金丝桃苷通过AMPK/mTOR/ULK1信号通路对肾病综合征大鼠自噬反应的影响

Influence of hyperoside on autophagy in rats with nephrotic syndrome through AMPK/mTOR/ULK1 signaling pathway

目的探究金丝桃苷(Hyp)对肾病综合征(nephrotic syndrome,NS)大鼠肾自噬及AMPK/mTOR/ULK1通路的影响。方法32只6周龄SD大鼠分为正常组(N组)、肾病综合征组(NS组)、金丝桃苷组(Hyp组,60 mg/kg Hyp)、金丝桃苷+AMPK抑制剂组(Hyp+CC组,60 mg/kg Hyp+0.2 mg/kg CC),每组8只。除N组外,其余各组大鼠采用一次性尾静脉注射阿霉素(6.5 mg/kg)建立NS模型,模型成功率为75.0%。Hyp组大鼠灌胃给予60 mg/kg的Hyp,Hyp+CC组给予60 mg/kg的Hyp灌胃和0.2 mg/kg CC腹腔注射,N组和NS组给予等量溶剂,每天1次,连续14 d。给药结束后,全自动分析仪检测24 h尿液总蛋白(UTP)、血尿素氮(BUN)、血清肌酐(Scr)和白蛋白(ALB)水平;HE染色观察肾组织病理形态;透射电子显微镜(TEM)观察肾组织超微结构变化;Western blot检测肾中自噬、足细胞及AMPK/mTOR/ULK1通路蛋白表达;免疫荧光染色观察自噬体和足细胞的定位。结果相比于N组,NS组肾小球体积变大、肾小管萎缩或部分消失、基底膜增厚;UTP、BUN、Scr、基底膜厚度、足突宽度及p-AMPK/AMPK比值显著增加(P<0.05),ALB、LC3-Ⅱ/Ⅰ、Beclin-1、Atg5、Atg7、NPHS2蛋白水平、NPHS2、Beclin-1相对荧光强度及p-AMPK/AMPK、p-ULK1/ULK1比值显著降低(P<0.05)。相比于NS组,Hyp治疗可改善肾小球形态,降低UTP、BUN、Scr、基底膜厚度、足突宽度及p-AMPK/AMPK比值(P<0.05),增加ALB、LC3-Ⅱ/Ⅰ、Beclin-1、Atg5、Atg7、NPHS2蛋白水平、NPHS2、Beclin-1相对荧光强度及p-AMPK/AMPK、p-ULK1/ULK1比值(P<0.05)。相比于Hyp组,Hyp+CC组肾小球体积变大、肾小管萎缩或部分消失、基底膜增厚;UTP、BUN、Scr、基底膜厚度、足突宽度及p-AMPK/AMPK比值显著增加(P<0.05),ALB、LC3-Ⅱ/Ⅰ、Beclin-1、Atg5、Atg7、NPHS2蛋白水平、NPHS2、Beclin-1相对荧光强度及p-AMPK/AMPK、p-ULK1/ULK1比值显著降低(P<0.05)。结论Hyp可能通过激活AMPK/mTOR/ULK1通路促进肾细胞自噬活性,减轻NS大鼠的足细胞损伤等肾病变。

ObjectiveTo investigate the influence of hypericin(Hyp)on renal autophagy and the AMPK/mTOR/ULK1 pathway in nephrotic syndrome(NS)rats.MethodsThirty-two 6-week-old SD rats were grouped into normal(N),NS,Hyp(60 mg/kg Hyp),and Hyp+CC(60 mg/kg Hyp+0.2 mg/kg CC AMPK inhibitor)groups,with eight rats per group.The NS model was established by one-time injection of adriamycin(6.5 mg/kg)through the tail vein,and the success rate of the model was 75.0%.Rats in the Hyp group were given 60 mg/kg Hyp intragastric administration;rats in the Hyp+CC group were given 60 mg/kg Hyp intragastric administration and 0.2 mg/kg CC intraperitoneal injection;and rats in the N and NS groups were given the same amount of solvent once a day for 14 days.After administration,an automatic analyzer was applied to detect the levels of 24-h urine total protein(UTP),blood urea nitrogen(BUN),serum creatinine(Scr),and albumin(ALB).HE staining was used to observe the pathological morphology of the kidney tissue.The ultrastructure of the renal tissue was observed by transmission electron microscope.Western blot was applied to detect the expression of autophagy,podocyte,and AMPK/mTOR/ULK1 pathway proteins in the kidney.Immunofluorescence staining was applied to visualize the localization of autophagosomes and podocytes.ResultsCompared with the N group,the NS group had increased glomerular volume;atrophied or partially disappeared renal tubules;a thickened basement membrane;and obviously increased UTP,BUN and Scr levels,basement membrane thickness,foot process width,and p-AMPK/AMPK ratio(P<0.05),while the levels of ALB,LC3-II/I,Beclin-1,Atg5,Atg7 and NPHS2;the relative fluorescence intensity of NPHS2 and Beclin-1;and p-AMPK/AMPK and p-ULK1/ULK1 ratios were obviously decreased(P<0.05).Compared with the NS group,the Hyp treatment group had improved glomerular morphology and decreased UTP,BUN,and Scr levels,basement membrane thickness,foot process width,and p-AMPK/AMPK(P<0.05)ratio,but there was an increase in the protein levels of ALB,LC3-II/I,Beclin-1,Atg5,Atg7,NPHS2;relative fluorescence intensity of NPHS2 and Beclin-1;and p-AMPK/AMPK and p-ULK1/ULK1 ratios(P<0.05).Compared with the Hyp group,the Hyp+CC group’s glomerular volume increased;renal tubules atrophied or partially disappeared;basement membrane thickened;and UTP,BUN,Scr levels,basement membrane thickness,foot process width,and p-AMPK/AMPK ratio were obviously increased(P<0.05),whereas the protein levels of ALB,LC3-II/I,Beclin-1,Atg5,Atg7,and NPHS2;relative fluorescence intensity of NPHS2 and Beclin-1;and p-AMPK/AMPK and p-ULK1/ULK1 ratios were obviously decreased(P<0.05).ConclusionsHyp may enhance the autophagic activity of renal cells and attenuate renal pathology,such as podocyte injury,in NS rats by activating the AMPK/mTOR/ULK1 pathway.