硫化氢对高脂诱导的非酒精性脂肪性肝炎大鼠肝脏炎性反应的作用

摘要

目的：观察硫化氢（ H2 S）对非酒精性脂肪性肝炎（ NASH）大鼠炎性因子水平的影响，探讨H2 S防治NASH的可能机制。方法2013年1月—2014年7月选用SPF级雄性SD大鼠60只，按照随机数字表法分为对照组、NASH模型组、硫氢化钠（ NaHS）干预组，各20只。对照组给予普通饲料， NASH模型组给予高脂饲料， NaHS干预组给予高脂饲料的同时腹腔注射H2S供体NaHS （56 mg・ kg-1・ d-1），对照组和NASH模型组每日腹腔注射等量0.9％氯化钠溶液。8周后处死所有大鼠，留取静脉血及肝脏组织。检测血清及肝脏生化指标〔血清丙氨酸氨基转移酶（ALT）﹑天冬氨酸氨基转移酶（AST）、三酰甘油（TG）、总胆固醇（TC）及肝脏组织TG、 TC〕﹑血浆及肝脏组织H2 S水平、肝脏组织氧化应激指标〔丙二醛（ MDA）、超氧化物歧化酶（ SOD）〕； HE染色观察肝脏组织病理学变化；检测肝脏组织肿瘤坏死因子α（TNF-α）、白介素6（IL-6） mRNA表达水平及肝脏组织TNF-α、 IL-6、核因子κB抑制因子α（IκBα）、核因子κB p65（NF-κB p65）表达水平。结果 NASH模型组和NaHS干预组血清ALT、 AST、 TG、TC水平及肝脏组织TG、 TC水平均高于对照组，且NaHS干预组以上指标均低于NASH模型组（ P＜0.05）。 NASH模型组血浆H2 S水平和肝脏组织H2 S水平均低于对照组及NaHS干预组（ P＜0.05）。与对照组比较， NASH模型组和NaHS干预组肝脏组织MDA水平升高， SOD水平降低（ P＜0.05）；与NASH模型组比较， NaHS干预组MDA水平降低， SOD水平升高（P＜0.05）。与对照组比较， NASH模型组肝脏组织结构紊乱，呈弥漫性脂肪变性、肝细胞灶性坏死，并可见淋巴细胞和中性粒细胞浸润； NaHS干预组肝脏组织脂肪变性、坏死及炎性反应程度较NASH模型组明显减轻。NASH模型组和NaHS干预组肝脏组织TNF-α、 IL-6 mRNA表达水平和TNF-α、 IL-6表达水平均高于对照组，且NASH模型组以上指标均高于NaHS干预组（ P＜0.05）。与对照组比较， NASH模型组和NaHS干预组细胞质IκBα表达水平均降低，细胞核NF-κB p65表达水平均升高（P＜0.05）；与NASH模型组比较， NaHS干预组细胞质IκBα表达水平升高，细胞核NF-κB p65表达水平降低（P＜0.05）。结论 H2S能够有效改善NASH大鼠肝脏组织病理变化，并抑制炎性反应，这可能是H2 S发挥治疗作用的机制之一。%Objective To observe the effect of hydrogen sulfide ( H2 S) on the levels of inflammatory cytokines in liver tissue of rats with nonalcoholic steatosishepatitis (NASH), and to explore the mechanisms of the anti-NASH effect of H2S. Methods 60 male SD rats at SPF level were randomly and equally divided into control group, NASH model group and NaHS intervention group.Rats in control group were fed with normal diet, rats in NASH model group were fed with high fat diet, rats in NaHS intervention group were fed with high fat diet and were injected intraperitoneally with NaHS (56 mg・ kg-1・ d-1 ) , rats in control group and NASH model group were injected intraperitoneally with 0.9% sodium chloride solution.At the end of the 8th week, rats in each group were killed to obtain venous blood and liver tissue samples.The serum level of alanine aminotransferase ( ALT) , aspartate aminotransferase ( AST ) , triglyceride ( TG ) , cholesterol ( TC ) and H2 S, the levels of TG, TC, malondialdehyde ( MDA) , superoxide dismutase ( SOD ) and H2 S in liver tissues were detected by kit.The histopathological changes of the liver tissues were observed under microscope with HE staining.The mRNA and protein expression levels of TNF-αand IL-6 in liver tissues were detected.The protein expression levels of IκBαand NF-κB p65 in the liver tissues were detected.Results The serum levels of ALT, AST, TG, TC, and the levels of TG, TC in hepatic tissues in NASH model group and NaHS intervention group were significantly higher than those in control group respectively, the above indicators in NaHS intervention group were significantly lower than those in NASH model group respectively ( P<0.05) .The levels of H2 S in blood and hepatic tissues in NASH model group were significantly lower than those in NaHS intervention group and control group respectively ( P <0.05 ) .The level of MDA in hepatic tissues in NASH model group and NaHS intervention group was significantly higher than that in control group respectively, whereas the level of SOD in hepatic tissues in NASH model group and NaHS intervention group was significantly lower than that in control group respectively ( P<0.05 ) .The MDA level in NASH model group was significantly lower than that in NaHS intervention group, and the SOD level in NASH model group was significantly higher than that in NaHS intervention group ( P<0.05 ) .Compared with the liver tissues of rats in control group, the liver tissues of rats in NASH model group showed structure disturbance, macrovesicular steatosis, localized necrosis, neutrephil and lymphocytes infiltration.The levels of macrovesicular steatosis, necrosis, and inflammatory reaction in hepatic tissues in NaHS intervention group were significantly lower than those in NASH model group respectively.The mRNA and protein expression levels of TNF-αand IL-6 in liver tissues in NASH model group and NaHS intervention group were significantly higher than those in control group respectively, and the above indicators in NaHS intervention group were significantly lower than those in NASH model group respectively ( P<0.05) .The protein expression level of IκBαin cytoplasm in NaHS intervention group and NASH model group was significantly lower than that in control group respectively, the protein expression level of NF-κB p65 in cell nucleus in NaHS intervention group and NASH model group was significantly higher than that in control group respectively (P<0.05) .The protein expression level of IκBαin cytoplasm in NaHS intervention group was significantly higher than that in NASH model group, the protein expression level of NF-κB p65 in cell nucleus in NaHS intervention group was significantly lower than that in NASH model group ( P <0.05 ) .Conclusion H2 S is effective in improving the pathological changes in hepatic tissues, and it can inhibit inflammatory reaction, which might be one of the mechanisms of the anti-NASH effect of H2 S.