miR-188-5p通过靶向调控丝裂原活化蛋白激酶信号通路对大鼠移植肾慢性排斥反应的作用

Effect of miR-188-5p on the chronic rejection of transplanted kidney through targeted regulation of MAPK signaling pathway

目的探讨miR-188-5p通过靶向调控丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号通路对大鼠移植肾慢性排斥反应的作用。方法SPF级F344大鼠20只和Lewis大鼠40只,其中以Lewis大鼠供体10只、受体10只进行肾移植为对照组;以F344大鼠20只为供体,Lewis大鼠20只为受体行肾移植建立大鼠慢性排斥反应模型,并分为模型组和过表达组。3组均于术后连续注射环孢素A 1.5 mg/kg 10 d。过表达组术后当天同时注射miR-188-5p高表达慢病毒颗粒0.1 mL,对照组与模型组分别注射等量生理盐水。术后12周观察3组大鼠存活情况,比较3组大鼠肾功能指标;观察3组大鼠移植肾组织病理变化情况,采用慢性移植肾损伤指数评分系统评价移植肾损伤情况;采用反转录PCR法检测移植肾组织miR-188-5p、p38MAPK、细胞外调节蛋白激酶1/2(extracellular regulated protein kinase 1/2,ERK1/2)、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)mRNA相对表达量,采用Western blot法检测p38MAPK、ERK1/2、JNK蛋白及其磷酸化蛋白相对表达量,并比较p-p38MAPK/p38MAPK、(p-ERK1/2)/(ERK1/2)、p-JNK/JNK。结果术后12周3组大鼠均存活。模型组和过表达组血清肌酐水平、24 h尿蛋白定量、慢性移植肾损伤指数评分高于对照组(P<0.05),模型组高于过表达组(P<0.05)。移植肾组织病理学观察,对照组存在交界性改变,仅少量炎症细胞浸润;模型组慢性排斥反应病理改变明显,过表达组较轻。过表达组移植肾组织miR-188-5p相对表达量(1.90±0.22)高于模型组(0.62±0.18)和对照组(1.02±0.25)(P<0.05),对照组高于模型组(P<0.05);模型组p38MAPK、ERK1/2、JNK mRNA相对表达量,p-p38MAPK、p38MAPK、p-ERK1/2、ERK1/2、p-JNK蛋白相对表达量及p-p38MAPK/p38MAPK、(p-ERK1/2)/(ERK1/2)、p-JNK/JNK高于过表达组和对照组(P<0.05),过表达组高于对照组(P<0.05)。结论miR-188-5p可有效抑制大鼠移植肾慢性排斥反应,其机制可能与靶向调控MAPK信号通路中相关基因的表达及蛋白磷酸化过程有关。

Objective To investigate the effect of miR-188-5 p on the chronic rejection of transplanted kidney through targeted regulation of mitogen activated protein kinase(MAPK)signaling pathway.Methods In 20 SPF-class F344 rats and 40 Lewis rats,10 Lewis rats were transplanted with the kidneys from 10 Lewis rats(control group),20 Lewis rats were transplanted with the kidneys from 20 F344 rats to establish the rat models of chronic rejection(model group and overexpression group).All these 3 groups were given continuous injection of cyclosporine A 1.5 mg/kg for 10 days.In overexpression group,0.1 mL of miR-188-5 p high-expressed lentiviral particles were injected simultaneously on the day after surgery,while control group and model group were injected with the same amount of normal saline.The survival of the rats was observed 12 weeks after surgery,and the renal function indexes were compared in three groups.The pathological changes of transplanted kidney tissues were observed,and chronic renal graft damage index scoring system was used to evaluate the transplanted kidney injury.The relative expressions of miR-188-5 p,p38 MAPK,extracellular regulated protein kinase 1/2(ERK1/2)and c-Jun amino terminal kinase(JNK)mRNA were detected by reverse transcription PCR.The relative expressions of p38 MAPK,ERK1/2,JNK and phosphorylated proteins were detected by Western blot,and p-p38 MAPK/p38 MAPK,p-ERK1/2/ERK1/2 and p-JNK/JNK were compared.Results All rats survived in 12 weeks after surgery.The levels of serum creatinine,24 h urine protein quantitation and the score of chronic graft injury index were higher in model group and overexpression group than those in control group(P<0.05),and higher in model group than those in overexpression group(P<0.05).The pathological examination results of transplanted kidney tissues showed a borderline change in control group,with a small amount of inflammatory cells infiltration;an obvious chronic rejection in model group;and a lighter pathological change in overexpression group.The relative expression of miR-188-5 p in transplanted kidney tissue was higher in overexpression group(1.90±0.22)than that in model group(0.62±0.18)and control group(1.02±0.25)(P<0.05),and higher in control group than that in model group(P<0.05).The relative expressions of p38 MAPK,ERK1/2 and JNK mRNA,the relative expressions of p-p38 MAPK,p38 MAPK,p-ERK1/2,ERK1/2 and p-JNK proteins,as well as the p-p38 MAPK/p38 MAPK,(p-ERK1/2)/(ERK1/2)and p-JNK/JNK protein ratios were the highest in model group(P<0.05),followed by overexpression group and control group in turn(P<0.05).Conclusion miR-188-5 p can effectively inhibit the chronic rejection of kidney transplantation,probably by targeted regulating the expressions of related genes and process of the related protein phosphorylation in MAPK signaling pathway.