曲克芦丁通过AMPK/mTOR通路介导自噬反应减轻阿霉素诱导的NS大鼠肾脏损伤

Troxerutin attenuates renal injury in rats with adriamycin-induced nephrotic syndrome through AMPK/mTOR pathway-mediated autophagy

目的探讨曲克芦丁(TRO)通过AMP活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)通路介导自噬反应减轻阿霉素(ADM)诱导的肾病综合征(NS)大鼠肾脏损伤的作用机制。方法建立ADM诱导的NS大鼠模型。将60只SPF级SD大鼠随机分为对照组(Control组)、模型组(Model组)、低剂量TRO组(TRO-L组,50 mg/kg)、高剂量TRO组(TRO-H组,100 mg/kg)、高剂量TRO+AMPK抑制剂Compound C组(TRO-H+CC组,100 mg/kg TRO+20 mg/kg CC),每组12只。收集造模前后大鼠24 h尿液检测尿蛋白含量。利用全自动生化分析仪检测大鼠血清中白蛋白(ALB)、总蛋白(TP)、三酰甘油(TG)、胆固醇(TC)、血尿素氮(BUN)和血清肌酐(Scr)的含量。HE染色观察各组大鼠肾组织病理学变化。ELISA法检测各组大鼠肾组织中SOD、MDA含量。TUNEL染色观察大鼠肾脏组织的细胞凋亡。实时荧光定量PCR(RT-qPCR)法检测大鼠肾脏组织中的AMPK和mTOR mRNA表达水平;Western Blot检测大鼠肾脏AMPK/mTOR通路相关蛋白的表达。结果与Control组相比,Model组大鼠24 h尿蛋白含量、TG、TC、BUN和Scr水平、肾脏组织细胞凋亡率、mTOR mRNA表达和p-mTOR/mTOR显著升高(均P<0.05),血清ALB、TP水平、肾脏SOD水平、AMPK mRNA表达、p-AMPK/AMPK、Beclin-1、LC3蛋白表达显著降低(均P<0.05),肾脏组织病理损伤严重;与Model组相比,TRO-L组和TRO-H组肾脏病理损伤有所减轻,细胞凋亡减少,相关指标变化趋势与上述相反(P<0.05)。Compound C减弱了TRO对NS大鼠肾脏功能的保护作用(P<0.05)。结论TRO通过激活AMPK/mTOR通路介导的自噬反应,减轻ADM诱导的NS大鼠肾脏损伤。

Objective To investigate the mechanism of troxerutin(TRO)alleviating renal injury in rats with adriamycin(ADM)-induced nephrotic syndrome(NS)through autophagy mediated by AMP-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)pathway.Methods ADM-induced NS rat model was established.Sixty SPF SD rats were randomly grouped into Control group,Model group,low-dose TRO group(TRO-L group,50 mg/kg),high-dose TRO group(TRO-H group,100 mg/kg),and high-dose TRO+AMPK inhibitor Compound C group(TRO-H+CC group,100 mg/kg TRO+20 mg/kg CC),12 animals in each group.The 24-hour urine of the rats was collected before and after modeling to detect the urinary protein content.The contents of serum albumin(ALB),total protein(TP),triacylglycerol(TG),cholesterol(TC),blood urea nitrogen(BUN)and serum creatinine(Scr)in rat serum were detected by automatic biochemical analyzer.HE staining was applied to observe the histopathological changes of the rat kidneys.ELISA method was applied to detect the content of SOD and MDA in kidney tissue of rats.TUNEL staining was applied to observe apoptosis in rat kidney tissue.RT-qPCR method was applied to detect the mRNA expression levels of AMPK and mTOR in rat kidney tissue;Western Blot was applied to detect the expression of AMPK/mTOR pathway-related proteins in rat kidney tissue.Results Compared with the control group,the 24-hour urine protein content,TG,TC,BUN and Scr levels,renal tissue apoptosis rate,mTOR mRNA expression and p-mTOR/mTOR were obviously increased,the serum ALB,TP levels,renal SOD level,AMPK mRNA expression and p-AMPK/AMPK were obviously decreased in the Model group(P<0.05),renal tissue pathological damage was severe.Compared with the Model group,the TRO-L and TRO-H groups had reduced renal pathological damage and decreased cell apoptosis,and the trends of related indicators were opposite to the above(P<0.05).Compound C attenuated the protective effect of TRO on kidney function in NS rats(P<0.05).Conclusion TRO alleviates ADM-induced kidney injury in NS rats by activating the autophagy response mediated by the AMPK/mTOR pathway.