Protective Effects Of Dihydromyricetin On LPS-induced Chicken Liver Injury Based On The Dynamic Balance Of Mitochondrial Fusion And Fission

Endotoxin is the main component of the cell wall of Gram-negative bacteria,also known as Lipopolysaccharide（LPS）.When bacteria die,large amounts of LPS are released into the liver through the portal vein,causing liver damage.Mitochondria are highly dynamic organelles,which play an important role in maintaining the REDOX balance of organisms by regulating their own fusion and fission.In diseases,the dynamic balance of mitochondrial fusion and fission may be disturbed,but there are few studies on this mechanism in chicken liver injury.Dihydromyricetin（DHM）is a flavonoid extracted from rattan tea,which has anti-inflammatory,antioxidant,antibacterial and other pharmacological effects.However,the effect of DHM on mitochondrial fission/fusion in chicken liver injury was still unclear.Therefore,this study was conducted to investigate the protective effect of DHM on LPS-induced liver injury in chicks and primary hepatocytes based on the dynamic balance of mitochondrial fusion and fission,aiming to provide data support for liver injury treatment and the development and utilization of DHM.The liver injury model of Hy-line white chicks was established in this experiment.The chicks were divided into control group,LPS group,DHM+LPS group and DHM group.The pathological changes of liver were observed by HE staining and transmission electron microscopy.The activities of ALT,AST and LDH in serum and the levels of MDA,SOD,H₂O₂and NO in liver were detected by the kit.The expression of Mfn1,Mfn2,Opa1,Drp1 and Fis1were detected by real-time fluorescence quantitative PCR and Western Blot to study the protective effect of DHM on LPS-induced liver injury in chickens.Chicken primary hepatocytes were isolated and cultured to replicate the hepatocyte injury model.Mitochondrial membrane potential（MMP）was detected by JC-1 staining,and the optimal concentration of DHM was determined.The cells were divided into control group,LPS group,DHM+LPS group and DHM group.The activities of ALT,AST and LDH in supernatant and the levels of MDA and SOD in cells were detected.ROS content was detected by immunofluorescence assay.Mitochondrial morphology was observed by Mito-Tracker Green staining,and ATP content and MMP level in hepatocytes were detected by kit.The expression of Mfn1,Mfn2,Opa1,Drp1and Fis1 were detected by real-time fluorescence quantitative PCR and Western Blot to study the protective effect of DHM on LPS-induced liver cell injury.The test results are as follows:（1）Protective effects of DHM on liver injury:After 12 h of LPS administration,the activities of ALT and AST in serum of chicks in LPS group were significantly increased compared with those in control group（p<0.01）,indicating that the model was successfully replicated.Compared with the control group,the activities of ALT,AST and LDH in serum of LPS group were significantly increased（p<0.01）,the levels of MDA,H₂O₂ and NO in liver were significantly increased,and the activity of SOD was significantly decreased（p<0.01）.Compared with LPS group,the activities of ALT,AST and LDH in DHM+LPS group were significantly decreased（p<0.01）,the levels of MDA,H₂O₂ and NO were significantly decreased,and the activity of SOD was significantly increased（p<0.01）.Histopathological results showed that the liver cords in LPS group were disordered,the nucleus was pyknotic and dissolved,and the cell morphology was not clear.In DHM+LPS group,hepatic cord arrangement tended to be clear and cell morphology tended to be normal.The results of ultrastructural pathology showed that endoplasmic reticulum and mitochondria were vacuolated in LPS group,and the nuclear membrane space was enlarged.The morphology of nucleus and mitochondria in DHM+LPS group tended to be normal,indicating that DHM can alleviate LPS-induced liver injury and improve the antioxidant capacity of liver.Compared with the control group,the m RNA and protein expressions of mitochondrial fusion proteins Mfn1,Mfn2 and Opa1 in LPS group were significantly decreased（p<0.01）,while the m RNA and protein expressions of fission proteins Drp1 and Fis1 were significantly increased（p<0.01）.Compared with LPS group,the m RNA and protein expressions of mitochondrial fusion proteins Mfn1,Mfn2 and Opa1 in DHM+LPS group were significantly increased（p<0.01）,and the m RNA and protein expressions of mitochondrial mitotic protein Drp1 and Fis1 were significantly decreased（p<0.01）.These results indicated that DHM could promote the expression of mitochondrial fusion protein and inhibit the expression of mitochondrial mitotic protein in liver injury of chickens.（2）Protective effect of DHM on injury of chicken primary hepatocytes:After 12 h of LPS treatment,the activities of ALT and AST in supernatant of LPS group were significantly increased compared with control group（p<0.01）,indicating that the model was successfully replicated.The results showed that compared with the control group,the MMP level of cells in LPS group was significantly decreased（p<0.01）,and the MMP level of cells in 80μM DHM+LPS group was significantly increased（p<0.01）.Therefore,this concentration was selected for subsequent experiments.Compared with control group,the activities of ALT,AST and LDH in supernatant of LPS group were significantly increased（p<0.01）,the content of MDA and ROS in cells were significantly increased（p<0.01）,and the activity of SOD in cells was significantly decreased（p<0.05）.Compared with LPS group,the activities of ALT,AST and LDH in supernatant of DHM+LPS group were significantly decreased（p<0.01）,the content of MDA and ROS in cells were significantly decreased（p<0.01）,and the activity of SOD in cells was significantly increased（p<0.05）,suggesting that DHM can reduce liver cell injury and improve its antioxidant capacity.The results of fluorescence microscopy showed that mitochondria in the LPS group were fragmented into short rods or spots,and mitochondria in the DHM+LPS group tended to be reticulated.Compared with control group,MMP level and ATP content in LPS group were significantly decreased（p<0.01）.Compared with LPS group,MMP level and ATP content in DHM+LPS group were significantly increased（p<0.01）,suggesting that DHM could alleviate LPS-induced mitochondrial fragmentation changes and reduce mitochondrial function damage.Compared with the control group,the m RNA and protein expressions of mitochondrial fusion proteins Mfn1,Mfn2 and Opa1 in LPS group were significantly decreased（p<0.01）,while the m RNA and protein expressions of fission proteins Drp1 and Fis1 were significantly increased（p<0.01）.Compared with LPS group,the m RNA and protein expressions of fusion proteins Mfn1,Mfn2 and Opa1 in DHM+LPS group were significantly increased（p<0.01）,and the m RNA and protein expressions of fission proteins Drp1 and Fis1 were significantly decreased（p<0.01）,suggesting that DHM can promote mitochondrial fusion and inhibit mitochondrial fission to maintain its dynamic equilibrium.In this study,the effects of DHM on the dynamic balance of mitochondrial fusion-fission in liver injury were studied on the basis of liver injury model and primary hepatocyte injury model.The results showed that there were oxidative stress and mitochondrial fusion-fission imbalance in LPS-induced liver injury and hepatocyte injury,DHM intervention can maintain its dynamic balance,strengthen the function of mitochondria,alleviate oxidative stress and protect chicken liver damage.