技术领域technical field
本发明涉及医药技术领域，更具体地，涉及甲基巴多索隆在制备抑制呼吸道合胞病毒感染药物中的应用。The invention relates to the technical field of medicine, and more specifically, relates to the application of bardoxolone methyl in the preparation of drugs for inhibiting respiratory syncytial virus infection.
背景技术Background technique
呼吸道合胞病毒(Respiratory Syncytial Virus，简称RSV)是呼吸道最常见的病毒病原体之一，在全球范围内广泛传播。RSV是导致早产儿和婴幼儿毛支炎和肺炎的主要原因，约占全球学龄前儿童的60％，估计全球每年有94600-149400名5岁以下儿童死于RSV感染。RSV也是65岁以上老年人和免疫力低下人群呼吸道感染的重要病原体。据世界卫生组织统计，全球每年约3300万感染者，其中340多万严重感染者需要住院治疗，住院患者中10-31％需入住重症监护病房，3-17％需机械通气。RSV感染目前尚缺乏成熟的疫苗和有效治疗措施。因此，深入研究治疗RSV感染的药物和机制显得尤为重要。前期通过临床标本及体外实验发现：RSV感染介导了Toll样受体3(TLR3)和Toll样受体7(TLR7)的先天性免疫损伤，通过刺激NF-κB信号通路，产生大量炎症介质，如IL-6、IL-8、IL-10、IL-13、IL-32、TNF-α等，炎症因子增加，肺部损伤加重。Respiratory syncytial virus (RSV) is one of the most common viral pathogens in the respiratory tract and is widely spread around the world. RSV is the main cause of bronchitis and pneumonia in premature infants and infants, accounting for about 60% of preschool children worldwide. It is estimated that 94600-149400 children under the age of 5 die from RSV infection every year worldwide. RSV is also an important pathogen of respiratory tract infection in the elderly over 65 years old and the immunocompromised population. According to the statistics of the World Health Organization, there are about 33 million infected people in the world every year, of which more than 3.4 million severely infected people need hospitalization, 10-31% of hospitalized patients need to be admitted to the intensive care unit, and 3-17% need mechanical ventilation. RSV infection is still lack of mature vaccine and effective treatment. Therefore, it is particularly important to study the drugs and mechanisms for the treatment of RSV infection. Early clinical specimens and in vitro experiments found that RSV infection mediated the innate immune injury of Toll-like receptor 3 (TLR3) and Toll-like receptor 7 (TLR7), and stimulated the NF-κB signaling pathway to produce a large number of inflammatory mediators, such as IL-6, IL-8, IL-10, IL-13, IL-32, TNF-α, etc., increased inflammatory factors, and aggravated lung damage.
RSV虽已发现60余年，但治疗手段非常有限。到目前为止，被美国食品药品监督管理局(Food and Drug Administration，FDA)认证的RSV防治药物仅两种：吸入型利巴韦林和帕利珠单抗。利巴韦林为一种核苷类似物，是广谱的抗病毒药物。早期研究表明，它可快速地清除RSV感染，减轻患者症状和缩短住院时间，但有其它观点认为利巴韦林并没有降低RSV感染后死亡率和减少重症患者机械通气时间。此外，利巴韦林会导致溶血、先天致畸、心血管毒性等药物毒副作用，这使得它的使用受到了越来越多的质疑。帕利珠单抗是针对呼吸道合胞病毒融合蛋白(RSV F)的人源小鼠单克隆抗体，仅适用于预防RSV所致儿童严重下呼吸道感染，它能明显减少住院次数、住院时间及死亡率，但因代谢快，每半年需重新注射，价格昂贵，而且在我国没有上市。Although RSV has been discovered for more than 60 years, the treatment options are very limited. So far, there are only two RSV prevention and treatment drugs certified by the US Food and Drug Administration (FDA): inhaled ribavirin and palivizumab. Ribavirin, a nucleoside analog, is a broad-spectrum antiviral drug. Early studies have shown that it can quickly clear RSV infection, relieve symptoms and shorten hospital stays, but there are other views that ribavirin does not reduce mortality after RSV infection and reduce the duration of mechanical ventilation in critically ill patients. In addition, ribavirin can cause hemolysis, congenital teratogenicity, cardiovascular toxicity and other drug side effects, which makes its use more and more questioned. Palivizumab is a humanized mouse monoclonal antibody against the fusion protein of respiratory syncytial virus (RSV F). It is only suitable for the prevention of severe lower respiratory tract infection in children caused by RSV. It can significantly reduce the number of hospitalizations, length of hospitalization and mortality. However, due to its rapid metabolism, it needs to be re-injected every six months. It is expensive and is not available in my country.
目前RSV感染尚缺乏成熟的疫苗和有效治疗措施，因此，亟需开发一种有效的治疗RSV感染的方法。At present, RSV infection still lacks a mature vaccine and effective treatment measures. Therefore, it is urgent to develop an effective method for treating RSV infection.
发明内容Contents of the invention
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出甲基巴多索隆在制备抑制呼吸道合胞病毒感染药物中的应用，本发明通过用倒置显微镜观察BEAS-2B细胞感染RSV后的病变效应，通过CCK8方法检测甲基巴多索隆的细胞毒性和抗病毒效应，逆转录聚合酶链式反应(RT-PCR)检测RSV、IL-6、IL-8、IKKβ和NF-κB mRNA表达，蛋白免疫印迹(Western blot)检测RSV F、p-IKKβ、p-NF-κB、IL-6蛋白的表达发现甲基巴多索隆抗RSV感染的分子机制与IKKβ/NF-κB通路相关，证实了甲基巴多索隆通过抑制IKKβ/NF-κB通路来抑制呼吸道合胞病毒感染以及抑制呼吸道合胞病毒炎症因子表达。The present invention aims to solve at least one of the technical problems in the above-mentioned prior art. For this reason, the present invention proposes the application of bardoxolone methyl in the preparation of the medicine for inhibiting respiratory syncytial virus infection. The present invention observes the pathological effect after BEAS-2B cells are infected with RSV with an inverted microscope, detects the cytotoxicity and antiviral effect of bardoxolone methyl by the CCK8 method, detects the expression of RSV, IL-6, IL-8, IKKβ and NF-κB mRNA by reverse transcription polymerase chain reaction (RT-PCR), and detects RSV F, p-IKK by Western blot. The expression of β, p-NF-κB, and IL-6 proteins found that the molecular mechanism of bardoxolone methyl against RSV infection was related to the IKKβ/NF-κB pathway, which confirmed that bardoxolone methyl inhibited RSV infection and the expression of respiratory syncytial virus inflammatory factors by inhibiting the IKKβ/NF-κB pathway.
