目的 基于网络药理学探讨金藤清痹颗粒对大鼠膝骨关节炎（knee osteoarthritis，KOA）的影响及与促进线粒体自噬改善铁死亡的相关性。方法 通过中药系统药理数据库和分析平台（traditional Chinese medicine systems pharmacology database and analysis platform，TCMSP）数据库搜集金藤清痹颗粒的活性成分及靶点，利用人类基因数据库（Gene Cards）、在线人类孟德尔遗传数据库（online Mendelian inheritance in Man，OMIM）等数据库搜集KOA及金藤清痹颗粒药物作用靶点，构建蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络和“中药-活性成分-靶点”网络。应用Autodock软件对筛选出的有效成分和关键靶点进行分子对接。将大鼠随机分为对照组、假手术组、模型组、双氯芬酸钠（5 mg/kg）组和金藤清痹颗粒高、中、低（5.4、2.7、1.4 g/kg）剂量组，各组ig相应药物，1次/d，给药4周后取材。ELISA检测白细胞介素-1β（interleukin-1β，IL-1β）、IL-6、肿瘤坏死因子α（tumornecrosisfactor-α，TNF-α）的含量；苏木素-伊红（hematoxylin-eosin，HE）染色评估软骨病理变化；蛋白免疫印迹（Western blotting，WB）法检测半胱氨酸天冬氨酸蛋白酶-1（cystein-asparate protease-1，Caspase-1）、核因子-κB（nuclear factor-κB，NF-κB）、磷酸酶及张力蛋白同源物诱导的蛋白激酶1（PTEN-induced putative kinase 1，Pink1）、帕金抗体（Parkinson disease，Parkin）、泛素结合蛋白（sequestosome 1，SQSTM1/p62）、膜铁转运蛋白1（ferroportin 1，FPN1）、谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）、酰基辅酶A合成酶长链家族成员4（acyl-CoA synthetase long chain family member 4，ACSL4）、铁蛋白（ferritin）、转铁蛋白受体蛋白1（transferrin receptor protein 1，TFR1）、过氧化还原酶3重组蛋白（recombinant peroxiredoxin 3，PRDX3）的表达。结果 数据库共检索到金藤清痹颗粒116种活性成分、268个作用靶点，共有靶点107个，关键靶点涉及NF-κB信号通路、线粒体自噬信号通路等；分子对接结果提示靶蛋白与小分子之间存在结合位点且结合能较低，提示有较强的结合活性。动物实验结果表明，与对照组比较，模型组大鼠IL⁃1β、IL⁃6、TNF⁃α水平显著升高（P＜0.01、0.001）；膝关节软骨病理组织增生明显；Pro-Caspase-1、p-NF-κB、NF-κB、p62、ASL4、TFR1、PRDX3表达量显著上升（P＜0.05）；Pink1、Parkin、FPN1、GPX4、ferritin表达量显著下降（P＜0.05），自噬蛋白Beclin、LC3BII/I的值显著下降（P＜0.05）；与模型组比较，金藤清痹颗粒各组病理形态损伤减轻；IL-1β、IL-6、TNF-α，Pro-Caspase-1、p-NF-κB、NF-κB、p62、ASL4、TFR1、PRDX3、NCOA4显著降低（P＜0.05）；Pink1、Parkin、FPN1、GPX4、ferritin及自噬蛋白表达量Beclin、LC3BⅡ/Ⅰ显著升高（P＜0.05）。结论 金藤清痹颗粒可能与增强线粒体自噬，改善铁死亡，抑制NF-κB炎症信号等实现对膝骨关节炎的治疗。

Objective To explore the effect of Jinteng Qingbi Granules on knee osteoarthritis (KOA) in rats based on network pharmacology and its correlation with promoting mitophagy to improve ferroptosis. Methods The active components and targets of Jinteng Qingbi Granules were collected through the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) database. The targets of KOA and Jinteng Qingbi Granules were collected using databases such as the GeneCards and the online Mendelian inheritance in Man (OMIM). Protein-protein interaction (PPI) network and “traditional Chinese medicine-active component-target” networks were constructed. The Autodock software was used for molecular docking of the screened effective components and key targets. Rats were randomly divided into control group, sham group, model group, diclofenac sodium (5 mg/kg) group and high-, medium- and low-dose (5.4, 2.7, 1.4 g/kg) Jinteng Qingbi Granule groups. Each group was administered the corresponding drug by intragastric administration once a day. After four weeks of administration, samples were collected. The contents of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were detected by ELISA. The pathological changes of cartilage were evaluated by hematoxylin-eosin (HE) staining. The expressions of cystein-asparate protease-1 (Caspase-1), nuclear factor-κB (NF-κB), PTEN-induced putative kinase 1 (Pink1), Parkinson disease (Parkin), sequestosome 1 (SQSTM1/p62), ferroportin 1 (FPN1), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long chain family member 4 (ACSL4), ferritin, transferrin receptor protein 1 (TFR1), and recombinant peroxiredoxin 3 (PRDX3) were detected by Western blotting (WB). Results A total of 116 active components and 268 action targets of Jinteng Qingbi Granules were retrieved from the database, with 107 common targets. The key targets were involved in the NF-κB signaling pathway, mitophagy signaling pathway, etc. Molecular docking results suggested that binding sites existed between the target proteins and small molecules, with low binding energy, indicating strong binding activity. The results of animal experiments showed that compared with the control group, the levels of IL-1β, IL-6, and TNF-α in the model group were significantly increased (P < 0.01, 0.001), the pathological tissue hyperplasia of knee joint cartilage was obvious, the expression levels of Pro-Caspase-1, p-NF-κB, NF-κB, p62, ASL4, TFR1 and PRDX3 were significantly increased (P < 0.05), the expression levels of Pink1, Parkin, FPN1, GPX4, and ferritin were significantly decreased (P < 0.05), and the values of autophagy proteins Beclin and LC3BII/I were significantly decreased (P < 0.05). Compared with the model group, the pathological morphological damage in each group of Jinteng Qingbi Granules was alleviated, the levels of IL-1β, IL-6, TNF-α, Pro-Caspase-1, p-NF-κB, NF-κB, p62, ASL4, TFR1, PRDX3 and NCOA4 were significantly decreased (P < 0.05), and the expression levels of Pink1, Parkin, FPN1, GPX4, ferritin, and autophagy proteins Beclin and LC3BⅡ/Ⅰ were significantly increased (P < 0.05). Conclusion Jinteng Qingbi Granules may treat knee osteoarthritis by enhancing mitophagy, improving ferroptosis, and inhibiting NF-κB inflammatory signaling pathway.

R285.5

山东省自然科学基金项目（ZR2020MH127）;山东省自然科学基金项目（ZR2020MH412）