扯根菜对肝星状细胞分泌转化生长因子-β1及I型胶原的影响

目的 观察扯根菜浸膏对活化型肝星状细胞(HSC)增殖分泌转化生长因子-β1(TGF-131)及I型胶原的影响.方法 雄性SD大鼠20只分为2组,分别以0.9%氯化钠溶液、扯根菜浸膏灌胃,3 d后采血并分离血清.HSC-T6细胞常规培养后分为对照组和扯根菜浸膏组,分别以空白大鼠血清、体积分数为0.1扯根菜浸膏药物大鼠血清的改良Eagle培养基(DMEM)培养.AlamarBlue法检测细胞活力.噻唑蓝(MTT)法检测药物细胞毒性,实时PCR检测TGF-β1及I型胶原mRNA表达,Western印迹法观察细胞TGF-β1蛋白及I型胶原表达.应用单因素方差分析,两两比较用q检验.结果 不同浓度扯根菜浸膏血清均能抑制细胞增殖.尤以体积分数为0.1扯根菜浸膏血清在24 h时对细胞的增殖抑制率最为明显(P＜0.01).与相应浓度正常血清比较,不同浓度扯根菜浸膏血清无明显细胞毒性(P＞0.05).体积分数为0.1扯根菜浸膏血清作用于HSC-T6 24 h后,其TGF-β1及I型胶原mRNA为2.790±0.174和1.213±0.099,正常血清组分别为9.827±1.429和4.053±1.005,差异有统计学意义(P＜0.01);10%扯根菜浸青血清作用于HSC-T6 24 h,TGF-β1及I型胶原蛋白(210×103,90× 103)表达分别为0.432±0.066、0.567±0.012和0.450±0.085,正常血清组分别为1.595±0.061、1.483±0.020和1.636±0.147,差异有统计学意义(P＜0.01).结论 扯根菜浸膏能明显抑制肝星状细胞增殖及TGF-β1、I型胶原分泌.为临床治疗肝纤维化提供了实验依据.

The experimental study on the effects of Penthorum chinense Pursh on transforming growth factor-β1 and collagen type I secretion in hepatic stellate cells

Objective To observe the effects of serum with drug Penthorum chinense Pursh extractum on transforming growth factor (TGF)-β1 and collagen I secretions of activated hepatic stellate cells. Methods Twenty male SD rats were divided into 2 groups, and were administered with 0.9% sodium chloride solution and Penthorum chinense Pursh extraetum via gastrogavage for 3 days respectively and then sacrificed. Serum samples of these rats were collected. HSC-T6 cells were divided into the normal group and the treatment group. The cells of the normal group were incubated in Dulbecco's modified eagle medium (DMEM)with sera of normal rats, while those of the treatment group were incubated in DMEM with sera from Penthorum chinense Pursh extractum treated rats. The HSC-T6 viability was observed by AlamarBlue assay, while the toxicity of Penthorum ehinense Pursh extractum was measured by 3-(4, 5-Dimethyhhiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The expression of collagen I and TGF-β1 mRNA were determined by real timepolymerase chain reaction (real time PCR). The protein expressions of collagen I and TGF-β1 were analyzed by Western blotting. The data were analyzed by single factor analysis of variance and pairwise comparison was done by q test. Results Different concentrations of sera from Penthorum chinense Pursh extractum treated rats could all inhibit HSC-T6 proliferation, especially when the sera concentration were 10% and the HSC-T6 cells were incubated for 24 h (P＜0.01 ). MTT assay indicated that sera from Penthorum chinense Pursh extractum treated rats showed no obvious toxicity to HSC-T6 compared with those from normal rats (P ＞0.05). After 24 h incubation, 10% sera from Penthorum chinense Pursh extractum treated rats could significantly down-regulate mRNA expression of TGF-β1 and collagen I compared with normal group (TGF-β1 2.790±0.174 vs 9. 827 ± 1.429, P＜0.01 ; collagen I 1.213 ± 0.099 vs 4.053 ± 1.005, P＜0.01 ). Mcanwhile, the protein expressions of TGF-β1 and collagen I were also obviously inhibited in drug treated group compared with normal group (P＜0.01). Conclusions Serum from Penthorum chinense Pursh extractum treated rats can significantly decrease TGF-β1 and collagen I secretions of activated hepatic stellate cells, which provides the experimental evidence for liver fibrosis treatment.