Abstract:

Objective: To explore the target of anti-knee osteoarthritis (KOA) in the effective chemical compounds of piper longum L based on network pharmacological methods.

Methods: The active chemical compounds of piper longum L were collected employing database retrieval on TCMSP, TCM-PTD, and literature mining. The Swiss Target Prediction service predicts the targets of active chemical compounds, and at the same time, the targets of the drugs treating knee osteoarthritis were collected by retrieving the OMIM and CTD databases. The targets were subjected to an alignment analysis to screen out piperine and we simulated the binding sites in vivo of compounds and proteins via AutoDock. After that, the rat models of knee osteoarthritis were established. The rats in model groups were given piperine treatment. The verification of the anti-KOA target PPARG and MAPK1 was done by Western blot and co-immunoprecipitation.

Results: Nine active ingredients were predicted. According to Lipinski's rule, piperine was speculated as a possible active ingredient. According to the possible targets of piperine and the KOA's possible targets, three co-targets of them were confirmed, PPARG and MAPK1 were related to knee osteoarthritis (KOA). Molecular docking results show that piperine can hinder the binding of PPARG protein ARG-212 and GLN-420 amino-acid residues to each other. After 20 weeks of piperine treating, Western blot found that piperine can significantly increase the expression level of PPARG and reduce the expression level of MAPK1 in model rats. The endogenous interaction between PPARG and MAPK1 was verified by co-immunoprecipitation.

Conclusion: Piper longum L can regulate the progression of knee osteoarthritis (KOA) by its active ingredient piperine，can affect the expression of PPARG and MAPK1 proteins, and PPARG and MAPK1 proteins have endogenous interactions.

摘要：

目的：基于网络药理学方法，探讨长L有效成分中抗膝骨关节炎的目标。

方法：利用TCMSP，TCM-PTD数据库检索和文献挖掘等方法，收集长材L的活性化学成分。Swiss Target Prediction服务可预测活性化合物的目标，同时，可通过检索OMIM和CTD数据库来收集治疗膝盖骨关节炎的药物的目标。对靶标进行比对分析以筛选出胡椒碱，并且我们通过AutoDock模拟了体内化合物和蛋白质的结合位点。此后，建立了大鼠膝骨关节炎的模型。模型组大鼠给予胡椒碱治疗。抗KOA靶标PPARG和MAPK1的验证通过蛋白质印迹和免疫共沉淀进行。

结果：预测了九种有效成分。根据Lipinski的规则，推测胡椒碱是一种可能的活性成分。根据胡椒碱的可能靶标和KOA的可能靶标，确定了它们的三个共同靶标，PPARG和MAPK1与膝骨关节炎（KOA）有关。分子对接结果表明，胡椒碱可阻碍PPARG蛋白ARG-212和GLN-420氨基酸残基彼此结合。胡椒碱处理20周后，Western blot发现，胡椒碱可显着提高模型大鼠PPARG的表达水平并降低MAPK1的表达水平。通过共免疫沉淀法验证了PPARG和MAPK1之间的内源性相互作用。

结论：胡椒提取物有效成分胡椒碱可以调节膝关节骨性关节炎的发生，影响PPARG和MAPK1蛋白的表达，PPARG和MAPK1蛋白具有内源性相互作用。