Dihydromyricetin Alleviates LPS Induced Liver Injury In Chicks By Inhibiting JNK Signaling Pathway

Recently,the abuse of antibiotics has caused an increasing incidence rate of animal liver injury,leading to huge economic losses to poultry industry.Chickens are susceptible to Gram-negative bacteria.A large amount of endotoxin（lipopolysaccharides,LPS）will be released after bacterial infection,which could cause liver damage.c-Jun N-terminal kinase（c-Jun N-terminal kinase,JNK）is an important pathway in the pathogenesis of many diseases.It can participate in various biological reactions such as apoptosis.Dihydromyrice tin（DHM）is a kind of natural flavonoids compound from Ampelopsis grossedentata.It has many pharmacological activities,such as anti-inflammatory,anti-oxidation and hepatic protection.This study aims to investigate the effect of dihydromyricetin on JNK signaling pathway and apoptosis in LPS induced liver injury in chicks,and to provide a theoretical foundation for the prevention and treatment of liver injury and the development of DHM.In this experiment,12 chicks（22-day-old）were randomly divided into two groups:control group and LPS group.Chicks in LPS group were intraperitoneally injected with LPS(60 mg·kg^-1)after fasting for 12 h,while chicks in control group were injected with saline.After 12 hours,blood samples were collected from the heart,and serum transaminase（ALT,AST）activities were detected by commercial kits.On the basis of this model,40 chicks（1-day-old）were randomly divided into 6 groups after adaptive feeding for 7 days:control group,LPS group,0.025%DHM+LPS group,0.05%DHM+LPS group,0.1%DHM+LPS group and DHM control group.The control group and LPS group were fed with basal diet,and other groups were fed with diets mixed with different doses of DHM for 14 days.Then chicks were fasted for12 h,and chicks in LPS groups intraperitoneally injected with LPS(60 mg·kg^-1),while chicks in control group and DHM control group were injected with saline.After 12 hours,blood was collected from the heart.Liver samples（1 cm³）were fixed with 10%formalin,and the rest part of liver were stored at-80°C.The activity of serum transaminase in serum was detected by commercial kits,and the liver was observed by H&E staining.The protein expression and phosphorylation levels of ASK1,MKK4and MKK7 in the upstream of JNK signaling pathway,JNK protein expression and phosphorylation levels,and c-jun protein expression and phosphorylation levels in the downstream were determined by Western blot.The apoptosis was test by Hoechst 33258,Western blot method was used to detect the relative expression of anti-apoptotic protein Bcl-2 and the relative expression of pro-apoptotic protein Bax and caspase-3.The results are as follows:（1）The protective effects of DHM on chicken liver injury.Compared with the control group,the activities of serum transaminase in serum in LPS group were significantly increased（p<0.01）,indicating that the model of liver injury induced was successfully replicated.Compared with LPS group,the activities of serum transaminase in 0.05%DHM group were markedly increased（p<0.05）,and the activities of serum transaminase in 0.1%DHM group were significantly decreased（p<0.01）.The results of liver histopathology showed that the morphology of hepatocytes in the control group and DHM control group were normal,the structure was complete,and the hepatic cord was clear.There were lots of inflammatory cell infiltration and bleeding points,hepatic cord disorder,nuclear pyknosis in the LPS group.In 0.025%and 0.05%DHM group,the infiltration of inflammatory cells and bleeding point decreased.The bleeding points and inflammatory cells decreased significantly,and the morphology of hepatic cells was normal in 0.1%DHM group.These results suggested that DHM could protect the liver injury of chicks induced by LPS.（2）The effect of DHM on JNK signaling pathway in LPS-induced liver injury in chicks.The expressions and the phosphorylation levels of proteins in the upstream of JNK signaling pathway（ASK1,MKK4 and MKK7）in the LPS group were significantly decreased（p<0.01）compared with control group.Compared with LPS group,the expressions and the phosphorylation levels of ASK1,MKK4 and MKK7 were significantly decreased（p<0.01）in 0.1%DHM group.Compared with control group,the expressions and the phosphorylation levels of ASK1,MKK4 and MKK7 in0.1%DHM group did not change significantly.Compared with the control group,the expression and phosphorylation levels of protein JNK and downstream protein c-Jun in LPS group increased significantly（p<0.01）.Compared with LPS group,the expressions of JNK and c-Jun in 0.05%DHM group decreased markedly（p<0.05）.Compared with LPS group,the expressions of JNK and c-Jun in 0.1%DHM group significantly decreased（p<0.01）.The above results indicated that JNK signaling pathway could be activated in LPS-induced liver injury of chicks,and DHM could effectively inhibit the activation of JNK signaling pathway.（3）The effect of DHM on apoptosis in LPS-induced liver injury in chicks.The results showed that the nuclear morphology of control group and DHM control group was complete and the fluorescence was relatively balanced,while the cells in LPS group had nuclear pyknosis and the fluorescence intensity was significantly enhanced.The fluorescence intensity of DHM g roups decreased gradually,and nuclear pyknosis decreased.Compared with control group,the relative fluorescence intensity in LPS group was significantly increased（p<0.01）,the relative fluorescence intensity of DHM control group had no significant cha nge.Compared with LPS group,the relative fluorescence intensity of 0.05%DHM group decreased markedly（p<0.05）,and the relative fluorescence intensity of 0.1%DHM group decreased significantly（p<0.01）.Compared with the control group,the expression of anti-apoptotic protein Bcl-2 was significantly decreased（p<0.01）,the expression of Pro-apoptotic proteins Bax and caspase-3 in LPS group were significantly increased（p<0.01）.Compared with LPS group,the expressions of Bax and caspase-3 in 0.05%DHM group were markedly decreased（p<0.05）,and the expression of Bcl-2was markedly increased（p<0.05）.Compared with LPS group,the expression of Bax and caspase-3 protein were significantly decreased（p<0.01）,and the expression of Bcl-2 protein was significantly increased（p<0.01）in 0.1%DHM group.Compared with control group,the expressions of Bcl-2,Bax and caspase-3 in 0.1%DHM group did not change significantly.The above results showed that apoptosis occurred in the liver of chickens induced by LPS,and DHM reduced the level of hepatocyte apoptosis in a dose-dependent manner,and reduce the number of apoptotic cells,reduce the expression of apoptosis related proteins.In conclusion,the model of chicken liver injury was successfully reproduced by LPS.Under the treatment of LPS,the upstream JNK signaling pathway,such as ASK1,MKK4 and MKK7,were activated,the expression and phosphorylation levels of JNK were significantly increased,and the downstream c-Jun,was activated to increase the expression of apoptosis related proteins,resulting in liver injury.Dihydromyricetin can effectively inhibit the activation of ASK1,MKK4and MKK7,the activation of JNK,the phosphorylation of c-Jun,the expressions of apoptosis-related proteins,and eventually alleviate the liver injury induced by LPS.This study provides a theoretical basis for the development and utilization of dihydromyricetin.