基于AMPK/mTOR信号通路研究补骨膏对膝骨关节炎大鼠软骨细胞自噬与凋亡的影响*

作者：万文渊1,2,3,4，唐 雄1,2,3,4，袁尚锋1,2,4，彭真灵1,2,4，陈泽华1,2,3，彭铮磊1,2，胡 伟1,2

单位：1.株洲市中医伤科医院，湖南 株洲 412007； 2.湖南中医药高等专科学校附属医院，湖南 株洲 412007； 3.湖南省中西医结合诊治慢性骨关节病临床医疗技术示范基地，湖南 株洲 412007； 4.袁尚锋国家名老中医药专家传承工作室，湖南 株洲 412007

引用：引用：万文渊，唐雄，袁尚锋，彭真灵，陈泽华，彭铮磊，胡伟.基于AMPK/mTOR信号通路研究补骨膏对膝骨关节炎大鼠软骨细胞自噬与凋亡的影响[J].中医药导报,2025,31(4):60-66.

DOI：10.13862/j.cn43-1446/r.2025.04.010

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摘要：

目的：探究补骨膏对膝骨关节炎（KOA）大鼠腺苷酸激活蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路的调控作用，以及对软骨细胞自噬与凋亡的影响。方法：将40只大鼠随机分为正常组、KOA组、激动剂组和补骨膏组，采用双膝关节腔内注射木瓜蛋白酶及持续抽屉样运动建立KOA大鼠模型，然后予相应药物灌胃8周。苏木精-伊红（HE）染色及番红O-固绿染色观察软骨组织病理形态；TUNEL染色观察软骨细胞凋亡情况；透射电镜观察软骨细胞自噬情况；酶联免疫吸附试验（ELISA）检测关节液中白细胞介素-1β（IL-1β）、IL-6、肿瘤坏死因子-α（TNF-α）、基质金属蛋白酶-3（MMP-3）、MMP-13水平；免疫组化法检测软骨组织中磷酸化腺苷单磷酸激活的蛋白激酶（p-AMPK）、MMP-13阳性表达；蛋白免疫印迹法（Western blotting）检测软骨组织中p-AMPK/AMPK、微管相关蛋白1轻链3（LC3）-Ⅱ/Ⅰ及mTOR、自噬效应蛋白1（Beclin1）、胱天蛋白酶-3（Caspase-3）相对表达量。结果：正常组大鼠软骨组织形态正常，潮线完整，番红O-固绿染色正常；KOA组大鼠软骨组织可见裂隙形成，潮线被破坏，细胞排列紊乱，关节面软骨局部或全层缺失，番红O-固绿染色不均或失染；激动剂组和补骨膏组大鼠潮线基本完整，可见少量增生，番红O-固绿染色稍显不均或轻染，软骨破坏程度低于KOA组。KOA组大鼠软骨细胞自噬小体数量低于正常组（P＜0.01），软骨细胞凋亡率高于正常组（P＜0.01）；激动剂组及补骨膏组大鼠软骨细胞自噬小体数量均高于KOA组（P＜0.01），软骨细胞凋亡率均低于KOA组（P＜0.01）。KOA组大鼠关节液MMP-3、MMP-13、IL-1β、IL-6、TNF-α水平高于正常组（P＜0.01）；激动剂组及补骨膏组大鼠关节液中MMP-3、MMP-13、IL-1β、TNF-α水平均低于KOA组（P＜0.01）；补骨膏组大鼠关节液中MMP-13水平高于激动剂组（P＜0.05），IL-6、TNF-α水平均低于激动剂组（P＜0.01）。KOA组大鼠软骨组织p-AMPK蛋白阳性表达水平低于正常组（P＜0.01），MMP-13蛋白阳性表达水平高于正常组（P＜0.01）；激动剂组及补骨膏组大鼠软骨组织p-AMPK蛋白阳性表达水平高于KOA组（P＜0.01），MMP-13蛋白阳性表达水平低于KOA组（P＜0.01）；补骨膏组大鼠软骨组织MMP-13蛋白阳性表达水平高于激动剂组（P＜0.05）。KOA组大鼠软骨组织p-AMPK/AMPK、LC3Ⅱ/Ⅰ低于正常组，mTOR、Beclin1、Caspase-3蛋白相对表达量高于正常组（P＜0.01或P＜0.05）；激动剂组及补骨膏组大鼠软骨组织p-AMPK/AMPK、LC3Ⅱ/Ⅰ均高于KOA组（P＜0.01），mTOR、Caspase-3蛋白相对表达量均低于KOA组（P＜0.01）；激动剂组大鼠软骨组织Beclin1蛋白相对表达量高于KOA组（P＜0.05）；补骨膏组大鼠软骨组织p-AMPK/AMPK、LC3Ⅱ/Ⅰ低于激动剂组（P＜0.01或P＜0.05）。结论：补骨膏可能通过激活AMPK/mTOR信号通路增强软骨细胞自噬能力，从而抑制软骨细胞凋亡，减少促炎因子释放及软骨基质降解，减轻KOA软骨组织病理损伤。

