Establishment Of Animal Model Of Isoniazid Induced Liver Injury And Correlative Study On Immunoregulatory Mechanism Of Isoniazid Induced Liver Injury And Repair

Aims: 1)To clarify the goal of prevention and treatment of drug-induced liver injury and the direction of future research,by analyzing the major pathogenic drugs and clinical features of drug-induced liver injury in Xinjiang in recent years.2)To provide the basis for further study on the pathogenesis of isoniazid induced drug-induced liver injury,by establishing the animal model of isoniazid induced liver injury in C57BL/6 mice.3)To find the biomarkers of early diagnosis and prevention of isoniazid induced liver injury,and to provide the theoretical basis for the prevention and treatment of isoniazid induced liver injury.Methods: 1)All patients who were hospitalized for the clinical diagnosis of drug-induced liver injury in the first affiliated hospital of Xinjiang Medical University from 2012.1 to 2014.12,were recruited with screening RUCAM score ≥ 3 points.2)Seven-week-old male C57BL/6 mice were assigned to two types of feeding condition.One was fed ad libitum(AL)on commercial chow and another was starved for 8h during the day then ad libitum.In each feeding condition,all animals were administered INH100mg/kg every day by gavage for 8 weeks.Sera were collected for measurement of biochemical parameters.Liver sections were stained with HE and Masson to evaluate the hepatic histology.3)Seven-week-old male C57BL/6 mice were divided into the following four groups: blank control group,starvation group,IMH group,INH+starvation group,and were treated for 6 weeks.Starvation group was fasted for 12 hours every day;isoniazid group was given isoniazid(100mg/kg)daily;starvation + isoniazid group were fasted daily at 12 h and 1h after the administration of isoniazid(100mg/kg).The mice were sacrificed at 3d,2W,4W,6W after treatment,and the liver tissue and blood samples were collected.Flow cytometry was used to detect T cells and dendritic cell subsets.Cytometric Bead Array was applicated to measure the expression levels of cytokines.Reverse transcription quantitative polymerase chain reaction(RT-PCR)and western blot analysis were performed to measure the expression levels of HSP70.2 、HGF、Tgf-β、α-SMA、collagen I and collagen III.Results: 1)Anti-tuberculosis drugs are the main drugs that cause drug-induced liver injury in Xinjiang.Anti-tuberculosis drug-induced liver injury is the main goal of future drug-induced liver injury prevention and research.2)INH administration(100mg/kg)daily for 8 weeks did not induce any histological damage in INH group.In addition,no significant difference on serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST).But in starvation group,serum ALT and AST elevated for 4 weeks,reached the peak at the 3rd day,and gradually returned to normal after 4 weeks.Liver sections showed liver cell edema at the 3rd day.The performance of the central vein around the liver cells with significant edema with inflammatory cell infiltration and necrosis at 1w,then gradually improved,after 6w has been basically normal.3)The percentage of CD4 + T lymphocytes in the group of starvation + isoniazid group was higher than that in the isoniazid group and the starvation group,and the percentage of CD8 + T lymphocytes was lower than that of the isoniazid group and the starvation group.The changes of CD11c+ and CD86+ in dendritic cells of starvation + isoniazid group,isoniazid group and starvation group were consistent at each treatment time point.In peripheral blood,the levels of IL-5,TNF-α,IL-2,IL-6,IL-17 A and IL-4 in isoniazid + starvation group were higher than those in starvation group and isoniazid group at injury phase(2w),with significant difference(P < 0.05).The concentration of IL-10 and IL-21 in isoniazid + starvation group was higher than that in starvation group and INH group at acute injury phase(3d),with significant difference(P<0.01).In the liver tissue,IL-22 concentration in INH + starvation group was higher than the starvation group and INH group,at acute injury phase(3d).The amount of relative expression of HGF and HSP70.2 m RNA in mice liver tissue of isoniazid +starvation group is the highest at injury phase(2w).HSP70.2 protein in mice liver tissue increased gradually,and higher than those in INH group and starved group(P<0.05)at acute injury phase(3d)and stable phase(6w).The expression of HGF protein in liver tissue showed two peak at injury phase(2w)and stable phase(6w).TGF-β increased gradually with the prolongation of administration.Conclusions: We conclude that: 1)Antituberculosis drugs are the main causes of drug-induced liver injury in Xinjiang;The main clinical types of drug-induced liver injury is hepatocellular-type liver injury,and there was no significant difference in the severity of liver injury between the three clinical types and no significant difference in prognosis.The body weight,glutamyl transpeptidase was positively correlated with RUCAM score,with the higher of the weight or the glutamyl transpeptidase,the RUCAM score is higher.2)Starvation increases the hepatotoxity of INH in mouse,and this is a validated animal model to study INH-induced liver injury.3)In the early stage,the increase of proinflammatory cytokines(IL-5、TNF-α、IL-2、IL-6、IL-17A、IL-21),anti-inflammatory factor(IL-4、IL-10)in peripheral blood,and IL-22 in liver tissue suggest proinflammatory cytokines and antiinflammatory cytokines compete with each other,during liver injury and repair,eventually lead to liver injury or repair.IL-4,IL-10,IL-22 may be the key indicators for liver recover.The increase of HGF and HSP70.2m RNA and protein expression in the early stage of liver injury may indicate that the liver cells can repair after injury.