红景天苷通过抑制PI3K/AKT/mTOR信号通路对大鼠脓毒症急性肾损伤的保护作用

Protective effect of salidroside on septic acute kidney injury in rats by inhibiting phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin signal pathway

目的探讨红景天苷(SLDS)调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对大鼠脓毒症急性肾损伤(SAKI)的保护作用。方法采用随机数字表法将40只Sprague-Dawley大鼠分为空白对照组(Control组)、假手术组(Sham组)、盲肠结扎穿孔术组(CLP组)和红景天苷预处理组(CLP+SLDS组),每组10只。评估各组大鼠肾组织病理损伤情况,并采用酶联免疫吸附测定(ELISA)法检测大鼠血清肌酐(Scr)、血浆中性粒细胞明胶酶相关脂蛋白(pNGAL)和血浆肾损伤分子1(pKIM-1)水平,同时检测血浆中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、IL-4和IL-10的表达水平。采用TUNEL法观察肾组织细胞凋亡情况,实时荧光定量PCR(RT-qPCR)法检测含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)、B细胞淋巴瘤2相关X(Bax)和B细胞淋巴瘤2(Bcl-2)信使RNA(mRNA)表达水平。最后,采用western-blotting法检测磷酸化PI3K(p-PI3K)、磷酸化AKT(p-AKT)和磷酸化mTOR(p-mTOR)蛋白表达。结果4组大鼠肾组织病理损伤评分、Scr、pKIM-1、pNGAL、TNF-α、IL-1β、IL-4、IL-10、肾组织细胞凋亡数量、Caspase-3 mRNA、Bax mRNA、Bcl-2 mRNA及肾组织中p-PI3K、p-AKT和p-mTOR蛋白水平比较,差异均有统计学意义(F=132.603、626.719、216.573、335.719、368.219、403.612、169.901、181.281、169.312、960.912、1479.000、32.221、178.346、137.560、136.241,P均<0.001)。与Sham组相比,CLP组大鼠肾组织病理损伤加重,Scr、pKIM-1、pNGAL、TNF-α、IL-1β、Caspase-3 mRNA、Bax mRNA以及p-PI3K、p-AKT和p-mTOR蛋白表达水平均明显升高,肾组织细胞凋亡数量显著增多,而IL-4、IL-10和Bcl-2 mRNA表达均降低(P均<0.05);与CLP组相比,CLP+SLDS组大鼠肾组织病理损伤减轻,Scr、pKIM-1、pNGAL、TNF-α、IL-1β、Caspase-3 mRNA、Bax mRNA以及p-PI3K、p-AKT和p-mTOR蛋白表达水平均显著降低,肾组织细胞凋亡数量显著减少,而IL-4、IL-10和Bcl-2 mRNA表达均增加(P均<0.05)。结论SLDS通过抑制PI3K/AKT/mTOR信号通路对SAKI具有显著的保护作用。

Objective To explore the protective effect of salidroside(SLDS)on septic acute kidney injury(SAKI)in rats by regulating the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signaling pathway.Methods Forty Sprague-Dawley rats were randomly divided into four groups,namely the control group,the sham group,the cecal ligation and perforation group(CLP group)and the SLDS pretreatment group(CLP+SLDS group),10 rats in each group.We assessed the pathological damage of kidney tissue in each group,and used enzyme-linked immunosorbent assay(ELISA)to detect the levels of serum creatinine(Scr),plasma neutrophil gelatinase-associated lipocalin(pNGAL)and plasma kidney injury molecule 1(pKIM-1)in rats.Meanwhile,we detected the expression levels of interleukin-1 beta(IL-1β),tumor necrosis factor-alpha(TNF-α),IL-4 and IL-10 in plasma.We used the TUNEL method to observe renal cell apoptosis,real-time fluorescence quantitative PCR(RT-qPCR)method to detect cysteinyl aspartate specific proteinase-3(Caspase-3),B-cell lymphoma-2 associated X(Bax)and B-cell lymphomato-2(Bcl-2)messenger RNA(mRNA)expression,and western-blotting method to detect phosphorylated PI3K(p-PI3K),phosphorylated AKT(p-AKT)and phosphorylated mTOR(p-mTOR)protein expression.Results There were significant differences in the pathological damage score,Scr,pKIM-1,pNGAL,TNF-α,IL-1β,IL-4,IL-4,IL-10,number of apoptosis,Caspase-3 mRNA,Bax mRNA,Bcl-2 mRNA,p-PI3K protein,p-AKT protein and p-mTOR protein in kidney tissue among the four groups(F=132.603,626.719,216.573,335.719,368.219,403.612,169.901,181.281,169.312,960.912,1479.000,32.221,178.346,137.560,136.241;all P<0.001).Compared with the sham group,the pathological damage of kidney tissue was worsened,the levels of Scr,pKIM-1,pNGAL,TNF-α,IL-1β,Caspase-3 mRNA,Bax mRNA,p-PI3K protein,p-AKT protein and p-mTOR protein in kidney tissue and the number of renal cell apoptosis were significantly increased,and the levels of IL-4,IL-10 and Bcl-2 mRNA were decreased in the CLP group(all P<0.05).Compared with the CLP group,the pathological damage of kidney tissue was reduced,the levels of Scr,pKIM-1,pNGAL,TNF-α,IL-1β,Caspase-3 mRNA,Bax mRNA,p-PI3K protein,p-AKT protein and p-mTOR protein in kidney tissue and the number of renal cell apoptosis were significant decreased,and the levels of IL-4,IL-10 and Bcl-2 mRNA were increased in the CLP+SLDS group(all P<0.05).Conclusion SLDS has a significant protective effect on SAKI by inhibiting the PI3K/AKT/mTOR signaling pathway.