CCl4诱发galectin-3基因敲除鼠急性肝损伤的实验研究

目的研究四氯化碳(CCl4)诱发急性肝损伤中半乳凝素3(galectin-3)作用及相关线粒体抗凋亡途径。方法利用PCR和Northern印迹杂交确认galectin-3 / 和galectin-3-/-遗传背景及galectin-3表达,HE组织切片和ELISA分别检测肝细胞形态和谷草转氨酶(AST)活性,采用Western印迹杂交检测CYP2E1、CYP1A2、Bid和PUMA蛋白表达。结果遗传背景正确galectin-3 / 小鼠肝脏经CCl4作用后galectin-3mRNA表达和血清AST水平升高而后下降、肝脏表现水肿和坏死等形态学改变,galectin-3 / 小鼠肝细胞坏死和血清AST水平高于galectin-3-/-小鼠。细胞色素氧化酶CYP2E1和CYP1A2主要存在于微粒体中,CCl4毒性作用降低其表达水平。Bid蛋白主要定位在细胞浆和线粒体中,CCl4诱导下线粒体中22kd和15kdBid升高,galectin-3 / 鼠表现升高显著。PUMA蛋白主要定位于细胞浆,galectin-3 / 鼠中表达水平高于对应基因缺陷鼠,CCl4降低其表达。结论CCl4毒性对galectin-3 / 和galectin-3-/-小鼠可产生以细胞色素氧化酶降低、AST水平升高、细胞水肿或坏死等肝损伤,galectin-3基因缺损可减轻CCl4肝毒性,该生物学现象可能与galectin-3抗凋亡作用有关,急性肝细胞损伤中galectin-3作用与线粒体途径中Bid和PUMA表达无显著相关。

Acute Liver Injury Induced by CCl4 and Related Mechanisms in Galdectin-3 Knockout Mice

Objective To investigate the roles of galectin-3 in acute liver injury induced by carbon tetrachloride (CCl4) and clarify the related anti-apoptotic mechanisms from mitochondrial pathways. Methods Galectin-3 / and -/- mice were genetically confirmed by PCR,RT-PCR,and Northern blot. Morphological changes and serum aspartate aminotransferase (AST) level were examined to indicate liver damage degree during acute mice liver injury induced by CCl4 using HE staining and an autoanalyzer,respectively. CYP2E1,CYP1A2,Bid,and PUMA were detected by Western blot in the liver of both galectin-3 / and galectin-3 -/- mice. Results Galectin-3 knockout mice showed that the part of exon 5 had been knocked out and no expression of full-length galectin-3 mRNA. Serum AST level reached a sharp peak in galectin-3 / mice after 24-hour post-treatment of CCl4,compared with galectin-3 -/- mice,and subsequently decreased until 120 h in both kinds of mice. CYP2E1 and CYP1A2 protein expression significantly decreased in CCl4-injured liver of galectin-3 -/- and / mice. The expressions of 22 kd and 15 kd Bid were up-regulated in mitochondria fraction of both mice treated with CCl4,even more significantly in galectin-3 / ones although mainly localized in cytosol. PUMA expression in cytosolic fraction decreased 24 hours after CCl4 treatment and was stronger in galecin-3 / mice than in galecin -/- ones. Conclusion CCl4 hepatoxicity might induce the reduction in cytochrome P540 oxidases,increase in AST level,hepatocytic ballooning,and necrosis in galectin-3 / and -/- mice. Galectin-3 deficiency might attenuate CCl4 hepatoxicity,closely linked to anti-apoptotic effect of galectin-3. In acute liver injury,biological functions of galectin-3 are not closely correlated with Bid and PUMA in mitochrondrial pathway of apoptosis.