本发明的第一方面提供甲基巴多索隆在制备抑制呼吸道合胞病毒感染药物中的应用。The first aspect of the present invention provides the use of bardoxolone methyl in the preparation of a medicament for inhibiting respiratory syncytial virus infection.
具体地，甲基巴多索隆在制备抑制呼吸道合胞病毒感染的药物中的应用。Specifically, the application of bardoxolone methyl in the preparation of a drug for inhibiting respiratory syncytial virus infection.
甲基巴多索隆(Bardoxolone Methyl，简称BXM)是一种新型小分子化合物，是半合成的三萜类齐墩果酸衍生物，其CAS号为218600-53-4，具有抗氧化应激、抗炎、抗增殖、抗癌等作用。甲基巴多索隆直接作用靶点有Keap1和IKKβ，它与Keap1结合破坏了半胱氨酸残基，导致Nrf2释放，Nrf2释放可出现抗氧化、抗炎反应。甲基巴多索隆还能直接与IKKβ激活环中的Cys-179结合，抑制NF-κB激活，从而抑制下游促炎通路。Bardoxolone Methyl (BXM for short) is a new type of small molecule compound, a semi-synthetic triterpenoid oleanolic acid derivative, with a CAS number of 218600-53-4, which has anti-oxidative stress, anti-inflammatory, anti-proliferation, anti-cancer effects. The direct targets of bardoxolone methyl are Keap1 and IKKβ. It binds to Keap1 and destroys cysteine residues, leading to the release of Nrf2. The release of Nrf2 can lead to antioxidant and anti-inflammatory reactions. Bardoxolone methyl can also directly bind to Cys-179 in the IKKβ activation loop to inhibit NF-κB activation, thereby inhibiting downstream pro-inflammatory pathways.
甲基巴多索隆在制备通过抑制IKKβ/NF-κB通路来抑制呼吸道合胞病毒感染的药物中的应用。Application of bardoxolone methyl in the preparation of a medicament for inhibiting respiratory syncytial virus infection by inhibiting the IKKβ/NF-κB pathway.
甲基巴多索隆在制备通过抑制IKKβ/NF-κB通路来抑制呼吸道合胞病毒炎症因子表达的药物中的应用。Application of bardoxolone methyl in the preparation of a medicament for inhibiting the expression of respiratory syncytial virus inflammatory factors by inhibiting the IKKβ/NF-κB pathway.
甲基巴多索隆在制备通过抑制IKKβ/NF-κB通路来抑制呼吸道合胞病毒感染以及呼吸道合胞病毒炎症因子表达的药物中的应用。Application of bardoxolone methyl in preparing a drug for inhibiting respiratory syncytial virus infection and expression of respiratory syncytial virus inflammatory factors by inhibiting IKKβ/NF-κB pathway.
优选地，所述甲基巴多索隆的浓度为0.01-20mg/Kg/d。Preferably, the concentration of bardoxolone methyl is 0.01-20 mg/Kg/d.
进一步优选地，所述甲基巴多索隆的浓度为0.1-10mg/Kg/d。Further preferably, the concentration of bardoxolone methyl is 0.1-10 mg/Kg/d.
进一步优选地，所述甲基巴多索隆的浓度为1-5mg/Kg/d。Further preferably, the concentration of bardoxolone methyl is 1-5 mg/Kg/d.
更优选地，所述甲基巴多索隆的浓度为1-3mg/Kg/d。More preferably, the concentration of bardoxolone methyl is 1-3 mg/Kg/d.
更优选地，所述甲基巴多索隆的浓度为2.5-3mg/Kg/d。More preferably, the concentration of bardoxolone methyl is 2.5-3 mg/Kg/d.
优选地，所述药物的剂型为片剂、胶囊剂、散剂、颗粒剂、注射剂、口服液体中的至少一种。Preferably, the dosage form of the medicine is at least one of tablet, capsule, powder, granule, injection and oral liquid.
相对于现有技术，本发明的有益效果如下：Compared with the prior art, the beneficial effects of the present invention are as follows:
(1)本发明提出甲基巴多索隆可用于制备抑制呼吸道合胞病毒感染的药物，目前尚无甲基巴多索隆对RSV感染治疗及炎性损伤分子机制的相关研究，本发明通过建立RSV感染细胞和小鼠模型，用倒置显微镜观察BEAS-2B细胞感染RSV后的病变效应，通过CCK8试剂(Cell Counting Kit-8)检测甲基巴多索隆的细胞毒性和抗病毒效应，逆转录聚合酶链式反应(RT-PCR)检测RSV、IL-6、IL-8、IKKβ和NF-κB mRNA表达，蛋白免疫印迹(Western blot)检测RSV F、p-IKKβ、p-NF-κB、IL-6蛋白的表达，首次证实甲基巴多索隆通过IKKβ/NF-κB通路缓解RSV感染所致肺部炎症损伤的药效学，甲基巴多索隆可用于制备抑制呼吸道合胞病毒感染的药物，以期为临床诊治RSV提供有希望的药物、治疗靶点及策略；(1) The present invention proposes that bardoxolone methyl can be used to prepare drugs for inhibiting respiratory syncytial virus infection. At present, there is no related research on bardoxolone methyl for the treatment of RSV infection and the molecular mechanism of inflammatory injury. The present invention establishes RSV infected cells and mouse models, observes the lesion effect of BEAS-2B cells infected with RSV with an inverted microscope, detects the cytotoxicity and antiviral effect of bardoxolone methyl by CCK8 reagent (Cell Counting Kit-8), and detects RS by reverse transcription polymerase chain reaction (RT-PCR). V, IL-6, IL-8, IKKβ and NF-κB mRNA expression, western blot detection of RSV F, p-IKKβ, p-NF-κB, IL-6 protein expression, confirmed for the first time the pharmacodynamics of bardoxolone methyl in alleviating lung inflammatory injury caused by RSV infection through the IKKβ/NF-κB pathway, bardoxolone methyl can be used to prepare drugs to inhibit respiratory syncytial virus infection, in order to provide promising drugs for clinical diagnosis and treatment of RSV, Therapeutic targets and strategies;
(2)本发明利用甲基巴多索隆制备通过抑制IKKβ/NF-κB通路来抑制呼吸道合胞病毒感染以及呼吸道合胞病毒炎症因子表达的药物，通过对甲基巴多索隆的浓度控制可调控治疗效果，其中甲基巴多索隆施加量为1mg/Kg/d的治疗效果可达到与利巴韦林施加量40mg/Kg/d相当的水平，甲基巴多索隆施加量为3mg/Kg/d时对RSV感染小鼠的治疗效果最佳。(2) The present invention utilizes bardoxolone methyl to prepare a medicine that inhibits respiratory syncytial virus infection and expression of respiratory syncytial virus inflammatory factors by inhibiting the IKKβ/NF-κB pathway. The therapeutic effect can be regulated by controlling the concentration of bardoxolone methyl, wherein the therapeutic effect of bardoxolone methyl is 1 mg/Kg/d, which can reach a level equivalent to that of ribavirin 40 mg/Kg/d. When bardoxolone methyl is 3 mg/Kg/d, the treatment of RSV-infected mice Works best.
