桑白皮提取物桦木酸对TGF-β1诱导的人肺泡上皮细胞HPAEpiC上皮间质转化的抑制作用

Inhibitory Effect of Betulinic Acid from Mori Cortex on TGF-β1-induced Epithelial-mesenchymal Transformation of HPAEpiC cells

目的探讨桑白皮提取物桦木酸对生长转化因子(TGF-β1)诱导的人肺泡上皮细胞HPAEpiC上皮间质转化的抑制作用及其可能机制。方法以HPAEpiC细胞为研究对象,采用0.5 ng·mL^-1 TGF-β1诱导的上皮间质转化模型进行实验。设置空白对照组、模型组、桦木酸给药组。MTT法检测桦木酸对HPAEpiC细胞存活率的影响;Real-time PCR分析桦木酸对诱导后的HPAEpiC细胞内miR-200b-5p、miR-200c-5p表达水平及上皮间质转化标志物(Coll、E-cad、α-SMA mRNA)表达水平的影响。酶联免疫吸附法(ELISA)检测桦木酸对诱导后HPAEpiC细胞内羟脯氨酸(HYP)分泌水平的影响。结果桦木酸对HPAEpiC细胞增殖活性呈单向抑制趋势,且增殖抑制率与药物浓度呈依赖关系。与模型组比较,桦木酸可有效抑制诱导后细胞内miR-200b-5p(P<0.05)、miR-200c-5p(P<0.01,P<0.05)表达,同时降低细胞内ColⅠ(P<0.01)、α-SMA mRNA(P<0.05)表达,促进E-cadherin mRNA表达(P<0.01,P<0.05),降低细胞内HYP含量(P<0.01,P<0.05)。结论桑白皮提取物桦木酸对TGF-β1诱导的人肺泡上皮细胞HPAEpiC上皮间质转化有一定的抑制作用,为特发性肺纤维化潜在的治疗药物,其可能的作用机制为抑制miR-200b-5p、miR-200c-5p表达;下调ColⅠ和α-SMA mRNA,上调E-cad mRNA。

Objective To study the effect of betulinic acid from Mori cortex on inhibition of TGF-β1-induced epithelial mesenchymal transition in HPAEpiC cells and its possible mechanism.Methods HPAEpiC cells were used as the research object,the experiment was performed on epithelial mesenchymal transition model induced with0.5 ng·mL^-1 TGF-β1,and blank control group,model group and betulinic acid group were set up.MTT assay was carried out to detect the effect of betulinic acid on the survival of HPAEpiC cells.HPAEpiC cells induced by TGF-β1(0.5 ng·mL^-1) was intervened by betulinic acid,Real-time quantitative polymerase chains reaction(RT-qPCR) was taken to detect the expression of miR-200 b-5 p,miR-200 c-5 p and the epithelial mesenchymal transition markers such as Col Ⅰ,α-SMA mRNA and E-cadherin.Enzyme linked immunosorbent assay(ELISA) was used to detect hydroxyproline(HYP) level.Results The betulinic acid could inhibit the growth of HPAEpiC cells and showed a concentration dependence.Compared with the model group,betulinic acid can effectively inhibit the expression of miR-200 b-5 p(P<0.05) and miR-200 c-5 p(P<0.01,P<0.05),reduce the expression of Col Ⅰ(P<0.01) and α-SMA(P<0.05) mRNA but promote E-cad mRNA(P<0.01,P<0.05).Content of HYP in betulinic acid group was significantly lower than that in model group(P<0.01,P<0.05).Conclusion The betulinic acid can effectively inhibit the occurrence of epithelial mesenchymal transition in HPAEpiC cells induced by TGF-β1,which is a potential therapeutic agent for idiopathic pulmonary fibrosis.Its possible mechanism maybe inhibiting the expression of miR-200 b-5 p,miR-200 c-5 p,down-regulating Col Ⅰ and α-SMA mRNA expression,and up-regulating Ecadherin mRNA expression.