基于Akt/mTOR信号通路探讨补肾活血方对去卵巢大鼠软骨细胞自噬的影响

Effect of Bushen Huoxue(补肾活血)recipe on autophagy of ovariectomized rat chondrocytes based on Akt/mTOR signaling pathway

目的:研究补肾活血方是否可以通过调控Akt/mTOR信号通路促进去卵巢大鼠软骨细胞自噬反应来保护关节软骨。方法:选取30只SPF级12周龄雌性SD大鼠,体重(247.0±7.0)g,先随机选择6只为空白对照组,剩余大鼠采用卵巢切除术结合右膝关节腔注射碘乙酸钠(monosodium iodoacetate,MIA)建立绝经后膝骨关节炎模型,随机分为模型组、BSHXR-L组、BSHXR-M组、BSHXR-H组,每组6只。运用肉眼观察评分、番红O-固绿染色、免疫组化等方法明确补肾活血方对大鼠关节软骨损伤的保护作用,Western-blot检测自噬相关蛋白的表达,qPCR检测Akt、mTOR及下游自噬基因的相对表达。结果:造模后BSHXR(L、M、H)各组均可减轻软骨组织形态学损伤,免疫组化示Collagen-Ⅱ、Aggrecan表达逐渐升高,基质金属蛋白酶-13(matrix metallopriteinase-13,MMP-13)表达逐渐降低,其中BSHXR-M组和BSHXR-H组与模型组相比差异均有统计学意义(P<0.05)。Western-blot结果示BSHXR(L、M、H)各组自噬通路蛋白p-Akt/Akt、p-mTOR/mTOR受到抑制,下游蛋白Beclin-1、LC3Ⅱ表达逐渐升高,而p62逐渐降低,呈剂量效应。qPCR结果示BSHXR(L、M、H)各组均可促进Beclin-1、LC3ⅡmRNA的相对表达,抑制p62、Akt、mTOR mRNA的相对表达,与模型组相比差异有统计学意义(P<0.05)。结论:补肾活血方可以通过抑制Akt/mTOR信号通路增强软骨细胞自噬反应,进而发挥保护软骨的作用。

Objective To investigate whether Bushen Huoxue recipe can protect articular cartilage by regulating Akt/mTOR signaling pathway to promote the autophagy of chondrocytes in ovariectomized rats.Methods Among 30 SPF 12-week-old female SD rats weighing(247.0±7.0)g,6 were randomly selected as the blank control group,and the remaining rats were randomly divided into model group,BSHXR-L group,BSHXR-M group and BSHXR-H group,with 6 rats in each group.The protective effect of Bushen Huoxue recipe on articular cartilage injury in rats was determined by visual observation score,muscovine O-solid green staining and immunohistochemistry.The expression of autophagy related proteins was detected by Western-blot,and the relative expression of Akt,mTOR and downstream autophagy genes was detected by qPCR.Results After modeling,BSHXR(L,M,H)groups could alleviate the histological damage of cartilage.Immunohistochemistry showed that the expression of Collagen-Ⅱand Aggrecan gradually increased,and the expression of MMP-13 gradually decreased,and the differences between BSHXR-M and BSHXR-H groups and model group were statistically significant(P<0.05).The results of Western-blot showed that the autophagy pathway proteins p-Akt/Akt and p-mTOR/mTOR were inhibited in the BSHXR(L,M,H)groups,and the expressions of downstream proteins Beclin-1 and LC3Ⅱwere gradually increased,while p62 was gradually decreased,showing a dose effect.QPCR results showed that BSHXR(L,M,H)groups could promote the relative expression of Beclin-1 and LC3ⅡmRNA,and inhibit the relative expression of p62,Akt,mTOR mRNA,and the differences were statistically significant compared with model group(P<0.05).Conclusion Bushen Huoxue recipe can enhance the cartilage autophagy response by inhibiting the Akt/mTOR signaling pathway,and then protect the cartilage.