附图说明Description of drawings
图1为体外实验的实验方法示意图；Fig. 1 is the experimental method schematic diagram of in vitro experiment;
图2为体内实验的分组及技术路线示意图；Figure 2 is a schematic diagram of the grouping and technical route of the in vivo experiment;
图3为通过扫描电镜观察RSV感染Hep-2细胞图；Fig. 3 is to observe RSV to infect Hep-2 cell figure by scanning electron microscope;
图4为通过显微镜观察RSV感染Hep-2和BEAS-2B细胞图；Fig. 4 is to observe RSV to infect Hep-2 and BEAS-2B cell figure by microscope;
图5为甲基巴多索隆在BEAS-2B细胞中通过抑制IKKβ/NF-κB通路对RSV的抑制效应图；Figure 5 is a graph showing the inhibitory effect of bardoxolone methyl on RSV by inhibiting the IKKβ/NF-κB pathway in BEAS-2B cells;
图6为TPCA-1对BEAS-2B细胞中的RSV感染的作用效果图；Figure 6 is a diagram showing the effect of TPCA-1 on RSV infection in BEAS-2B cells;
图7为不同分组中小鼠体重变化图；Figure 7 is a graph showing the change in body weight of mice in different groups;
图8为不同分组中小鼠肺组织的苏木精-伊红染色(HE染色)图；Figure 8 is a diagram of hematoxylin-eosin staining (HE staining) of mouse lung tissue in different groups;
图9为不同分组中小鼠肺组织病理评分图；Fig. 9 is the pathological scoring diagram of mouse lung tissue in different groups;
图10为不同分组中小鼠肺组织病毒滴度测定结果图；Figure 10 is a graph showing the results of the determination of mouse lung tissue virus titers in different groups;
图11为甲基巴多索隆在小鼠中通过抑制IKKβ/NF-κB通路抑制RSV感染及相关炎症因子产生图。Figure 11 is a graph showing that bardoxolone methyl inhibits RSV infection and the production of related inflammatory factors by inhibiting the IKKβ/NF-κB pathway in mice.
具体实施方式Detailed ways
为了让本领域技术人员更加清楚明白本发明所述技术方案，现列举以下实施例进行说明。需要指出的是，以下实施例对本发明要求的保护范围不构成限制作用。In order to make those skilled in the art understand the technical solution of the present invention more clearly, the following examples are listed for illustration. It should be pointed out that the following examples do not limit the protection scope of the present invention.
以下实施例中所用的原料、试剂或装置如无特殊说明，均可从常规商业途径得到，或者可以通过现有已知方法得到。Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels, or can be obtained by existing known methods.
一、实验方法1. Experimental method
1、细胞实验：1. Cell experiments:
以探究甲基巴多索隆是否可抑制RSV感染，以及甲基巴多索隆是否通过IKKβ/NF-κB通路抑制RSV感染导致的炎症损伤为目的，建立了RSV感染细胞模型，研究甲基巴多索隆通过IKKβ/NF-κB通路抗RSV感染的作用及分子机制。In order to explore whether bardoxolone methyl can inhibit RSV infection, and whether bardoxolone methyl inhibits the inflammatory injury caused by RSV infection through the IKKβ/NF-κB pathway, a RSV infected cell model was established to study the effect and molecular mechanism of bardoxolone methyl against RSV infection through the IKKβ/NF-κB pathway.
(1)BEAS-2B(人支气管上皮细胞)、Hep-2(人喉癌上皮细胞，RSV扩增载体)细胞均购于American Type Culture Collection(ATCC)，细胞株为第2-3代。(1) BEAS-2B (human bronchial epithelial cells), Hep-2 (human laryngeal carcinoma epithelial cells, RSV amplified vector) cells were purchased from American Type Culture Collection (ATCC), and the cell lines were 2-3 passages.
(2)细胞RSV感染模型的制备：(2) Preparation of cell RSV infection model:
复苏BEAS-2B和Hep-2细胞并进行传代，在Hep-2细胞中进行RSV扩增，测定扩增的TCID50，然后以500TCID50 RSV感染BEAS-2B。BEAS-2B and Hep-2 cells were revived and passaged, RSV was amplified in Hep-2 cells, and the amplified TCID50 was measured, and then BEAS-2B was infected with 500 TCID50 RSV.
