The Effect And Mechanism Of Tumor Necrosis Factor-? In Different Types Of Liver Injury

Background and ObjectThe liver is the largest parenchymal organ in the abdominal cavity and plays an important physiological role in the human body.It has been showed that viral infection,microorganisms or related components,drug toxicity and other factors could cause liver injury.In recent years,the incidence of liver injury caused by various liver diseases is also increasing year by year.Liver diseases have become one of the common diseases threatening human health.In China,about 300 million people suffer from various degrees of liver diseases,such as viral hepatitis,drug-induced liver injury,endotoxin-induced liver injury and alcoholic liver injury.Various types of liver injury have become an important factor affecting people's health in China,which has brought a heavy burden to people's lives.Sustained liver injury can lead to cirrhosis and hepatocellular carcinoma.In China,90%of patients with hepatocellular carcinoma?HCC?have a history of liver disease.Most patients with hepatocellular carcinoma experience the evolution of liver injury-hepatitis-cirrhosis-HCC.Therefore,it is of great significance to study the mechanism of different types of liver injury for the prevention and treatment of diseases.Inflammatory injury is considered to be one of the main factors leading to the occurrence and development of HCC.It may be an important strategy to control the occurrence of HCC to slow down the progression of liver injury by regulating inflammatory microenvironment.Studies have shown that liver injury caused by different causes has relatively specific inflammatory microenvironment characteristics,such as viruses,bacteria and so on,which could induce rapid immune response,infiltration of inflammatory cells and production of inflammatory factors in the liver,thus destroying the original immune balance in the liver and inducing a series of liver pathological damage.However,some drugs or chemicals?such as APAP,CCl₄,etc.?induced hepatic injury,which started with hepatocytes injury.A large number of intermediate metabolites trigger hepatocyte necrosis through a series of metabolic reactions,release intracellular contents,and subsequently induce inflammation.At present,there is no clear conclusion about the role of inflammation in this kind of liver injury,and there is even a view that inflammation does not play a role in APAP-induced liver injury.Therefore,the mechanisms and roles of inflammation in different types of liver injury are different.Tumor necrosis factor-??TNF-??is an important member of inflammatory microenvironment.It is not clear whether TNF-?has the same function in these two types of liver injury.A thorough study of the role and mechanism of TNF-?in different types of liver injury will not only help us to deepen our understanding of the mechanism of liver injury,but also provide an indication for the prevention and treatment of different types of liver injury in clinic.Meanwhile,it is conducive to the early prevention of HCC.In addition,liver is one of the organs with gender differences.Clinical data show that the incidence of HCC is about 2-8:1 in males and females.In the animal models of hepatocellular carcinoma induced by carbon tetrachloride?CCl₄?and diethylnitrosamine?DEN?,the liver injury in males is more serious than that in females.Researching the influence of gender on liver injury could provide theoretical basis for the treatment of different gender patients with corresponding diseases,and it is conducive to targeted prevention of liver cancer.At present,more and more studies have shown that gender differences in liver injury are closely related to immune function.However,the role of TNF-?in gender differences of liver injury is still unclear.Here,we constructed animal models of immune liver injury induced by lipopolysaccharide?LPS?and chemical liver injury induced by acetaminophen?APAP?using TNF-?or receptor knockout rats,in order to clarify the function of TNF-?in these two types of liver injury and the corresponding receptors.Further,we studied the level of TNF-?secretion during liver injury and the effect of different concentrations of TNF-?on the survival of hepatocytes,in order to explore the influencing factors and possible mechanisms of different functions of TNF-?.Finally,we used CCl₄-induced rat liver injury model to study the role of TNF-?and estrogen in different gender liver injury,in order to clarify the gender differences and possible mechanisms of this type of liver injury.Part?:Comparison of the role of tumor necrosis factor-?involved in lipopolysaccharide and acetaminophen-induced liver injury in ratsObjective:Some studies suggest that TNF-?participates in the occurrence of liver injury.However,it was also argued that TNF-?has a protective effect in liver injury.The role of TNF-?in liver injury is controversial.This part aims to clarify the role of TNF-?in LPS-induced immunological liver injury and APAP-induced chemical liver injury by using TNF-?and its receptor knockout rat model.Method:Wild type rats,TNF-?