技术领域Technical field
本发明属于脂肪肝治疗技术领域，尤其涉及一种防治非酒精性脂肪肝的药物。The invention belongs to the technical field of fatty liver treatment, and in particular relates to a medicine for preventing and treating non-alcoholic fatty liver.
背景技术Background technique
非酒精性脂肪肝病（NAFLD）是指除酒精和其他明确的损肝因素（如药物，病毒感染或自身免疫等）所导致的以肝脏细胞内脂质过度沉积为主要特征的病理综合征。根据发展过程划分，非酒精性脂肪肝病可以分为单纯性脂肪肝、非酒精性脂肪性肝炎以及肝硬化。目前，全球范围内非酒精性脂肪肝病的患病率呈现出迅速上升的情况，而且，由于非酒精性脂肪肝病的发病机理复杂多样，目前尚没有专门用于治疗非酒精性脂肪肝的特效药物，因此，探究非酒精性脂肪肝的治疗方法和治疗药物是当前研究的热点。Non-alcoholic fatty liver disease (NAFLD) refers to a pathological syndrome mainly characterized by excessive lipid deposition in liver cells caused by alcohol and other clear liver-damaging factors (such as drugs, viral infection or autoimmunity, etc.). According to the development process, non-alcoholic fatty liver disease can be divided into simple fatty liver, non-alcoholic steatohepatitis and cirrhosis. Currently, the prevalence of non-alcoholic fatty liver disease is rising rapidly worldwide. Moreover, due to the complex and diverse pathogenesis of non-alcoholic fatty liver disease, there are currently no specific drugs specifically designed to treat non-alcoholic fatty liver disease. , Therefore, exploring the treatment methods and drugs for non-alcoholic fatty liver disease is a hot topic in current research.
大花旋覆花内酯（Britannin）也被称之为大花旋覆花素，其是从大花旋覆花中提取出来的一种植物提取物，其分子式为C19H26O7，其CAS号为33627-28-0。现有的研究表明，大花旋覆花内酯对于正常细胞的毒性较低，且能够产生有效的抗癌效果。除此之外，现有的研究表明大花旋覆花内酯还具有改善脑缺血再灌注损伤的作用。然而，目前尚无关于大花旋覆花内酯在脂肪肝治疗方面的相关内容，因此本发明对大花旋覆花内酯在脂肪肝治疗中的作用效果和机制进行研究。Britannin is also called Inula grandiflorum. It is a plant extract extracted from Inula grandiflorum. Its molecular formula is C19 H26 O7 . Its CAS number is 33627-28-0. Existing research shows that Inula grandiflorum has low toxicity to normal cells and can produce effective anti-cancer effects. In addition, existing research shows that inulalide can also improve cerebral ischemia-reperfusion injury. However, there is currently no relevant content on the treatment of fatty liver by inulalide. Therefore, the present invention studies the effect and mechanism of inulalide in the treatment of fatty liver.
发明内容Contents of the invention
本发明要解决的技术问题是提供一种大花旋覆花内脂的新应用，并提供一种可以有效治疗非酒精性脂肪肝的药物。The technical problem to be solved by the present invention is to provide a new application of inulatus grandiflorum and to provide a medicine that can effectively treat non-alcoholic fatty liver disease.
本发明的第一个目的是提供大花旋覆花内酯在制备治疗非酒精性脂肪肝的药物中的应用。The first object of the present invention is to provide the application of inulalide in the preparation of medicines for treating non-alcoholic fatty liver disease.
优选地，所述药物通过降低肝细胞中甘油三酯含量来发挥治疗非酒精性脂肪肝的作用。Preferably, the drug acts to treat non-alcoholic fatty liver disease by reducing triglyceride content in liver cells.
优选地，所述药物通过降低肝细胞的衰老来发挥治疗非酒精性脂肪肝的作用。Preferably, the drug plays a role in treating non-alcoholic fatty liver disease by reducing the senescence of liver cells.
优选地，所述药物通过降低肝细胞的脂质积累来发挥治疗非酒精脂肪肝的作用。Preferably, the drug acts to treat non-alcoholic fatty liver disease by reducing lipid accumulation in hepatocytes.
优选地，所述非酒精性脂肪肝为游离脂肪酸导致的非酒精性脂肪肝。Preferably, the non-alcoholic fatty liver disease is non-alcoholic fatty liver disease caused by free fatty acids.
优选地，所述大花旋覆花内脂的浓度为31.25nM-1000nM。Preferably, the concentration of Inula grandiflorum is 31.25nM-1000nM.
优选地，所述大花旋覆花内脂的最佳浓度为125nM。Preferably, the optimal concentration of Inula grandiflorum is 125 nM.
