基于TLRs/MyD88信号通路探究温针灸干预兔膝骨性关节炎软骨退变机制  

Exploring the mechanism of warm acupuncture intervention on cartilage degeneration in rabbit knee osteoarthritis based on TLRs/MyD88 signaling pathway

作　　者：吴琦 阮金新 陈朝霞 吴福春[2] 余德标 WU Qi;RUAN Jinxin;CHEN Zhaoxia;WU Fuchun;YU Debiao(Medical Health Department,Meizhouwan Vocational and Technical College,Putian,Fujian,351119,China;Provincial Clinical Medical College,Fujian Medical University/Rehabilitation Department,Fujian Provincial Hospital,Fuzhou,Fujian,350001,China)

机构地区：[1]湄洲湾职业技术学院医学健康系,福建建莆田351119 [2]福建医科大学省立临床医学院,福建省立医院康复医学科,福建福州350001

出　　处：《甘肃中医药大学学报》2024年第1期1-7,共7页Journal of Gansu University of Chinese Medicine

基　　金：福建省自然科学基金项目(2021J01391);福建省卫生健康科技计划项目(2020GGA001);莆田市科技项目(2018SM02)。

摘　　要：目的基于Toll样受体(TLRs)/髓样分化因子88(MyD88)信号通路探究温针灸干预兔膝骨性关节炎(KOA)软骨退变的作用机制。方法将30只6月龄清洁级新西兰雄兔采用随机数字表法分为空白对照组、模型组、温针灸组3组,每组10只。除空白对照组外,其余2组通过木瓜蛋白酶关节腔注射建立KOA模型。实验干预后处死各组兔并取膝关节液和关节软骨组织,采用酶联免疫吸附试验(ELISA)法检测各组关节液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)的含量;采用HE染色镜下观察各组关节软骨病理改变,并比较关节软骨组织Mankin’s评分;采用TUNEL法检测关节软骨细胞凋亡情况,并比较各组软骨细胞凋亡率;通过免疫组化检测各组关节软骨Ⅱ型胶原蛋白表达水平,实时荧光定量聚合酶链式反应(qPCR)检测各组软骨细胞中TLR2、TLR4、MyD88、基质金属蛋白酶(MMP)-1、MMP-13 mRNA表达水平。结果HE染色镜下观察结果显示,空白对照组软骨层结构正常,潮线清晰完整;模型组软骨表面不光滑,有纤维组织形成,细胞排列无序、层次紊乱,潮线模糊;温针灸组较模型组明显改善,软骨表面不光滑,部分潮线模糊,表层软骨细胞较模型组增多。与空白对照组比较,模型组关节软骨组织Mankin’s评分,软骨细胞凋亡率,关节液中TNF-α、IL-1β含量,软骨细胞中TLR2、TLR4、MyD88、MMP-1、MMP-13 mRNA相对表达量明显升高,差异均有统计学意义(P<0.05);与模型组比较,温针灸组以上指标明显降低,差异均有统计学意义(P<0.05)。与空白对照组比较,模型组关节软骨Ⅱ型胶原蛋白相对表达量明显降低(P<0.05);与模型组比较,温针灸组关节软骨Ⅱ型胶原蛋白相对表达量明显升高(P<0.05)。结论温针灸可能是通过抑制TLRs/MyD88信号通路的激活,下调炎症因子TNF-α、IL-1β、MMP-1、MMP-13的表达水平,促进软骨细胞Ⅱ型胶原蛋白合成,从而抑制软骨细胞凋亡,延�Objective To investigate the mechanism of warm acupuncture intervention on cartilage degeneration of rabbit knee osteoarthritis(KOA)based on toll-like receptors(TLRs)/myeloid differentiation factor 88(MyD88)signaling pathway.Methods 30 clean New Zealand male rabbits of six months old were divided into blank control group,model group,and warm acupuncture group by random number table method,with 10 rabbits in each group.Except for blank control group,the KOA model was established by intraarticular injection of papain in the other two groups.After experimental intervention,the rabbits in each group were sacrificed and knee synovial fluid and articular cartilage tissue were collected.The contents of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in synovial fluid of each group were detected by enzyme-linked immunosorbent assay(ELISA).The pathological changes of articular cartilage of each group were observed under microscope by HE staining,and the Mankin's score of articular cartilage tissue was compared between the groups.The apoptosis of articular cartilage cells was detected by TUNEL method,and the apoptosis rate of articular cartilage cells was compared between the groups.The expression level of type Ⅱ collagen in articular cartilage of each group was detected by immunohistochemistry,and the mRNA expression levels of TLR2,TLR4,MyD88,matrix metalloproteinase(MMP)-1 and MMP-13 mRNA in articular cartilage cells of each group were detected by real-time fluorescent quantitative polymerase chain reaction(qPCR).Results The results of HE staining showed that the structure of cartilage layer in the blank control group was normal,and the tidemark was clear and complete;the cartilage surface in the model group was not smooth,with fibrous tissue formation,cell disordered arrangement,hierarchical disorder,and blurred tidemark;the surface of cartilage in the warm acupuncture group was not smooth,part of the tidemark was blurred,and the surface chondrocytes were increased compared with the model group.Compared wit

关 键 词：膝骨性关节炎 温针灸 Toll样受体/髓样分化因子88信号通路 炎症因子 Ⅱ型胶原蛋白 软骨细胞凋亡率 兔 实验研究 

分 类 号：R-332[医药卫生] R246.9