Lipopolysaccharide/Amoxicillin-clavul Anate Potassium-induced Chicken Primary Hepatocyte Injury In Vitro And The Hepatoprotective Effect Of Ammonium Glycyrrhetate

Poultry farming often accompanied by bacterial disease,however,antibacterial can not only kill the bacteria,but promote lipopolysaccharide from gram-negative bacteria as well.The reason and the way how lipopolysaccharides(LPS)and antibacterial can induce liver injury and the solutions should be explored.In this research,we isolated the chicken primary hepatocyte to detect injury induced by lipopolysaccharides(LPS)/amoxicillin clavulanate potassium(AC)and investigate the damage mechanisms of oxidative stress and apoptosis and utilized the model to estimate the cytoprotective effects of the compound ammonium glycyrrhizin(CAG)with isolated in vitro.The main contents and results are as follows:1 Establishing a model of liver injury induced by LPS combined with ACHepatocytes were isolated from male Hailan chickens weighing 1.0-1.5 kg by a two-step collagenase perfusion method.Cells were seeded and cultured well for 24 h.Then the hepatocytes were incubated with LPS/AC at concentrations of 30+60,30+80,30+100,30+120,and 30+140 ?g/ml for 24 h to screen for the concentration and use MTT to detct livability incubated with CAG of the concentration 1,10,and 100 ?g/ml for 24 h.The supernatant was discarded and cells were incubated with LPS/AC at concentration that death of 50%of the hepatocytes for 24 h.Measured cell viability and the levels of AST and ALT in the cell culture supernatant.The results show that:Most of the hepatocytes adhered to the bottom of the culture plate after the cells had been isolated for 6 h.The cells had fused and differentiated to form the shape of islands at 24 h.Cells were treated with 30+60 ?g/ml and 30+80 ?g/ml LPS/AC,tycal morphology did not changed,and the relative cell viability was 81.52±2.35%and 75.54±2.79%respectively.At a concentration of 30 + 100 ?g/ml,the cell viability was 53.56±6.17%and the cell shape was irregular,with disruption of the cell membrane.Exposure of the cells to 30 + 140 ±g/ml LPS/AC injury led to significant reduction in cell numbers.These cells exhibited morphological changes such as disruption of the cell membrane and nuclear fragmentation.A concentration of(30+100 p.g/ml)LPS/AC caused the death of 50%of the cells,so it was deemed the most appropriate concentration in the model groups.2 The effects of the LPS/AC induced oxidative stress and apoptosisThe 'control' group,which was neither administered CAG nor exposed to LPS/AC;the 'model' group,given LPS/AC at a dose of(30+100 ±g/ml)for 24 h;the 'combined'group,treated with LPS/AC(30+100 ±g/ml)for 24 h after the addition of CAG 1,10,and 100 ±g/ml.Dected SOD,MDA,GSH,GSH-PX and ROS of three groups.The results indicated that:administration of LPS/AC(30+100 ±g/ml)for 24 h led to a significant decrease(P<0.01)in the activity of GSH and the antioxidant enzymes SOD while raised(P<0.01)the MDA level compared with control group.When treated with CAG(1,10,100 ±g/ml)for 24 h it generated significant increases in SOD and GSH activity(P<0.05)and a decrease MDA(P<0.01).Suggested that CAG treatment protects hepatocytes effectively from LPS/AC-induced acute liver injury in vitro.3 Effect on the primary chicken liver cells apoptosis with LPS/AC3.1 Apoptosis rates of LPS/AC group and the protective effect on CAGThe 'control' group,which was neither administered CAG nor exposed to LPS/AC;the 'model' group,given LPS/AC at a dose of(30 + 100 ±g/ml)for 24 h;the 'combined'group,treated with LPS/AC(30 + 100 ±g/ml)for 24 h after the addition of CAG 1,10,and 10±g/ml.After treating with LPS/AC and CAG,hepatocytes were isolated by the 0.25%trypsin without EDTA and collected the cells after washed by PBS.Dyeing with Annexin V-FITC and PI and dected by flow cytometer.When compared with control group,percentages of apoptotic cells of LPS/AC group were markedly increased(P<0.05).The percentages of apoptotic cells of CAG were significantly decreased(P<0.01)with the model group.The results suggest that it possibly induce apoptosis on cells to injury chicken liver with LPS/AC.3.2 Measurement of caspase-3 activityCaspase-3 is a key protein in apoptosis.When apoptosis occurred,caspase-3 were cleaved.To some extent caspase-3 can be on behalf of apoptosis.According to the method to collect and wash the cells and then basis on the kit to extract total protein and BCA to determinate concentration.The model group showed higher activity of caspase-3 than the control group(P<0.01).Similarly,when treated with GAG it induced a decrease on the activity.3.3 Ultrastructural changes in hepatocytes under transmission electron microscopyAccording to the group,dealed with cells,collection,washed cells with PBS,fixed with glutaraldehyde,prepared with sliced electron microscopy and observed under a Hitachi transmission electron microscope.The cells of the control group showed normal ultrastructural features,including smooth round nuclei,intact nuclear membrane,and integrated mitochondria with normal crista.The mitochondria of liver cells of the LPS/AC-treated group were swollen,vacuolated,and exhibited disintegrated or the loss of cristae.Which reveals how LPS/AC damage cells through mitochondria.3.4 Dectect hepatocytes mRNA and protein expression in apoptosisExtracted total RNA and protein of hepatocytes and quantified mRNA and protein expressions of caspase-3,bax,bcl-2,fas,and actin using real-time PCR(RT-PCR)and Western blot respectively.The results show that,mRNA expression of caspase-3,caspase-9,bax,fas and cyt-c significantly increased in model group compared with control group.However,treated with CAG can significantly inhibit the mRNA expression compared with the model group which induced similarly changes on protein.It has a possibility that LPS/AC can induce apoptosis of hepatocytes by the mitochondria pathway.