关键词：膝骨关节炎；关节软骨；补骨膏；AMPK/mTOR信号通路；细胞自噬；细胞凋亡；大鼠

Abstract：

Objective: To investigate the regulatory effects of Bugu ointment on the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in knee osteoarthritis (KOA) rats, and to evaluate its impact on chondrocyte autophagy and apoptosis. Methods: Totally 40 rats were randomly divided into normal group, KOA group, agonist group, and Bugu ointment group. KOA rat model was established through bilateral knee joint cavity injections of papain combined with persistent drawer-like motion, followed by 8-week oral gavage administration of corresponding medications. Histopathological morphology was assessed using Hematoxylin-eosin (HE) staining and Safranin O-Fast Green staining. Chondrocyte apoptosis was evaluated by TUNEL staining, and autophagy was observed via transmission electron microscopy (TEM). Synovial fluid levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3) and MMP-13 were quantified by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry (IHC) detected the positive expression of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) and MMP-13 in cartilage tissues. Western blotting analyzed the relative expression levels of AMPK/p-AMPK, microtubule-associated protein 1 light chain 3 (LC3)-Ⅰ/Ⅱ, mTOR, autophagy effector protein 1 (Beclin1), and Caspase-3 in cartilage tissues. Result: In the normal group, the morphological structure of rat cartilage tissue was normal, and the tidemark was intact. The safranin and fast green staining results were normal in normal group. In the KOA group, the cartilage tissue of rats showed formation of fissures, and the tidemark was damaged. The cell arrangement was disordered, and there was local or full-thickness loss of articular surface cartilage in KOA group. The safranin staining was uneven or lost in KOA group. In the agonist group and Bugu ointment group, the tidemark of rat cartilage was basically intact, with a small amount of hyperplasia visible. The safranin staining was slightly uneven or lightly stained in agonist group and Bugu ointment group, and the degree of cartilage damage was lower than that in the KOA group. The KOA group showed lower number of autophagic vacuoles in chondrocytes than normal group (P<0.01), while higher apoptosis rate of chondrocytes than normal group (P<0.01). The agonist group and the Bugu ointment group showed higher number of autophagic vacuoles in chondrocytes than KOA group (P<0.01), while lower apoptosis rate of chondrocytes than KOA group (P<0.01). The KOA group showed higher levels of MMP-3, MMP-13, IL-1β, IL-6 and TNF-α in the synovial fluid than normal group (P<0.01). The agonist group and the Bugu ointment group showed lower levels of MMP-3, MMP-13, IL-1β, IL-6 and TNF-α in the synovial fluid than KOA group (P<0.01). The Bugu ointment group showed higher level of MMP-13 in the synovial fluid than agonist group (P<0.05), while lower levels of IL-6 and TNF-α than agonist group (P<0.01). The KOA group showed lower positive expression level of p-AMPK protein in the cartilage tissue than normal group (P<0.01), while higher positive expression level of MMP-13 protein than normal group (P<0.01). The agonist group and Bugu ointment group showed higher positive expression level of p-AMPK protein in the cartilage tissue than KOA group (P<0.01), while lower positive expression level of MMP-13 protein than KOA grou (P<0.01). The Bugu ointment group showed higher positive expression level of MMP-13 protein in the cartilage tissue than agonist group (P<0.05). The KOA group showed lower ratios of p-AMPK/AMPK and LC3Ⅱ/Ⅰ than normal group, while higher relative expression levels of mTOR, Beclin1 and Caspase-3 proteins than normal group (P<0.01 or P<0.05). The agonist group and the Bugu ointment group showed higher ratios of p-AMPK/AMPK and LC3Ⅱ/Ⅰ than KOA group (P<0.01), while lower relative expression levels of mTOR and Caspase-3 proteins than KOA group (P<0.01). The agonist group showed higher relative expression level of Beclin1 protein in the cartilage tissue than KOA group (P<0.05). The Bugu ointment group showed lower ratios of p-AMPK/AMPK and LC3Ⅱ/Ⅰ than agonist group (P<0.01 or P<0.05). Conclusion: Bugu ointment may enhance the autophagic ability of chondrocytes by activating the AMPK/mTOR signaling pathway, thereby inhibiting the apoptosis of chondrocytes, reducing the release of pro-inflammatory factors and the degradation of the cartilage matrix, and alleviating the pathological damage of the cartilage tissue in knee osteoarthritis.

Key words：knee osteoarthritis; articular cartilage; Bugu ointment; AMPK/mTOR signaling pathway; autophagy; apoptosis; rat

发布时间：2025-12-20

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