六味地黄汤含药血清对CoCl_(2)诱导HK-2细胞后E-cadherin、α-SMA表达的影响

Influence of Liuwei Dihuang Decoction durg-containing serum on expression of E-cadherin and α-SMA in HK-2 cells induced by CoCl_(2)

目的 观察六味地黄汤含药血清对CoCl_(2)缺氧后HK-2细胞中的上皮-间充质转化(epithelial-mesenchymal transition,EMT)标志物E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的影响。方法 体外培养HK-2细胞,分为3组:正常组(10%空白血清)、模型组(10%空白血清+200μmol·L^(-1)CoCl_(2))、含药血清组(10%含药血清+200μmol·L^(-1)CoCl_(2))。CCK-8法检测不同浓度空白血清、含药血清(2.5%、5%、10%、20%、40%)在24、36 h对HK-2细胞存活率的影响;以及不同浓度CoCl_(2)(150、200、300、600μmol·L^(-1))在24 h的最佳干预浓度。qRT-PCR法和Western blot法分别检测各组E-cadherin、α-SMA的mRNA和蛋白表达水平;免疫荧光观察各组E-cadherin、α-SMA蛋白表达情况。结果 CCK-8法确定含药血清和空白血清的最佳干预浓度和干预时间分别为10%和24 h;CoCl_(2)的最佳干预浓度为200μmol·L^(-1)。与正常组比较,模型组E-cadherin的mRNA和蛋白表达水平均明显降低(P<0.01)、α-SMA明显升高(P<0.05);与模型组比较,含药血清组E-cadherin的mRNA和蛋白表达水平均明显升高(P<0.01)、α-SMA明显降低(P<0.01)。结论 缺氧能诱导HK-2细胞发生EMT,六味地黄汤含药血清能够降低α-SMA的表达,升高Ecadherin的表达,从而缓解肾小管EMT。

Objective To observe the biomarkers E-cadherin and α-smooth muscle actin(α-SMA) of epithelial-mesenchymal transition(EMT) in HK-2 cells after CoCl_(2) hypoxia with Liuwei Dihuang Decoction drug-containing serum.Methods HK-2 cells were cultured in vitro and divided into 3 groups:normal group(10% blank serum),model group(10% blank serum+200 μmol·L^(-1)CoCl_(2)),and drug-containing serum group(10% drug-containing serum+200 μmol·L^(-1)CoCl_(2)).The CCK-8 method was used to detect the effects of different concentrations of blank serum and drug-containing serum(2.5%,5%,10%,20%,40%) on the survival rate of HK-2 cells at 24 and 36 h;and the best intervention concentration of different concentrations of CoCl_(2)(150,200,300,600 μmol·L^(-1))in 24 h.qRT-PCR and Western blot method were used to detect the expression of E-cadherin and α-SMA protein and mRNA in each group.The expression of E-cadherin and α-SMA protein in each group was observed by immunofluorescence.Results CCK-8 method determined that the best intervention concentration and time of drug-containing serum and blank serum was 10%and 24 h,and the best intervention concentration of CoCl_(2)was 200 μmol·L^(-1).Compared with normal group,the mRNA and protein expression level of E-cadherin in model group were significantly decreased(P<0.01),and α-SMA expression was increased(P<0.05).Compared with model group,the mRNA and protein expression level of E-cadherin were significantly increased(P<0.01),and α-SMA expression was significantly decreased(P<0.01) in drug-containing serum group.Conclusion Hypoxia can induce EMT in HK-2 cells.Liuwei Dihuang Decoction drug-containing serum can reduce the expression of α-SMA and increase the expression of Ecadherin,thus alleviating EMT in renal tubule.