肾复康对TGF-β1诱导的肾小管上皮细胞HK-2纤维化模型的影响及其机制研究

Effect and Mechanism of Shenfukang (SFK) on Fibrogenesis in Renal Tubular Epithelial HK-2 cells Induced by TGF-β1

目的:探讨肾复康含药血清对转化生长因子-β1(TGF-β1)诱导人肾小管上皮细胞株HK-2间质转化的影响及其分子机制。方法:以HK-2细胞作为研究对象,常规培养后将细胞进行如下分组及刺激处理:空白对照组、模型对照组(10 ng/ml TGF-β1刺激)、32 g/kg尿毒清30%含药血清组(30%尿毒清含药血清+TGF-β1刺激)、18.32 g/kg肾复康10%、30%含药血清组(10%或30%肾复康含药血清+TGF-β1刺激)。MTT法检测肾复康含药血清对HK-2细胞活力的影响;显微镜下观察各组细胞间形态学的差异;ELISA法检测各组细胞上清液中I型胶原(collagen I)和纤维连接蛋白(fibronectin)的水平;q-PCR法检测各组细胞内E-钙黏蛋白(E-cadherin)、波形纤维蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA)和整联蛋白连接激酶(ILK)基因的表达水平;蛋白免疫印迹法检测Smad2/3总蛋白、核蛋白表达及其磷酸化水平;蛋白免疫印迹法检测下游的ILK蛋白表达水平。结果:肾复康含药血清对HK-2细胞的存活率无抑制作用;与空白对照组比较,模型对照组细胞形态由上皮样的铺路石状变成间质样的长梭形,E-cadherin的mRNA水平显著下调(P<0.01),Vimentin、α-SMA和ILK的mRNA水平显著上调(P<0.01),Smad2/3的蛋白磷酸化及核内水平显著上调(P<0.01);与模型对照组比较,肾复康各含药血清组E-cadherin的mRNA水平显著上调(P<0.01),Vimentin、α-SMA和ILK的mRNA水平显著下调(P<0.05或P<0.01),Smad2/3的蛋白磷酸化及核内水平明显下调(P<0.05或P<0.01)。结论:肾复康能逆转TGF-β1诱导的肾小管上皮细胞HK-2纤维化模型的上皮间质转化,其作用机制可能与其抑制胞内Smad2/3-ILK信号通路的活化有关。

Objective:To explore the effect and molecular mechanism of serum containing Shenfukang(SFK) on epithelial-mesenchymal transition(EMT) of HK-2 cells induced by TGF-β1. Methods: HK-2 cells were used in the experiment. After the conventional culture, the cells were grouped and stimulated as follows:the serum group,the model group(10 ng/ml TGF-β1 stimulation), serum containing 30% Niaoduqing group(30% Niaoduqing +TGF-β1 stimulation), serum containing 10% SFK group(10% SFK+ TGF-β1 stimulation) and serum containing 30% SFK group(30% SFK+ TGF-β1 stimulation). The effects of serum containing SFK on the viability of HK-2 cells were determined by MTT assay. Morphological differences among cells were observed under microscope. The levels of collagen I and fibronectin in the supernatant were detected by ELISA. The mRNA expression levels of E-cadherin, Vimentin, α-SMA and ILK in each group were determined by q-PCR assay. The levels of total protein expression, nucleoprotein expression and phosphorylation of smad2/3 were detected by western blot assay. The protein expression level of ILK downstream was detected by western blot. Results: Serum containing SFK had no inhibitory effect on the viability of HK-2 cells. Compared with the control group, the cell morphology of HK-2 cells changed from pavement stone to long spindle type, the mRNA level of E-cadherin was significantly decreased(P<0.01), and the mRNA levels of Vimentin, α-SMA and ILK were significantly increased(P<0.01), also the intranuclear protein level and phosphorylation of smad2/3 were increased(P<0.01). Compared with the model group,the mRNA level of E-cadherin wAS significantly increased(P<0.01), the mRNA levels of Vimentin, α-SMA and ILK were significantly decreased(P<0.05,P<0.01), also the smad2/3 protein phosphorylation and intranuclear level were decreased(P<0.05,P<0.01) in serum containing SFK groups. Conclusion:SFK can significantly inhibit the EMT of HK-2 cells in hepatic fibrosis model induced by TGF-β1, and the mechanism may be related to its inhibitory effect on the activation of intracellular smad2/3-ILK signaling pathway.