丹参酮ⅡA通过抑制信号通路延缓膝骨关节炎大鼠软骨退变及抑制局部炎症的研究

TanshinoneⅡA Alleviates Cartilage Degeneration and Local Inflammation in Rats with Knee Osteoarthritis by Inhibiting Pathway

目的探究丹参酮ⅡA(TanⅡA)通过抑制相关信号通路延缓膝骨关节炎(KOA)大鼠模型软骨退变和抑制局部炎症的作用机制。方法将60只SD大鼠随机分为假手术组、KOA组、阳性药物塞来昔布20 mg·kg^(-1)组、TanⅡA不同剂量处理组(TanⅡA10、20、50 mg·kg^(-1)),每组各12只。采用前交叉韧带切断术(ACLT)建立KOA大鼠模型,造模成功次日,各实验组腹腔注射不同剂量TanⅡA,假手术组和KOA组给予等体积生理盐水,共给药28 d。通过步态行为分析、机械刺激缩足反应阈值(PWMT)和热刺激缩足反应潜伏期(PWTL)来评价大鼠疼痛行为;改良的Mankin's评分和OARSI评分评价软骨病理损伤程度;酶联免疫吸附测定法检测关节组织和血清中炎症因子、骨代谢生化指标水平;免疫组化法检测软骨组织中核因子κB p65(NF-κB p65)和Ⅱ型胶原(COL2A1)蛋白表达;Western blot法检测TLR4/Myd88/NF-κB信号通路相关蛋白Toll样受体4(TLR4)、髓样分化蛋白(Myd88)及NF-κB p65表达水平;分离胞核和胞浆蛋白,检测NF-κB p65核转位。结果实验结束时,与假手术组相比,KOA组和各实验组大鼠步态行为评分、PWTL、软骨组织COL2A1蛋白表达MOD值降低,同时PWMT、Mankin's评分和OARSI评分、关节组织和外周血清白细胞介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α)、Ⅰ型胶原交联C-末端肽(CTX-Ⅰ)、Ⅱ型胶原交联C-末端肽(CTX-Ⅱ)水平升高,软骨组织中TLR4、Myd88和NF-κB p65蛋白表达上调,同时NF-κB p65核转位明显(均P<0.05)。此外,与KOA组相比,塞来昔布组和TanⅡA不同剂量处理组大鼠步态行为评分、PWTL、软骨组织COL2A1蛋白表达MOD值升高,同时PWMT、Mankin's评分和OARSI评分、关节组织和外周血清IL-1β、IL-6、TNF-α、CTX-Ⅰ、CTX-Ⅱ水平降低,软骨组织中TLR4、Myd88和NF-κB p65蛋白表达下调,同时NF-κB p65核转位被抑制(均P<0.05)。尤其是TanⅡA 50 mg·kg^(-1)组上述指标改善更显著。结论TanⅡA通过抑制TLR4/Myd88/NF-κB信号通路相关蛋白表达,减少细胞炎症因子释放,进而抑制关节软骨退变,达到改善KOA大鼠膝关节功能的目的。

OBJECTIVE To study the mechanism of tanshinoneⅡA(TanⅡA)on ameliorating the cartilage degeneration and inhibiting local inflammation in knee osteoarthritis(KOA)rats by inhibiting the related pathway.METHODS Sixty rats were divided into sham-operation group,KOA model group,positive drug celecoxib 20 mg·kg^(-1) group and experimental groups(TanⅡA10,20,50 mg·kg^(-1)),with 12 rats in each.Except the sham-operation group,other rats were operated by anterior cruciate ligament transection(ACLT)to construct the KOA models.On the second day after successful modeling,the experimental groups were intraperitoneally injected with different doses of TanⅡA,the sham-operated group and the KOA model group were given the same volume of normal saline for 28 days.Pain behavior was evaluated by Gait behavior analysis,paw withdrawal mechanical threshold(PWMT)and paw withdrawal thermal latency(PWTL),the degree of cartilage injury was evaluated by modified Mankin's score and OARSI score,and the levels of inflammatory factors and biochemical markers of bone metabolism in joint tissue and serum were detected by enzyme-linked immunosorbent assay.The expression of nuclear factor kappa B p65(NF-κB p65)and typeⅡcollagen(COL2A1)in cartilage was detected by immunohistochemistry.The expression of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88)and NF-κB p65 proteins were detected by Western blot.Nuclear and cytoplasmic proteins were extracted and detected for the nuclear translocation of NF-κB p65.RESULTS At the end of the experiment,compared with the sham-operation group,the gait behavior scores,PWTL,COL2A1 expression MOD values of the cartilage in KOA model group and the experimental groups were decreased,while the levels of interleukin(IL)-1β,IL-6,tumor necrosis factor alpha(TNF-α),C-terminal crosslinking telopeptide of typeⅠcollagen(CTX-Ⅰ)and C-terminal crosslinking telopeptide of typeⅡcollagen(CTX-Ⅱ)in joint tissues or serum samples increased,and the expressions of TLR4,MyD88 and NF-κB p65 proteins in cartilage tissues were up-regulated,while the nuclear translocation NF-κB p65 was significantly increased(P<0.05).In addition,compared with KOA model group,the gait behavior score,PWTL,COL2A1 protein expression MOD value in cartilages were increased in experimental groups,while PWMT,Mankin's score and OARSI score,the levels of IL-1β,IL-6,TNF-α,CTX-Ⅰ,CTX-Ⅱin joint tissues or serum samples were decreased,and the expressions of TLR4,MyD88 and NF-κB p65 proteins in cartilage were down-regulated,and the nuclear translocation NF-κB p65 was inhibited(P<0.05).In particular,the improvement of the above-mentioned indicators in TanⅡA mg·kg^(-1) group were more significant.CONCLUSION TanⅡA can inhibit the expression of TLR4/Myd88/nfb signaling pathway-related proteins,decrease the release of inflammatory cytokines,and then inhibit the degeneration of articular cartilage,so as to improve the knee joint function of KOA rats.