目的 基于網(wǎng)絡藥理學探討金藤清痹顆粒對大鼠膝骨關節(jié)炎（knee osteoarthritis，KOA）的影響及與促進線粒體自噬改善鐵死亡的相關性。方法 通過中藥系統(tǒng)藥理數(shù)據(jù)庫和分析平臺（traditional Chinese medicine systems pharmacology database and analysis platform，TCMSP）數(shù)據(jù)庫搜集金藤清痹顆粒的活性成分及靶點，利用人類基因數(shù)據(jù)庫（Gene Cards）、在線人類孟德爾遺傳數(shù)據(jù)庫（online Mendelian inheritance in Man，OMIM）等數(shù)據(jù)庫搜集KOA及金藤清痹顆粒藥物作用靶點，構(gòu)建蛋白質(zhì)-蛋白質(zhì)相互作用（protein-protein interaction，PPI）網(wǎng)絡和“中藥-活性成分-靶點”網(wǎng)絡。應用Autodock軟件對篩選出的有效成分和關鍵靶點進行分子對接。將大鼠隨機分為對照組、假手術組、模型組、雙氯芬酸鈉（5 mg/kg）組和金藤清痹顆粒高、中、低（5.4、2.7、1.4 g/kg）劑量組，各組ig相應藥物，1次/d，給藥4周后取材。ELISA檢測白細胞介素-1β（interleukin-1β，IL-1β）、IL-6、腫瘤壞死因子α（tumornecrosisfactor-α，TNF-α）的含量；蘇木素-伊紅（hematoxylin-eosin，HE）染色評估軟骨病理變化；蛋白免疫印跡（Western blotting，WB）法檢測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）、磷酸酶及張力蛋白同源物誘導的蛋白激酶1（PTEN-induced putative kinase 1，Pink1）、帕金抗體（Parkinson disease，Parkin）、泛素結(jié)合蛋白（sequestosome 1，SQSTM1/p62）、膜鐵轉(zhuǎn)運蛋白1（ferroportin 1，F(xiàn)PN1）、谷胱甘肽過氧化物酶4（glutathione peroxidase 4，GPX4）、?；o酶A合成酶長鏈家族成員4（acyl-CoA synthetase long chain family member 4，ACSL4）、鐵蛋白（ferritin）、轉(zhuǎn)鐵蛋白受體蛋白1（transferrin receptor protein 1，TFR1）、過氧化還原酶3重組蛋白（recombinant peroxiredoxin 3，PRDX3）的表達。結(jié)果 數(shù)據(jù)庫共檢索到金藤清痹顆粒116種活性成分、268個作用靶點，共有靶點107個，關鍵靶點涉及NF-κB信號通路、線粒體自噬信號通路等；分子對接結(jié)果提示靶蛋白與小分子之間存在結(jié)合位點且結(jié)合能較低，提示有較強的結(jié)合活性。動物實驗結(jié)果表明，與對照組比較，模型組大鼠IL⁃1β、IL⁃6、TNF⁃α水平顯著升高（P＜0.01、0.001）；膝關節(jié)軟骨病理組織增生明顯；Pro-Caspase-1、p-NF-κB、NF-κB、p62、ASL4、TFR1、PRDX3表達量顯著上升（P＜0.05）；Pink1、Parkin、FPN1、GPX4、ferritin表達量顯著下降（P＜0.05），自噬蛋白Beclin、LC3BII/I的值顯著下降（P＜0.05）；與模型組比較，金藤清痹顆粒各組病理形態(tài)損傷減輕；IL-1β、IL-6、TNF-α，Pro-Caspase-1、p-NF-κB、NF-κB、p62、ASL4、TFR1、PRDX3、NCOA4顯著降低（P＜0.05）；Pink1、Parkin、FPN1、GPX4、ferritin及自噬蛋白表達量Beclin、LC3BⅡ/Ⅰ顯著升高（P＜0.05）。結(jié)論 金藤清痹顆?？赡芘c增強線粒體自噬，改善鐵死亡，抑制NF-κB炎癥信號等實現(xiàn)對膝骨關節(jié)炎的治療。

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

山東省自然科學基金項目（ZR2020MH127）;山東省自然科學基金項目（ZR2020MH412）