(3)探讨IKKβ/NF-κB通路相关的分子机制：(3) Explore the molecular mechanism related to the IKKβ/NF-κB pathway:
①以BEAS-2B细胞为实验对象，按图1分为：正常对照组、500TCID50 RSV感染组、500TCID50 RSV+0.1μM甲基巴多索隆组、500TCID50 RSV+0.3μM甲基巴多索隆组，共4组。用倒置显微镜观察BEAS-2B细胞感染RSV后的病变效应；通过CCK8方法检测甲基巴多索隆的细胞毒性和抗病毒效应；通过逆转录聚合酶链式反应(RT-PCR)检测RSV、IL-6、IL-8、IKKβ和NF-κB mRNA表达；通过蛋白免疫印迹(Western blot)检测RSV F、p-IKKβ、p-NF-κB、IL-6蛋白的表达。①Take BEAS-2B cells as the experimental objects, and divide them into 4 groups according to Figure 1: normal control group, 500TCID50 RSV infection group, 500TCID50 RSV+0.1μM bardoxolone methyl group, 500TCID50 RSV+0.3μM bardoxolone methyl group, a total of 4 groups. The pathological effect of BEAS-2B cells infected with RSV was observed with an inverted microscope; the cytotoxicity and antiviral effect of bardoxolone-methyl was detected by CCK8 method; the expression of RSV, IL-6, IL-8, IKKβ and NF-κB mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR); the expression of RSV F, p-IKKβ, p-NF-κB and IL-6 protein was detected by Western blot.
②以IKKβ特异性抑制剂TPCA-1与甲基巴多索隆进行对照实验，实验分为：正常对照组、500TCID50 RSV感染组、500TCID50 RSV+0.3μM甲基巴多索隆组、500TCID50 RSV+0.3μMTPCA-1组，通过RT-PCR检测RSV mRNA表达，证明不是所有的IKKβ抑制剂都可以通过IKKβ/NF-κB通路抑制RSV感染。②The IKKβ-specific inhibitor TPCA-1 and bardoxolone-methyl were used for the control experiment. The experiment was divided into: normal control group, 500TCID50 RSV infection group, 500TCID50 RSV+0.3μM bardoxolone-methyl group, 500TCID50 RSV+0.3μM MTPCA-1 group, RSV mRNA expression was detected by RT-PCR, which proved that not all IKKβ inhibitors could inhibit RS through the IKKβ/NF-κB pathway V infection.
2、动物实验2. Animal experiments
(1)小鼠RSV感染模型制备：(1) Preparation of mouse RSV infection model:
①将健康、雌性SPF级BALB/c小鼠(6-8周龄，体重20-25g)按图2随机分为：野生型组(即未经任何处理的小鼠，为空白对照)、RSV感染组、BXM(3mg/Kg/d)组、RSV感染+低剂量BXM(1mg/Kg/d)组、RSV感染+高剂量BXM(3mg/Kg/d)组、RSV感染+利巴韦林(40mg/Kg/d)阳性对照组(利巴韦林RIB，作为阳性对照)，共6组，每组小鼠6只，用苦味酸给每组小鼠编号。适应性饲养5天后开始实验，小鼠用乙醚麻醉后开始滴鼻造模，RSV感染组、RSV感染+低剂量BXM组、RSV感染+高剂量BXM组、RSV感染+利巴韦林阳性对照组中每只小鼠滴入RSV病毒原液100μL，野生型组及BXM组滴入等体积的生理盐水，连续滴鼻3日，从第4日开始给予腹腔注射药物，连续腹腔注射给药3日后，第7日处理小鼠。每天同一时间检测小鼠体重，密切观察小鼠精神、进食、饮水及活动状态(炸毛、弓背、流涕、咳嗽等)。① Healthy, female SPF grade BALB/c mice (6-8 weeks old, body weight 20-25g) were randomly divided according to Figure 2 into: wild-type group (that is, mice without any treatment, which is the blank control group), RSV infection group, BXM (3mg/Kg/d) group, RSV infection+low-dose BXM (1mg/Kg/d) group, RSV infection+high-dose BXM (3mg/Kg/d) group, RSV infection+ribavirin (4 0 mg/Kg/d) positive control group (ribavirin RIB, as positive control), totally 6 groups, 6 mice in each group, number each group of mice with picric acid. The experiment was started after 5 days of adaptive feeding. The mice were anesthetized with ether and started to make nasal models. In the RSV infection group, RSV infection + low-dose BXM group, RSV infection + high-dose BXM group, RSV infection + ribavirin positive control group, each mouse was dripped with 100 μL of RSV virus stock solution. Handle mice. The weight of the mice was detected at the same time every day, and the spirit, eating, drinking and activity status of the mice were closely observed (fried hair, arched back, runny nose, coughing, etc.).
②肺组织进行匀浆，利用TCID50检测病毒的载量。②The lung tissue was homogenized, and the virus load was detected by TCID50 .
③取出每只小鼠的右肺，进行充气，并用4％中性缓冲甲醛固定。将固定的组织包埋在石蜡中并切成5μm的切片。然后将玻片用HE染色，通过光学显微镜检查组织学变化，根据组织炎症、水肿和细支气管周围炎症的程度评估组织病理学变化。③ The right lung of each mouse was taken out, inflated, and fixed with 4% neutral buffered formaldehyde. Fixed tissues were embedded in paraffin and cut into 5 μm sections. The slides were then stained with HE and examined for histological changes by light microscopy, which were evaluated according to the degree of tissue inflammation, edema, and peribronchiolar inflammation.
④肺组织病理评分④Lung histopathological score
根据肺组织病理评分体系对肺组织HE病理切片进行评分(如表1)，总分＝A+3×(B+C)+D+E，总分0-26分。According to the scoring system of lung tissue pathology, HE pathological sections of lung tissue were scored (Table 1), total score=A+3×(B+C)+D+E, total score 0-26 points.
表1.肺组织病理评分Table 1. Lung histopathological score
(2)探讨IKKβ/NF-κB通路相关的分子机制：(2) Explore the molecular mechanism related to the IKKβ/NF-κB pathway:
利用Western blot检测RSV F、p-IKKβ、p-NF-κB、IL-6蛋白的表达。The expressions of RSV F, p-IKKβ, p-NF-κB and IL-6 proteins were detected by Western blot.
3、实验结果3. Experimental results
(1)扫描电镜验证RSV在Hep-2细胞中扩增(1) Scanning electron microscopy verifies that RSV amplifies in Hep-2 cells
Hep-2细胞适用于多种病毒的培养和增殖。因此，通过扫描电镜观察RSV感染Hep-2细胞后的变化情况，以评估RSV感染后的病毒增殖模型是否构建成功(图3)。结果显示：图3A为正常Hep-2细胞，图3B是RSV感染后的Hep-2细胞，图3B中RSV感染后的Hep-2细胞可见融合病变形成(箭头所指)，证明RSV感染细胞模型成功。Hep-2 cells are suitable for the cultivation and propagation of various viruses. Therefore, the changes of RSV-infected Hep-2 cells were observed by scanning electron microscope to evaluate whether the virus proliferation model after RSV infection was successfully constructed ( FIG. 3 ). The result shows: Fig. 3 A is normal Hep-2 cell, and Fig. 3 B is the Hep-2 cell after RSV infection, and the Hep-2 cell after RSV infection among Fig. 3 B shows fusion lesion formation (pointed by arrow), proves that RSV infects cell model success.
