The Double Effects And Mechanism Research Of Different Concentrations Of Tumor Necrosis Factor-α In Acute Liver Injury

Background and Aims:Acute liver injury is more common form of the disease in clinic,with its characters of pathogenetic urgency,rapid progression,severe illness,which needs urgent diagnosis and treatment.However,acute liver injury is a special state between deterioration and rehabilitation.If it is treated timely,the injury will tend to be controlled,and gradually be recovered.On the contrary,if it is not treated properly,it will continue to be deteriorated,even develope to liver failure and cause irreversible damage to the health.Acute liver injury could be evolved by a variety of diseases,such as viral hepatitis,drug-induced liver injury,liver resection,liver transplantation,endotoxin-induced liver injury and hepatic ischemia-reperfusion etc.These diseases often cause changes of blood flow of hepatocytes,resulting in various degrees of hepatocellular hypoxia,and causing damage to liver tissue.Inflammatory microenvironment is important for the occurrence and development of injured tissues.It is paid more and more attention to cure injured diseases by regulating the inflammatory microenvironment.The pathogenesis of liver injury is extremely complex.Its occurrence and development is closely related with the change of inflammatory microenvironment.At the early stage,Kupffer cells and macrophages can be activated and promoted to release reactive oxygen species and some inflammatory factors including interleukin and tumor necrosis factor alpha.However,TNF-α plays an important role in acute liver injury.TNF-α is a cytokine with polymorphisms,which can regulate inflammation,apoptosis and metabolism etc.The biological activities of TNF-α are associated with acute liver injury.Relate researches show that,in the process of liver injury,a large number of TNF-α are generated and regulate regeneration of other cytokines;Meanwhile,TNF-α quickly move into hepatocytes and induce the expression of nuclear factor kappa B（NF-κB）of signaling pathways,participate in the process of liver cell repair together.However,some literatures have reported that TNF-α can aggravate the deterioration of liver damage in the process of acute liver injury,it activated a series of signaling pathways in the hepatocytes,and ultimately triggers the program of apoptosis to accelerate hepatocellular death.But other literatures have reported that the increased degree of TNF-α in patients’ serum have positive correlation with deterioration of illness.Although,at present,the studies of TNF-α has a more understanding,but the detailed role of TNF-α in acute liver injury is not clear.Based on the above research background,we applied carbon tetrachloride（CCl₄）to build the acute liver injury model in rats;and injected intraperitoneally different doses of TNF-α inhibitor TNFR2-Fc variants（TNFR2-Fcv,a gene synthesis of soluble protein can bind and inhibit its activity of TNF-α）to observe the effect of different concentrations of TNF-α for acute liver injury and furtherly explored the possible mechanism.Research methods:（1）Preparation of liver injury model: CCl₄ was dissolved in corn oil to prepare a 40% concentration.TNFR2-Fcv were intraperitoneal injected into SD male rats.15 minutes later,1ml/kg CCl₄ were injected subcutaneously into the rats of the respective groups.24 hours later,all the rats were sacrificed,and blood were collected and fresh liver tissue were separated for subsequent experiments.（2）Grouping and treatment: 90 SD male male rats weighing between 200 to 210 g were randomly divided into 9 groups of 10.These rats were managed with CCl₄（-）+ 0mg / kg TNFR2-Fcv,CCl₄（-）+ 8mg/kg TNFR2-Fcv,CCl₄（+）+ 0mg/kg TNFR2-Fcv,CCl₄（+）+ 0.25 mg/kg TNFR2-Fcv,CCl₄（+）+ 0.5mg/kg TNFR2-Fcv,CCl₄（+）+ 1mg/kg TNFR2-Fcv,CCl₄（+）+ 2mg/kg TNFR2-Fcv,CCl₄（+）+ 4mg/kg TNFR2-Fcv,CCl₄（+）+ 8mg/kg TNFR2-Fcv.The TNF-α-/-rats were only injected with 1ml/kg of CCl₄.All of these rats were kept in the SPF animal breeding room,and feed with clean drinking water The room temperature was 25 ± 3 ° C,and with 12 hours of light alternating day and night.（3）Detect serum TNF-α levels: Enzyme-linked immunosorbent assay（enzyme linked immunosorbent assay,ELISA）was used to detect the levels of TNF-α in acute liver injured rats.Each group was administrated by different doses of TNFR2-Fcv,to ascertain the roles of different concentrations of TNF-α in the liver injury.