Study On The Protective Effect And Mechanism Of Resveratrol Against Toxoplasma Gondii Infection-induced Liver Injury

Objective:Toxoplasma gondii(T.gondii)RH strain tachyzoites were infected with murine liver cell line(NCTC-1469 hepatocytes)in vitro and BALB/c mice in vivo to establish an in vitro and in vivo liver injury model to investigate the protective effects and mechanism of resveratrol(RSV)on T.gondii infection-induced liver injury,and to provide a scientific basis for the prevention and treatment of T.gondii liver disease.Methods:1)In vitro experiment:MTS method was used to detect the safe concentration of different concentrations of RSV(12.5～50 μM)on NCTC-1469 hepatocytes.NCTC-1469 hepatocytes at logarithmic growth stage were randomly divided into 5 group:Normal group(N,uninfected+untreated),model group(CN,T.gondii infected+0.1%DMSO treatment),RSV-12.5 μM group[(T.gondii infection+RSV(12.5 μM)treatment)],RSV-25 μM group[(T.gondii infection+RSV(25 μM)treatment)]and SD group[positive control group,(T.gondii infection+SD(25 μM))].Except group N,other groups were infected with the ratio of T.gondii trophozoite number to cell number=5:1.After 4 h of infection,free T.gondii was removed.Each group was given corresponding drug treatment for 36 h,and cell supernatant and precipitation were collected for subsequent experiments.2)In vivo experiment:40 female BALB/c mice were randomly divided into 5 groups(n=8 mice/group):Normal group(N,uninfected+untreated),model group(CN,T.gondii infection+0.5%CMC-Na),RSV-50 group(T.gondii infection+50 mg/kg RSV treatment),RSV-100 group(T.gondii infection+100 mg/kg RSV treatment)and positive control group(SD-Na,T.gondii infection+100 mg/kg SD-Na).Except the N group,3 × 103 tachyzoites were injected intraperitoneally in each group to establish acute T.gondii infection liver injury model.After 4 h of T.gondii infection,except N group,other groups were administered either 0.2 mL of solvent or different concentrations of drugs,once a day,for 6 days,respectively.The disease symptoms of mice were observed and recorded daily,and the survival of mice was recorded,and the survival rate of mice was counted.One hours after the last administration,blood was collected from the eyeballs of mice and serum was prepared by conventional methods.After euthanizing the mice,the liver tissue was stripped,the right lobe of the liver was used for pathological sections,and the left lobe of the liver was used for the detection of relevant indicators.3)The proliferation of T.gondii in the liver of mice in each group was quantitatively detected by quantitative competitive-polymerase(QC-PCR).The pathological changes of liver tissue were observed by hematoxylin-eosin(HE)staining.The expression levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in mouse serum and NCTC-1469 hepatocytes supernatant were detected by Reisler’s method.The expression levels of TLR4,MyD88,p-NF-KB p65,NF-κB p65,p-IκBα and IκBα,TNF-α,iNOS,HMGB1 proteins in each group of NCTC-1469 hepatocytes and mouse liver tissues were detected by Western blotting.The nuclear translocation of HMGB1 and NF-κB P65 in each group of NCTC-1469 hepatocytes was detected by immunofluorescence staining.Results:1)RSV can significantly improve the disease symptoms of T.gondii infection mice,prolonged the survival time of T.gondii infection mice.Therefore,RSV has a good protective effect on T.gondii infection mice.2)RSV can effectively inhibit T.gondii proliferation in the liver tissue of T.gondii infection mice.3)RSV significantly inhibits the expression levels of ALT and AST in mouse serum and supernatant NCTC-1469 hepatocytes of T.gondii infected mice.In addition,HE staining results of liver tissue showed that RSV could improve the pathological changes of liver tissue of T.gondii infected mice.4)RSV inhibited the expression levels of HMGB1,iNOS and TNF-α in mouse liver tissue and NCTC-1469 hepatocytes of T.gondii infected mice,and prevent the transfer of HMGB1 from the nucleus to the cytoplasm.5)RSV significantly inhibited the over-expression of TLR4 and MyD88 proteins,and inhibited the phosphorylation of NF-κB and IκBα,and prevented the transfer of NF-KBp65 from cytoplasm to nucleus.Conclusion:1)RSV has an anti-T.gondii effect,and can effectively inhibit the proliferation of T.gondii in mouse liver;2)RSV has a good protective effect on liver injury induced by T.gondii infection;3)The possible mechanism is to inhibit the excessive inflammatory response induced by T.gondii infection by interfering with the activation of HMGB1/TLR4/NF-κB signaling pathway in liver cells.