(2)RSV感染Hep-2和BEAS-2B细胞后产生细胞病变效应(2) RSV produces cytopathic effect after infection of Hep-2 and BEAS-2B cells
Hep-2细胞为RSV病毒扩增时所需的细胞，BEAS-2B细胞为实验对象，两种均为贴壁生长的细胞。采用显微镜观察Hep-2和BEAS-2B细胞，结果如图4所示，正常Hep-2细胞呈不规则多角形(图4A)，而正常BEAS-2B细胞呈长梭形(图4B)。RSV感染Hep-2及BEAS-2B细胞后均产生了细胞病变效应(Cytopathic Effect，CPE)，其中Hep-2表现为细胞变圆、脱落、聚集等，部分细胞边界消失，细胞膜发生融合(图4C)；而BEAS-2B表现为细胞被拉长、伸展，部分细胞被拉长后成丝状，细胞与细胞之间间隙增宽(图4D)。Hep-2 cells are the cells required for RSV virus amplification, and BEAS-2B cells are the experimental objects, both of which are adherent growth cells. The Hep-2 and BEAS-2B cells were observed under a microscope. As shown in Figure 4, normal Hep-2 cells were irregularly polygonal (Figure 4A), while normal BEAS-2B cells were long spindle-shaped (Figure 4B). Both Hep-2 and BEAS-2B cells infected by RSV produced cytopathic effects (Cytopathic Effect, CPE). In Hep-2, the cells became round, detached, aggregated, etc., some cell boundaries disappeared, and the cell membrane fused (Figure 4C); while BEAS-2B showed that the cells were elongated and stretched, some cells were elongated and became filamentous, and the gap between cells was widened (Figure 4D).
(3)甲基巴多索隆在BEAS-2B细胞中通过抑制IKKβ/NF-κB通路抑制RSV感染及相关炎症因子产生(3) Bardoxolone methyl inhibits RSV infection and the production of related inflammatory factors by inhibiting the IKKβ/NF-κB pathway in BEAS-2B cells
为了验证甲基巴多索隆(Bardoxolone Methyl，简称BXM)在BEAS-2B细胞中毒性作用，用不同浓度药物分别处理BEAS-2B细胞72小时。结果如图5所示，图5A为甲基巴多索隆在BEAS-2B细胞中的细胞毒性及对RSV抑制效应图，图5B和图5C分别为BXM通过IKKβ/NF-κB通路抑制RSV感染图及感染导致的相关炎症因子表达图。其中，图5中，Cytoxicity为细胞毒性，Inhibition为抑制作用，Relative expression of mRNA为mRNA的相对表达，Relativeprotein expression为相对蛋白质表达。In order to verify the toxic effect of Bardoxolone Methyl (BXM) on BEAS-2B cells, BEAS-2B cells were treated with different concentrations of the drug for 72 hours. The results are shown in Figure 5. Figure 5A is the cytotoxicity of bardoxolone methyl in BEAS-2B cells and its inhibitory effect on RSV. Figure 5B and Figure 5C are the pictures of BXM inhibiting RSV infection through the IKKβ/NF-κB pathway and the expression of related inflammatory factors caused by infection, respectively. Wherein, in Fig. 5, Cytoxicity is cytotoxicity, Inhibition is inhibitory effect, Relative expression of mRNA is relative expression of mRNA, Relative protein expression is relative protein expression.
通过CCK8检测发现，当甲基巴多索隆浓度为0.4μM以下时，BEAS-2B细胞存活率达85％以上，而甲基巴多索隆浓度大于0.8μM以上时，显微镜下观察贴壁细胞有不同程度的皱缩、变形及脱落，计算得到CC50(图5A)，根据上述测得安全范围，选取0.025、0.05、0.1、0.2和0.3μM的甲基巴多索隆，来测定不同剂量的甲基巴多索隆对RSV感染BEAS-2B细胞的EC50(图5A)。RSV感染介导了多条免疫调节通路，通过激活NF-κB信号通路，产生大量炎症介质，引起反复感染加重疾病进程，而甲基巴多索隆是IKKβ抑制剂，因此本发明探讨了甲基巴多索隆是否可通过抑制IKKβ/NF-κB通路抑制RSV感染。Through CCK8 detection, it was found that when the concentration of bardoxolone methyl was below 0.4 μM, the survival rate of BEAS-2B cells was over 85%, and when the concentration of bardoxolone methyl was above 0.8 μM, the adherent cells were observed under the microscope to have different degrees of shrinkage, deformation and shedding, and the CC50 was calculated (Figure 5A). The EC50 of different doses of bardoxolone methyl on RSV-infected BEAS-2B cells was determined ( FIG. 5A ). RSV infection mediates multiple immune regulation pathways. By activating the NF-κB signaling pathway, a large number of inflammatory mediators are produced, causing repeated infections and aggravating the disease process. Bardoxolone methyl is an IKKβ inhibitor. Therefore, the present invention explores whether bardoxolone methyl can inhibit RSV infection by inhibiting the IKKβ/NF-κB pathway.
通过RT-PCR检测发现，RSV感染BEAS-2B细胞24小时后RSV、NF-κB、IKKβ、IL-6、IL-8mRNA表达增加，但当加入甲基巴多索隆后，RSV、NF-κB、IKKβ、IL-6、IL-8mRNA表达下降(*p＜0.05，**p＜0.01为500TCID50RSV感染组与正常对照组相比，#p＜0.05，##p＜0.01为500TCID50RSV+(0.1/0.3μM)甲基巴多索隆组与500TCID50RSV感染组相比)(图5B)。RT-PCR detection found that RSV, NF-κB, IKKβ, IL-6, IL-8mRNA expression increased 24 hours after RSV infected BEAS-2B cells, but after adding bardoxolone methyl, RSV, NF-κB, IKKβ, IL-6, IL-8mRNA expression decreased (*p<0.05, **p<0.01 is 500TCID50 RSV infection group compared with normal control group,# p<0.05,# # p<0.01 for the 500 TCID50 RSV + (0.1/0.3 μM) bardoxolone methyl group compared to the 500 TCID50 RSV infected group) ( FIG. 5B ).
