尖叶假龙胆有效成分雏菊叶龙胆酮抑制TGF-β1诱导心肌成纤维细胞表型转化的研究

Bellidifolin from the Active Ingredient of Gentianella Acuta Preventing TGF-β1-induced Phenotypic Transformation of Cardiac Fibroblasts

目的:探讨尖叶假龙胆有效成分雏菊叶龙胆酮(BEL)对转化生长因子β1(TGF-β1)诱导的心肌成纤维细胞(CFs)表型转化的作用。方法:采用SD乳大鼠,利用胰蛋白酶联合Ⅱ型胶原酶消化法和差速贴壁法提取原代CFs,细胞经传代、纯化,行波形蛋白(Vimentin)鉴定,给予TGF-β1(5、10、20 ng/mL)刺激,倒置显微镜下观察细胞形态并拍照;给予10 ng/mL的TGF-β1刺激0、6、12、24、48、72 h,Western blot检测α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(CollagenⅠ)和Ⅲ型胶原蛋白(CollagenⅢ)的表达;给予10 ng/mL的TGF-β1刺激24 h,细胞免疫荧光染色和Western blot检测α-SMA、CollagenⅠ和CollagenⅢ的变化;给予BEL预处理0.5 h,再用TGF-β1刺激24 h,采用细胞免疫荧光染色观察α-SMA、CollagenⅠ和CollagenⅢ的改变。结果:提取的细胞经形态观察和Vimentin鉴定为CFs,10 ng/mL的TGF-β1刺激CFs可显著诱导其表型转化;BEL预处理可抑制TGF-β1诱导α-SMA、CollagenⅠ和CollagenⅢ表达。结论:BEL能抑制TGF-β1诱导CFs表型转化,从而改善心肌纤维化。

Objective:To study the effect of Bellidifolin(BEL)on the phenotypic transformation of Cardiac fibroblasts(CFs)induced by TGF-β1.Methods:Primary CFs were extracted from SD neonatal rats’hearts by trypsin combined with typeⅡcollagenase digestion and differential adhesion.The cells were cultured,purified,and identified by immunofluorescence staining with vimentin.TGF-β1(5,10,20 ng/mL)was used to stimulate CFs,and cell morphology was observed under an inverted microscope and photographed.The expressions ofα-SMA,CollagenⅠ,and CollagenⅢwere detected by Western blot after being stimulated with 10 ng/mL TGF-β1 for 0,6,12,24,48,and 72 h.After being subjected to 10 ng/mL TGF-β1 for 24 h,the changes ofα-SMA,CollagenⅠ,and CollagenⅢin CFs were detected by immunofluorescence staining and Western blot.BEL was pretreated for 0.5 h and then administrated with TGF-β1 for 24 h.The expression ofα-SMA,CollagenⅠ,and CollagenⅢwere observed by immunofluorescence staining.Results:The extracted cells were identified as CFs by morphological observation and identified with vimentin.The phenotypic transformation was significantly induced by 10 ng/mL TGF-β1 in CFs.BEL pretreatment inhibited TGF-β1-induced expression ofα-SMA,CollagenⅠ,and CollagenⅢ.Conclusion:BEL could inhibit the phenotypic transformation of CFs induced by TGF-β1 to improve myocardial fibrosis.