摘要
目的:观察膝痹宁Ⅱ及脂肪干细胞外泌体(ADSC-Exos)单独及联合应用对软骨退变及线粒体自噬的影响,探讨其改善膝骨关节炎(KOA)的作用机制。方法:将40只SD大鼠分为假手术组、模型组、ADSC-Exos组(4×10^(8)个/周)、膝痹宁组(8 g·kg^(-1)·d^(-1))和ADSC-Exos(4×10^(8)个/周)+膝痹宁(8 g·kg^(-1)·d^(-1))组。采用用前交叉韧带切断术(ACLT)建立膝骨关节炎模型。评估大鼠痛敏状态、微计算机断层扫描技术(Micro-CT)及病理染色检测膝关节及软骨组织退变程度、免疫荧光观察p62和微管相关蛋白1轻链3(LC3)B表达情况、酶联免疫吸附测定法(ELISA)检测大鼠血清肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6、IL-15水平、蛋白免疫印迹法(Western blot)检测基质金属蛋白酶(MMP)-3、MMP-13、含血小板结合蛋白基序的解聚素金属5(ADAMTS5)、Ⅱ型胶原(ColⅡ)、基质金属蛋白酶组织抑制剂(TIMP)、软骨蛋白聚糖抗体(ACAN)、PTEN诱导激酶1(PINK1)、帕金蛋白(Parkin)、p62、LC3A/B蛋白表达水平。结果:与假手术组比较,模型组大鼠冷刺激缩足阈值及机械痛敏阈值逐渐下降,软骨组织出现不同程度的磨损及缺失,软骨组织退变,血清IL-1β、IL-6、IL-15和TNF-α含量明显升高(P<0.01),软骨组织中MMP-3、MMP-13和ADAMTS5蛋白表达升高,ColⅡ、TIMP1和ACAN蛋白表达明显降低(P<0.01);与模型组比较,各治疗组大鼠冷刺激缩足阈值及机械痛敏阈值升高,软骨组织退变程度减轻,血清IL-1β、IL-6、IL-15和TNF-α含量降低(P<0.05,P<0.01),软骨组织中MMP-3、MMP-13和ADAMTS5蛋白表达降低,ColⅡ、TIMP1和ACAN蛋白表达升高(P<0.05,P<0.01);且以Exos+膝痹宁组改变最为明显。结论:ADSCs-Exos及膝痹宁Ⅱ均可以通过促进PINK1/Parkin信号通路表达,提高软骨细胞线粒体自噬水平,延缓软骨组织退变,且二者联用可以增强疗效。
Objective:To observe the effects of Xibining(XBN)and adipose stem cell exosome(ADSC-Exos)in the cases of separate or joint application on cartilage degeneration and mitochondrial autophagy and explore its mechanism of action to improve knee osteoarthritis(KOA).Method:SD rats were divided into a sham operation group(sham group),a model group,an ADSC-Exos group(Exos group),an XBN group,and an ADSC-Exos+XBN group(Exos+XBN group).KOA model was established by using anterior cruciate ligament transection(ACLT).The pain sensitivity status of rats was evaluated,and the degeneration degree of the knee joint and cartilage tissue was detected by Micro-CT and pathological staining.The expression of p62 and LC3B was observed by immunofluorescence,and the serum levels of TNF-α,IL-1β,IL-6,and IL-15 in rats were detected by ELISA.The Western blot was used to detect the protein expression levels of MMP-3,MMP-13,ADAMTS5,Col Ⅱ,TIMP,ACAN,PINK1,Parkin,p62,and LC3A/B.Result:Compared with the sham group,rats in the model group showed decreased cold-stimulated foot-shrinkage thresholds and mechanical pain sensitivity thresholds,varying degrees of abrasion and loss of cartilage tissue,degeneration of cartilage tissue,elevated serum IL-1β,IL-6,IL-15,and TNF-αlevels(P<0.01),and increased protein expression of MMP-3,MMP-13,and ADAMTS5 in cartilage tissue.In addition,the protein expression of Col Ⅱ,TIMP1,and ACAN was decreased(P<0.01).Compared with the model group,rats in each treatment group showed higher cold-stimulated foot-shrinkage thresholds and mechanical pain sensitivity thresholds,reduced cartilage tissue degeneration,lower serum levels of IL-1β,IL-6,IL-15,and TNF-α(P<0.05,P<0.01),decreased protein expression of MMP-3,MMP-13,and ADAMTS5,and higher protein expression of Cold,TIMP1,and ACAN in cartilage tissue(P<0.05,P<0.01).Moreover,the changes were the most obvious in the Exos+XBN group.Conclusion:Both ADSCs-Exos and XBN can increase the level of mitochondrial autophagy in chondrocytes and delay cartilage tissue degeneration by promoting the expression of the PINK1/Parkin signaling pathway,and the combination of the two can enhance the therapeutic effect.
作者
康俊峰
揭立士
伏厚宇
廖太阳
施蕾
苏子珊
于利凯
魏义保
刘德仁
田地
茆军
王培民
KANG Junfeng;JIE Lishi;FU Houyu;LIAO Taiyang;SHI Lei;SU Zishan;YU Likai;WEIYibao;LIU Deren;TIAN Di;MAO Jun;WANG Peimin(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210004,China;Affiliated Hospital of Shanxi University of Chinese Medicine,Taiyuan 030024,China;First Clinical Medical College,Nanjing University of Chinese Medicine,Nanjing 210023,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第11期111-119,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(82274545)
江苏省中医院高峰学术人才项目(y2021rc02)
江苏省医学重点学科/实验室建设单位项目(js2252)
山西省自然科学研究面上项目(202303021221242)
山西省中医药管理局课题(2022ZYYC277)
山西中医药大学科技创新能力培育计划项目(2022PY-ZBK-002)。
关键词
脂肪干细胞外泌体
膝痹宁Ⅱ
线粒体自噬
软骨退变
膝骨关节炎
adipose stem cell exosomes
XibiningⅡ
mitochondrial autophagy
cartilage degeneration
kneeosteoarthritis
作者简介
第一作者:康俊峰,在读博士,副主任医师,硕士生导师,从事膝骨关节炎基础与临床研究,E-mail:36171696@qq.com;通信作者:王培民,博士,教授,主任医师,从事膝骨关节炎基础与临床研究,E-mail:drwpm@163.com。