通过Western blot检测发现，BEAS-2B细胞经RSV感染后，RSV F、IL-6、p-NF-κB和p-IKKβ蛋白表达增加，但当加入甲基巴多索隆后，RSV F、IL-6、p-NF-κB和p-IKKβ蛋白表达降低。说明甲基巴多索隆可能通过抑制IKKβ/NF-κB通路来达到抑制RSV感染及炎症因子表达的效果(*p＜0.05，**p＜0.01为500TCID50RSV感染组与正常对照组相比，#p＜0.05，##p＜0.01为500TCID50RSV+(0.1/0.3μM)甲基巴多索隆组与500TCID50RSV感染组相比)(图5C)。By Western blot detection, it was found that the expression of RSV F, IL-6, p-NF-κB and p-IKKβ proteins increased after BEAS-2B cells were infected with RSV, but the expressions of RSV F, IL-6, p-NF-κB and p-IKKβ proteins decreased after adding bardoxolone methyl. It shows that bardoxolone methyl may inhibit RSV infection and expression of inflammatory factors by inhibiting the IKKβ/NF-κB pathway (*p<0.05, **p<0.01 is compared with the normal control group with 500TCID50 RSV infection,# p<0.05,## p<0.01 is 500TCID50 RSV+(0.1/0.3μM) bardoxolone methyl group and 500TCID50 RSV infection group) (Fig. 5C).
(4)其他IKKβ特异性抑制剂对经RSV感染后BEAS-2B细胞的作用(4) Effects of other IKKβ-specific inhibitors on BEAS-2B cells infected by RSV
以IKKβ特异性抑制剂TPCA-1与甲基巴多索隆进行对照实验，处理BEAS-2B细胞24小时。Control experiments were performed with IKKβ-specific inhibitor TPCA-1 and bardoxolone-methyl, and BEAS-2B cells were treated for 24 hours.
结果表明：BEAS-2B细胞经RSV感染后，RSV mRNA表达明显增加(**P＜0.01)，甲基巴多索隆作为IKKβ抑制剂可以抑制RSV mRNA表达(##P＜0.01)(图6，其中图6中Relativeexpression of mRNA为mRNA的相对表达)，而IKKβ特异性抑制剂TPCA-1不能抑制RSV mRNA表达(P＞0.05)。以上结果表明并非所有IKKβ抑制剂都可抑制RSV mRNA表达。The results showed that: after BEAS-2B cells were infected with RSV, the expression of RSV mRNA was significantly increased (** P<0.01), and bardoxolone methyl, as an IKKβ inhibitor, could inhibit the expression of RSV mRNA (## P<0.01) (Figure 6, where the Relative expression of mRNA in Figure 6 is the relative expression of mRNA), while the IKKβ-specific inhibitor TPCA-1 could not inhibit the expression of RSV mRNA (P>0.05). The above results indicated that not all IKKβ inhibitors could inhibit RSV mRNA expression.
(5)小鼠体重变化(5) Changes in body weight of mice
将SPF级BALB/c雌性小鼠按图2方法进行分组(共6组，分别为野生型组(即未经任何处理的小鼠，为空白对照)、RSV感染组、BXM(3mg/Kg/d)组、RSV感染+低剂量BXM(1mg/Kg/d)组、RSV感染+高剂量BXM(3mg/Kg/d)组、RSV感染+利巴韦林(40mg/Kg/d)阳性对照组)及处理。每日同一时间将小鼠进行称重，结果如图7所示，观察发现，野生型组及BXM组小鼠毛色顺滑、行为活泼、进食及饮水正常，BXM组小鼠起初体重略有下降，而后体重逐渐保持平稳，但精神行为和饮食无明显变化。RSV感染组小鼠进行滴鼻实验后均出现体重逐渐下降、毛发干枯紊乱、精神差、不活泼、喜欢聚堆，而且进食量和饮水量均减少。RSV感染+低/高剂量BXM组小鼠起初表现与RSV感染组相似，但从第5天开始，经甲基巴多索隆和利巴韦林处理的RSV感染组小鼠的体重逐渐上升，精神行为异常逐渐改善，在RSV感染+高剂量BXM组(3mg/Kg/d)改善最明显，RSV感染+低剂量BXM组(1mg/Kg/d)与RSV感染+利巴韦林组(40mg/Kg/d)结果较为接近。说明甲基巴多索隆能抑制RSV感染小鼠，当BXM的剂量为3mg/Kg/d时效果最为显著，当BXM的剂量为1mg/Kg/d时的效果可以达到与阳性对照利巴韦林相当的水平。其中图7中，Virus为RSV感染，Drugs为施用甲基巴多索隆，Treat为治疗，Weight of Mice为小鼠的重量。SPF grade BALB/c female mice were grouped according to the method in Figure 2 (6 groups in total, respectively wild-type group (i.e. mice without any treatment, as blank control), RSV infection group, BXM (3mg/Kg/d) group, RSV infection+low dose BXM (1mg/Kg/d) group, RSV infection+high dose BXM (3mg/Kg/d) group, RSV infection+ribavirin (40mg/Kg/d) positive control group) and processing. The mice were weighed at the same time every day, and the results are shown in Figure 7. It was observed that the mice in the wild-type group and the BXM group had smooth fur, lively behavior, and normal eating and drinking. The mice in the BXM group initially lost weight slightly, and then gradually maintained a stable body weight, but there was no significant change in mental behavior and diet. After the nasal drop test, the mice in the RSV-infected group showed gradual weight loss, dry and disordered hair, poor energy, inactivity, like to gather together, and decreased food intake and water intake. The mice in the RSV infection+low/high dose BXM group behaved similarly to the RSV infection group at first, but from the 5th day, the body weight of the RSV infection group mice treated with bardoxolone methyl and ribavirin gradually increased, and the abnormal mental behavior gradually improved. The improvement was the most obvious in the RSV infection+high dose BXM group (3mg/Kg/d). relatively close. Explain that bardoxolone methyl can inhibit RSV infection mice, and when the dose of BXM is 3mg/Kg/d, the effect is the most significant, and when the dose of BXM is 1mg/Kg/d, the effect can reach a level equivalent to that of the positive control ribavirin. Among them, in Fig. 7, Virus is RSV infection, Drugs is the administration of bardoxolone methyl, Treat is treatment, and Weight of Mice is the weight of the mice.
