电针对内毒素性急性肾损伤兔肾组织Nrf2表达的影响：与p38MAPK信号通路的关系

Effect of electro-acupuncture on Nrf2 expression in renal tissues of rabbits with endotoxic shock-induced acute kidney injury： relationship with p38MAPK signaling pathway

目的 探讨电针对内毒素性急性肾损伤兔肾组织NF-E2相关因子2（Nrf2）表达的影响及其与p38丝裂原活化蛋白激酶（p38MAPK）信号通路的关系.方法 健康雄性新西兰大白兔70只,体重1.5- 2.0 kg,2月龄,采用随机数字表法分为7组（n=10）：正常对照组（C组）、内毒素性急性肾损伤组（AKI组）、电针＋内毒素性急性肾损伤组（EA组）、非穴位＋内毒素性急性肾损伤组（SA组）、电针＋内毒素性急性肾损伤＋ p38MAPK特异性阻断剂SB203580组（EAS组）、SB203580组（S组）和无水乙醇组（A组）.EA组和EAS组电针双侧足三里穴和肾俞穴15 min（刺激强度以兔下肢微颤为宜,疏密波,频率2/100 Hz,刺激电流1-2 mA,波宽0.2 - 0.6 ms）,1次/d,连续5 d;SA组以相同电针参数刺激足三里穴及肾俞穴旁开0.5 cm处.最后一次电针后24 h,AKI组、EA组、SA组和EAS组静脉注射脂多糖5 mg/kg制备内毒素性急性肾损伤模型,其余各组给予等容量生理盐水.模型制备前30 minEAS组及S组静脉注射SB203580 5μmol/kg（溶于0.5ml无水乙醇）,A组静脉注射0.5 ml无水乙醇,其余组给予等容量生理盐水.给予脂多糖或生理盐水后6h时,采集动脉血样,测定血清BUN和Cr浓度,处死兔取肾组织,进行肾组织损伤评分,采用Western blot法测定肾组织Nrf2蛋白表达及p38MAPK磷酸化水平,采用荧光定量PCR法测定Nrf2 mRNA的表达.结果 与C组比较,AKI组、EA组、SA组及EAS组肾组织损伤评分及血清BUN和Cr浓度升高,肾组织Nrf2 mRNA及蛋白表达上调,AKI组、EA组及SA组p38MAPK磷酸化水平升高（P＜0.05）,S组及A组上述各指标差异无统计学意义（P＞0.05）;与AKI组比较,EA组及EAS组肾组织损伤评分及血清BUN和Cr浓度降低,肾组织Nrf2 mRNA及蛋白表达上调,EA组p38MAPK磷酸化水平升高,EAS组p38MAPK磷酸化水平降低（P＜0.05）,SA组上述各指标差异无统计学意义（P＞ 0.05）;与EA组比较,EAS组肾组织损伤评分及血清BUN和Cr浓度升高,肾组织Nrf2 mRNA及蛋白表达下调,p38MAPK磷酸化水平降低（P＜0.05）.结论 电针减轻兔内毒素性急性肾损伤的机制可能与激活p38MAPK信号通路从而上调肾组织Nrf2表达的有关.

Objective To investigate the effect of electro-acupuncture （EA） on nuclear factor E2-related factor 2 （Nrf2） expression in the renal tissues of rabbits with endotoxic shock-induced acute kidney injury （AKI） and the relationship with p38 mitogen-activated protein kinase （p38MAPK） signaling pathway.Methods Seventy male New Zealand white rabbits,weighing 1.5-2.0 kg,aged 2 months,were randomized into 7 groups （n =10 each） using a random number table：normal control group （C group）,endotoxic shock-induced AKI group （AKI group）,EA ＋ endotoxic shock-induced AKI group （EA group）,non-acupoints ＋ endotoxic shock-induced AKI group （SA group）,EA ＋ endotoxic shock-induced AKI ＋ specific p38MAPK blocker SB203580 group （EAS group）,SB203580 group （S group）,and ethanol group （A group）.EA （intensity 1-2 mA,frequency 2/100 Hz,wave length 0.2-0.6 ms） of Zusanli and Shenyu lasting for 15 min was performed once a day for 5 consecutive days in EA and EAS groups.In SA group,EA was performed at the points 0.5 cm lateral to the acupoints of bilateral Zusanli and Shenyu using the parameters of EA mentioned above.At 24 h after the last EA,endotoxic shock-induced AKI was induced by injection of lipopolysaccharide （LPS） 5 mg/kg （in 2 ml normal saline） in AKI,EA,SA and EAS groups,while the equal volume of normal saline was given in the other groups.At 30 min before the model was established,5/μmol/kg SB203580 （in 0.5 ml ethanol） was injected intravenously in EAS and S groups,while ethanol 0.5 ml was given in A group and the equal volume of normal saline was given in the other groups.Blood samples were obtained at 6 h after administration of LPS or normal saline for determination of serum urea nitrogen （BUN） and creatinine （Cr） concentrations.The animals were sacrificed and kidney specimens were obtained for microscopic examination of pathological changes which were scored and for measurement of Nrf2 protein expression and phosphorylation of p38MAPK （by Western blot） and Nrf2 mRNA expression （using fluorescent quantitative PCR）.Results Compared with C group,the pathological score and serum BUN and Cr concentrations were significantly increased,and Nrf2 mRNA and protein expression was up-regulated in AKI,EA,SA and EAS groups,the phosphorylation of p38MAPK was increased in AKI,EA and SA groups,and no significant changes were found in the parameters mentioned above in S and A groups.Compared with AKI group,the pathological score and serum BUN and Cr concentrations were significantly decreased,and Nrf2 mRNA and protein expression was up-regulated in EA and EAS groups,the phosphorylation of p38MAPK was increased in EA group,the phosphorylation of p38MAPK was decreased in EAS group,and no significant changes were found in the parameters mentioned above in SA group.Compared with EA group,the pathological score and serum BUN and Cr concentrations were significantly increased,Nrf2 mRNA and protein expression was down-regulated,and the phosphorylation of p38MAPK was decreased in EAS group.Conclusion The mechanism by which EA mitigates endotoxic shock-induced AKI may be related to activation of p38MAPK signaling pathway and up-regulation of Nrf2 expression in renal tissues of rabbits.