硫化氢对大鼠肝纤维化的抑制作用及机制探讨

Inhibitory effect of hydrogen sulfide on liver fibrosis in rats and its mechanism

目的观察外源性硫化氢(H2S)对四氯化碳(CCl4)诱导大鼠肝纤维化的抑制作用,并探讨其机制。方法将50只雄性SD大鼠随机分为5组,空白对照组皮下注射花生油2.0 mL/kg体质量,肝纤维化组注射同剂量40%CCL4花生油,NaHS低剂量组注射同剂量40%CCL4花生油+NaHS 7μmol/kg,NaHS高剂量组注射同剂量40%CCL4花生油+NaHS 14μmol/kg,秋水仙碱组注射相同剂量40%CCL4花生油+秋水仙碱0.25 mg/kg灌胃,每周2次,共8周。采用全自动生化检测仪检测各组大鼠血清ALT、AST,放射免疫法检测肝脏组织透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原(PCⅢ)及Ⅳ型胶原(CⅣ),羟胺法检测肝组织超氧化物歧化酶(SOD),比色法检测肝组织丙二醛(MDA)和谷胱甘肽过氧化酶(GSH-Px),HE染色观察肝组织学改变,网状纤维和masson染色观察肝纤维组织增生状况,免疫组化法检测肝组织Ⅰ型、Ⅲ型胶原。结果与空白对照组比较,肝纤维化组ALT、AST、HA、LN、PCⅢ、CⅣ、MDA升高,SOD、GSH-Px降低(P均<0.05);与肝纤维化组比较,NaHS低剂量组、NaHS高剂量组、秋水仙碱组ALT、AST、HA、LN、PCⅢ、CⅣ、MDA降低,SOD、GSH-Px升高(P均<0.05)。与肝纤维化组比较,NaHS低剂量组、NaHS高剂量组、秋水仙碱组肝纤维化程度减轻,肝组织胶原纤维异常分布明显减少,Ⅰ型、Ⅲ型胶原明显减少。结论外源性H2S能有效抑制大鼠肝纤维化的发生、发展,其机制可能与其清除自由基、提高抗氧化物酶的活性、抑制HSC的胶原合成、减少细胞外基质在肝脏中的沉积有关。

Objective To observe the inhibitive effect of hydrogen sulfide on liver fibrosis in rats and to discuss its mechanism of action. Methods Fifty male rats were randomly divided into 5 groups ： the blank control group with subcuta- neous injection of peanut oil 2.0 mL/kg, liver fibrosis group which was injected with the same dose of 40% CCL4 peanut oil, low dose NariS group which was injected with the same dose of 40% CCL4 peanut oil ＋ NariS 7 μmol/kg, high dose NariS group which was injected with the same dose of 40% CCL4 peanut oil ＋ NariS 14 μmol/kg, and the colchicine group which was injected with the same dose of 40% CCL4 peanut oil ＋ 0.25 mg/kg colchieine. All rats except those in the con- trol group were subcutaneously injected with CCIA twice a week for 8 weeks to test liver fibrosis. The activities of transami- nase in serum, malondiadehyde （MDA） content, superoxide dismutase （SOD）, and glutathione peroxidase （GSH-Px） ac- tivities in liver homogenate were assayed, and levels of serum hyaluronic acid （HA）, laminin （LN）, collegen type Ⅳ （C Ⅳ ）, procollagen Ⅲ （PC Ⅲ ） were tested. The pathological changes of liver were observed by HE, reticular fiber and Mas- son stain. The expression of type I and type Ⅲ collagen was detected by immunohistochemical method. Results The re- suits showed that the serum ALT and AST decreased by NariS treatment. Pathological examination showed that H2 S could remarkably alleviate the hepatic fibrosis. H2S not only decreased the HA and LC content in liver homogenates,but also ele- vated the levels of PC Ⅲ and C IV in serum. H2 S also ameliorated the oxidative stress state of hepatic fibrosis rats, decreased the production of MDA and enhanced the activities of antioxidative enzyme including SOD and GSH-Px （ all P 〈 0.05 ）. Conclusions H2S has protective effect on liver fibrosis rats induced by CCL4. The mechanisms of anti-fibrotic effects may be associated with its action of ameliorating the oxidative stress in liver and inhibiting the production of collagen in HSC.