山芋丸治“痹症”模型大鼠机制的网络药理及实验验证

Network Pharmacology and Experimental Validation of the Mechanism of ShanYuWan in Treating "Arthromyodynia" Model Rats

关键词:  山芋丸  痹症  类风湿关节炎  网络药理学  实验验证

Key words:  ShanYuWan  arthromyodynia  rheumatoid arthritis  network pharmacology  experimental verification

摘要:

摘 要：目的 通过网络药理学和动物实验初步探究山芋丸对佐剂大鼠踝关节痹症的治疗作用及机制，并从中医“痹症”与“扶正祛邪”的角度探其理论基础。方法 通过TCMSP数据库对山药、白术、人参活性成分进行筛选与靶点预测。在GeneCards、OMIM、DrugBank中进行类风湿关节炎（RA）靶点的检索及筛选。利用Cytoscape 3.9.1构建“药物-活性成分-疾病”网络图。将交集靶点输入到STRING数据库分析获得蛋白互作网络，导入Cytoscape 3.9.1软件进行可视化处理。使用Metascape对交集靶点进行GO及KEGG富集分析。通过AutoDock软件进行分子对接，并用Pymol进行结果可视化。采用弗氏完全佐剂（CFA）造佐剂性关节炎大鼠模型，将36只SD大鼠随机分为正常对照组、模型组、阳性药组、山芋丸低剂量组（4.61 g/kg）、山芋丸中剂量组（9.22 g/kg）、山芋丸高剂量组（18.43 g/kg）；阳性药组大鼠每周灌胃一次1.5 mg/kg的甲氨蝶呤，山芋丸每天灌胃。测量大鼠的踝关节直径，计算大鼠的踝关节肿胀度；取大鼠的踝关节进行组织病理学观察，使用TUNEL细胞凋亡试剂检测大鼠滑膜细胞的凋亡程度，并测定血清中IL-6，TNF-α，MDA的含量，取大鼠脾脏并计算脾脏指数，采用qRT-PCR验证滑膜组织中MAPK3、Caspase3 mRNA的表达，蛋白免疫印迹法检测踝关节组织中MAPK3表达水平。结果 筛选出54个有效成分，对应于山芋丸的325个靶标。此外，845个基因与RA密切相关，其中有86个与山芋丸的靶点重叠。功能富集分析表明，山芋丸通过调节多种途径发挥其在类风湿关节炎中的治疗作用。给药组肿胀度相较于模型组明显降低（P＜0.05）。HE染色结果显示山芋丸能抑制滑膜肿胀程度。TUNEL细胞凋亡的结果显示，给药山芋丸后能够抑制滑膜细胞凋亡。与模型组相比，给药组的IL-6，TNF-α，MDA水平以及脾脏指数均降低(P＜0.05)；大鼠踝关节组织中的MAPK3、Caspase 3 mRNA表达和MAPK3蛋白表达也明显降低(P＜0.05)。结论 山芋丸能减轻佐剂模型大鼠的踝关节破坏，降低关节炎症，可能通过“扶正祛邪”进而治疗痹症。

Abstract:

Abstract Objective: To investigate the therapeutic effects of ShanYuWan on ankle arthrogryposis in adjuvant rats through animal experiments, and to explore the theoretical basis of using ShanYuWan to treat RA from the perspective of “arthromyodynia” and “Therapy of Health Strengthening and Pathological Factors Resistanc” in traditional Chinese medicine (TCM). Methods:Screening and target prediction of active ingredients from Dioscorea oppositifolia L., Atractylodes macrocephala, and Panax ginseng C. A. Mey through the TCMSP database.Search and screen rheumatoid arthritis (RA) targets in GeneCards, OMIM, and DrugBank. Construct a "drug-active ingredient-disease" network diagram using Cytoscape 3.9.1. Input the intersection targets into the STRING database for analysis to obtain protein interaction networks, and import them into Cytoscape 3.9.1 software for visualization processing. Use Metascape to perform GO and KEGG enrichment analysis on intersecting targets, and then use a microbiome platform for enrichment analysis visualization.Perform molecular docking using AutoDock software and visualize the results using Pymol. A rat model of adjuvant arthritis was established using Freund's complete adjuvant. Thirty six SD rats were randomly divided into a normal control group, a model group, a positive drug group, a low dose group of ShanYuWan (4.61 g/kg), a medium dose group of ShanYuWan (9.22 g/kg), and a high dose group of ShanYuWan (18.43 g/kg); Positive drug group rats were given 1.5 mg/kg of methotrexate by gavage once a week, while ShanYuWan were given daily. Measure the diameter of the ankle joint of rats and calculate the swelling degree of the ankle joint; Take the ankle joint of rats for histopathology observation, use TUNEL reagent to detect the degree of apoptosis of synovial cells of rats, and determine IL-6, TNF-α, The content of MDA was measured by taking rat spleen and calculating spleen index. The expression of MAPK3, and Caspase3 mRNA in synovial tissue was detected using qRT PCR. Result: 45 effective ingredients were selected, corresponding to 325 targets of ShanYuWan. In addition, 845 genes are closely related to RA, of which 86 overlap with the target of ShanYuWan. Functional enrichment analysis indicates that ShanYuWan exerts its therapeutic effect in rheumatoid arthritis by regulating multiple pathways. The swelling degree of the medication group was significantly reduced compared to the model group (P<0.05). HE staining results showed that ShanYuWan can inhibit the degree of synovial swelling. The results of TUNEL staining showed that administration of ShanYuWan can inhibit synovial cell apoptosis. Compared with the model group, IL-6, TNF-α, MDA level and spleen index decreased (P<0.05);The expression of MAPK3, Caspase 3 mRNA and MAPK3 protein in the ankle joint tissue of rats were also significantly reduced (P<0.05). Conclusion: ShanYuWan can attenuate ankle joint destruction in adjuvant model rats, which may help “Therapy of Health Strengthening and Pathological Factors Resistanc” and then treat arthromyodynia.