柴胡皂苷D对人肾小管上皮细胞转分化的影响及机制

Effect and mechanism of saikosaponin D on transdifferentiation of human renal tubular epithelial cells

目的探讨柴胡皂苷D(SSD)对人肾小管上皮细胞转分化的影响及其作用机制。方法将人肾小管上皮细胞HK-2细胞分为对照组、空白溶剂对照组、肾间质纤维化(RIF)组(5μg/L TGF-β1)、阳性对照组(5μg/L TGF-β1+10μmol/L贝那普利)、SSD组(5μg/L TGF-β1+10.00μmol/L SSD)、SSD+DKK-1组(5μg/L TGF-β1+10.00μmol/L SSD+100μg/L DKK-1)。光学显微镜观察各组细胞形态变化,免疫荧光检测细胞E-钙黏素(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)表达,Western blotting检测细胞基质金属蛋白酶(MMP)-7、Snail、E-cadherin、α-SMA、β-catenin蛋白表达,qRT-PCR检测细胞胶原蛋白Ⅰ(CollagenⅠ)相对表达量。结果对照组、空白溶剂对照组细胞有序、紧密排列,RIF组细胞呈现出长梭形且细胞排列分散;阳性对照组、SSD组和SSD+DKK-1组中长梭形细胞较RIF组减少且细胞分散程度降低。RIF组E-cadherin荧光斑低于对照组(P<0.05),α-SMA荧光斑和MMP-7、Snail、E-cadherin、α-SMA、β-catenin蛋白表达、CollagenⅠ基因相对表达量高于对照组(P<0.05)。阳性对照组、SSD组E-cadherin荧光斑和蛋白表达高于RIF组(P<0.05),α-SMA荧光斑和MMP-7、Snail、α-SMA、β-catenin蛋白表达、CollagenⅠ基因相对表达量低于RIF组(P<0.05)。SSD+DKK-1组E-cadherin荧光斑和蛋白表达高于SSD组(P<0.05),α-SMA荧光斑和MMP-7、Snail、α-SMA、β-catenin蛋白表达、CollagenⅠ基因相对表达量低于SSD组(P<0.05)。结论SSD可抑制人肾小管上皮细胞转分化,可能与抑制Wnt/β-catenin途径活化有关。

Aim To explore the effects and action mechanism of saikosaponin D(SSD)on the transdifferentiation of human renal tubular epithelial cells.Methods The human renal tubular epithelial cells HK-2 were divided into control group,blank solvent control group,RIF group(5μg/L TGF-β1),positive control group(5μg/L TGF-β1+10μmol/L benazepril),SSD group(5μg/L TGF-β1+10.00μmol/L SSD)and SSD+DKK-1 group(5μg/L TGF-β1+10.00μmol/L SSD+100μg/L DKK-1).The morphological changes of cells were observed by optical microscope.Expressions of E-cadherin andα-smooth muscle actin(α-SMA)were detected by immunofluorescence,expressions of MMP-7,Snail,E-cadherin,α-SMA andβ-catenin proteins were detected by Western blotting,and relative gene expression level of collagenⅠ(CollagenⅠ)in cells was detected by qRT-PCR.Results The cells were orderly and tightly arranged in control group and blank solvent control group,while cells were long,stringy,and were dispersedly arranged in RIF group.The number of long and stringy cells in positive control group,SSD group and SSD+DKK-1 group was less than that in RIF group,and the degree of cells dispersion was reduced.The fluorescence spots of E-cadherin in RIF group were lower than those in control group(P<0.05),while fluorescence spots ofα-SMA,expressions of MMP-7,Snail,Ecadherin,α-SMA andβ-catenin,and relative gene expression level of CollagenⅠwere higher than those in control group(P<0.05).The fluorescence spots and expressions of E-cadherin in positive control group and SSD group were higher than those in RIF group(P<0.05),while fluorescence spots ofα-SMA,expressions of MMP-7,Snail,α-SMA andβ-catenin,and relative gene expression level of CollagenⅠwere lower than those in RIF group(P<0.05).The fluorescence spots and expressions of E-cadherin in SSD+DKK-1 group were higher than those in SSD group(P<0.05),while fluorescence spots ofα-SMA,expressions of MMP-7,Snail,α-SMA andβ-catenin,and relative gene expression level of CollagenⅠwere lower than those in SSD group(P<0.05).Conclusion SSD can inhibit the transdifferentiation of human renal tubular epithelial cells,which may be related to the inhibition of activation of Wnt/β-catenin pathways.