Protective Effects Of Dihydromyricetin On LPS-induced Liver Injury In Chickens

Endotoxin,also known as lipopolysaccharide?LPS?,is a major component of the cell wall of Gram-negative bacteria and one of the pathogenic factors of Gram-negative bacteria.Endotoxin that comes from the hepatic portal system will be phagocytized by Kupffer cells,but when endotoxin is excess it would cause liver injury.Liver injury is one of the major problems in poultry and it has brought sever economic loss to poultry industry.Dihydromyricetin?DHM?is a flavonoid compound with a range of pharmacological effects,including anti-tumor,anti-inflammatory,antioxidation,anti-alcohol,liver protection,and lipid-regulating activities,and it could be widely used in poultry industry.In this study,different concentrations of DHM were given to chickens and primary chicken hepatocytes to explore the protective effect of DHM based on LPS-induced chicken liver injury models in vitro and in vivo.The research aims to explore the potential use of DHM in poultry industry and provide a theoretical basis for the protection of poultry liver.In this experiment,primary hepatocytes were divided into 6 groups:control group,model group(LPS 40?g·m L^-1),DHM groups?20,40,80?M?and DHM control group?80?M DHM?.When the confluence of primary hepatocytes reached 80%,DHM groups and DHM control group were incubated with different concentrations of DHM?20,40,80?M?for 2 h,and then model group and DHM groups were given 40?g·m L^-1 LPS.After 12 h,cells and cell supernatant were collected to detect the activities of ALT,AST and LDH.The antioxidant indexes?T-AOC,CAT,NO,LPO?were detected,and the m RNA expressions of inflammatory factors?TNF-?,IL-6,IL-12,i NOS?in the cells were detected by RT-PCR.7-day-old chickens were divided into 6 groups:control group,model group(LPS 60 mg·kg^-1),DHM groups?0.025%,0.05%,0.1%DHM?and DHM control group?0.1%DHM?.Different doses?0.025%,0.05%,0.1%?of DHM was added to the feed of chickens in DHM groups and DHM control group.,while control group and model group were given normal feed and chickens were fed with relative feed for 14 days.LPS was given to chickens from model group and DHMs group by intraperitoneal injection,and control group and DHM control group only received an injection of same amount of saline.After12 h,the blood and liver tissues were collected to detect changes of ALT,AST and LDH activities in serum,liver antioxidant indexes?T-AOC,CAT,NO,LPO?,and m RNA expressions of inflammation factors?TNF-?,IL-6,IL-12,i NOS?in liver.Liver sections were fixed in formalin for histopathological examination.The results are as follows:?1?the protective effect of DHM on LPS-induced primary hepatocyte injury:After 12 h administration of LPS,the activity of LDH significantly increased in40,60,80,and 100?g·m L^-1 group?P<0.01?.The results of MTT showed that the cell viabilities significantly increased in 80,160,320,640,1280?M DHM groups compared with the control group after 12 h,?P<0.01?,the cell vitality group significantly increased in 1280?M DHM after24 h and 48 h?P<0.01?,The above results showed that DHM had no cytotoxicity to chicken primary hepatocytes.Compared with control group,the activities of AST,ALT and LDH in culture supernatants significantly increased?P<0.01?,and the activities of AST,ALT and LDH from 40?M DHM group significantly decreased?P<0.01?.The results showed that 40?M DHM could attenuate LPS-induced hepatocytes injury.Compared to control group,the levels of CAT and T-AOC significantly decreased?P<0.01?,and the contents of LPO and NO significantly increased?P<0.01?.Compared with model group,the activity of CAT and the level of T-AOC significantly increased in 40?M DHM group?P<0.01?.Compared with model group,the contents of NO and LPO in 40?M DHM groups significantly decreased?P<0.01?.Compared with control group,the expression levels of TNF-?,IL-6,IL-12 and i NOS m RNA significantly increased in model group?P<0.01?.The expression level of TNF-?,IL-6,IL-12 and i NOS m RNA significantly decreased in 40?M DHM groups?P<0.01?relative to model group,these data showed that 40?M DHM could improve the anti-inflammatory ability of hepatocytes by regulating the expressions of inflammatory factors.?2?The protective effect of DHM on LPS-induced liver injury in chickens:compared with control group,the activities of AST,ALT and LDH in serum significantly increased after LPS treatment?P<0.01?,and the activities of ALT,AST and LDH significantly decreased in 0.05%DHM group?P<0.01?relative to model group.The results showed that 0.05%DHM reduced the activities of transaminase and lactate dehydrogenase in serum.Compared with control group,the levels of CAT and T-AOC significantly decreased?P<0.01?,and the contents of LPO and NO significantly increased?P<0.01?.Compared with model group,the levels of CAT and T-AOC significantly increased in 0.05%DHM group?P<0.01?,and the contents of LPO and NO significantly decreased?P<0.01?,which indicated that 0.05%DHM could improve the antioxidant capacity of chickens by increasing the activities of antioxidant enzyme in liver and decreasing the contents of peroxide products.The expression levels of TNF-?,IL-6,IL-12 and i NOS m RNA significantly increased?P<0.01?compared with the control group.Compared with model group,the m RNA expression of TNF-?significantly decreased in 0.05%and0.1%DHM groups?P<0.05 or P<0.01?,and the expressions of IL-6,IL-12 and i NOS m RNA significantly decreased in 0.05%and 0.1%DHM groups?P<0.01?.The results showed that DHM could modulate the expressions of inflammatory factors in the liver.Histopathological results showed that liver from control group showed clear hepatic lobule structure and normal cell morphology,but liver from model group had many swelling cells.Compared with model group,the hepatocytes in 0.025%DHM group showed less swelling and infiltration of inflammatory cells.The structure of lobules in the liver from 0.05%and 0.1%DHM groups tended to be normal,and inflammatory cell infiltration decreased.These data showed DHM reduced LPS-induced liver injury in chickens.In this study,the LPS-induced primary hepatocytes injury model and LPS-induced chicken liver injury were replicated successfully.It was clear that DHM enhanced the levels of T-AOC and CAT,reduced the contents of NO and LPO,enhanced the anti-oxidation ability of primary hepatocyte and liver.DHM alleviated LPS induced liver injury and primary hepatocytes injury by regulating the m RNA expressions of IL-6,TNF-?,IL-12 and i NOS in primary hepatocytes and chicken liver.Therefore,dihydromyricetin could alleviate LPS-induced liver injury by regulating antioxidant capacity and anti-inflammatory capacity.