四氯化碳诱导小鼠内质网应激所致急性肝损伤的作用研究

Study of carbon tetrachloride induced acute liver injury by endoplasmic reticulum stress in mice

目的研究四氯化碳(CCl_4)诱导小鼠内质网应激(ERS)所致急性肝损伤的作用及机制。方法将50只昆明种小白鼠随机分为对照组10只和实验组40只。实验组按0.01 L·kg^(-1)腹腔注射10%CCl_4(豆油稀释),对照组注射同体积生理盐水,分别在注射后16,24,48,72 h处死动物并取材。检测血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)的变化;计算肝、脾指数;以苏木精-伊红(HE)染色法观察肝病理学变化,以原位末端标记法(TUNEL)检测肝组织细胞凋亡情况,以免疫组化法检测ERS标志性蛋白DNA损伤诱导因子3(DDIT3/CHOP)和葡萄糖调节蛋白(GRP78)的表达情况。结果实验组16,24,48,72 h时ALT水平分别为(278.38±6.44),(261.52±10.52),(124.46±13.82)和(42.67±12.74)U·L^(-1),AST水平分别为(35.01±7.84),(31.93±11.33),(24.05±1.11)和(19.66±3.21)U·L^(-1),与对照组的(25.48±7.16)和(17.60±2.14)U·L^(-1)相比,实验组小鼠血清中ALT、AST水平均有所上升,在16 h达到顶峰(P<0.01),16 h后有所下降,并逐渐恢复至接近正常水平;实验组肝指数显著增高(P<0.01),24 h后逐渐恢复,脾指数与之相反。HE染色结果显示,实验组小鼠中肝肿大,胞质疏松,出现明显的片状坏死和气球样变。TUNEL结果显示,实验组16,24,48,72 h时的平均凋亡率分别为(64.00±5.69)%,(81.00±3.61)%,(45.00±3.21)%和(31.00±1.53)%,与对照组的(8.00±1.01)%相比,差异均有统计学意义(均P<0.01)。免疫组化结果显示,与对照组相比,CHOP和GRP78表达量显著升高(P<0.01),并在24 h达到峰值。结论以0.01 L·kg^(-1)的10%CCl_4能够诱导小鼠ERS,其所致急性肝损伤的最佳时间为24 h。

Objective To study the mechanism of carbon tetrachloride（CCl4）induced endoplasmic reticulum stress resulted in acute liver injury in mice.Methods Fifty Kunming mice were randomly divided into control group（n=10）and experimental group（n=40）.The experimental group was injected intraperitoneally with 10%CCl4（Dilution of soybean oil）.The same volume of normal saline was injected into the control group.Animals were sacrificed at 16,24,48,and 72 h after injection.Detection of aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）and malondialdehyde（MDA）in serum.Calculating the lover index and spleen index.Hematoxylin and eosin（HE）staining for liver pathological changes.Terminal-deoxynucleoitidyl transferase mediated nick end labeling（TUNEL）was used to detect apoptosis in liver tissue.Immunohistochemistry was used to detect the expression of the ERS-marker proteins DDIT3/CHOP and GRP78.Results The levels of ALT in experimental groups at 16,24,48 and 72 h were（278.38±6.44）,（261.52±10.52）,（124.46±13.82）and（42.67±12.74）U·L^-1,the levels of AST were（35.01±7.84）,（31.93±11.33）,（24.05±1.11）and（19.66±3.21）U·L^-1,compared with（25.48±7.16）and（17.60±2.14）U·L^-1in control group,they were all increased,and reached the peak at 16 h（P〈0.01）,then gradually decreased to normal level.The liver index of experimental group increased significantly（P〈0.01）,gradually recovered after 24 h,and the spleen index was opposite.The results of HE staining showed that hepatomegaly,loose cytoplasm,obvious flaky necrosis and balloon-like change in experimental group.The results of TUNEL showed that the apoptosis rates of experimental groups at 16,24,48 and 72 h were（64.00±5.69）%,（81.00±3.61）%,（45.00±3.21）%,（31.00±1.53）%,had significant difference with（8.00±1.01）%in control group（P〈0.01）.Immunohistochemical results showed that the expression of CHOP and GRP78 significantly increased compared with control group（P〈0.01）,and reached the peak at 24 h.Conclusion The mice ERS could be induced by 0.01 L·kg^-1of 10%CCl4.The best time for acute liver injury was 24 h.