本发明的第二个目的是提供一种用于治疗非酒精性脂肪肝的药物，其特征在于，所述药物的有效成分为大花旋覆花内脂。The second object of the present invention is to provide a medicine for treating non-alcoholic fatty liver disease, characterized in that the active ingredient of the medicine is inuladin grandiflorum.
优选地，所述药物通过降低肝细胞中甘油三酯含量来发挥治疗非酒精性脂肪肝的作用；Preferably, the drug plays a role in treating non-alcoholic fatty liver disease by reducing triglyceride content in liver cells;
所述药物通过降低肝细胞的衰老来发挥治疗非酒精性脂肪肝的作用；The drug plays a role in treating non-alcoholic fatty liver disease by reducing the senescence of liver cells;
所述药物通过降低肝细胞的脂质积累来发挥治疗非酒精脂肪肝的作用。The drug works by reducing lipid accumulation in liver cells to treat non-alcoholic fatty liver disease.
优选地，所述非酒精性脂肪肝为游离脂肪酸导致的非酒精性脂肪肝。Preferably, the non-alcoholic fatty liver disease is non-alcoholic fatty liver disease caused by free fatty acids.
优选地，所述大花旋覆花内脂的浓度为31.25nM-1000nM。Preferably, the concentration of Inula grandiflorum is 31.25nM-1000nM.
优选地，所述大花旋覆花内脂的最佳浓度为125nM。Preferably, the optimal concentration of Inula grandiflorum is 125 nM.
本发明的第三个目的是提供大花旋覆花内脂在制备降低游离脂肪酸导致的肝细胞中甘油三酯含量升高的药物中的应用。The third object of the present invention is to provide the application of inulatus grandiflorum in the preparation of medicines for reducing the increase in triglyceride content in liver cells caused by free fatty acids.
优选地，所述大花旋覆花内脂的浓度为31.25nM-1000nM。Preferably, the concentration of Inula grandiflorum is 31.25nM-1000nM.
优选地，所述大花旋覆花内脂的最佳浓度为125nM。Preferably, the optimal concentration of Inula grandiflorum is 125 nM.
本发明的第四个目的是提供大花旋覆花内脂在制备降低游离脂肪酸导致的肝细胞衰老的药物中的应用。The fourth object of the present invention is to provide the application of Inula grandiflorum in the preparation of medicines for reducing liver cell senescence caused by free fatty acids.
优选地，所述大花旋覆花内脂的浓度为31.25nM-1000nM。Preferably, the concentration of Inula grandiflorum is 31.25nM-1000nM.
优选地，所述大花旋覆花内脂的最佳浓度为125nM。Preferably, the optimal concentration of Inula grandiflorum is 125 nM.
本发明的第五个目的是提供大花旋覆花内脂在制备降低游离脂肪酸导致的肝细胞脂质积累的药物中的应用。The fifth object of the present invention is to provide the application of Inula grandiflorum in the preparation of medicines for reducing lipid accumulation in liver cells caused by free fatty acids.
优选地，所述大花旋覆花内脂的浓度为31.25nM-1000nM。Preferably, the concentration of Inula grandiflorum is 31.25nM-1000nM.
优选地，所述大花旋覆花内脂的最佳浓度为125nM。Preferably, the optimal concentration of Inula grandiflorum is 125 nM.
本发明具有以下有益效果：The invention has the following beneficial effects:
本发明提供了大花旋覆花内脂在制备非酒精性脂肪肝治疗药物中的新应用，本发明发现大花旋覆花内脂能够通过降低肝细胞中甘油三酯含量，降低肝细胞的衰老和降低肝细胞的脂质积累来发挥治疗非酒精脂肪肝的作用。因此，本发明不仅扩宽了大花旋覆花内脂的应用范围，还为非酒精性脂肪肝的治疗提供了一种新的安全可靠的药物。The present invention provides a new application of inulatus grandiflorum in the preparation of non-alcoholic fatty liver treatment drugs. The present invention finds that inula inflorescence can reduce the triglyceride content in hepatocytes and reduce hepatocyte dysfunction. Aging and reducing lipid accumulation in liver cells play a role in the treatment of non-alcoholic fatty liver disease. Therefore, the present invention not only broadens the application range of Inula grandiflorum, but also provides a new safe and reliable drug for the treatment of non-alcoholic fatty liver disease.