(6)小鼠肺组织病理变化及评分(6) Pathological changes and scoring of mouse lung tissue
将SPF级BALB/c雌性小鼠按图2中的实验方法进行分组(野生型组、RSV感染组、BXM(3mg/Kg/d)组、RSV感染+低剂量BXM(1mg/Kg/d)组、RSV感染+高剂量BXM(3mg/Kg/d)组、RSV感染+利巴韦林(40mg/Kg/d)阳性对照组)及处理。第7天处死小鼠，取小鼠右肺下叶以4％多聚甲醛固定48h后进行HE染色，结果如图8所示，其中图8A为正常对照组，图8B为RSV感染组，图8C为RSV感染+低剂量BXM组(1mg/Kg/d)，图8D为RSV感染+高剂量BXM组(3mg/Kg/d)，图8E为RSV感染+利巴韦林阳性对照组(40mg/Kg/d)，图8F为BXM组(3mg/Kg/d)，各组上层图片放大倍数为200，下层图片放大倍数为400。SPF grade BALB/c female mice were grouped according to the experimental method in Figure 2 (wild type group, RSV infection group, BXM (3mg/Kg/d) group, RSV infection+low dose BXM (1mg/Kg/d) group, RSV infection+high dose BXM (3mg/Kg/d) group, RSV infection+ribavirin (40mg/Kg/d) positive control group) and treatment. The mice were sacrificed on the 7th day, and the lower lobe of the right lung of the mice was fixed with 4% paraformaldehyde for 48h and carried out HE staining. The results are shown in Figure 8, wherein Figure 8A is the normal control group, Figure 8B is the RSV infection group, Figure 8C is the RSV infection+low dose BXM group (1mg/Kg/d), Figure 8D is the RSV infection+high dose BXM group (3mg/Kg/d), Figure 8E is the RSV infection+ribavirin positive control group (40mg/Kg/d) ), Fig. 8F is the BXM group (3mg/Kg/d), the magnification of the upper picture of each group is 200, and the magnification of the lower picture is 400.
图8结果显示：野生型组和BXM组小鼠肺组织、肺泡间隔正常(图8A、图8F)。RSV感染组与野生型组相比，RSV感染组肺组织明显炎症细胞浸润、有核细胞增多、肺泡间隔增厚、肺泡壁断裂损伤、支气管小血管炎症细胞浸润、支气管伴渗出(图8B)。而RSV感染+低/高剂量BXM组与RSV感染组相比，肺组织炎症细胞浸润减少、肺泡壁完整、肺泡间隔增厚减少、支气管小血管炎症浸润减少，在RSV感染+高剂量BXM组(3mg/Kg/d)缓解效果最为明显(图8D)，而RSV感染+低剂量BXM组(1mg/Kg/d)(图8C)较RSV感染+高剂量BXM组(3mg/Kg/d)(图8D)效果欠佳。在RSV感染+利巴韦林阳性对照组(40mg/Kg/d)也存在炎症缓解表现(图8E)，但较RSV感染+高剂量BXM组(3mg/Kg/d)效果稍欠佳。The results in Fig. 8 show that the lung tissues and alveolar septa of mice in the wild-type group and BXM group are normal (Fig. 8A, Fig. 8F). Compared with the wild-type group, the RSV-infected group had obvious inflammatory cell infiltration, increased nucleated cells, thickened alveolar septum, broken alveolar wall, inflammatory cell infiltration in small bronchial vessels, and bronchi with exudation in the RSV-infected group (Fig. 8B). Compared with the RSV infection group, the RSV infection + low/high dose BXM group had less inflammatory cell infiltration, alveolar wall integrity, less alveolar septal thickening, and less bronchovascular inflammatory infiltration. The RSV infection + high dose BXM group (3mg/Kg/d) had the most obvious relief effect (Fig. /d) (Fig. 8D) was less effective. In the RSV infection + ribavirin positive control group (40mg/Kg/d), there was also inflammation relief (Figure 8E), but the effect was slightly less than that of the RSV infection + high-dose BXM group (3mg/Kg/d).
此外，还进行了肺组织病理评分(表1、图9)，统计可见RSV感染+低/高剂量BXM组和RSV感染+利巴韦林阳性对照组一样，可明显降低小鼠肺组织炎症情况(感染组与正常组比较，**p＜0.01；药物组与感染组比较，##p＜0.01)，而且RSV感染+高剂量BXM组(3mg/Kg/d)较RSV感染+低剂量BXM组(1mg/Kg/d)效果更加明显(▲▲p＜0.01)。其中图9中Histology scoreof lung tissue为肺组织的组织学评分。In addition, lung histopathological scores were also carried out (Table 1, Figure 9). Statistics showed that the RSV infection+low/high dose BXM group was the same as the RSV infection+ribavirin positive control group, which could significantly reduce the inflammation of the mouse lung tissue (compared with the normal group, **p<0.01; compared with the drug group and the infection group,## p<0.01), and the RSV infection+high dose BXM group (3mg/Kg/d) was significantly lower than the RSV infection+low dose BXM group (1mg/Kg /d) The effect is more obvious (▲▲ p＜0.01). Wherein the Histology score of lung tissue in Fig. 9 is the histological score of the lung tissue.
