桂枝去桂加茯苓白术汤含药血清对TGF-β_(1)诱导的人肾小管上皮细胞EMT的抑制作用及其机制

Inhibitory effect of Guizhi Qugui plus Fuling Baizhu decoction-containing serum on TGF-β_(1)-induced EMT in human renal tubular epithelial cells and its mechanism

目的观察桂枝去桂加茯苓白术汤含药血清对TGF-β_(1)诱导的人肾小管上皮细胞上皮—间充质转化(EMT)的影响,并探讨其作用机制。方法取雄性SD大鼠40只,随机分为低剂量组、中剂量组、高剂量组和空白对照组各10只,其中低剂量组、中剂量组、高剂量组分别给予桂枝去桂加茯苓白术汤5、10、20 g/(kg·d)灌胃,空白对照组给予等体积生理盐水灌胃,连续7 d。腹主动脉采血,取上清。将HK-2细胞分为5组,空白对照组加入空白对照组大鼠血清;TGF-β_(1)组加入5 ng/mLTGF-β_(1);低浓度含药血清干预组先加入低剂量组大鼠含药血清后,再加入5 ng/mLTGF-β_(1);中浓度含药血清干预组先加入中剂量组大鼠含药血清后,再加入5 ng/mLTGF-β_(1);高浓度含药血清干预组先加入高剂量组大鼠含药血清后,再加入5 ng/mLTGF-β_(1);各组均培养48 h。采用Westernblotting法检测各组细胞EMT标志蛋白E-cadherin、α-SMA、Vimintin蛋白表达,MAPK信号通路相关蛋白ERK1/2、p38、JNK及其磷酸化水平,Smad3信号通路中Smad3及p-Smad3蛋白表达。结果与空白对照组比较,TGF-β_(1)组α-SMA、Vimintin蛋白表达升高,E-cadherin蛋白表达降低(P<0.05或<0.01);与TGF-β_(1)组比较,低浓度含药血清干预组E-cadherin及α-SMA蛋白表达差异无统计学意义;中浓度及高浓度含药血清干预组α-SMA、Vimintin蛋白水平降低,E-cadherin蛋白表达升高(P<0.05或<0.01)。与空白对照组比较,TGF-β_(1)组ERK1/2、JNK、P38、Smad3蛋白磷酸化水平均升高(P均<0.05);与TGF-β_(1)组比较,低浓度含药血清干预组p38蛋白磷酸化水平降低,中浓度含药血清干预组JNK、p38、Smad3蛋白磷酸化水平降低,高浓度含药血清干预组ERK1/2、JNK、p38、Smad3蛋白磷酸化水平均降低(P<0.05或<0.01)。结论桂枝去桂加茯苓白术汤能够抑制TGF-β_(1)诱导的HK-2细胞EMT过程,其作用机制可能与抑制TGF-β_(1)/Smad3、TGF-β_(1)/MAPK信号通路的激活有关。

Objective To investigate the effects of Guizhi Qugui plus Fuling Baizhu decoction-containing serum on TGF-β_(1)-induced epithelial-mesenchymal transition(EMT)of human renal tubular epithelial cells and to explore its mecha⁃nism.Methods Forty male rats were randomly divided into four groups:the blank control group,low-dose,medium-dose and high-dose groups,which were treated with 5,10,20 g/(kg·d)Guizhi Qugui plus Fuling Baizhu decoction,and the same volume of normal saline,for continuous administration of 7 days.Then,blood was taken from abdominal aortic in asepsis condition,and then the serum was separated.HK-2 cells were divided into into five groups:the blank control group(by adding serum of the blank control group rats),TGF-β_(1)group(by adding TGF-β_(1)of 5 ng/mL),and low-dose,medium-dose,and high-dose drug-containing serum groups(by adding different concentrations of drug-containing serum and then adding TGF-β_(1)of 5 ng/mL).All groups were cultured for 48 hours.Western blotting was used to detect the ex⁃pression of EMT marker proteins E-cadherin,α-SMA and Vimintin,MAPK signaling pathway-related proteins ERK1/2,p38,JNK and their phosphorylation levels,and Smad3 and p-Smad3 proteins in Smad3 signaling pathway.Results The relative protein expression ofα-SMA and Vimintin in the TGF-β_(1)group was significantly higher than that in the blank con⁃trol group,the protein expression of E-cadherin was significantly lower(P<0.05 or P<0.01).Compared with the TGF-β_(1)group,there was no significant difference in the expression of E-cadherin orα-SMA proteins in the low-dose group;the pro⁃tein expression ofα-SMA and Vimintin significantly decreased,and E-cadherin expression increased in the medium-dose group and high-dose group(P<0.05 or P<0.01).Compared with the blank control group,the relative expression levels of ERK1/2,JNK and p38,and phosphorylation levels of Smad3 were significantly higher in the TGF-β_(1)group(all P<0.05).Compared with the TGF-β_(1)group,the phosphorylation level of p38 protein decreased in the low-dose group,the phosphory⁃lation levels of JNK,p38,and Smad3 proteins decreased in the medium-dose group;and the phosphorylation levels of ERK1/2,JNK,p38 and Smad3 decreased in the high-dose group(P<0.05 or P<0.01).Conclusion Guizhi Qugui plus Fuling Baizhu decoction could significantly inhibit TGF-β_(1)-induced renal tubular EMT in HK-2 cells,whose mecha⁃nism may be related to the inhibition of the activation of TGF-β_(1)/Smad3 and TGF-β_(1)/MAPK signal pathways.