羟基喜树碱对大鼠肝星状细胞转化生长因子β1、α-平滑肌肌动蛋白和I型胶原表达的影响

Effects of hydroxycamptothecin on β1、α--SMA and collagen I expression in rat hepatic satellite cells

目的研究羟基喜树碱（HCPT）对大鼠肝星状细胞（HSC）-T6增殖、转化生长因子D1、α-平滑肌肌动蛋白、I型胶原表达的影响，探讨HCPT抑制肝纤维化的机制。方法体外培养肝星状细胞-T6，设立空白对照组和HCPT低剂量组（0．25μg／L）、HCPT中剂量组（0．50μg／L）、HCPT高剂量组（0．75μg／L）。四甲基偶氮唑盐比色实验测定细胞增殖的情况，RT-PCR检测转化生长因子β1、α-平滑肌肌动蛋白、I型胶原基因表达；Westem blot检测转化生长因子D1、α-平滑肌肌动蛋白表达；酶联免疫吸附法检测培养细胞上清液中的I型胶原的含量。组间差异用方差分析，两两比较用q检验。结果（1）HCPT低剂量组、中剂量组、高剂量组4值分别为0．631±0．074、0．469±0．012、0．204±0．001，均较空白对照组（0．793±0．098）降低（F=82．86，P〈0．01）；（2）与空白对照组比较，HCPT低剂量组、中剂量组、高剂量组I型胶原（0．716ziz0．064、0．611±0．040、0．510±0．014、0．403±0．026）、α-平滑肌肌动蛋白（0．696±0．075、0．579±0．037、0．470±0．024、0．299±0．017）、转化生长因子D1（1．019±0．056、0．835±0．022、0．696±0．055、0．322±0．104）mRNA表达均较显著下降∽值分别为133．304，244．501，100．164，P值均〈0．01）；（3）与空白对照组比较，HCPT低剂量组、中剂量组、高剂量组α-平滑肌肌动蛋白（0．858±0．050、0．620±0．045、0．525±0．042、0．434±0．052）、TGFD1蛋白（0．872±0．053、0．654±0．047、0．545±0．042、0．436±0．039）表达均下降（F值分别为234．56，312．34，P值均〈0．01）；（4）HCPT低剂量组、中剂量组、高剂量组I型胶原含量分别为（168．367±16．453）ng／ml、（141．284±11．731）ng／ml、（132．910±10．048）ng／ml，均较空白对照组（188．733±18．299）ng／ml下降（F=15．49，P〈0．01）。结论HCPT能下调肝星状细胞-T6TGFp1表达，抑制肝星状细胞-T6增殖、活化以及I型胶原的合成和分泌，这可能是HCPT抗肝纤维化的作用机制之一。

Objective To investigate the molecular mechanism of hydroxycamptothecin （HCPT）- mediated anti-hepatic fibrosis by evaluting its effects on expression of tumor growth factor-beta 1 （TGFI31）, alpha-smooth muscle actin （α-SMA） and collagen I （Col I） in hepatic satellite cells （HSCs）. Methods Cultured HSCs were treated with different concentrations of HCPT： low-dose group, 0.25 mg/L; middle-dosegroup, 0.5 mg/L; high-dose group, 0.75 mg/L; and control group, 0 mg/L. Cell proliferation was assessed by the MTT assay. The mRNA expressions of TGF[31, α-SMA and Col I were determined by reverse transcription- polymerase chain reaction. The protein expressions of TGF[31 and α-SMA were detected by Western blot. The content of Col I in the cultured HSCs＇ supematant was measured by enzyme-linked immunosorbent assay. Results The MTT absorbance values of the low-dose group （0.631 ± 0.074）, middle-dose group （0.469 ± 0.012） and high-dose group （0.204＋ 0.001） were significantly lower than that of the control group （0.793 ＋0.098; F= 82.86, P〈0.01）. Compared with the control group, the HCPT-treated groups showed significantly down- regulated gene expressions of TGF[31 （control： 0.716±0.064 vs. low： 0.611 ±0.040, middle： 0.510±0.014, high： 0.403 ±0.026）, ±t-SMA （control： 0.696±0.075 vs. low： 0.579±0.037, middle： 0.470±0.024, high： 0.299±0.017）, and Col I （control： 1.019-4-0.056 vs. low： 0.835±0.022, middle： 0.696±0.055, high： 0.322±0.104） （all, P〈 0.01）. Meanwhile, HCPT-treated HSCs showed significantly reduced protein expressions of TGF]31 （control： 0.872 ± 0.053 vs. low： 0.654 ± 0.047, middle： 0.545 ± 0.042, high： 0.436 ± 0.039） and α-SMA （control： 0.858 ± 0.050 vs. low： 0.620＋ 0.045, middle： 0.525 ± 0.042, high： 0.434 ＋ 0.052） （all, P〈 0.01）. The Col I levels secreted by HSCs were significantly lower in the HCPT-treated groups （low： 168.367 ± 16.453 ng/mi; middle： 141.284 ± 11.731 ng/ml; high： 132.910± 10.048 ng/ml） than in the control group （188.733 ±18.299ng/ml） （all, P〈0.01）. Conclusions The mechanism of HCPT-mediated anti-hepatic fibrosis may involve down-regulation of TGF[31 expression to inhibit HSC proliferation and activation, as well as reduction of Col I synthesis and secretion.