附图说明Description of the drawings
图1为大花旋覆花内酯对于肝细胞LO-2的细胞毒性检测结果；Figure 1 shows the cytotoxicity test results of Inula grandiflorum on hepatocyte LO-2;
图2为大花旋覆花内酯对于非酒精性脂肪肝细胞模型中TG含量影响的检测结果；Figure 2 shows the detection results of the effect of inuladin on TG content in the non-alcoholic fatty liver cell model;
图3为大花旋覆花内酯对于非酒精性脂肪肝细胞模型的细胞衰老影响的检测结果；Figure 3 shows the detection results of the effect of inuladin on the cell senescence of non-alcoholic fatty liver cell model;
图4为大花旋覆花内酯对于非酒精性脂肪肝细胞模型中脂质积累影响的检测结果。Figure 4 shows the test results of the effect of inuladin on lipid accumulation in non-alcoholic fatty liver cell model.
具体实施方式Detailed ways
实施例1：选择大花旋覆花内酯在浓度梯度：1000nM，500nM，250nM，125nM，62.5nM，31.25nM，0nM下进行下列步骤：Example 1: Select Inula grandiflorum and carry out the following steps under the concentration gradient: 1000nM, 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 0nM:
（1）将复苏后培养的LO-2细胞进行消化并计数，将细胞悬液的浓度调整为5×104个/ml；(1) Digest and count the cultured LO-2 cells after recovery, and adjust the concentration of the cell suspension to 5×104 cells/ml;
（2）将100ul细胞悬液加入细胞培养孔中培养24h后逐一弃去培养基，PBS缓冲液反复冲洗2次；(2) Add 100ul of cell suspension into the cell culture well and culture for 24 hours, discard the culture medium one by one, and rinse twice with PBS buffer;
（3）用1640完全培养基稀释大花旋覆花内酯（购自上海源叶生物科技有限公司）至所需浓度，向细胞培养板中加入200ul相应梯度的含药培养基；(3) Use 1640 complete culture medium to dilute Inula grandiflorum (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) to the required concentration, and add 200ul of the corresponding gradient drug-containing culture medium to the cell culture plate;
（4）将细胞培养板置于培养箱中培养24h后进行CCK-8染色，测定OD值并计算各组别的细胞活性。(4) Place the cell culture plate in the incubator for 24 hours, then perform CCK-8 staining, measure the OD value, and calculate the cell activity of each group.
首先，本发明检测了大花旋覆花内酯对于肝细胞LO-2是否存在毒性，从图1的结果中能够看出，在0-1000nM的浓度范围内，大花旋覆花内酯对于肝细胞LO-2的细胞活性没有明显的影响，说明大花旋覆花内酯对于肝细胞无毒性可以用于药物的制备。First, the present invention detects whether inulalide is toxic to liver cells LO-2. From the results in Figure 1, it can be seen that in the concentration range of 0-1000nM, inulalide is toxic to liver cells LO-2. There was no obvious effect on the cell activity of liver cell LO-2, indicating that inula lactone has no toxicity to liver cells and can be used for the preparation of drugs.
实施例2：（1）使用分析天平称取0.0609g油酸和0.0278g棕榈酸，依次加入15ml25%牛血清蛋白的PBS溶液中，70℃加热后，放入超声中至完全溶解；Example 2: (1) Use an analytical balance to weigh 0.0609g oleic acid and 0.0278g palmitic acid, add them sequentially to 15ml of 25% bovine serum albumin PBS solution, heat at 70°C, and put into ultrasonic until completely dissolved;
（2）将配置好的20mM的母液稀释为1mM 游离脂肪酸混合液，使用0.22um过滤器过滤除菌后备用；(2) Dilute the prepared 20mM stock solution into a 1mM free fatty acid mixture, filter and sterilize with a 0.22um filter before use;
（3）将LO-2细胞均匀接种到培养皿中，待细胞贴壁24h后按照如下的分组进行处理：(3) Inoculate LO-2 cells evenly into the culture dish. After the cells have adhered for 24 hours, process them according to the following groupings:
正常组：更换1640完全培养基；Normal group: replace with 1640 complete culture medium;
NAFLD模型组：更换为含有1mM 游离脂肪酸混合液的1640完全培养基；NAFLD model group: replaced with 1640 complete medium containing 1mM free fatty acid mixture;
低剂量大花旋覆花内酯组：更换为含有1mM 游离脂肪酸混合液＋31.25nM 大花旋覆花内酯的1640完全培养基；Low-dose inuladin group: replaced with 1640 complete culture medium containing 1mM free fatty acid mixture + 31.25nM inulalide;
