miR-145-5p对肾缺血/再灌注损伤大鼠的肾脏保护作用及其机制

Renal protective effect of miR-145-5p on rats with renal ischemia-reperfusion injury and its underlying mechanism

目的 观察miR-145-5p对肾缺血/再灌注(I/R)损伤大鼠肾损伤的影响,并探讨其肾脏保护作用是否与通过蛋白激酶B/雷帕霉素靶蛋白(AKT/mTOR)信号通路而调控自噬水平有关。方法 将40只SD大鼠随机分成Sham组、I/R组、I/R+agomir组、I/R+agomir+LY组,每组10只;除Sham组外均建立肾I/R模型,I/R+agomir组和I/R+agomir+LY组在建模操作前3 d经尾静脉注射miR-145-5p agomir 10 nmol/L,I/R+agomir+LY组同时腹腔注射AKT特异性抑制剂TLY294002 50 mg/kg,Sham组和I/R组经尾静脉注射等量PBS溶液。各组分组处理2 d,比较血清尿素氮(BUN)、肌酐(Scr)水平及肾组织损伤评分,肾组织miR-145-5p表达,肾组织细胞凋亡率及促凋亡蛋白Bax、抗凋亡蛋白Bcl-2表达,肾组织自噬相关蛋白LC3B荧光表达强度、LC3Ⅱ/LC3Ⅰ、肌球蛋白样Bcl-2结合蛋白(Beclin1)、自噬相关蛋白5(Atg5)表达,肾组织AKT/mTOR通路相关蛋白p-AKT/AKT、p-mTOR/mTOR。结果 与Sham组比较,I/R组大鼠肾组织miR-145-5p表达明显降低,I/R+agomir组和I/R+agomir+LY组均明显升高(P均<0.01)。与Sham组比较,其他三组肾组织细胞凋亡率及损伤评分均升高,且I/R组和I/R+agomir+LY组升高更明显(P均<0.05)。与Sham组、I/R+agomir组比较,I/R组、I/R+agomir+LY组大鼠血清BUN、Scr水平及肾组织Bax表达、LC3B荧光表达强度、LC3Ⅱ/LC3Ⅰ、Beclin1表达均升高,Bcl-2、Atg5蛋白表达及p-AKT/AKT、p-mTOR/mTOR均降低(P均<0.05);Sham组、I/R+agomir组上述指标比较,I/R组、I/R+agomir+LY组上述指标比较均无统计学差异(P均>0.05)。结论 miR-145-5p可以通过激活AKT/mTOR信号通路来抑制晚期自噬的过度激活,从而减轻肾I/R大鼠的肾损伤,发挥肾脏保护作用。

Objective To observe the effect of mi R-145-5p on renal injury in rats with renal ischemia/reperfusion(I/R)injury,and to explore whether its renal protective effect is related to the regulation of autophagy through protein kinase B/target of rapamycin(AKT/m TOR)signaling pathway.Methods Forty SD rats were randomly divided into the Sham group,I/R group,I/R+agomir group,and I/R+agomir+LY group,with 10 rats in each group.Renal I/R models were established in all groups except the Sham group.The mi R-145-5p agomir(10 nmol/L)was injected via tail vein 3days before I/R operation in the I/R+agomir and I/R+agomir+LY groups.LY294002(50 mg/kg)was simultaneously intraperitoneally injected in the I/R+agomir+LY group,while the same volume of PBS was injected through tail vein in the Sham and I/R groups.The levels of serum urea nitrogen(BUN)and creatinine(Scr),renal tissue injury score,mi R-145-5p expression,apoptosis rate of renal tissue,and the expression levels of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 were compared.The fluorescence intensity of autophagy-related protein LC3B,the expression levels of LC3Ⅱ/LC3Ⅰ,Beclin1 and Atg5,and the values of p-AKT/AKT and p-m TOR/m TOR were compared.Results Compared with the Sham group,the expression of mi R-145-5p in the I/R group decreased,and the expression levels of mi R-145-5p in the I/R+agomir and I/R+agomir+LY groups increased(all P<0.01).Compared with the Sham group,apoptosis rate and renal tissue injury score in the other three groups increased,and the increase was more obvious in the I/R+agomir and I/R+agomir+LY groups(all P<0.05).Compared with the Sham and I/R+agomir groups,serum BUN and Scr levels,Bax expression,LC3B fluorescence intensity,LC3Ⅱ/LC3Ⅰand Beclin1 expression in the I/R and I/R+agomir+LY groups significantly increased,while the expression levels of Bcl-2 and Atg5,p-AKT/AKT and p-m TOR/m TOR significantly decreased(all P<0.05).There were no significant differences in the above indexes between Sham group and I/R+agomir group,and between I/R group and I/R+agomir+LY group(all P>0.05).Conclusion Mi R-145-5p plays a protective role in rats with renal ischemia/reperfusion injury by inhibiting late autophagy through activating AKT/m TOR signaling pathway.