(7)小鼠肺组织病毒载量(7) Viral load in mouse lung tissue
进一步检测了小鼠肺组织中RSV病毒滴度变化情况，通过TCID50方法测定肺组织病毒滴度。结果显示：RSV感染组中肺组织RSV病毒滴度最高，经甲基巴多索隆和利巴韦林药物处理后病毒滴度明显下降(药物组包括：RSV感染+低剂量BXM(1mg/Kg/d)组、RSV感染+高剂量BXM(3mg/Kg/d)组、RSV感染+利巴韦林(40mg/Kg/d)阳性对照组，药物组与RSV感染组对比，#p＜0.05，##p＜0.01)，RSV感染+高剂量BXM组(3mg/Kg/d)的病毒滴度比RSV感染+低剂量BXM组(1mg/Kg/d)下降更为明显(**p＜0.01)(图10，其中Virus Titer为病毒滴度)。The change of RSV virus titer in mouse lung tissue was further detected, and the virus titer of lung tissue was determined by TCID50 method.结果显示：RSV感染组中肺组织RSV病毒滴度最高，经甲基巴多索隆和利巴韦林药物处理后病毒滴度明显下降(药物组包括：RSV感染+低剂量BXM(1mg/Kg/d)组、RSV感染+高剂量BXM(3mg/Kg/d)组、RSV感染+利巴韦林(40mg/Kg/d)阳性对照组，药物组与RSV感染组对比，# p＜0.05，## p＜0.01)，RSV感染+高剂量BXM组(3mg/Kg/d)的病毒滴度比RSV感染+低剂量BXM组(1mg/Kg/d)下降更为明显(**p＜0.01)(图10，其中Virus Titer为病毒滴度)。
(8)甲基巴多索隆在小鼠中通过抑制IKKβ/NF-κB通路抑制RSV感染及相关炎症因子产生(8) Bardoxolone methyl inhibits RSV infection and the production of related inflammatory factors by inhibiting the IKKβ/NF-κB pathway in mice
病毒入侵机体后产生大量炎性介质使机体损伤，在细胞实验中本发明已验证甲基巴多索隆可抑制RSV感染并通过抑制IKKβ/NF-κB通路抑制炎性因子产生。因此，本发明进一步在RSV感染小鼠模型中提取小鼠肺组织通过Western blot检测RSV F、p-IKKβ、p-NF-κB及IL-6蛋白的表达情况。结果显示：RSV感染组的RSV F蛋白表达水平较野生型组升高(图11A和11C，**p＜0.01)；RSV感染+低剂量BXM(1mg/Kg/d)组和RSV感染+高剂量BXM(3mg/Kg/d)组能明显抑制RSV F蛋白表达水平(图11A和11C，##p＜0.01)，而RSV感染+利巴韦林(40mg/Kg/d)阳性对照组虽较RSV感染组RSV F蛋白表达有所下降，但无统计学意义(图11A和11C，p＞0.05)，表明甲基巴多索隆在体内也能抑制RSV感染。本发明进一步检测IKKβ/NF-κB通路相关蛋白表达，结果显示：RSV感染组与野生型组相比，p-IKKβ、p-NF-κB及IL-6的蛋白表达水平明显升高(图11A-11B，图11D-11F，*p＜0.05，**p＜0.01)，野生型组和BXM组的p-IKKβ、p-NFκB及IL-6蛋白表达水平一致，但感染后RSV感染+低剂量BXM(1mg/Kg/d)组、RSV感染+高剂量BXM(3mg/Kg/d)组和RSV感染+利巴韦林(40mg/Kg/d)阳性对照组处理的p-IKKβ、p-NF-κB及IL-6的蛋白表达水平较RSV感染组明显下降(图11A-11B，图11D-11F，#p＜0.05，##p＜0.01)。以上结果说明，甲基巴多索隆在小鼠中通过抑制IKKβ/NF-κB通路以达到抑制RSV感染及相关炎症因子表达的效果(图11中，Relative protein expression为相对蛋白质表达)。其中图11中Relative protein expression为相对蛋白质表达。After the virus invades the body, a large number of inflammatory mediators are produced to damage the body. In cell experiments, the present invention has verified that bardoxolone methyl can inhibit RSV infection and inhibit the production of inflammatory factors by inhibiting the IKKβ/NF-κB pathway. Therefore, the present invention further extracts mouse lung tissue in the RSV-infected mouse model and detects the expressions of RSV F, p-IKKβ, p-NF-κB and IL-6 proteins by Western blot. The result shows: the expression level of RSV F protein of RSV infection group is higher than that of wild type group (Fig. 11A and 11C,** p＜0.01); /d) Although the expression of RSV F protein in the positive control group was lower than that in the RSV infection group, there was no statistical significance (Figure 11A and 11C, p>0.05), indicating that bardoxolone methyl can also inhibit RSV infection in vivo.本发明进一步检测IKKβ/NF-κB通路相关蛋白表达，结果显示：RSV感染组与野生型组相比，p-IKKβ、p-NF-κB及IL-6的蛋白表达水平明显升高(图11A-11B，图11D-11F，*p＜0.05，**p＜0.01)，野生型组和BXM组的p-IKKβ、p-NFκB及IL-6蛋白表达水平一致，但感染后RSV感染+低剂量BXM(1mg/Kg/d)组、RSV感染+高剂量BXM(3mg/Kg/d)组和RSV感染+利巴韦林(40mg/Kg/d)阳性对照组处理的p-IKKβ、p-NF-κB及IL-6的蛋白表达水平较RSV感染组明显下降(图11A-11B，图11D-11F，# p＜0.05，## p＜0.01)。 The above results indicate that bardoxolone methyl inhibits RSV infection and the expression of related inflammatory factors by inhibiting the IKKβ/NF-κB pathway in mice (in Figure 11, Relative protein expression is the relative protein expression). The Relative protein expression in Figure 11 is the relative protein expression.
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| CN202310407222.4ACN116473979A (en) | 2023-04-14 | 2023-04-14 | Application of Bardoxolone Methyl in the Preparation of Drugs for Inhibiting Respiratory Syncytial Virus Infection |
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| CN202310407222.4ACN116473979A (en) | 2023-04-14 | 2023-04-14 | Application of Bardoxolone Methyl in the Preparation of Drugs for Inhibiting Respiratory Syncytial Virus Infection |
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| Title |
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| ANTONIO CUADRADO 等: "Can Activation of NRF2 Be a Strategy against COVID-19?", TRENDS IN PHARMACOLOGICAL SCIENCES, vol. 41, no. 9, 30 September 2020 (2020-09-30), pages 605, XP093141586, DOI: 10.1016/j.tips.2020.07.003* |
| HYE-YOUN CHO 等: "Antiviral Activity of Nrf2 in a Murine Model of Respiratory Syncytial Virus Disease", AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, vol. 179, 31 December 2009 (2009-12-31), pages 146* |
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