Establishment Of The Chicken Liver Injury Model Induced By Enrofloxacin And Lps And Protective Effects Of Cag Soluble Powder

Following the treatment of bacterial infections in poultry raising, antibiotics induce Gram-negative organisms to liberate amounts of endotoxin. LPS and antibiotics are considered as common causes of liver injury. It is significant to investigate the pathogenesis of hepatocyte injury, caused by the LPS cooperate with antibiotics, and study the effects and mechanism of the liver protective drugs. The novel model of chicken liver injury induced by LPS and enrofloxacin was established. Above this foundation, we investigated the process of chicken liver injury generation and development and the preventive and thrapeutic effects and mechanism of CAG.1Establishment of chicken liver injury model induced by enrofloxacin and LPS1.1Selection of the dosage of enrofloxacin and LPS The Hyline egg-type chickens were divided randomly into3groups:enrofloxacin groups (EⅠ～Ⅶ, n=10) received enrofloxacin at the doses of50,80,100,200,300,400,500mg/kg by gavage, once a day, for three days; LPS groups (LⅠ～Ⅶ, n=10) received Lipopolysaccharide crude extract (LPS) once, at the doses of1,2,4,6,8,10,15ml/kg by intraperitoneal injection, Control group(n=10) were served as normal control. At48th h post-treatment with drugs, the mental status, livability and characteristics of histopathology of chickens was observed,serum levels of AST, ALT were determined.EⅣ～Ⅶand LⅤ～Ⅶgroups’ livability≤100%, the serum levels of AST, ALT were significantly increased, and the mental status was low in Em and LⅡ～Ⅳ groups, Extensive hepatocyte tumefaction was observed in Em group and necrosis, vacuolation and inflammatory infiltration was observed in LⅡ～Ⅳ groups. So,determined the optimal doses of enrofloxacin and LPS were100mg/kg and2,4,6ml/kg.1.2Establishment of the chicken liver injury model by enrofloxacin cooperate with LPS The chickens were treated with enrofloxacin(100mg/kg) once a day,for three days,and on the third day administered LPS at the doses of2,4,6ml/kg in ELⅠ～Ⅲgroups, At48th h post-treatment with LPS, the livability and characteristics of histopathology of chickens was observed,serum levels of AST, ALT, TP, ALB, GLB and A/G were determined. ELⅢ groups’ livability≤100%,the serum levels of AST, ALT and GLB were more significantly increased, TP, ALB and A/G were more significantly decreased in ELⅡ group than others, and characteristics of histopathology of liver were more visible. The novel model of chicken liver injury induced by LPS and enrofloxacin was successfully established by enrofloxacin (100mg/kg) and LPS (4ml/kg). It is a comparatively ideal animal model for further studying of the chickens liver injury and the thrapeutic effect of liver protective drugs.2Protective effect of CAG in chicken liver injury induced by enrofloxacin and LPS2.1Animal grouping, model establishment, CAG administration and sampling A total of104Hyline egg-type chickens were divided randomly into5groups:Group I(n=8) served as normal control. Group Ⅱ, Ⅲ, Ⅳ,&Ⅴ (n=24) were administered enrofloxacin and LPS with the established method. Group Ⅱ was maintained as liver injury control. Group Ⅲ received prior administration of CAG at the dosage of40mg/L (by Monoammonium glycyrrhizinate, the same as follows) drinking water for three days before enrofloxacin and LPS treatment, till to the liver injury models were successfully established. Group IV and V received CAG at a dose of22.5and7.5mg/kg, twice a day for three days after enrofloxacin and LPS treatment. Eight chickens were sacrificed at6th,24th,48th h post-treatment with LPS and their blood and livers samplewere harvested.2.2Protective effects of CAG on liver function The levels of serum ALT, AST, TP, ALB, TBIL, DBIL, γ-GT were detected by automatic biochemical analyzer.The histomorphological changes were evaluated. At6th,24th,48th h post-treatment with LPS, the levels of serum ALT, AST, γ-GT and TBIL were significantly higher, and TP and ALB were significantly lower in group Ⅱ than group Ⅰ. When compared with group Ⅱ, in group Ⅲ, there was significantly fall in ALT at6th h, AST, γ-GT and TBIL at6th h and24th h, rise in TP at6th h, ALB at6th h,24h and48th h. In group IV, there was significantly fall in ALT, AST, γ-GT and TBIL, rise in TP and ALB at24th h and48th h. There was no obvious alteration in group V. There was significantly rise in DBIL at24h and48th h in group IV, no obvious alteration in others.The results of pathological study also support the results of biochemical findings. The results of the present study indicate that CAG possess hepatoprotective effect and it might be related to dosage and treatment time.2.3Protective effects of CAG againsts oxygen free radical damage The levels of serum MDA, NO, SOD and GSH were detected by enzyme-labeling instrument. At6th,24th,48th h post-treatment with LPS, the levels of serum MDA and NO were significantly higher, and SOD and GSH were significantly lower in group Ⅱ than group Ⅰ. When compared with group Ⅱ, in group Ⅲ there was significantly fall in MDA and NO at6th h and24th h, MDA at48th h, rise in SOD at6th h, GSH at6th h,24h and48th h. In group IV, there was significantly fall in MDA and NO, rise in SOD and GSH at24th h and48th h. There was no obvious alteration in group V. The results of the present study indicate that the liver injury effects might be related to oxygen free radicals, and the hepatoprotective effect of CAG probably is due to it’s antioxidant property.2.4Immunoregulatory effects of CAG on chicken liver injury The levels of serum TNF-a, IL-1, IL-6were detected by ELISA methods, the number of CD3(+), CD4(+) and CD8(+) T-lymphocytes by Flow Cytometer. The levels of serum TNF-a, IL-1and IL-6were significantly higher at6th,24th,48th h, and CD3(+), CD4(+) and CD8(+) T-lymphocytes were significantly higher at6th and24th, and lower at48th h post-treatment with LPS in group Ⅱ than group Ⅰ. When compared with group Ⅱ, in group Ⅲ, there was significantly fall in the levels of serum TNF-a, IL-6at6th h, rise in the number of CD3(+), CD4(+) and CD8(+) T-lymphocytes at48th h. In group IV, there was significantly fall in TNF-α, IL-6at6th h and24th h, IL-1and CD3(+), CD4(+) and CD8(+) T-lymphocytes at48th h, rise in CD3(+) T-lymphocytes at24th h and48th h, CD4(+) and CD8(+) T-lymphocytes at48th h. In group Ⅴ, there was significantly fall in CD8(+) T-lymphocytes at48th h, no obvious alteration in others. The results indicate that the novel liver injury effects might be related to proinflammatory cytokines production and T-cell subsets deviation in peripheral blood. CAG may inhibit proinflammatory cytokines production, and regulating the number of T-cell subsets in peripheral blood deviation by bi-directional effects.