肝爽颗粒对HSC-T6细胞ColⅠ、ColⅢ、TIMP1基因及蛋白表达的影响

Effects of Ganshuang Keli on gene and protein expression of collagenⅠ,collagenⅢ and tissue inhibitor of metalloproteinase 1 in HSC-T6 cells

目的:观察肝爽颗粒对HSC-T6细胞Ⅰ型胶原(CollagenⅠ,ColⅠ)、Ⅲ型胶原(CollagenⅢ,ColⅢ)、基质金属蛋白酶组织抑制因子1(Tissue Inhibitor of Metalloproteinase1,TIMP1)基因及蛋白表达的影响。方法:用浓度0.025、0.05、0.1、0.15、0.2、0.35、0.5、1.0、1.5、2.0、4.0、8.0、16.0mg/ml肝爽颗粒作用于HSC-T6细胞48小时,采用MTT比色法观察其对HSC-T6细胞生长的影响;以0.05、0.1、0.2mg/ml肝爽颗粒作用于HSC-T6细胞48小时,采用逆转录PCR与ELISA方法分别测定其对HSC-T6细胞ColⅠ、ColⅢ、TIMP1基因及蛋白表达的影响。结果:空白对照组ColⅠ、ColⅢ、TIMP1基因表达水平分别为0.91±0.11、1.54±0.09、1.83±0.13辉度值,蛋白表达水平分别为(187.63±4.11)、(7.59±1.04)、(23.85±2.13)ng/ml,以0.05、0.1、0.2mg/ml肝爽颗粒作用于HSC-T6细胞48小时,浓度0.05mg/ml肝爽颗粒仅能降低ColⅠ蛋白的表达;当浓度升为0.10mg/ml时不仅可显著降低ColⅠ蛋白的表达,而且对ColⅢmRNA与蛋白的表达亦产生明显抑制作用;当浓度达到0.20mg/ml时作用更明显,可显著降低ColⅠ、ColⅢmRNA,ColⅠ、ColⅢ、TIMP1蛋白的表达。结论:肝爽颗粒能明显抑制HSC-T6细胞ColⅠ、ColⅢ基因表达,抑制ColⅠ、ColⅢ、TIMP1蛋白表达,从而可抑制Ⅰ、Ⅲ型胶原的合成,减弱TIMP1对基质金属蛋白酶的抑制作用,这可能是其抗纤维化的作用机制之一。

Objective： To investigate the in vitro effects of Ganshuang Keli on gene and protein expression of collagen Ⅰ （Col Ⅰ）, collagen Ⅲ（ Col Ⅲ ） and tissue inhibitor of metalloproteinase 1 （ TIMP1 ） in HSC-T6 cell. Methods： Cultured HSC-T6 cells were exposed to Ganshuang Keli at a different concentration ranging from 0.025mg/ml to 16. 0mg/ml for 48 hours. MTT colormetric assay was used to evaluate the effect of Ganshuang Keli on HSC-T6 cell proliferation. After incubation with 0. 05, 0. 1, 0. 2mg/ml Ganshuang Keli for 48 hours, HSC-T6 cells were harvested to detect Col Ⅰ , Col Ⅲ, TIMP1 steady state mRNA and protein levels by reverse-transcription polymerase chain reaction （RT-PCR） and enzymelinked immunosorbent assy （ELISA）. Results： The Col Ⅰ, Col Ⅲ and TIMP1 mRNA relative levels of control group were （0.91 ± 0.11 ）, （ 1.54 ± 0. 09）, （ 1.83 ± 0. 13 ） brilliance quantity, protein levels were （ 187. 63 ± 4. 11 ）, （7. 59 ± 1.04）, （ 23.85 ± 2. 13 ） ng/ml, respectively. Treated by 0. 05mg/ml Genshuang Keli for 48 hours, only Col Ⅰ protein expression was downregulated compared with controls. At the concentration of 0.10 mg/ml it could reduce col Ⅰ protein, Col Ⅲ mRNA and Col Ⅲ protein expression obviously. With its concentration rising to 0. 20mg/ml, Ganshuang Keli played more powerful role, it could significantly inhibit Col Ⅰ , Col Ⅲ mRNA expression and Col Ⅰ , Col Ⅲ, TIMP1 protein expression. Conclusion： Ganshuang Keli can suppress HSC-T6 cells Col Ⅰ , Col Ⅲ gene expression and Col Ⅰ , Col Ⅲ, TIMP1 protein expression. The antifibrotic mechanism of Ganshuang Keli might be partly due to its down-regulation of Col Ⅰ , Col Ⅲ mRNA levels and Col Ⅰ , Col Ⅲ, TIMP1 protein levels in HSC-T6 cells.