^-/-rats,TNFR1^-/-rats and TNFR2^-/-rats were used to prepare different types of liver injury models by LPS or APAP.The survival status of rats in each group were observed and recorded.liver injury was assessed by detecting liver function indicators?ALT,AST?in plasma,HE staining and TUNEL staining at 6h after establishment of animal model.Immunohistochemical staining was used to detect the changes of hepatic macrophages in rats after LPS injection.Furthermore,macrophage scavengers were used to scavenge hepatic macrophages to prepare corresponding hepatic injury models,and to evaluate the role of macrophages in LPS-induced immune liver injury.The effects of TNF-?or receptor on oxidative stress indicators induced by APAP in the liver,malondialdehyde?MDA?and glutathione?GSH?,were further detected by the biochemistry kits.Result:Detetion of TNF-?gene or TNFR1 receptor increased the survival rate of LPS-induced rats compared with wild type rats.The levels of plasma ALT and AST were decreased significantly in TNF-?^-/-rats and TNFR1^-/-rats.The pathological damage of livers were alleviated significantly and the apoptotic cells in livers were decreased significantly in TNF-?^-/-rats and TNFR1^-/-rats.However,TNFR2 knockout did not affect the degree of acute liver injury induced by LPS,and the number of apoptotic cells in livers had no significant difference compared with wild type rats.The number of CD68⁺Kupffer cells in livers increased significantly after LPS injection.LPS-induced liver injury in rats could be alleviated,when LPS-induced macrophages?Kupffer cells?were eliminated by GdCl3.When deletion of TNF-?or TNFR1 receptor,the survival rate of APAP-induced rats decreased,the levels of ALT and AST in plasma increased significantly,and the liver pathological damage were aggravated significantly compared with wild type rats.However,the acute liver injury induced by APAP were not affected by TNFR2 knockout.After APAP injection,the levels of MDA in livers of TNF-?^-/-rats and TNFR1^-/-rats were significantly higher than those in wild type rats,while the levels of GSH were significantly lower than those in wild type rats.There was no significant difference in the levels of hepatic MDA and GSH in between TNFR2^-/-rats and wild type rats.Conclusion:TNF-?mediated death of rat hepatocytes and played a protective role in LPS-induced immune liver injury.While,TNF-?could reduce the oxidative stress induced by APAP and played a protective role in APAP-induced drug-induced liver injury.In addition,TNF-?mediated liver injury and protection through TNFR1.Part?:Molecular mechanisms of tumor necrosis factor-?involved in hepatic damage and protective effectObjective:TNF-?play different roles in LPS-induced immunological liver injury and APAP-induced chemical liver injury.This part aims to study the influence factor of TNF-?function and its mechanism.Method:The levels of TNF-?in livers during LPS and APAP-induced liver injury were detected by the kit and the changes of hepatic macrophages were detected by immunohistochemical staining.Different doses of TNF-?were injected into TNF-?^-/-rats to detect the effects of TNF-?on plasma ALT and AST levels.Rat liver cell line BRL were treated with different concentrations of TNF-?,and the effects of TNF-?on cell survival were detected.Western blot and immunofluorescence were used to detect the changes of Yap activity.Result:TNF-?were significantly increased when LPS induced liver injury occurred and the number of CD68⁺macrophages increased significantly.However,the concentration of TNF-?in the liver did not increase when APAP induced liver injury occurred and the number of CD68⁺macrophages in the liver did not increase.Low dose of TNF-??0 to 20 pg/100g?could decrease the levels of plasma ALT and AST in TNF-?^-/-rats,but the levels of plasma ALT and AST increased with increasing of TNF-?concentration?higher than 20 pg/100g?.Meanwhile,low concentration of TNF-??10 ng/ml?could promote the proliferation of BRL cells,while high concentration of TNF-??500ng/ml?could increase the apoptotic rate of BRL hepatocytes.The expression of p-Yap^S127,p-MST1/2^T183,p-LATS1/2^T1079/1041 were decreased by stimulation of low concentration TNF-??10 ng/ml?.The total expression of Yap was increased,and the fluorescence intensity of Yap in the nucleus was enhanced.This indicated that Hippo signaling pathway was inhibited and Yap was activated.While the expression of p-Yap^S127,p-MST1/2^T183,p-LATS1/2^T1079/1041 were increased,the expression of total Yap was decreased and the fluorescence intensity of Yap in the nucleus was decreased by high concentration of TNF-??500 ng/ml?,which indicated that Hippo pathway was activated