组织蛋白酶B抑制剂对小鼠急性肝功能衰竭的保护作用及机制

Protective effect and its mechanisms of cathepsin B inhibitor on acute liver failure in mice

目的探讨组织蛋白酶B抑制剂对小鼠急性肝功能衰竭的保护作用及机制。方法将雄性昆明种小鼠45只分为对照组、模型组和保护组,每组各15只。模型组和保护组一次性腹腔注射脂多糖/D-氨基半乳糖(LPS/D-GalN),保护组于造模前30min予组织蛋白酶B抑制剂(CA-074me)腹腔注射,而模型组、对照组分别注射同等体积的0.5%氯化钠溶液作为对照,给药后6 h分别取血清、肝组织标本。检测肝功能变化及凝血酶原时间(PT),观察肝脏病理改变,末端转移酶标记技术(TUNEL)检测肝细胞凋亡,免疫组织化学分析细胞色素c表达,RT-PCR检测肝组织中天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)的表达。另取75只小鼠按上述方法分组处理,每组25只,比较各组小鼠24 h存活率。统计学处理采用t检验和x^2检验。结果保护组小鼠24 h存活率明显高于模型组分别为76%(19/25)和36%(9/25)(x^2=8.12,P<0.05);保护组小鼠血清ALT[(568.50±45.68)U/L比(1394.55±78.58)U/L]、AST[(755.16±51.14)U/L比(1 488.72±64.62)U/L]、TBil[(22.82±2.04)μmol/L比(52.08±4.10)μmol/L]和PT[(14.26±.32)s比(17.40±0.30)s]水平均明显低于模型组(均P<0.05);与模型组比较,保护组小鼠肝组织炎性细胞浸润明显减少,肝细胞坏死程度明显减轻,凋亡指数明显下降[(18.4±2.6)%比(30.4±2.8)%,P<0.05];保护组小鼠肝组织中细胞色素c[(0.19±0.02)比(0.33±0.05)]和Caspase-3[(0.18±0.03)比(0.34±0.05)]表达水平明显低于模型组(均P<0.05)。结论 CA-074me对小鼠急性肝功能衰竭具有保护作用,可减轻肝细胞炎性反应、坏死和凋亡,降低急性肝功能衰竭的病死率,其机制可能与CA-074me下调细胞色素c和Caspase-3的表达有关。

Objective To investigate the protective effect and its mechanisms of cathepsin B inhibitor （CA-074me） on acute liver failure （ALF） in mice. Methods Male mice were divided into three groups： control, model and protected group. The model and protected group were intraperitoneally injected by lipopolysacchride （LPS） and D-galactosamine （D-GaIN）, the protected group was administered CA-074me for 30 min before LPS/D-GalN treatment, the control group was given 0.5% sodium chloride. All biochemical and histological indexes were determined at 6 h after administration. The 24 hours survival rate, the serum levels of alanine aminotransferase （ALT）, aspartate aminotransferase fAST）, total bilirubin（TBil） and prothrombin time （PT） of different groups were observed. The liver pathologic changes were observed by light microscopy by using hematoxylin and eosin （H. E） staining. Hepatoeyte apoptosis were measured by TdT-mediated UTP nick end labeling （TUNEL）. The expression of cytochrome c and Caspase-3 of the liver were measured by immunohistochemical staining and Reversed Transcript Polymerase Chain Reaction （RT-PCR）. Results The 24 hours survival rate of the protected group was significantly higher than that of the model group （76% vs 36%, 2 = 8. 12, P〈 0.05）. The serum levels of ALT [（568.50±45.68）U/L vs （1 394. 55 ±78.58）U/L3, AST ]-（755. 16 ± 51. 14）U/L vs （1 488.72 ± 64.62）U/L3, TBIL [-（22.82 ±2.04）μmol/L vs （52.08±4. 10）μmol/L] and PT [（ 14.26 ± 0.32）s vs （17.40 ± 0.30）s3 in the protected group were significantly lower than those in the model group （all P〈0.05）. Massive hepatocyte apoptosis and necrosis were obviously increased in the model group; the apoptosis index （AI） in the protected group was obviously lower than that in the model group [（18.4 ±2.6） % vs （30.4 ±2.8 ） %, P〈0.05]. The levels of Cytochrome e [（0.19 ± 0.02）vs （0, 33±0. 05）] and Caspase-3 [（0.18 ± 0.03）vs （0. 34± 0.05 ）] in the liver tissue of the protected group were significantly reduced compared with the model group （all P〈0. 05）. Conclusion CA-074me plays an effective protective role in acute liver failure in mice. CA-074me can relieve the inflammation, necrosis and apoptosis of hepatocyte, decrease the mortality of the acute liver failure. The possible mechanism is that CA-074me may reduce the expression of Cytoehrome c and Caspase-3.