollout planning in least developed/developing countries
- What is the role of plasma cell in the lamina propria of terminal ileum in Good’s syndrome patient?
- Case Report
- Rivaroxaban triggered multifocal intratumoral hemorrhage of the cabozantinib-treated diffuse brain metastases: A case report and review of literature
- CTU findings of duplex kidney in kidney: A rare duplicated renal malformation
- Synchronous primary malignancy of colon cancer and mantle cell lymphoma: A case report
- Sonazoid-enhanced ultrasonography and pathologic characters of CD68 positive cell in primary hepatic perivascular epithelioid cell tumors: A case report and literature review
- Persistent SARS-CoV-2-positive over 4 months in a COVID-19 patient with CHB
- Pulmonary parenchymal involvement caused byTropheryma whipplei
- Mediastinal mixed germ cell tumor: A case report and literature review
- Ovarian female adnexal tumor of probable Wolffian origin – Case report
- Rare paratesticular aggressive angiomyxoma mimicking an epididymal tumor in an 82-year-old man: Case report
- Perimenopausal giant hydatidiform mole complicated with preeclampsia and hyperthyroidism: A case report and literature review
- Primary orbital ganglioneuroblastoma: A case report
- Primary aortic intimal sarcoma masquerading as intramural hematoma
- Sustained false-positive results for hepatitis A virus immunoglobulin M: A case report and literature review
- Peritoneal loose body presenting as a hepatic mass: A case report and review of the literature
- Chondroblastoma of mandibular condyle: Case report and literature review
- Trauma-induced complete pacemaker lead fracture 8 months prior to hospitalization: A case report
- Primary intradural extramedullary extraosseous Ewing’s sarcoma/peripheral primitive neuroectodermal tumor (PIEES/PNET) of the thoracolumbar spine: A case report and literature review
- Computer-assisted preoperative planning of reduction of and osteosynthesis of scapular fracture: A case report
- High quality of 58-month life in lung cancer patient with brain metastases sequentially treated with gefitinib and osimertinib
- Rapid response of locally advanced oral squamous cell carcinoma to apatinib: A case report
- Retrieval of intrarenal coiled and ruptured guidewire by retrograde intrarenal surgery: A case report and literature review
- Usage of intermingled skin allografts and autografts in a senior patient with major burn injury
- Retraction
- Retraction on “Dihydromyricetin attenuates inflammation through TLR4/NF-kappa B pathway”
- Special Issue Computational Intelligence Methodologies Meets Recurrent Cancers - Part I
- An artificial immune system with bootstrap sampling for the diagnosis of recurrent endometrial cancers
- Breast cancer recurrence prediction with ensemble methods and cost-sensitive learning
