补肾活血方调控NF-κB信号通路对膝骨关节炎小鼠软骨组织炎症及细胞凋亡的影响

Effect of Bushen Huoxue formula regulating NF-κB signaling pathway on inflammation and apoptosis of cartilage tissue in mice with knee osteoarthritis

目的观察补肾活血方(BSHXF)对膝骨关节炎(KOA)小鼠软骨组织炎症及细胞凋亡的影响,探讨BSHXF治疗KOA的作用机制。方法将24只雄性BALB/c小鼠按体重随机分为假手术组、KOA模型组、KOA模型+BSHXF组,每组8只。造模、灌胃干预后,小鼠取血,ELISA检测血清IL-6、iNOS、COX2水平;提取小鼠膝关节软骨组织,HE染色及番红O-固绿染色观察软骨组织病理学情况,TUNEL染色检测软骨组织细胞凋亡情况,Western blot检测Cleaved-Caspase-9、Bcl-2、Bax蛋白表达,qRT-PCR检测CollagenⅡ、Aggrecan、ADAMTS-5 mRNA表达,Western blot检测核转录因子-κB(NF-κB)信号通路关键蛋白p-IκBα、IκBα、p-p65、p65的表达。结果与假手术组相比,KOA模型组小鼠膝关节软骨细胞病理改变明显;血清炎症细胞因子IL-6、iNOS和COX2的水平升高,软骨组织细胞凋亡率增加,Cleaved-Caspase-9、Bax蛋白表达水平升高,Bcl-2蛋白表达降低,CollagenⅡ、Aggrecan的mRNA表达水平降低,ADAMTS-5 mRNA表达升高,p-IκBα/IκBα、p-p65/p65比值升高。与KOA模型组相比,KOA模型+BSHXF组膝关节软骨损伤改善、病理变化减轻,炎症细胞因子IL-6、iNOS和COX2的水平降低,软骨组织细胞凋亡率降低,Cleaved-Caspase-9、Bax蛋白表达降低,Bcl-2蛋白表达升高,CollagenⅡ、Aggrecan的mRNA表达升高,ADAMTS-5 mRNA表达降低,p-IκBα/IκBα、p-p65/p65比值降低。结论BSHXF可抑制NF-κB信号通路,减少炎性因子释放、抑制软骨细胞凋亡及细胞外基质降解,从而缓解KOA疾病进展。

Objective To determine the mechanism of Bushen Huoxue formula(BSHXF)for treatment of knee osteoarthritis(KOA)and to observe the effect of BSHXF on the apoptosis and inflammation of KOA in mice.Methods Totally 24 male BALB/c mice were stratified by body weight and randomly divided into a sham group,a KOA group and a KOA+BSHXF group(8 mice in each group).After the intervention of model building and intragastric administration,the serum was collected and the levels of IL-6,iNOS and COX2 were detected by ELISA.The cartilage tissues of the mice were collected,and the histopathology of the knee cartilage was detected by HE staining and saffranine O-solid green staining.TUNEL staining was used to detect the apoptosis of the cartilage tissues.The protein expression of Cleaved-Caspase-9,Bcl-2,Bax and p-IκBα,IκBα,p-p65 and p65,namely the key proteins in NF-κB signaling pathway,were detected by Western blot,and the mRNA expressions of CollagenⅡ,Aggrecan and ADAMTS-5 were detected by qRT-PCR.Results Compared with the sham group,the pathological changes in the knee cartilage cells in the KOA group were obvious.The serum levels of inflammatory cytokines IL-6,iNOS and COX2 were increased.The apoptosis rate in the cartilage tissue,Cleaved-Caspase-9 and Bax protein expression levels were also increased.Bcl-2,CollagenⅡand Aggrecan mRNA levels were decreased,while ADAMTS-5 mRNA,the ratios of p-IκBα/IκBαand p-p65/p65 were increased.Compared with the KOA group,injurying the knee joint cartilage and pathological changes were improved the levels of inflammatory cytokines IL-6,iNOS and COX2,apoptosis rate in the cartilage tissue,the protein expression levels of Cleaved-Caspase-9 and Bax were all decreased,but Bcl-2,the mRNA levels of CollagenⅡand Aggrecan were increased,while ADAMTS-5 mRNA and the ratios of p-IκBα/IκBαand p-p65/p65 were decreased in the KOA+BSHXF group.Conclusion BSHXF may inhibit NF-κB signaling pathway,reduce the release of inflammatory factors,inhibit chondrocyte apoptosis and extracellular matrix degradation,to alleviate the progression of KOA.