and Yap pathway was inhibited.Conclusion:Low concentration of TNF-?could inhibit Hippo signaling pathway,promote Yap activation and maintain the survival of hepatocytes,while high concentration of TNF-?could activate Hippo signaling pathway,result in phosphorylation of Yap and induce hepatic cell death.Part?:The protective effect of lipopolysaccharide on drug-induced liver injury by appropriate amount of tumor necrosis factor-alphaObjective:Based on the study that the concentration of TNF-?could affect its biological function,this part aims to explore the role and mechanism of TNF-?in the protection of LPS against APAP-induced hepatotoxicity,and provide effective interventions for the control of APAP-induced liver injury.Method:Wild type rats were injected with different doses of LPS?0.05 mg/kg,0.25mg/kg,0.5 mg/kg,2.5 mg/kg,5 mg/kg?.Then,rats were injected with APAP.The changes of plasma TNF-?levels were detected.And the levels of plasma ALT and AST induced were detected by APAP injection,in order to explore the appropriate dosage of LPS.A certain dose of LPS?0.5 mg/kg?was used to pretreat wild type rats,TNF-?^-/-rats,TNFR1^-/-rats or TNFR2^-/-rats.Then APAP-induced liver injury models were prepared to verify the effect of TNF-?induced by LPS on APAP-induced liver injury,and to further detect the levels of MDA and GSH in the liver.Result:The concentration of TNF-?in plasma increased with increasing of LPS dose,which indicated that LPS stimulated TNF-?production in a dose-dependent manner.Appropriate dose of LPS?0.25 mg/kg or 0.5 mg/kg?pretreatment could improve the survival rate of rats induced by APAP,and reduce the levels of ALT and AST in plasma and liver pathological damage.Excessive or low dose LPS pretreatment could not alleviate APAP-induced liver injury.The protective effect of LPS pretreatment on APAP-induced liver injury disappeared,when deletion of TNF-?or TNFR1 receptor.LPS pretreatment could alleviate the oxidative stress in liver of wild type rats and TNFR2^-/-rats induced by APAP,while the oxidative stress in liver of TNF-?^-/-rats and TNFR1^-/-rats induced by APAP did not decrease,which indicated that LPS pretreatment could reduce oxidative stress through TNF-?/TNFR1 signaling pathway.Conclusion:LPS pretreatment could alleviate APAP-induced liver injury through TNF-?/TNFR1 signaling pathway.Part?:The role and mechanism of tumor necrosis factor-?in sex difference of liver injury.Objective:More and more attention has been paid to the gender differences of liver diseases such as liver injury and liver cancer.This part aims to clarify the role of TNF-?in the gender differences of liver injury.Method:Models of liver injury were established by subcutaneous injection of CCl₄ at the same dose in wild type rats and TNF-?^-/-rats of different sexes.Survival status of rats in each group were observed and recorded.Plasma levels of ALT and AST were detected.Liver injury was evaluated by HE staining.Hepatocytes proliferation and apoptosis were analyzed by immunohistochemical staining.Then,female rats were ovariectomized or male rats were injected with 17?-estradiol to study the effect of estrogen on liver injury.TNF-?and 17?-estradiol were used to treat the hepatocyte lines,and their effects on cell survival were detected.The Yap activity were detected by western blot and immunofluorescence.Result:CCl₄-induced liver injury in male wild type rats were significantly more severe than that in female wild type rats,suggesting that female rats were more tolerant to CCl₄-induced liver injury than male rats.CCl₄-induced liver injury in male TNF-?^-/-rats was significantly worse than that in male wild type rats.However,the liver injury of female TNF-?^-/-rats was not aggravated compared with female wild type rats,suggesting that the protective effect of male rats on CCl₄ was mediated by TNF-?,while that of female rats did not depend solely on TNF-?.Exogenous E₂ could alleviate CCl₄-induced liver injury in male rats,while ovariectomy in female rats did not aggravate CCl₄-induced liver injury.CCl₄-induced liver injury were significantly aggravated when female TNF-?^-/-rats were ovariectomized,indicating that both TNF-?and estrogen have liver protection function,and both of them could resist CCl₄-induced liver injury.Both TNF-??10 ng/ml?and E₂?100 nM?could stimulate the proliferation of hepatocytes in vitro.Further study showed that both TNF-?and E₂ could decrease the expression of p-Yap?S127?and enhance the fluorescence intensity of Yap in the nucleus,indicating that both TNF-?and E₂ could activate the Yap pathway.Conclusion:There are double protective effects of TNF-?and estrogen in female rats.Both of them could induce the activation of Yap pathway,thus resisting CCl₄-induced apoptosis and promoting the proliferation of hepatocytes.There are only protective effect of TNF-?in male rats,which are more vulnerable to CCl₄-induced injury.