Establishment Of The Chicken Liver Cells Injury Induced By LPS And Envrofloxacin 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 combine with antibiotics, and study the effects and mechanism of the liver protective drugs. The novel model of chicken liver injury in vitro induced by LPS and enrofloxacin was established. Above this foundation, we investigated the process of chicken liver injury generation and the therapy mechanism of CAG.1 Development of isolation and culture methods for primary chicken hepatocytes.Chicken hepatocytes were obtained from Hailan chicken(40-50 day old) by a half-step in situ perfusion. Hepatocytes were seeded into a 6-well microplate at a density of 5×105cell/ml, each well contains 3ml, cultured in CO2 with Williams’medium E containing 10% FBS. Morphological assessment of hepatocyte monolayers and determination of LDH activity in the culture supernatant were performed. The survival rate of isolated hepatocytes was 95%. Cultured in this condition, the LDH activity decreased gradually from 48h to 144h after seeding and the chicken hepatocytes could maintain good state up to 7 days.2 Established LPS combine with enrofloxacin induced liver injury model in vitro2.1 Choose the optimal dose of LPS and enrofloxacin separately induced liver injury in vitro Chicken hepatocytes were cultured for 48h, the cells were divided into three groups(n=3), Control group:The supernatant was removed and 200μl of medium with non-treated was added; LPS group:The supernatant was removed and 200μl of medium containing 10,20,30,40,50,60,80,100μg/ml of LPS was added; Enrofloxacin group: The supernatant was removed and 200μl of medium containing 100,120,140,160,180, 200,220,240 μg/ml of enrofloxacin was added, the cells were incubated at 37℃ for 24 h. Cell viability and activities of ALT, AST in culture media were measured. The results showed that the cells treated with LPS, enrofloxacin at doses of 60-100μg/ml and 180-240 μg/ml, the cell viability were significantly lower and the activities of ALT, AST in the culture media were significantly higher than those of controls (p<0.01). LPS at a dose of 10-50μg/ml and enrofloxacin at a dose of 100-160 μg/ml is not obvious to induced liver injury. LPS and enrofloxacin at a dose of 60 μg/ml and 180 μ g/ml was determined to be the most appropriate concentration to induced liver injury in vitro.2.2 Established LPS combined with enrofloxacin-induced liver injury model in vitro Chicken hepatocytes were cultured for 48h, on the basis of the optimal dose of LPS and enrofloxacin separately induced liver injury in vitro, the cells were divided into two groups(n=3), Control group:The supernatant was removed and 200μl of medium with non-treated was added; Model group:The supernatant was removed and 200μl of medium containing 30+40,30+60,30+80,30+100,30+120 μg/ml of LPS+enrofloxacin was added, the cells were incubated at 37℃ for 24 h. Cell viability and activities of ALT, AST in culture media were measured. The results showed that LPS(30μg/ml) combined with enrofloxacin (80-120 μg/ml) the cell viability were significantly lower and the activities of ALT, AST in the culture media were significantly higher than those of controls (p<0.01). When LPS(30 μg/ml) combined with enrofloxacin (80μg/ml) the cell viability and ALT, AST release were more appropriate, so this dose was determined to be the most appropriate concentration which would be used in the model groups.3 Protective effect of CAG in chicken hepatocytes liver injury induced by LPS and enrofloxacinChicken hepatocytes were cultured for 48h, the cells were divided into three groups (n=3):The control group,which was neither administered CAG nor exposed to LPS and enrofloxacin; The model group which was given LPS+enrofloxacin at a dose of (30+80μg/ml) for 24h; The treatment group, the cells were treated with CAG at concentrations ranging from 25 to 400 μg/ml for 24h after the addition of LPS/Enrofloxacin(30+80μg/ml). Each group were incubated at 37℃ for 24 h.3.1 Protective effects of CAG on hepatocytes and againsts oxygen free radical damage To investigate the protective effects of CAG on chicken liver injury in vitro, the level of ALT, AST, SOD, GSH, MDA in culture media and in homogenates were detected by enzyme-labeling instrument. The results showed that CAG can improved cell viability and inhibited the elevation of ALT, AST, MDA (p<0.05), significantly increased the reduced level of SOD, GSH (p<0.05). The results of the present study indicate that CAG possess hepatoprotective effect and the hepatoprotective effect probably due to it’s antioxidant property.3.2 Apoptosis effects of CAG on chicken hepatocytes injury Each groups were detected by flow cytometric analysis early apoptosis ratio of hepatocytes, real time PCR for their mRNA expression levels of Caspase-3, Bax, Bcl-2 and Fas. The results showed that the numbers of early apoptotic cells were markedly reduced following treatment with various concentrations of CAG in comparison to cells in model group; The model group reduced the mRNA expression level of anti-apoptosis gene Bcl-2, but increased the mRNA expression level of apoptosis associated gene Caspase-3, Bax, Fas compared with control group. CAG can increased the mRNA expression level of gene Bcl-2 and reduced the mRNA expression level of gene Caspase-3, Bax, Fas in hepatocytes. The result indicated that CAG inhibit apoptosis of hepatocytes and thus play a protective effect on chicken liver injury, the molecular mechanisms may be related it’s ability to regulate apoptosis-related genes expression.