火把花根片对高糖诱导肾小管上皮间充质转化的影响及机制探讨

Effect and mechanism of colquhounia root tablet on renal tubular epithelial mesenchymal transition induced by high glucose

目的研究火把花根片(CRT)对高糖诱导的肾小管上皮细胞(HK-2)上皮-间充质转化(EMT)的影响并探讨其可能的作用机制。方法体外培养HK-2,将HK-2分为以下5组:对照组(CON组)、高渗组(MA组)、高糖组(HG组)、高糖+火把花根片组(HG+CRT组)、高糖+PI3K抑制剂组(HG+LY29400组)、高糖+火把花根片+PI3K抑制剂组(HG+CRT+LY29400组)。采用实时荧光定量PCR(qPCR)检测各组E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、磷酸酶和张力蛋白同系物(PTEN)mRNA表达水平;采用Western blot检测各组PTEN、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、E-cadherin、α-SMA的蛋白表达水平。结果与CON组比较,HG组细胞α-SMA的蛋白及mRNA表达水平、p-Akt的蛋白表达水平、p-Akt/Akt比值升高,E-cadherin、PTEN的蛋白及mRNA表达水平降低,差异有统计学意义(P<0.05)。与HG组比较,HG+CRT组细胞中α-SMA的蛋白及mRNA表达水平降低,E-cadherin的蛋白及mRNA表达水平相对增加,差异有统计学意义(P<0.05);与HG组比较,HG+LY29400组PI3K、p-Akt、α-SMA蛋白表达水平和p-Akt/Akt比值降低,PTEN的蛋白及mRNA表达水平、E-cadherin的蛋白表达水平升高,差异有统计学意义(P<0.05)。与HG+CRT组比较,HG+CRT+LY29400组E-cadherin、α-SMA、PTEN、PI3K、Akt的蛋白表达水平及p-Akt/Akt比值差异均无统计学意义(P>0.05),而p-Akt蛋白表达升高(P<0.05)。结论在体外,CRT可以通过PTEN/PI3K/Akt信号通路逆转高糖诱导的肾小管上皮细胞EMT。

Objective To investigate the effect of colquhounia root tablet(CRT)on hyperglucose-induced epithelial-mesenchymal transition(EMT)in renal tubular epithelial cells(HK-2),and to explore its possible action mechanism.Methods HK-2 was cultured in vitro,and HK-2 was divided into the following five groups:control group(CON group),hyperosmolar group(MA group),high glucose group(HG group),high sugar+CRT group(HG+CRT group),high sugar+phosphatidylinositol 3 kinase inhibitor group(HG+LY29400 group),high sugar+CRT+phosphatidylinositol 3 kinase inhibitor group(HG+CRT+LY29400).The real time immunofluorescence quantitative PCR(qPCR)was used to detect the mRNA expression levels of E-cadherin,α-smooth muscle actin(α-SMA)and phosphatase and tensin homolog(PTEN)in each group.Western-blot was used to detect the protein expression levels of PTEN,phosphatidylinositol 3 kinase(PI3K),protein kinase B(Akt),phosphorylated protein kinase B(p-Akt),E-cadherin andα-SMA in each group.Results Compared with the CON group,the protein and mRNA expression levels ofα-SMA,p-Akt protein expression level and p-Akt/Akt ratio in the HG group were increased,the protein and mRNA expression levels of E-cadherin and PTEN were decreased,and the differences were statistically significant(P<0.05).Compared with the HG group,theα-SMA protein and mRNA expression levels in the HG+CRT group were decreased,while the E-cadherin protein and mRNA expression levels were increased,and the differences were statistically significant(P<0.05).Compared with the HG+CRT group,there was no significant difference in the E cadherin,αSMA,PTEN,PI3K and Akt protein expression levels and p-Akt/Akt ratio in the HG+CRT+LY29400 group had no significant differences(P>0.05),while the expression level of p-Akt protein was increased,and the difference was statistically significant(P<0.05).Conclusion In vitro,CRT could reverse hyperglucose-induced renal tubular epithelial cell EMT via the PTEN/PI3K/Akt signaling pathway.