摘要
目的观察针刀对膝关节骨关节炎(KOA)大鼠膝关节软骨细胞单磷酸腺苷活化蛋白激酶(AMPK)/Unc-51样激酶1(ULK1)信号通路的影响,探究针刀治疗KOA的潜在机制。方法将36只SD大鼠随机分为正常、模型和针刀组,每组12只。采用碘乙酸钠(MIA)法建立KOA大鼠模型。造模后,针刀组给予针刀干预,每周1次;正常组及模型组仅抓取和固定,连续4周。采用HE染色法观察大鼠软骨细胞病理改变;电镜观察大鼠软骨细胞线粒体结构形态;ELISA法检测各组大鼠软骨组织白细胞介素(IL)-1β、肿瘤坏死因子(TNF)-α水平;Western Blot法检测大鼠软骨组织自噬蛋白ULK1、p-ULK1、AMPK、p-AMPK、微管相关蛋白轻链3(LC3Ⅱ/Ⅰ)、苄氯素1(Beclin-1)、p62蛋白表达水平;流式细胞术检测大鼠软骨组织线粒体膜电位和活性氧(ROS)水平。结果与正常组比较,模型组大鼠膝关节软骨细胞病理损伤严重,自噬体、线粒体数量减少,软骨组织IL-1β、TNF-α、p62蛋白表达上升(P<0.01),软骨组织ULK1、p-ULK1、AMPK、p-AMPK、LC3Ⅱ/Ⅰ、Beclin-1蛋白表达水平降低(P<0.01),软骨细胞线粒体膜电位水平下降、ROS荧光强度加强(P<0.01)。与模型组比较,针刀组大鼠膝关节软骨细胞病理损伤得到明显改善,线粒体较多,自噬体略有增加,软骨组织IL-1β、TNF-α、p62蛋白降低(P<0.01),软骨组织ULK1、p-ULK1、AMPK、p-AMPK、LC3Ⅱ/Ⅰ、Beclin-1蛋白表达水平上升(P<0.01),软骨细胞线粒体膜电位水平上升、ROS荧光强度减弱(P<0.05,P<0.01)。结论针刀能够有效改善KOA模型大鼠膝关节软骨组织损伤,其机制可能与调控AMPK/ULK1信号通路,进而提升KOA大鼠软骨细胞自噬水平有关。
Objective To observe the effects of needle knife therapy on the signaling pathway of adenosine monophosphate-activated protein kinase(AMPK)/Unc-51-like kinase 1(ULK1)in knee chondrocytes of rats with knee osteoarthritis(KOA),and to explore the potential mechanisms underlying the therapeutic effects of needle knife treatment on KOA.Methods Thirty-six SD rats were randomly divided into three groups:normal,model and needle knife group,with 12 rats in each group.The KOA rat model was established using the sodium iodoacetate(MIA)method.After modeling,the needle knife group received weekly needle knife intervention,while the normal and model groups were only grasped and fixed for 4 consecutive weeks.Pathological changes of rat chondrocytes were observed using HE staining.Mitochondrial structure and morphology were examined by electron microscopy.The levels of interleukin(IL)-1βand tumour necrosis factor(TNF)-αin cartilage tissues were detected by ELISA.The expression of autophagy proteins,including ULK1,p-ULK1,AMPK,p-AMPK,microtubule-associated protein light chain 3(Lc3Ⅱ/Ⅰ),Beclin-1,and p62 were assessed by Western Blot.Additionally,mitochondrial membrane potential and reactive oxygen species(Ros)levels in cartilage tissues were measured using flow cytometry.Results Compared with the normal group,rats in the model group showed severe pathological damage in knee joint chondrocytes,a reduction in the number of autophagosomes and mitochondria,increased expression of IL-1β,TNF-αand p62 proteins(P<0.01),and decreased expression of ULK1,p-ULK1,AMPK,p-AMPK,LC3II/I and Beclin-1(P<0.01),mitochondrial membrane potential was decreased,and ROs fluorescence intensity was enhanced(P<0.01).In contrast,compared with the model group,the needle knife group exhibited significant improvements in pathological changes of knee joint chondrocytes,mitochondrial numbers increased,autophagosomes were slightly elevated.Expression levels of IL-1β,TNF-α,and p62 proteins were reduced(P<0.01),expression of ULK1,p-ULK1,AMPK,p-AMPK,Lc3Ⅱ/Ⅰ,and Beclin-1 protein in cartilage tissues were increased(P<0.01),mitochondrial membrane potential was elevated,and ROs fluorescence intensity was decreased(P<0.05,P<0.01).Conclusions Needle knife therapy effectively ameliorates cartilage tissue damage in the knee joints of KOA model rats.The underlying mechanism may be related to the regulation of the AMPK/ULK1 signaling pathway,which enhances autophagy levels in chondrocytes of KOArats.
作者
李蔚然
杨琪琪
周欣华
王克坡
李飞
LI Wei-ran;YANG Qi-qi;ZHOU Xin-hua;WANG Ke-po;LI Fei(Graduate School of Anhui University of Chinese Medicine,Hefei,230038;Rehabilitation Department II,Second Affiliated Hospital of Anhui University of Chinese Medicine,Hefei,230061)
出处
《中国中西医结合杂志》
北大核心
2025年第10期1215-1221,共7页
Chinese Journal of Integrated Traditional and Western Medicine
基金
国家青年岐黄学者支持项目[No.国中医药人教函(2022)256号]
安徽省自然科学基金面上项目(No.2308085MH297)
安徽高校自然科学重大研究项目(No.2023AH040099)
安徽高校自然科学重点研究项目(No.KJ2021A0549)。
