葫芦巴总皂苷对心肌成纤维细胞转分化的影响及机制的实验研究

目的探讨葫芦巴总皂苷（Trigonellafoenumgreacum．LSaponin，TFGs）对尾加压素Ⅱ（UrotensinII，UII）诱导体外培养的大鼠心肌间质成纤维细胞（CFs）发生肌成纤维细胞转分化的干预作用及相关分子机制．方法体外培养大鼠心肌间质成纤维细胞，随机分为正常对照组、UⅡ刺激组和TFGs干预组．正常对照组在整个培养过程中未加任何刺激；uⅡ刺激组加入10“mol／L的尾加压素Ⅱ培养，并分别终止培养于12，24和48h；TFGs干预组将UⅡ作为刺激因素，分别加入终浓度为0，25，50，100和200mg／L的TFGs．应用免疫荧光和免疫组化方法检测显示d—SMA的表达，应用RT—PCR和免疫印迹法分别检测结缔组织生长因子（CTGF）的基因及蛋白表达．结果免疫荧光检测显示，培养24h后，正常对照组细胞胞浆中仅有少量仅．SMA表达，而仅．SMA表达量在经uⅡ作用后明显增多．尾加压素Ⅱ干预CFs不同时间后（12，24，48h），免疫组化显示仪-SMA表达量逐渐增加．UⅡ的这种诱导CFs发生肌成纤维细胞转分化作用可被含TFGs的培养液所抑制，且呈浓度和时问依赖性．与对照组比较，UⅡ刺激组的CTGF的基因及蛋白表达均明显增高（P〈0．01）．而TFGs则可以抑制UⅡ诱导的CTGF基因及蛋白表达量的上调（P〈0．01）．结论尾加压素Ⅱ具有诱导大鼠心肌问质成纤维细胞发生肌成纤维细胞转分化的作用，此作用可能与其促进CTGF的基因及蛋白表达有关．葫芦巴总皂苷能有效地抑制大鼠CFs的转分化，并且下调UⅡ诱导的CTGF的过表达．这进一步明确UⅡ在心肌纤维化发生、发展中的作用，也为临床应用葫芦巴总皂苷防治心肌纤维化提供了实验依据．

Experimental Study on Effects of TFGs on Cardiac Fibroblast Transdifferentiation and Its Mechanisms

Objective To investigate the intervention of Trigonella foenum greacum. L Saponin （TFGs） on urotensin Ⅱ-induced cardiac fibroblasts （CFs）transdifferentiation cultured rats and its related molecular mechanism. Method Cultured CFs were randomly divided into control group, U Ⅱ stimulation group and TFGs intervention group. CFs of the control group were not given any stimulation; CFs of U Ⅱ stimulation group werecultured with 10 -8 mol/L U Ⅱ and the cultures were terminated at 12,14 and 48 hours ; CFs of TFGs intervention group were added U Ⅱ as a stimulus ,whose final concentrations were 0,25,50,100 and 200 mg/L respectively. The expression of a-SMA was observed with immunofluorescence and immunohistochemistry. The expression of connective tissue growth factor （CTGF） was assessed with RT-PCR and Western blotting. Results The results from immunofluorescence showed that there was a little expression of a-SMA in the cytoplasm of cells of the control group after 24-hour culture, but a-SMA expression after the U Ⅱstimulation increased significantly. The results from immunohistochemistry showed that a-SMA expression increased gradually with the intervention of U H at the different times （ 12,24 and 48 hours）. The CFs transdifferentiation induced by U Ⅱ could be inhibited by the culture solution containing TFGs, and the inhibition showed a dose-dependent and time-dependent manner. Compared with those in control group, the expressions of CTGF gene and its protein in U Ⅱ stimulation group increased significantly （ P 〈 0.01 ）, but the regulation of the expressions induced by U Ⅱ could be inhibited by TFGs （P 〈 0.01 ）. Conclusion The transdifferentiation of cardiac interstitial fibroblasts into a-SMA ＋ myofibroblasts of rats may be induced by U Ⅱin vitro. The transdifferentiation may be partially related to the increased CTGF expression. TFGs can effectively inhibit the transdifferentiation of CFs and reduce U Ⅱ -induced overexpression of CTGF. This study should further clarify the role of U Ⅱ in myocardial fibrosis, and also provide an experimental basis for the application of TFGs in the clinical prevention and treatment of myocardial fibrosis.