中剂量大花旋覆花内酯组：更换为含有1mM 游离脂肪酸混合液＋125nM 大花旋覆花内酯的1640完全培养基；Medium-dose inuladin group: replaced with 1640 complete culture medium containing 1mM free fatty acid mixture + 125nM inulalide;
高剂量大花旋覆花内酯组：更换为含有1mM 游离脂肪酸混合液＋250nM 大花旋覆花内酯的1640完全培养基；High-dose inuladin group: replaced with 1640 complete culture medium containing 1mM free fatty acid mixture + 250nM inulalide;
（4）处理24h后，吸去培养液，PBS清洗2次，加入1ml的PBS并使用细胞刮板将细胞刮下，使用移液器将细胞收集到离心管中，1000rpm，4℃离心5min；(4) After 24 hours of treatment, aspirate the culture medium, wash twice with PBS, add 1 ml of PBS and use a cell scraper to scrape off the cells, use a pipette to collect the cells into a centrifuge tube, and centrifuge at 1000 rpm and 4°C for 5 minutes;
（5）弃去上清，加入200ul 2%的Triton X-100裂解液，冰上裂解40min，参照甘油三酯（TG）试剂盒检测细胞内TG含量。(5) Discard the supernatant, add 200ul of 2% Triton
甘油三酯升高是导致非酒精性脂肪肝的主要原因，因此在实施例2中，本发明检测了对于非酒精性脂肪肝细胞模型中TG含量的影响，结果如图2所示。Elevated triglycerides are the main cause of non-alcoholic fatty liver disease. Therefore, in Example 2, the present invention detected the impact on TG content in the non-alcoholic fatty liver cell model. The results are shown in Figure 2.
由图2可知，与正常组相比，NAFLD模型组的TG含量出现了明显的升高，而相较于NAFLD模型组，低，中，高剂量大花旋覆花内酯组的TG含量均发生了一定程度的下降，差异具有统计学意义，说明使用大花旋覆花内酯处理非酒精性脂肪肝细胞模型时，能够有效的抑制细胞内的TG含量，且在125nM左右的浓度达到最佳效果。As can be seen from Figure 2, compared with the normal group, the TG content of the NAFLD model group increased significantly. A certain degree of decline occurred, and the difference was statistically significant, indicating that when using inuladin to treat non-alcoholic fatty liver cell models, it can effectively inhibit the intracellular TG content, and reach the maximum concentration at about 125nM. Best results.
实施例3：（1）将LO-2细胞均匀接种到培养皿中，待细胞贴壁24h后按照如下的分组进行处理：Example 3: (1) Inoculate LO-2 cells evenly into a culture dish. After the cells have adhered for 24 hours, they are processed according to the following groupings:
正常组：更换1640完全培养基；Normal group: replace with 1640 complete culture medium;
NAFLD模型组：更换为含有1mM 游离脂肪酸混合液的1640完全培养基；NAFLD model group: replaced with 1640 complete medium containing 1mM free fatty acid mixture;
低剂量大花旋覆花内酯组：更换为含有1mM 游离脂肪酸混合液＋31.25nM 大花旋覆花内酯的1640完全培养基；Low-dose inuladin group: replaced with 1640 complete culture medium containing 1mM free fatty acid mixture + 31.25nM inulalide;
中剂量大花旋覆花内酯组：更换为含有1mM 游离脂肪酸混合液＋125nM 大花旋覆花内酯的1640完全培养基；Medium-dose inuladin group: replaced with 1640 complete culture medium containing 1mM free fatty acid mixture + 125nM inulalide;
高剂量大花旋覆花内酯组：更换为含有1mM 游离脂肪酸混合液＋250nM 大花旋覆花内酯的1640完全培养基；High-dose inuladin group: replaced with 1640 complete culture medium containing 1mM free fatty acid mixture + 250nM inulalide;
（2）处理24h后，吸去培养基，加入PBS清洗细胞2次，加入细胞固定液固定15min；(2) After 24 hours of treatment, aspirate the culture medium, add PBS to wash the cells twice, and add cell fixative to fix for 15 minutes;
（3）去除固定液，加入PBS清洗细胞2次，加入β-半乳糖苷酶染色液，使用保鲜膜封住后，置于37℃孵育12h；(3) Remove the fixative, add PBS to wash the cells twice, add β-galactosidase staining solution, seal with plastic wrap, and incubate at 37°C for 12 hours;
（4）去除染色液，进行阳性细胞的计数并计算阳性率。(4) Remove the staining solution, count the positive cells and calculate the positivity rate.
引起肝细胞的衰老也是游离脂肪酸导致非酒精性脂肪肝的重要原因，因此本发明检测了大花旋覆花内酯在非酒精性脂肪肝细胞模型中对于细胞衰老的影响，结果如图3所示。The senescence of liver cells is also an important reason why free fatty acids cause non-alcoholic fatty liver disease. Therefore, the present invention detects the effect of inulalide on cell senescence in the non-alcoholic fatty liver cell model. The results are shown in Figure 3 Show.
