Dihydromyricetin Attenuates LPS-induced Chicken Hepatic Injury By Inhibiting TRIF/RIPK3/MLKL Necroptosis Signal Pathway

Bacterial disease is a major problem troubling the healthy development of poultry industry.LPS is an important component of the cell wall of Gram-negative bacteria,which can invade the liver through blood or portal vein and cause injury.Liver injury seriously affects the physiological function of poultry,resulting in the decline of production performance and even death.Necroptosis is a new way of cell death which is regulated by signal molecules and has necrosis-like morphological characteristics.It plays an important role in liver injury and can trigger severe oxidative stress and inflammation.LPS can activate TRIF/RIPK3/MLKL necroptosis signal pathway,the key step of this pathway is that TRIF recruits its downstream RIPK3 to form necrotic body complex,and further recruits and phosphorylates downstream MLKL.Activated MLKL destroys cell membrane and leads cell to death,so TRIF,RIPK3 and MLKL are biomarkers for detection of necroptosis.Dihydromyricetin(DHM)is a kind of flavonoid,which has many pharmacological activities such as antioxidation,anti-inflammation and liver-protecting.It has potential application value in animal husbandry.At present,the study on the intervention of DHM on necroptosis in poultry liver injury has not been reported.Therefore,this study took chickens and chicken primary hepatocytes as the research object to explore the protective effects of DHM on chicken liver injury induced by LPS.Moreover,this study explored whether DHM can alleviate LPS-induced chicken primary hepatocyte injury by interfering with TRIF/RIPK3/MLKL necroptosis signal pathway,which will provide scientific basis for the development and utilization of DHM in veterinary clinic.The test contents and results are as follows:(1)The protective effects of DHM on chicken liver injury induced by LPS: The chicken model of liver injury in vivo was replicated.There were four groups,namely control group,model group,LPS+DHM group and DHM group.The observation of hepatic ultrastructure was conducted by using transmission electron microscope.The activities of ALT,AST and LDH in serum were detected by automatic biochemical analyzer.The activities of antioxidant enzymes(CAT,SOD,GSH-Px)and the contents of MDA and GSH in liver were detected by kit.The expression of IL-1β,IL-6,necroptosis related factors(TRIF,RIPK3,MLKL)m RNA in liver were detected by real-time fluorescence quantitative PCR.The expression of TRIF,RIPK3 protein and the phosphorylation of MLKL protein in liver were detected by Western blot.The results of transmission electron microscope observation displayed that the cytomembrane was ruptured in model group,the shape of nucleus was irregular,and the mitochondria were swollen or even vacuolated,showing typical morphological characteristics of necrosis.While the structure of hepatocytes in LPS+DHM group was basically normal.Compared with control group,the activities of ALT,AST and LDH in model group increased significantly(p < 0.01);the expression of TRIF,RIPK3,MLKL and the phosphorylation of MLKL protein increased significantly(p < 0.01), indicating that LPS induced necroptosis;the expression of IL-1β and IL-6 increased significantly(p < 0.01);CAT,SOD,GSH-Px,GSH and MDA changed significantly(p < 0.05 or p < 0.01).Compared with model group,the activities of ALT,AST and LDH in LPS+DHM group decreased significantly(p < 0.01);the expression of TRIF,RIPK3,MLKL and the phosphorylation of MLKL protein decreased significantly(p < 0.05 or p < 0.01);the expression of IL-1β and IL-6 decreased significantly(p < 0.01);the activities of SOD,CAT,GSH-Px and the contents of GSH increased significantly(p < 0.05 or p < 0.01),and the contents of MDA decreased significantly(p < 0.01).These results suggest that DHM can inhibit necroptosis,inflammation and oxidative stress to alleviate chicken liver injury induced by LPS.(2)The protective effects of DHM on chicken primary hepatocyte injury induced by LPS: The chicken model of liver injury in vitro was replicated.There were four groups,namely control group,model group,LPS+DHM group and DHM group.Cell necrosis was analyzed by Annexin V-FITC/PI double staining.The activities of ALT,AST and LDH in the supernatant,the expression of IL-1βand IL-6 m RNA,the expression of TRIF,RIPK3,MLKL m RNA and protein,and the phosphorylation of MLKL protein in the cells were detected.Results of Annexin V-FITC/PI staining showed that LPS caused a large number of necrotic chicken hepatocytes,while DHM could obviously reduce the number of necrotic cells.Compared with control group,the indexes of liver injury,the expression of inflammatory factors and necroptosis-related factors in model group changed significantly(p < 0.05 or p < 0.01).Compared with model group,DHM significantly decreased the expression of liver injury indexes,inflammatory factors and necroptosis-related factors(p < 0.05 or p < 0.01).These results suggest that DHM can inhibit necroptosis,inflammation and alleviate the chicken hepatocyte injury induced by LPS.(3)DHM interferes chicken hepatocyte injury by inhibiting TRIF/RIPK3/MLKL necroptosis signal pathway: RIPK3 is the hub of necroptosis signal pathway,and multiple upstream signals converge on RIPK3 to further induce downstream MLKL executive cell death.GSK’872 is a specifical inhibitor of RIPK3,so GSK’872 was used in this experiment.To screen the optimal concentration of GSK’872,the cell viability and liver injury indexes(ALT,AST and LDH)was detected.The results showed that 1 μM GSK ’872 had no obvious toxicity to chicken hepatocytes and significantly decreased the indexes of liver injury(p < 0.05 or p < 0.01).Therefore,the concentration was selected for the follow-up test.There were six groups,namely control group,model group,LPS+GSK’872 group,LPS+DHM group,LPS+DHM+GSK’872 group and GSK’872group.Liver injury indexes in supernatant,the expression of TRIF,RIPK3,MLKL m RNA and protein,and the phosphorylation of MLKL protein in cells were detected.The test results displayed that the liver injury indexes,the expression of RIPK3 and MLKL and the phosphorylation of MLKL protein in LPS+GSK’872 group were significantly decreased by comparing with model group(p <0.05 or p < 0.01),while the expression of TRIF was not significantly different,indicating that GSK’872 could inhibit the expression of RIPK3 and further reduce the expression of downstream MLKL and the phosphorylation of MLKL protein.However,the effect of GSK’872 on the signal transduction of TRIF upstream of RIPK3 was not obvious.Compared with model group,the indexes of liver injury,the expression of TRIF,RIPK3,MLKL and the phosphorylation of MLKL protein in LPS+DHM group and LPS+DHM+GSK’872 group were significantly decreased(p < 0.05 or p <0.01),indicating that DHM could inhibit the signal transduction of both upstream and downstream of RIPK3.These results suggest that DHM can interfere LPS-induced injury of chicken hepatocyte by inhibiting TRIF/RIPK3/MLKL necroptosis signal pathway.To sum up,in the chicken model of liver injury induced by LPS in vivo and in vitro,LPS caused necroptosis by activating TRIF/RIPK3/MLKL signal pathway.DHM could reduce oxidative stress and inflammation in chicken liver injury,and alleviate LPS-induced chicken liver injury by inhibiting TRIF/RIPK3/MLKL necroptosis signal pathway.This subject provides a data basis for further research on the protective mechanism of DHM on chicken liver injury,and also provides a scientific basis for the development and utilization of DHM.