基于mTOR通路的大黄素对肾小管上皮细胞转分化的干预作用机制研究

Intervention mechanism of emodin on renal tubular epithelial cells transdifferentiation based on mTOR pathway

目的:观察大黄素(EMO)对高糖诱导的人肾小管上皮细胞(HK-2)转分化(EMT)的干预效应及对mTOR信号通路的影响,探讨大黄素防治糖尿病肾病(diabetic nephropathy,DN)的可能作用机制。方法:将体外培养的人肾小管上皮细胞HK-2分为正常组(NG组,5.56 mmol·L-1葡萄糖)、高糖组(HG组,60 mmol·L-1葡萄糖)、大黄素组(EMO组,60 mmol·L-1葡萄糖+10μmol·L-1大黄素),在用MTT证实大黄素对正常培养的HK-2细胞无毒性的基础上,选择一个有效的药物浓度,以mTOR特异性抑制剂雷帕霉素(20 nmol·L-1)作为对照。各组细胞培养72 h时后,倒置显微镜观察HK-2细胞形态。Western blot法检测细胞中mTOR、P70S6K、4-EBP1蛋白磷酸化水平;免疫荧光法检测α-SMA以及E-cadherin蛋白分布及表达。结果:高糖可以诱导HK-2细胞形态由多边鹅卵石样发生纤维化样改变,激活mTOR、P70S6K、4-EBP1蛋白磷酸化,降低细胞中上皮表型蛋白E-cadherin的表达,并增加间质表型蛋白α-SMA的表达。大黄素可以改善高糖导致的HK-2细胞形态的变化,降低mTOR、P70S6K、4-EBP1蛋白磷酸化水平,E-cadherin表达的减少及α-SMA表达的增加,雷帕霉素也能达到与大黄素相同的效果。结论:大黄素可以改善高糖诱导的HK-2细胞上皮-间质转分化,此效应可能与抑制mTOR信号通路有关。

OBJECTIVE To observe the effect of emodin(EMO)on high glucose-induced human renal tubular epithelial cells(HK-2)transdifferentiation(EMT)and mTOR signaling pathway,and to explore the possible mechanism of emodin in preventing and treating diabetic nephropathy(DN).METHODS HK-2 cells were divided into normal group(NG,5.56 mmol·L-1 glucose),high glucose group(HG,60 mmol·L-1 glucose),and emodin group(10μmol·L-1 EMO+60 mmol·L-1 glucose).On the basis of the non-toxicity of emodin on normal cultured HK-2 cells comfirmed by MTT,an effective drug concentration was selected,and the mTOR-specific inhibitor rapamycin(20 nmol·L-1)was used as a control.After the cells were cultured for 72 h,the morphology of HK-2 cells was observed under an inverted microscope.Western blot was used to detect the phosphorylation levels of mTOR,P70 S6 K and 4-EBP1 in the cells;The expression and distribution ofα-SMA and E-cadherin protein were detected by immunofluorescence.RESULTS High glucose could induce HK-2 cells to undergo fibrosis-like changes in morphology,activate the phosphorylation of mTOR,P70 S6 K and 4-EBP1,decrease the expression of E-cadherin and increase the expression ofα-SMA.Emodin could ameliorate the morphological changes of HK-2 cells induced by high glucose,decrease the phosphorylation levels of mTOR,P70 S6 K,4-EBP1,decrease the expression of E-cadherin and increase the expression ofα-SMA,and rapamycin could achieve the same effect as emodin.CONCLUSION Emodin can improve the epithelial-mesenchymal transition of HK-2 cells induced by high glucose,which may be related to the inhibition of mTOR signaling pathway.