（4）To detect the function of liver: separated serum from the blood and detect the levels of serum alanine aminotransferase（ALT）aminotransferase（AST）in an automatic analyzer.Liver tissue were embedded with paraffin and made into the tissue sections.The steatosis of liver tissue of each group were analysed by HE staining;The expression of hepatocellular apoptosis were detected by the detection kit of Tunnel, and the expression of the Cleaved-caspase3 in tissue sections were detected by immunohistochemistry.（5）Detect inflammatory cytokines: the inflammatory cytokines such as IL-4,IL-6,IL-8,IL-1β,and IFN-γ in acute liver injury were detected by Real-Time quantitative PCR（RT-PCR）.（6）Detect the expression of NF-κB and anti-apoptotic signaling pathway: The expressions of NF-κB signaling pathway genes include IκBα（inhibitor of NF-kappa B-α）,p-IκBα（Phospho-IκBα）and NF-κB-p65 were detected by Western-Blotting.And anti-apoptotic gene such as BCL-2（B-cell leukemia lymphoma 2）,BCL-XL（B-cell lymphoma-extra large）,XIAP（X-linked Inhibitor of Apoptosis Protein）,and FHC（Ferritin heavy chain）at different concentrations of TNF-α were also detected in the acute liver injury by Western-Blotting and RT-PCR.（7）Statistical analysis: the data results were used with SPSS 20.0 statistical software for statistical analysis.The mean standard error between the groups using ANOVA（Analysis of Variance,ANOVA）to test analysis to P <0.05 was considered statistically significanceResearch results:（1）In the CCl₄ induced acute liver injury model of rats,the serum TNF-α were decreased as the applying of TNF-α inhibitors TNFR2-Fcv,which had a significantly dose-depended correlation.（2）The serum ALT and AST were significantly increased in the CCl₄ induced acute liver injured model,ALT increased from 36.75 ± 8.73 U / L to 193.27 ± 14.26 U / L;AST increased from 150.86 ± 12.34 U / L to 253.79 ± 12.90 U / L,p <0.01.The results from HE staining suggested a large degree of liver tissue steatosis,while the number of positive cells staining with the Cleaved-caspase3 were increased significantly.The results of Tunnel staining indicated that an increased number of apoptotic hepatocytes and strong expression of apoptotic antibodies in Western-Blotting detection.（3）No matter how much TNF-α inhibitors TNFR2-Fcv were used in the normal rats,the serum ALT and AST levels remained largely unchanged,which were still in the normal range.However,in the CCl₄ induced acute liver injured model,with the dose of TNFR2-Fcv was added gradually from 0mg/kg to 8mg/kg,TNF-α levels gradually decreased,and serum ALT and AST becomed an obvious " V " shape.As TNFR2-Fcv were increased from 0mg/kg to 1mg/kg,the serum ALT and AST were obviously decreased（P <0.01）,the results from HE staining also indicated that the degree of liver steatosis were decreased.And the number of apoptotic hepatocytes detected by immunohistochemistry and Tunnel detection were decreased significantly（P<0.05）.However,furtherly increased the dose of TNFR2-Fcv from 1mg/kg to 8mg/kg,the levels of TNF-α continued to decrease,ALT and AST in serum were rised instead,even at 8mg/kg dose of TNFR2-Fcv,the level of serum ALT and AST levels were higher than at 0mg/kg TNFR2-Fcv（P <0.05）.The results of HE staining suggested that the degree of hepatic steatosis actually worsened;the number of apoptotic hepatocytes were also significantly increased by the detecting of immunohistochemistry and Tunnel（P <0.05）.（4）In the CCl₄ induced acute liver injury of TNF-α-/-rats,serum ALT and AST levels were significantly increased（P <0.01）compared to wild-type rats.The results of HE staining revealed hepatocellular steatosis were very serious in a single field of vision.Compared with wild-type rats during the process of acute liver injury,higher level of ALT and AST were found（P <0.05）,hepatocellular steatosis were relatively more serious in the TNF-α-/-rats.（5）RT-PCR results suggested that in the course of acute liver injury with increasing doses of TNFR2-Fcv,the concentrations of TNF-α and levels of IL-6,IL-8,IL-1β,and IFN-γ had decreased,while the level of IL-4 gradually rised.（6）Western-Blotting and RT-PCR results showed that with the TNF-α levels decreased,the expressions of NF-κB signaling pathway include p-IκBα and NF-κB-p65 and the genes of anti-apoptosis genes including Bcl-XL,FHC XIAP and Bcl-2 were decreased gradually in the process of acute liver injury.In conclusion:（1）TNF-α plays an important role in the regulation of acute liver injury.Appropriately reduce the concentration of TNF-α in vivo can effectively alleviate liver damage.However,excessively reduce the concentration of TNF-α will increase the liver damage.（2）The dual role of TNF-α always exist in the development of acute liver injury.The main effect of TNF-α may depend on its concentration.