| The endotoxin(LPS)is released with killing bacteria by the antibiotics,and then induce liver injury.High doses of antibiotics also induce liver injury.The mechanism of poultry liver injury caused by LPS combined with antimicrobial agents and the repair and mechanism of CAG remain to be studied.Based on the model of primary hepatocyte injury induced by LPS and FFC in healthy chickens,the pathological mechanism of hepatocyte injury induced by LPS/FFC and the protective effect and mechanism of CAG were explored.1.The cell viability of quantitative LPS combined with each concentration of FFCChicken primary hepatocytes were isolated by modified two-step type Ⅳ collagenase perfusion method and cultured on 96-well cell culture plate for 48 hours.Screening of the best dose of LPS/FFC-induced liver injury:96-well plate containing LPS+FFC(0,30+20,30+40,30+60,30+80,30+100 μg/mL)was added respectively;LPS was in the front,FFC was in the back)growth medium(n=3),continued to culture for 24 hours,MTT method was used to determine the survival rate of cells,and the best dose of combined injury was screened.The results showed that when LPS/FFC concentration was 30+40 μg/mL,the cell survival rate was(49.57+2.16%).Therefore,the concentration of LPS/FFC at 30/40 μg/mL is the best dose to induce hepatocyte injury.Screening of CAG dosage:After 48 hours of isolation and culture of chicken primary hepatocytes by the above method,the growth medium containing CAG(0,1,10,50,100,200,400 μg/mL)and LPS+FFC(30+40 μg/mL)were added to 96-well plate respectively,for 24 hours.MTT assay was used to determine the cell survival rate and the kit were used to evaluate the levels of ALT and AST in the supernatant.The results showed that 1 μg/mL could significantly reduce LPS+FFC-induced injury,increase cell viability,and decrease ALT and AST levels.Therefore,the protection range is determined to be 0-1μg/mL.According to the results,the protective doses of low,medium and high doses were determined to be 0.01,0.1 and 1 μg/mL.2.Compound ammonium glycyrrhizin protects hepatocytes from injury induced by lipopolysaccharide/florfenicol through a mitochondrial pathwayAccording to the above conditions,chicken primary hepatocytes were isolated and cultured.The protective doses of CAG were 0.01,0.1 and 1 ug/mL.The following experiments were carried out at the concentration of LPS/FFC(30+40 ug/mL).2.1 Apoptosis rates of the LPS/FFC and CAG groups as assessed by FCMThe apoptosis rate of LPS/FFC-treated hepatocytes was 33.65±3.48%,the apoptosis rate of the control group was 8.54±0.32%and the apoptosis rates of CAG treated hepatocytes were 27.07±0.54%,22.73±2.45%and 15.08±1.28%,respectively.The bar graph of apoptosis rates indicated that LPS/FFC can induce apoptosis.Moreover,the apoptosis rate decreased significantly(p<0.01)when the concentration of CAG was higher than 0.01 μg/mL.It was suggested that CAG decreased LPS/FFC-induced apoptosis of chicken hepatocytes.2.2 The change of mitochondrial membrane potential(MMP)Mitochondria with normal membrane potential showed red fluorescence,while mitochondria with decreased membrane potential that showed green fluorescence.The green fluorescence of the model group was enhanced which compared with the control group.The red fluorescence which treat with CAG was enhanced with the increased concentration of CAG.This suggests that LPS/FFC can reduce mitochondrial membrane potential and CAG can prevent mitochondrial membrane potential from decreasing.2.3 Ultrastructural changes in mitochondria of hepatocytesThe control group showed a normal structure with normal cristae.However,the mitochondria of the LPS/FFC group was shown swelling and vacuolization,degeneration,while above changes were not found in the CAG group.The result indicated that mitochondrial ultrastructure was severely altered in the model group,while the CAG group protected mitochondrial from damage.2.4 The change of mRNA and proteins expression levelLPS/FFC significantly up-regulated the mRNA and proteins expression levels of caspase-3,caspase-9,bax and cytochrome c(cytc)(p<0.05),while the CAG down-regulated those mRNA and proteins expression levels.In addition,the mRNA and proteins expression levels of bcl-2 was down-regulated significantly by LPS/FFC,and the mRNA and proteins expression levels of bcl-2 was up-regulated significantly by CAG.That was demonstrated that CAG protected hepatocytes from damage through mitochondria-mediated apoptosis pathway.2.5 The change of cytochrome C protein expressionThe cytochrome c protein expression of the model group in cytoplasm was significantly higher than that in the control and CAG group,and the protein expression of cytochrome c in mitochondria was significantly lower in the model group than that in the another two groups.It was suggested that LPS/FFC induced liver injury was mediated by mitochondrial pathway and CAG protected liver cells through mitochondrial pathway.3.Compound ammonium glycyrrhizin protects hepatocytes from injury induced by LPS/FFC through oxidative stress and a MAPK pathwayAccording to the above conditions,chicken primary hepatocytes were isolated and cultured.The protective doses of CAG were 0.01,0.1 and 1 ug/mL.The following experiments were carried out at the concentration of LPS/FFC(30+40 ug/mL).3.1 The hepatocytes are dyed by Hoechst33342/PIThe result was shown that the LPS/FFC group was shown high blue/low red.And the control group was shown low blue/low red,while the CAG group(concentration of 0.01μg/mL,0.1μg/mL,1μg/mL,respectively)for 24 h,the cells was shown low blue/low red.It was suggested that CAG decreased LPS/FFC-induced apoptosis of chicken hepatocytes.3.2 The activity of GSH,MDA and SODThe activities of GSH and SOD in the model group were significantly(p<0.01)reduced than the control group,those of the CAG groups were gradually increased with the increase of the dose of CAG.And the MDA was significantly(p<0.01)up-regulated by LPS/FFC,the MDA was gradually decreased with the increase dose of CAG.It was suggested that LPS/FFC can induce liver injury by oxidative stress.And CAG can inhibit oxidative stress to pretect the hepatocytes from injury.3.3 The change of mRNA and proteins expression levelRT-qPCR was used to analyze the mRNA expression level of JNK,ERK and P38.LPS/FFC significantly up-regulated the mRNA and proteins expression levels of JNK,ERK and P38(p<0.01),while the CAG down-regulated those mRNA expressions.The protein expression levels of JNK,ERK and P38 were no difference in the five groups.But LPS/FFC significantly up-regulated the protein expression levels of p-JNK,p-ERK and p-P38.The protein expression levels of those were gradually decreased with the increased concentration of CAG.which compared with the LPS/FFC group.It was suggested that LPS/FFC induced liver injury was mediated by the MAPK pathway and CAG protected liver cells through the MAPK pathway.3.4 The activity of caspase-3The activity of caspase-3 in the LPS/FFC group was significantly up-regulated than that of the control group.The activities of caspase-3 in the CAG groups were down-regulated with the increase of the concentration of CAG which compared with the model group.It was proved that CAG protects hepatocytes from injury induced by LPS/FFC through the oxidative stress and the MAPK pathway. |
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