黄芪甲苷调节PI_(3)K/Akt/mTOR信号对慢性间歇性低氧大鼠颏舌肌损伤及自噬反应的影响

Effects of AstragalosideⅣon Genioglossus Musele Injury and Autophagy in Chronic Intermittent Hypoxia Rats by Regulating PI_(3)K/Akt/mTOR Signaling

目的探究黄芪甲苷(astragalosideⅣ,ASⅣ)对磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin,PI_(3)K/Akt/mTOR)信号通路的调控作用以及对慢性间歇性低氧(chronic intermittent hypoxia,CIH)大鼠颏舌肌损伤及自噬反应的影响。方法96只SD大鼠按照随机数字表法分为正常组,模型组,ASⅣ低、中、高剂量组,ASⅣ+PI_(3)K抑制剂(LY294002)组,每组各16只。除正常组外,其余5组建立CIH大鼠模型。实验结束后检测大鼠动脉血气指标[动脉血氧分压(arterial partial pressure of oxygen,PaO_(2))、二氧化碳分压(partial pressure of carbon dioxide,PaCO_(2))、血氧饱和度(saturation of blood oxygen,SaO_(2))]以及血清中白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量;苏木精-伊红(hematoxylin eosin,HE)染色观察大鼠颏舌肌组织病理变化;免疫组化法检测颏舌肌组织中自噬相关蛋白Beclin1、微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3B,LC3B)蛋白表达;蛋白印迹法检测颏舌肌组织中PI_(3)K/Akt/mTOR信号通路相关蛋白表达。结果与正常组比较,模型组大鼠动脉血PaCO_(2)、血清中IL-6、TNF-α含量以及颏舌肌组织中PI_(3)K蛋白表达和磷酸化Akt(p-Akt)/Akt、磷酸化mTOR(p-mTOR)/mTOR比值显著升高(P<0.05),PaO_(2)、SaO_(2)以及颏舌肌组织中Beclin1、LC3B蛋白表达显著降低(P<0.05);颏舌肌组织细胞肿胀变形,边界模糊,排列凌乱,细胞角度消失。与模型组比较,ASⅣ低、中、高剂量组大鼠动脉血PaCO_(2)、血清中IL-6、TNF-α含量以及颏舌肌组织中PI_(3)K蛋白表达和p-Akt/Akt、p-mTOR/mTOR比值依次降低(P<0.05),PaO_(2)、SaO_(2)以及颏舌肌组织中Beclin1、LC3B蛋白表达依次升高(P<0.05);颏舌肌组织病变逐渐减轻。ASⅣ+LY294002组上述指标改变效果优于ASⅣ高剂量组。结论ASⅣ可能通过抑制PI_(3)K/Akt/mTOR信号通路的激活,诱导自噬反应发生,减轻颏舌肌损伤,改善CIH大鼠病变。

Objective To explore the regulatory effect of astragalosideⅣ(ASⅣ)on phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI_(3)K/Akt/mTOR)signaling pathway,and its influences on genioglossus muscle injury and autophagy in chronic intermittent hypoxia(CIH)rats.Methods Ninty-six 96 SD rats were divided into normal group,model group,ASⅣlow,medium,high dose groups,ASⅣ+PI_(3)K inhibitor(LY294002)group according to random number table method,16 in each group.Except the normal group,the other 5groups established the CIH rat model.After the experiment,the arterial blood gas indexes[arterial partial pressure of oxygen(PaO_(2)),partial pressure of carbon dioxide(PaCO_(2)),saturation of blood oxygen(SaO_(2))]and interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)content in serum were detected;hematoxylin eosin(HE)staining was performed to observe the pathological changes of genioglossal muscle tissue in rats;immunohistochemical method was used to detect the expression of autophagy related protein Beclinl and microtubule associated protein 1 light chain 3B(LC3B)in genioglossal muscle tissue;Western blot was used to detect the expression of PI_(3)K/Akt/mTOR signal pathway related proteins in genioglossal muscle tissue.Results Compared with the normal group,the PaCO_(2)of arterial blood,IL-6 and TNF-αcontents of serum,PI_(3)K protein expression and phosphorylated-Akt(p-Akt)/Akt and phosphorylated-mTOR(p-mTOR)/mTOR ratios in the genioglossal muscle tissue of rats in the model group were obviously increased(P<0.05),the PaO_(2),SaO_(2)and protein expression of Beclinl and LC3B in genioglossal muscle tissue were obviously decreased(P<0.05);genioglossal muscle tissue cells were swollen and deformed,with blurred borders,disordered arrangement,and cell angles were disappeared.Compared with the model group,the PaCO_(2)of arterial blood,IL-6 and TNF-&contents of serum,PI_(3)K protein expression and p-Akt/Akt and p-mTOR/mTOR ratios in the genioglossal muscle tissue of rats in ASⅣlow,medium and high dose groups were decreased in turn(P<0.05),the PaO_(2),SaO_(2)and protein expression of Beclinl and LC3B in genioglossal muscle tissue were increased in turn(P<0.05);the genioglossal muscle tissue lesions gradually reduced.The change effect of the above indexes in ASⅣ+LY294002 group was better than that in ASⅣhigh dose group.Conclusion ASⅣmay induce autophagy by inhibiting the actIVation of PI_(3)K/Akt/mTOR signaling pathway,alleviate genioglossus muscle injury,and improve the pathological changes of CIH rats.