| Objective: To study the immunologic mechanism in drug-induced liver injury (DILI) process in DILI model rat generated with carbon tetrachloride administering and to investigate the role of diammonium glycyrrhizinate (DGL) and S-adenosylmethionine (SAMe) in DILI treament.Methods: 40 male and 40 female wistar rats weighting 200±20grams were purchased from the expremental animal center of Hebei medical university. Rats were acclimatized for 7 days and randomly divided into groups A, B, C, D, E, F, G and H with 10 animals each group.Group A was given normal saline alone by intragastric administration. Groups B,C,D and E were separately given 5ml/kg of 50% carbon tetrachloride by subcutaneous injection in first time, and then given 2.5ml/kg of 50% carbon tetrachloride by subcutaneous injection with 2 times per week for four weeks to create drug-induced liver injury animal model.Group C was given diammonium glycyrrhizinate 375mg/kg/d. Group D was given ademetionine 0.833g/kg/d and group E was given diammonium glycyrrhizinate 375mg/kg/d and ademetionine 0.833g/kg/d by intragastric administration simultaneously.Two rats dead in 4th week in group E. Groups F, G and H were given 5ml/kg of 50% carbon tetrachloride by subcutaneous injection in first time,and then given 2.5ml/kg of 50% carbon tetrachloride by subcutaneous injection with 2 times per week for two weeks to create drug-induced liver injury model. Group F was given diammonium glycyrrhizinate 375mg/kg/d. Group G was given ademetionine 0.833g/kg/d and group H was given diammonium glycyrrhizinate 375mg/kg/d and ademetionine 0.833g/kg/d by intragastric administration simultaneously.Two rats in groups A,B,C,D and E were fasted and sacrificed randomly at the end of the second week, the others were fasted and sacrificed at the end of the fourth week,and the serum were collected. The liver function marker of alanine aminotransferase (ALT) , aspartate amino transferase(AST) , albumin (ALB) and total bilirubin (TBIL) in serum were measured with automatic biochemical analyzer. Pathological examination was performed with hematoxylin eosin (HE) section The levels of T-lymphocyte subsets (including CD4+ and CD8+) were measured with Expo32ADC analysis. The levels of immunoglobulin A(IgA), immunoglobulin G(IgG), immunoglobulin M(IgM) and complement C3 were measured by immunohistochemical assay.Results:1 The status of Wister rats:The mental status of group B is weak and the fur is dry.The level of body weight in group B was lower significantly than that of other groups(p<0.05), The wister rats in group B is characterized with slow movements, drowsiness, slow growth and behavioral disorders such as rolled and restless moving. The mental status of group A is good, and the fur is thick and smooth. They have comfortable posture and normal body weight.the status of other groups are better than group B but worse than group A.2 Liver histopathological examination:The examination of hepatic pathology in groups A, B, C, D and E at the 2th week: the section of group A showed that the normal liver cells, hepatic plate, no infiltrating inflammatory cell. Obvious swelling of liver cells,crowding of hepatic plate,scattered steatosis and piecemeal necrosis,sinus hepaticus with mild hematoma expansion were showed in group B;Mild swelling of liver cells,scattered steatosis and piecemeal necrosis in group C. Swelling of liver cells, diffuse distribution of steatosis and piecemeal necrosis, crowding of hepatic plate in group D; While mild swelling of liver cells, diffuse distribution of steatosis, scattered piecemeal necrosis in group E.The examination of hepatic pathology in all groups at the 4th week: The section of group A showed normal liver cells with normal hepatic plate and no inflammatory cell infiltrating; Obvious swelling of liver cells, diffuse distribution of piecemeal necrosis, obvious capillary bile thrombus and activing mesenchymal reaction showed in group B. Swelling of liver cells, obvious piecemeal necrosis and scattered steatosis in group C. Mild swelling of liver cells, scattered steatosis and piecemeal necrosis, partial liver cytoplasm pigment deposition in group D. Obvious swelling of liver cells, scattered steatosis, piecemeal necrosis and partial liver cytoplasm pigment deposition in group E. Mild swelling of liver cells, scattered steatosis and piecemeal necrosis in group F. Scattered steatosis and piecemeal necrosis, capillary bile thrombus, partial liver cytoplasm pigment deposition and activing mesenchymal reaction in group G. Mild swelling of liver cells, scattered steatosis and piecemeal necrosis in group H.3 Immunization indicators:3.1 Indicators of cellular immunity(CD4+,CD8+and CD4+/CD8+)The percentage of CD8+ in groups A, B, C, D, E, F, G and H are: 17.18±2.58, 23.46±3.08, 16.31±6.19, 17.09±10.70, 19.91±7.09, 17.26±3.77, 15.77±3.54 and 16.00±3.76 respectively;The ratio of CD4+/CD8+ in groups A, B, C, D, E, F, G and H are: 2.41±0.57, 1.55±0.24, 2.47±0.72, 2.92±1.87, 2.25±0.59, 2.93±0.76, 3.30±1.04 and 3.33±0.78 respectively; Compared with group A,the levels of CD8+,CD4+/CD8+ in group B were significant differences (P<0.05); Compared with group B, no statistical difference exist for CD8+,CD4+/CD8+ levels in groups C,D and E(p>0.05).The percentage of CD4+ in groups A, B, C, D, E, F, G and H are: 40.78±5.37, 35.75±3.70, 37.01±7.33, 37.29±9.34, 42.27±9.77, 48.65±7.89, 49.35±9.68 and 50.87±6.22 respectively. Compared with group B, no statistical difference exist for CD4+ levels in groups A,C, D and E (p>0.05).3.2 Indicators of humoral immunity (IgA, IgG, IgM and complement C3)The serum levels of IgM in groups A, B, C, D, E, F, G, and H are:0.13±0.02, 0.45±0.17, 0.20±0.06 , 0.21±0.09, 0.18±0.05, 0.14±0.02, 0.14±0.04, 0.13±0.04 respectively;Compared with groups A, C and E ,the level of IgM in serum in group B was higher significantly (p<0.05); Compared with group B,no statistical difference exist for IgM levels in group D (p>0.05);Compared with group B , no statistical difference exist for IgA and IgG levels in groups A, C, D and E (p>0.05);The serum levels of complement C3 in groups A, B, C, D, E, F, G and H are: 0.30±0.14 , 0.18±0.13, 0.47±0.05, 0.36±0.16, 0.47±0.06, 0.26±0.13, 0.15±0.09 , 0.19±0.12 respectively; Compared with groups C and E, the levels of complement C3 in serum in group B was higher significantly (P<0.05); Compared with group B, no statistical difference exist for complement C3 levels in groups A and D (p>0.05).4 Liver function indicators(ALT,AST,ALB and TBIL):Compared with groups A, C, D and E, the levels of ALT, AST and TBIL in serum in Group B was significantly higher (p<0.05) respectively; Compared with groups A, C, D and E, the level of ALB in serum in group B was lower significantly (p<0.05); No statistical difference exist refered to ALT, AST, ALB and TBIL levels among groups A, C, D and E (p>0.05) as well as them in groups A, F, G and H (p>0.05) respectively.Conclusions:1 The model of drug-induced liver injury can be successfully generated by subcutaneous injection of carbon tetrachloride.2 Both cellular immunity and humoral immunity response are involved in the DILI process. CD8+ and CD4+/CD8+ levels in cell immunity response were associated with the pathogenesis of DILI, while only IgM in humoral immunity response involved in this process.3 S-adenosylmethionine and diammonium glycyrrhizinate can restore the DILI damage and liver function of DILI rat probably through their influnce on humoral immunity.
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