目的 基于网络药理学和分子对接探讨羽扇豆醇(Lupeol)治疗类风湿关节炎(Rheumatoid arthritis，RA)的作用机制。方法 通过Swiss和TCMSP分析平台获取羽扇豆醇的作用靶点，在GeneCards数据库检索"RA"获取其相关靶点，并转化为相应的标准化基因名。利用R包绘制羽扇豆醇和RA共同靶点的韦恩图，获取交集基因。将交集基因导入STRING数据库，构建蛋白-蛋白相互作用(Protein-protein interactions，PPI)网络。通过clusterProfiler数据库进行京都基因(Geneontology，GO)和基因组百科全书(Kyoto encyclopedia of genes and gnomes，KEGG)通路富集分析。通过分子对接评价羽扇豆醇与AR、CASP3和CCNB1的结合作用。构建RA小鼠模型，测量各组小鼠的足体积，HE染色检测病理学变化，ELISA试剂盒检测小鼠血清TNF-α、IL-1β和IL-6的表达水平，采用Western blot检测各组小鼠Bcl-2、Bax、Casp3、Casp9的蛋白表达水平。结果 获取羽扇豆醇40个作用靶点，RA疾病4734个相关靶点，羽扇豆醇-RA共同靶点27个，PPI网络自由度靠前的3个基因为AR、CASP3和CCNB1，GO富集结果是291个和KEGG通路富集为20条信号通路。分子对接显示羽扇豆醇与AR、CASP3和CCNB1亲和作用较好。第8、12、16、20天模型组小鼠足体积显著高于正常对照组(P<0。05)，相较于模型组，羽扇豆醇组在第8、12、16、20天时小鼠足体积显著降低(P<0。05)，双氯芬酸钠组在第12、16、20天时小鼠足体积显著降低(P<0。05)。HE染色结果表明，与模型组相比，羽扇豆醇药物组明显改善病理学状态。与模型组相比，羽扇豆醇药物组小鼠血清中TNF-α、IL-1β和IL-6水平降低(P<0。01)。Western blot结果显示，与正常组相比，模型组小鼠Bax、Casp3和Casp9蛋白表达显著降低(P<0。05)，Bcl-2的蛋白表达显著升高(P<0。05)。与模型组比较，双氯芬酸钠组和羽扇豆醇药物组小鼠Bax、Casp3和Casp9蛋白表达显著上调(P<0。05)，Bcl-2蛋白表达显著下调(P<0。05)。结论 羽扇豆醇可通过调控p53信号通路中的Bax、Bcl-2、Casp3和Casp9从而发挥对RA的治疗作用。
Study on the Mechanism of Lupeol in the Treatment of Rheumatoid Arthritis Based on Network Pharmacology and Molecular Docking
Objective To explore the mechanism of lupeol in the treatment of rheumatoid arthritis(RA)based on network pharmacology and molecular docking.Methods The target of lupeol was obtained by Swiss and TCMSP analysis platform,and the RA related target was obtained by searching"rheumatoid arthritis"in GeneCards database,which was transformed into the corresponding standardized gene name by UniProt database.The common targets of lupeol and RA were mapped by R-package,and the cross genes were obtained.The cross genes were introduced into the string database to construct protein protein interactions(PPI)network.The clusterProfiler database was used to analyze the enrichment of GO and KEGG pathways.The binding of lupeol with AR,CASP3 and CCNB1 was evaluated by molecular docking.The RA mouse model was established and the foot volume of each group was measured.HE staining for pathological changes,ELISA kit for detecting mouse serum TNF-α,IL-1β and IL-6 expression levels.The protein expression levels of Bcl-2,Bax,CASP3 and CASP9 were detected by Western blot.Results Forty targets of lupeol,4734 related targets of RA disease and 27 common targets of lupeol RA were obtained.The top three genes of PPI network were AR,CASP3 and CCNB1.291 GO and 20 KEGG pathways were enriched.Molecular docking showed that lupeol had good affinity with AR,CASP3 and CCNB1.The foot volume of the model group was significantly higher than that of the normal control group on the 8th,12th,16th and 20th day(P<0.05).Compared with the model group,the foot volume ofthe lupeol group was significantly lower on the 8th,12th,16th and 20th day(P<0.05),andthat ofthe diclofenac group was significantly lower onthe 12th,16th and 20th day(P<0.05).The HE staining results showed that compared with the model group,the lupine alcohol drug group significantly improved the pathological state.Compared with the model group,the level of TNF-α,IL-1β and IL-6 in serum of mice in the Lupeol drug group decreased(P<0.01).WB results showed that compared with the normal group,the protein expressions of Bax,CASP3 and CASP9 in the model group were significantly decreased(P<0.05),and the protein expression of Bcl-2 was significantly increased(P<0.05).Compared with the model group,the protein expression of Bax,CASP3 and CASP9 in the positive control group and drug group were significantly up-regulated(P<0.05),and the protein expression of Bcl-2 was significantly down regulated(P<0.05).Conclusion Lupeol plays a therapeutic role in RA by regulating Bax,Bcl-2,Casp3 and Casp9 in the p53 signaling pathway.
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