柴胡皂苷-b2抑制内质网应激信号通路减轻四氯化碳致小鼠急性肝损伤

Saikosaponin-b2 alleviates CCl4-induced acute liver injury in mice by inhibiting endoplasmic reticulum stress signal pathway

目的探讨柴胡皂苷-b2(SS-b2)对四氯化碳(CCl4)诱导的小鼠急性肝损伤的保护作用及其对内质网应激通路的影响。方法 60只雄性BALB/c小鼠分为正常对照组、模型组、SS-b2(5,10和20 mg·kg-1)组和阳性药(水飞蓟宾10 mg·kg-1)组。各组小鼠连续7 d每天ig给予不同剂量的SS-b2、水飞蓟宾或生理盐水;末次给药后2 h,除正常对照组外,各组分别一次性ip给予5%CCl4建立小鼠急性肝损伤模型。比色法检测血清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;HE染色检测肝组织病理变化,免疫组织化学和Western蛋白印迹法检测小鼠肝组织中内质网应激通路蛋白激酶R样内质网激酶(PERK)、磷酸化真核起始因子2α(p-eIF2α)、转录激活因子4(ATF4)、葡萄糖调节蛋白78(GRP78)和CCAAT/增强子结合蛋白同源蛋白(CHOP)的蛋白表达水平。结果与正常对照组相比,模型组小鼠血清中GPT和GOT的活性及MDA的含量明显升高(P<0.05,P<0.01),SOD活性明显降低(P<0.01),同时,肝组织内质网应激通路相关蛋白PERK,p-eIF2α,ATF4,GRP78和CHOP表达水平显著增高(P<0.01)。与模型组相比,不同浓度SS-b2组小鼠血清中GPT和GOT的活性及MDA的含量明显降低(P<0.05,P<0.01),SOD的活性明显升高(P<0.01);肝组织上述内质网应激通路相关蛋白的表达水平均明显降低(P<0.05,P<0.01)。HE染色结果显示,不同浓度的SS-b2明显改善CCl4引起的小鼠肝细胞肿胀、肝小叶周围胞核溶解和肝索排列紊乱,使细胞形态明显好转。结论 SS-b2对CCl4诱导的小鼠急性肝损伤具有保护作用,其机制可能与改善氧化应激损伤,抑制内质网应激通路的激活有关。

OBJECTIVE To investigate the protective effect of saikosaponin-b2(SS-b2) on acute liver injury induced by carbon tetrachloride(CCl4) and its inhibition on endoplasmic reticulum stress pathway in mice. METHODS Sixty male BALB/c mice were randomly divided into normal control, model,SS-b2(5, 10 and 20 mg·kg-1) and silymarin(10 mg·kg-1) groups. Mice were ig given different drugs or saline for 7 d. Two hours after the last administration, mice were ip given 5% CCl4 to induce acute liver injury, except those in normal control group. The activities of glutamic-pyruvic transaminase(GPT), glutamicoxaloacetic transaminase(GOT) and superoxide dismutase(SOD) and the content of malondialdehyde(MDA) in mouse serum were detected by colorimetric method. Pathological changes in liver tissue were detected by HE staining. The expression levels of protein kinase R-like ER kinase(PERK), phosphoeukaryotic initiation factory 2α(p-e IF2α), activating transcription factor-4(ATF4), glucose regulated protein78(GRP78) and CCAAT/enhancer-binding protein homologous protein(CHOP) were detected by immunohistochemistry and Western blotting. RESULTS Compared with the normal control group, the activities of GPT and GOT and the content of MDA in mouse serum in model group were significantly increased(P<0.05, P<0.01), but the activity of SOD was significantly decreased(P<0.01). Meanwhile, the expression levels of PERK, p-eIF2α, ATF4, GRP78 and CHOP in the liver tissue were significantly increased in the model group(P<0.01). Compared with the model group, SS-b2 groups significantly reduced the activity of GPT, GOT and the content of MDA in the serum of acute liver injury mice(P<0.05, P<0.01),but increased the activity of SOD(P<0.01), and the expression levels of endoplasmic reticulum stress pathway proteins mentioned above were significantly reduced(P<0.05, P<0.01). HE staining results showed that different concentrations of SS-b2 could significantly improve the swelling of hepatocytes,the dissolution of nuclei around hepatic lobules, and the arrangement disorder of hepatic cords induced by CCl4 in mice. The cell morphology was obviously improved. CONCLUSION SS-b2 has a significant protective effect on CCl4-induced acute liver injury in mice. The mechanism may be related to the inhibition of oxidative stress and down-regulation of the expressions of ERS related protein.