中医药导报2025，Vol.31Issue(4)：60-66,7.DOI:10.13862/j.cn43-1446/r.2025.04.010
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
Effect of Bugu Ointment(补骨膏)on Autophagy and Apoptosis of Chondrocytes in Rats with Knee Osteoarthritis Based on AMPK/mTOR Signaling Pathway
摘要
Abstract
Objective:To investigate the regulatory effects of Bugu ointment on the adenosine monophos-phate-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.Histopatho-logical 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/Ⅰthan 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.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Key words
knee osteoarthritis/articular cartilage/Bugu ointment/AMPK/mTOR signaling pathway/autophagy/apoptosis/rat分类
医药卫生引用本文复制引用
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):60-66,7.基金项目
国家中医药管理局2022年全国名老中医专家传承工作室建设项目(国中医药人教函[2022]75号) （国中医药人教函[2022]75号）
湖南省中医药管理局中医药科研计划项目(B2023153) （B2023153）
湖南省中医药管理局-局市(州)联合攻关科研项目(E2023016,E2023017) （州）