从图3可以看出，相较于对照组，模型组的β-半乳糖苷酶阳性细胞的比例显著升高，而相较于模型组，低，中，高剂量大花旋覆花内酯组的β-半乳糖苷酶阳性细胞的比例均出现了显著的下降，且与实施例2相同，在中剂量时，效果达到最佳。说明大花旋覆花内酯能够有效的抑制游离脂肪酸导致的肝细胞衰老。As can be seen from Figure 3, compared with the control group, the proportion of β-galactosidase-positive cells in the model group was significantly increased, and compared with the model group, low, medium, and high doses of inula lactone The proportion of β-galactosidase-positive cells in both groups decreased significantly, and the same as Example 2, the best effect was achieved at the medium dose. It shows that Inula grandiflorum can effectively inhibit the aging of liver cells caused by free fatty acids.
实施例4：（1）将LO-2细胞均匀接种到培养皿中，待细胞贴壁24h后按照如下的分组进行处理：Example 4: (1) Inoculate LO-2 cells evenly into a culture dish. After the cells have adhered for 24 hours, they are processed according to the following groupings:
正常组：更换1640完全培养基；Normal group: replace with 1640 complete culture medium;
NAFLD模型组：更换为含有1mM 游离脂肪酸混合液的1640完全培养基；NAFLD model group: replaced with 1640 complete medium containing 1mM free fatty acid mixture;
大花旋覆花内酯组：更换为含有1mM 游离脂肪酸混合液＋125nM 大花旋覆花内酯的1640完全培养基；Inuladin group: replaced with 1640 complete culture medium containing 1mM free fatty acid mixture + 125nM inulalide;
（2）处理24h后，吸去培养液，PBS清洗2次，加入4%多聚甲醛固定细胞15min；(2) After 24 hours of treatment, aspirate the culture medium, wash twice with PBS, and add 4% paraformaldehyde to fix the cells for 15 minutes;
（3）吸去多聚甲醛，使用PBS清洗细胞2次，加入油红O工作液，避光染色10min；(3) Absorb the paraformaldehyde, wash the cells twice with PBS, add Oil Red O working solution, and stain for 10 minutes in the dark;
（4）使用60%异丙醇漂洗数秒，去除多余的染料；(4) Rinse with 60% isopropyl alcohol for a few seconds to remove excess dye;
（5）加入苏木素染色液染色1.5min，双蒸水漂洗3次，置于倒置显微镜下进行拍照。(5) Add hematoxylin staining solution for staining for 1.5 minutes, rinse with double-distilled water three times, and take pictures under an inverted microscope.
肝细胞脂质积累是非酒精性脂肪肝的主要表现，因此本发明检测了大花旋覆花内酯对于非酒精性脂肪肝细胞模型中脂质积累的影响。由图4可知，相较于正常组，模型组中的脂滴数量明显增加，而相较于模型组，大花旋覆花内酯组的脂滴数量明显减少，说明大花旋覆花内酯能够有效的减少细胞中脂质积累。Lipid accumulation in liver cells is the main manifestation of non-alcoholic fatty liver disease. Therefore, the present invention detects the effect of inulalide on lipid accumulation in the non-alcoholic fatty liver cell model. As can be seen from Figure 4, compared with the normal group, the number of lipid droplets in the model group increased significantly, and compared with the model group, the number of lipid droplets in the Inula grandiflorum group decreased significantly, indicating that the number of lipid droplets in the Inula grandiflorum group was significantly reduced. Esters can effectively reduce lipid accumulation in cells.
综合上述实施例，我们可以得出大花旋覆花内酯可以有效的降低游离性脂肪酸导致的肝细胞中TG含量升高，肝细胞衰老和肝细胞脂质积累，从而可以有效的对游离性脂肪酸导致的非酒精性脂肪肝产生治疗作用。Based on the above examples, we can conclude that Inula grandiflorum can effectively reduce the increase in TG content in liver cells caused by free fatty acids, liver cell aging and liver cell lipid accumulation, thereby effectively reducing free fatty acids. Treatment of non-alcoholic fatty liver disease caused by fatty acids.
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| CN202310981801.XACN116687914B (en) | 2023-08-07 | 2023-08-07 | A drug to treat non-alcoholic fatty liver disease |
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| CN202310981801.XACN116687914B (en) | 2023-08-07 | 2023-08-07 | A drug to treat non-alcoholic fatty liver disease |
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