Ammonium Glycyrrhetate Counteracts Liver Injury Caused By Lipopolysaccharide/enrofloxacin

With the rise of intensive livestock and poultry breeding,antimicrobial drugs are being used more and more widely.Enrofloxacin is an animal-specific fluoroquinolone antibacterial drug.Because of its broad antibacterial spectrum and strong killing activity,enrofloxacin is widely used in the prevention of respiratory and intestinal infections in livestock and poultry.Medical clinics show that fluoroquinolone antibacterial drugs used for prolonged or overdose will produce hepatotoxicity.Lipopolysaccharide(LPS)is a component of the cell wall of Gram-negative bacteria.When the bacteria die and dissolve,they release a large amount of LPS and become toxic to the host,especially hepatotoxicity.In this study,primary chicken hepatocyte culture models established in vitro were used to induce primary hepatocyte injury in chickens with LPS and enrofloxacin,and to explore the mechanism of injury and the protective effect of compound ammonium glycyrrhizinate.The main findings and contents are as follows:1 The Optimum dose of primary hepatocyte injury caused by LPS combined with enrofloxacin and protection of compound ammonium glycyrrhizinateA modified two-step type IV collagenase perfusion method was used to separate the primary chicken hepatocytes,and cultured for 48 h on the culture plate.The cells adhered to the cells and then the cells were added 0,30+40,30+60,30+80,30+100,30+120 μg/mL LPS and enrofloxacin(ENR)broth(n=3).After culturing for 24 hours,cell morphology was observed by light microscopy and cell viability was measured by MTT assay.After the isolated chicken primary hepatocytes were cultured for 48 hours,25,50,100,200 and 400μg/mL compound glycyrrhizin monoamines were added for 24 h,then LPS plus enrofloxacin was added,and the cell culture solution was 24 h,MTT.Methods The cell viability was measured and the supernatant of each group of cells was collected to determine the contents of ALT and AST.The results showed that when the concentration of LPS plus enrofloxacin was 30+80 p.g/mL,the cell survival rate was(48.27±2.50)%,the cell morphology was irregular,and the cell membrane was destroyed.The hepatocytes of different concentrations of compound glycyrrhizin monoamine were added.The activity of cells increased significantly,and ALT and AST decreased accordingly(P<0.05,P<0.01).Therefore,we chose the concentration of LPS and ENR of 30+80 μg/mL as the optimal dose for hepatocyte injury,while the protective concentration of compound glycyrrhizin monoamine was 100,200,and 400 μg/mL.2 Relationship between LPS/enrooxacin-induced primary hepatocyte injury and oxidative stress in chickenUsing the modified two-step collagenase perfusion method of type Ⅳ,primary chicken hepatocy-tes were isolated,cultured for 48 h,and adhered to the cells for the following tests.In the control group.the culture medium without any drug was added.In the injury group.30+80 μg/mL LPS/ENR was added,and the protection group was pretreated with 25,50,100,200 and 400μg/mL compound glycyrrhizin monoamines for 24 h.Add 30+80 μg/mL LPS/enrofloxacin for 24 h.The cells in each group were collected and tested for SOD,MDA,GSH,GSH-Px and ROS levels.The results showed that the levels of SOD(P<0.01),GSH(P<0.01),and GSH-Px(P<0.01)were significantly decreased in the liver when compared with the control group(30+100μg/mL LPS and ENR).However,after the addition of CAG,these indicators have all increased.However,MDA(P<0.01)and ROS(P<0.01)increased significantly,and GAG also decreased its content.It shows that LPS/ENR can cause liver cell damage through oxidative stress.3 Effect of LPS/enrofloxacin on apoptosis of primary hepatocytes in chickens3.1 Effect of LPS/enrofloxacin on hepatocyte apoptosis and the protective effect of compound ammonium glycyrrhizinateEffects of LPS/Enrofloxacin on Hepatocyte Apoptosis and Protective Effects of Compound Glycyrrhizin Monoamines The control group was supplemented with a culture solution that did not contain any drug.The injury group was treated with 30+80 μg/mL LPS/ENR,and the protection group was Compound glycyrrhizin monoamines at 25,50,100,200 and 400 μg/mL were added for 24 h,followed by 30+80 μg/mL LPS/ENR for 24 h.Cells were harvested,stained with Annexin V-FITC and PI and Hoechst 33342/PI,and observed by flow cytometry and fluorescence microscopy.Flow cytometry results showed that apoptosis rate increased significantly in the LPS+ENR group(P<0.01).However,when treated with CAG in advance for 24 h,the apoptosis rate of hepatocytes increased with the increase of CAG concentration.The mortality rate decreased significantly(P<0.05,P<0.01).Fluorescence microscopy results showed that in the LPS/ENR group,the cells showed a high blue/low red color and were more numerous than the control,while the CAG group was basically the same as the control group.Therefore,LPS/ENR may cause chicken liver damage by promoting chicken primary hepatocyte apoptosis.CAG pretreatment can reduce early apoptosis.3.2 Detection of mitochondrial membrane potentialThe decrease of mitochondrial membrane potential to some extent represents early apoptosis,and the decrease of mitochondrial membrane potential is an important marker of mitochondrial apoptosis pathway.The cells were treated as described above and each group of cells was collected,stained according to the kit instructions,and observed under a fluorescence microscope.The results showed that in the model group,the mitochondrial membrane potential decreased.Similarly,CAG treatment must be able to protect the mitochondrial membrane potential.3.3 Observing the ultrastructure of cell mitochondriaThe cells were treated in the same manner as above,and the cells in each group were collected,washed twice with PBS,fixed with 2.5%glutaraldehyde,and sectioned by electron microscopy.The photographs were observed by transmission electron microscopy.The results showed that the control group showed the ultrastructure of normal cells,including intact nuclei and rod mitochondria.In the group treated with LPS/ENR,the mitochondria of hepatocytes swelled,vacuoles appeared in the mitochondria,and the interstitial space became larger.Therefore,it is suggested that the liver damage caused by LPS/ENR treatment may be related to the mitochondrial pathway.3.4 Determination of apoptosis related mRNA and protein expression in hepatocytesThe experimental group was extracted as shown above and the mRNA expression levels of caspase-3,caspase-9,bax,bcl-2,cyt-c,jnk,p38 and erk were determined by RT-PCR.Total cellular protein was extracted and the expression levels of caspase-3,caspase-9,cyt-c,jnk,p38,erk,p-jnk,p-p38 and p-erk proteins were determined by Western blot.The results showed that in the LPS/ENR treatment group,caspase-3(P<0.05),caspase-9(P<0.05),bax(P<0.01),cyt-c(P<0.01),jnk(P<0.01),p38 The mRNA expression levels of both(P<0.01)and erk(P<0.01)increased significantly,but the mRNA expression levels of the related mRNA decreased after CAG pretreatment for 24 h(P<0.05,P<0.01).The expression changes of caspase-3,caspase-9,cyt-c,jnk,p38,erk,p-jnk,p-p38,and p-erk proteins were basically the same as those of mRNA.It was suggested that LPS/ENR may mediate chicken hepatocyte apoptosis through mitochondrial pathway and MAPKs pathway.