育亨宾对肾移植中肾间质纤维化的抑制作用及其机制

Inhibitory effect of Yohimbine on renal interstitial fibrosis in renal transplantation and its mechanism

目的探索育亨宾对肾移植大鼠肾间质纤维化的抑制作用及其机制。方法在F344大鼠(供体)和Lewis大鼠(受体)之间进行原位左肾移植。对照组大鼠行右肾切除,其他组大鼠为肾移植大鼠。肾移植后,将48只Lewis大鼠随机分为4组:对照组、肾移植组、0.1 mg/kg育亨宾治疗组(低剂量组)和0.5 mg/kg育亨宾治疗组(高剂量组)。从肾移植后的第1天起,育亨宾治疗组每周2次大鼠腹腔分别注射0.1 mg/kg和0.5 mg/kg的育亨宾(2 ml),对照组和肾移植组大鼠腹腔注射2 ml的0.9%生理盐水,共治疗16周。通过苏木精-伊红(HE)染色和Masson三色染色检测肾形态和纤维化。通过肌氨酸氧化酶法检测大鼠血清肌酐(Scr),通过二乙酰肟比色法检测血尿素氮(BUN),通过酶联免疫吸附法(ELISA)检测大鼠血清肿瘤坏死因子-α(TNF-α)和转化生长因子-β1(TGF-β1)。通过免疫组化和Western blotting检测肾组织中α-SMA、E-cadherin、Smurf1、Akt、p-Akt、mTOR、p-mTOR、P70S6K和p-P70S6K的表达。结果肾移植组大鼠肾小管扩张、上皮萎缩、间质炎性细胞浸润、间质间隙明显扩张,胶原沉积明显。低剂量组和高剂量组大鼠的肾组织形态较肾移植组明显改善。与肾移植组比较,低剂量组和高剂量组大鼠肾组织纤维化面积明显减小(P<0.05),血清TNF-α和TGF-β1水平明显降低(P<0.05),Scr和BUN水平明显降低(P<0.05)。与肾移植组比较,低剂量组和高剂量组大鼠α-SMA和Smurf1的蛋白表达水平降低(P<0.05),p-Akt/Akt、p-mTOR/mTOR和p-P70S6K/P70S6K水平降低(P<0.05),而E-cadherin的蛋白表达水平升高(P<0.05)。结论育亨宾可改善肾移植大鼠的肾形态和功能,并减轻肾纤维化,育亨宾对移植肾的保护作用可能通过Smurf1和Akt-mTOR-P70S6K信号通路来介导。

Objective To explore the inhibitory effect of Yohimbine on renal interstitial fibrosis in renal transplantation rats and its mechanism.Methods Orthotopic left renal transplantation was performed between F344 rats(donor)and Lewis rats(recipient).Rats in control group underwent right nephrectomy.The rats in the other groups underwent kidney transplantion.After renal transplantation,48 Lewis rats were randomly divided into 4 groups:control group,renal transplantation group,0.1 mg/kg Yohimbine group(low dose group)and 0.5 mg/kg Yohimbine group(high dose group).From the first day after kidney transplantation,the rats were intraperitoneally injected with 0.1 mg/kg and 0.5 mg/kg of Yohimbine(2 ml)twice a week for 16 weeks in low dose group and high dose group,respectively.Rats were intraperitoneally injected with 2 ml of 0.9%normal saline in control group and kidney transplantation group,respectively.Renal morphology and fibrosis were detected by hematoxylin-eosin(HE)staining and Masson’s trichrome staining.Serum creatinine(Scr)was detected by sarcosine oxidase method,blood urea nitrogen(BUN)was detected by diacetyl oxime colorimetric method,serum levels of tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1)were detected by enzyme-linked immunosorbent assay(ELISA).The expression levels ofα-SMA,E-cadherin,Smurf1,Akt,p-Akt,mTOR,p-mTOR,P70S6K and p-P70S6K in renal tissue were detected by immunohistochemistry and Western blotting.Results In renal transplantation group,renal tubular dilatation,epithelial atrophy,interstitial inflammatory cell infiltration,interstitial space dilation and collagen deposition were obviously observed.The renal tissue morphology in low dose group and high dose group was significantly improved compared with renal transplantation group.Compared with renal transplantation group,the area of renal tissue fibrosis was significantly reduced in low dose group and high dose group(P<0.05),serum TNF-αand TGF-β1 levels were significantly reduced(P<0.05),Scr and BUN levels were significantly reduced(P<0.05).Compared with kidney transplantation group,the protein expression levels ofα-SMA and Smurf1 in low dose group and high dose group were decreased(P<0.05),the levels of p-Akt/Akt,p-mTOR/mTOR,and p-P70S6K/P70S6K were decreased(P<0.05),while the protein expression level of E-cadherin was increased(P<0.05).Conclusion Yohimbine can improve the renal morphology and the function of renal transplanted rats,and reduce the renal fibrosis.The protective effect of Yohimbine on transplanted kidney may be mediated through the Smurf1 and Akt-mTOR-P70S6K signaling pathways.