The Effect Of Inhibiting Heme Oxygenase-1 On Immune Hepatic-fibrosis In Rats

Objective:Heme oxygenases (HO) are ubiquitous oxygenases in organs and tissues of human and mammal that catalyze the initial and rate-limiting steps in the oxidative degradation of heme, having important biological effect. As an inducible isozyme of HO, HO-1 and its enzyme products mediate anti-inflammatory, antioxidant and anti-apoptosis effects according to the recent reports. HO-1 is regarded as an important endogenous protection against oxidative stress injure in many tissues and organs, including heart, brain, lung, liver and kidney.In recent years, there are accumulated evidences that HO-1 has protection effects on liver cells while liver transplantation, acute liver injure and ischemia/reperfusion injury. Zinc protoporphyrin (ZnPP), a selective heme oxygenase inhibitor, can depress the activity of HO-1 and restrict the transformation of heme to biliverdin. In this study, we aimed to observe the relationship between the expression of HO-1 protein and the changes in the function and morphology of liver, using the treatment of ZnPP on the immune hepatic-fibrosis rat models, and to investigate the effect of HO-1 on the pathogenesis of hepatic fibrosis, which may provide some experimental evidence for clinical treatment of hepatic fibrosis.Method: Studies were performed on 52 adult male Sprague-Dawley rats weighing 220-270g. All rats were divided into 3 groups at random: Control Group with 12 rats, Fibrosis Group with 20 rats and ZnPP Group with 20 rats. Hepatic fibrosis model was induced in Fibrosis Group and ZnPP Group by human serum albumin (HSA). ZnPP Group received intraperitoneal injection of ZnPP (5mg/kg) at the same time of attacking with HSA by vena caudalis. After 6 weeks of attacking, all rats were killed. The expressions of HO-1 protein in liver were measured by immunohistochemistry and western blotting methods. The levels of serum alanine aminotransferase (ALT), aspartate amino transferase (AST) and albumin (Alb) were assayed by the automatic blood biochemistry analyzer. Fibrosis markers in serum including hyaluronate acid (HA), laminin (LN), typeⅢprocollagen (PCⅢ) andⅣcollagen (Ⅳ-C) were assayed by the mean ofγ-radioimmunoassay. The changes of hepatocytes, the infiltration of inflammatory cells, the fibrous hyperplasia degree and the number of spindle cells in perisinusoid location beyond portal and septa area were detected using HE Staining. The proliferations of CollagenⅠandⅢwere observed by VG staining and Foot's reticular fiber staining.Results:1. Results of immunohistochemistry: Comparing with Control Group, the staining range and intensity of HO-1 expressions were significantly increased in Fibrosis Group. However in ZnPP Group, the staining range was significantly increased compared to Control Group, while the staining intensity of HO-1 expression was weakened compared to Fibrosis Group.2. Results of western blotting: Comparing with Control Group, the expressions of HO-1 protein in liver increased significantly in Fibrosis Group (P<0.01). They were depressed in ZnPP Group (P<0.05) which were still higher than those in Control Group (P<0.01).3. There were no statistical significances between three groups in serum Alb. Comparing with the Control Group, the level of ALT, AST increased significantly in both of Fibrosis Group and ZnPP Group (P<0.01), and were much more in ZnPP Group (P<0.05).4. Statistical significances were showed between Fibrosis Group and Control Group in serum HA, LN, PCⅢ, andⅣ-C (P<0.01). They were enhanced more in ZnPP Group than in Fibrosis Group (P<0.05).5. Comparing with the Control Group, the liver cells in Fibrosis Group and ZnPP Group presented hydropic degeneration. Although the changes in ZnPP Group appeared more seriously, necrosis was not found in both two groups. Though infiltration of leukomonocytes and the number of spindle cells were detected in Fibrosis Group, they were more visible in ZnPP Group. The proliferation extent of fibroblast, typeⅠand typeⅢcollagen were significant aggravated in Fibrosis Group (P<0.01), and much worse in ZnPP Group (P<0.01).Conclusions:1. The expressions of HO-1 protein increased significantly in immune hepatic fibrosis rat models. ZnPP may decrease the expressions.2. Injure of liver was slight, but the collagen fibers hyperplasia was significant in immune hepatic fibrosis rat models.3. In immune hepatic fibrosis rat models, ZnPP aggravated injure of liver and the degree of hepatic fibrosis.4. ZnPP may worsen the hydropic degeneration of liver cells, increase the infiltration of leukomonocytes and the number of spindle cells in perisinusoid location beyond portal and septa area in immune hepatic fibrosis rat models.5. HO-1 may be involved in the pathological progress of hepatic fibrosis and have protection effect on liver.