Dihydromyricetin Attenuates LPS-induced Chicken Liver Injury By Regulating STAT3/IL-17A Signaling Pathway

Lipopolysaccharide(LPS)is the main pathogenic factor of Gram-negative bacteria,it exists in intestinal tract in high concentration during bacterial infection and enters liver through portal vein,producing a large number of inflammatory factors and causing liver damage.Signal and activator of transcription 3(STAT3)is an inflammatory protein that transmits signals through the cytoplasm and acts as a transcription factor in the nucleus,mediating various inflammatory reactions.Interleukin-17a(IL-17A)is a cytokine involved in both acute and chronic inflammatory responses and plays a critical role in defense against microbial infection.STAT3/IL-17 A signaling pathway plays an important role in the occurrence and development of liver injury,and is associated with its overactivation in various liver diseases.Dihydromyricetin(DHM)is a flavonoid compound with anti-inflammatory,antioxidant,liver protection,nerve protection and other pharmacological effects.It is cheap,easy to extract,and has high potential application value in aquaculture.At present,studies on DHM focus on mammals and relatively little research has been done in avian species,while the role of DHM on STAT3/IL-17 A signaling pathway is still unclear.The aim of this thesis is to investigate the role of STAT3/IL-17 A signaling pathway in the mitigation of LPS-induced liver injury in chickens by DHM,to explore the protective mechanism of DHM against liver injury in chickens,and to provide a theoretical basis for the application of DHM in livestock and poultry farming and the development of liver-protective drugs.The test contents and results are as follows:(1)Protective effect of DHM on LPS-induced liver injury in chicken: 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.Automatic biochemical instrument was used to detect liver injury indexes(ALT,AST and LDH);H&E staining was used to observe the pathological changes of liver;ELISA kits were used to detect inflammatory factors(IL-1β,IL-6,TNF-α,TGF-β)and IL-17A;real-time fluorescence quantitative PCR to detect inflammatory factors,STAT3,IL-17 A,TRAF6 m RNA expression;Western blot detect STAT3 activation(p-STAT3/STAT3 ratio)and TRAF6 protein expression After 12 h of LPS treatment,the chicken liver injury model was successfully replicated.The pathological histological results showed that the LPS group had disordered liver cords,inflammatory cell infiltration and hemorrhagic spots;the DHM+LPS group had obvious liver cords,normal cell morphology and intact liver lobule structure.Compared with the control group,liver injury indicators,inflammatory factor,STAT3,IL-17 A and TRAF6 m RNA expression,inflammatory factor and IL-17 A content,and STAT3 activation and TRAF6 protein expression were highly significant higher in the LPS group(p < 0.01).Compared with the LPS group,liver injury indicators,inflammatory factor,STAT3,IL-17 A and TRAF6 m RNA expression,inflammatory factor and IL-17 A content,and STAT3 activation and TRAF6 protein expression were all highly significantly decreased in the DHM+LPS group(p < 0.01).The above results indicated that STAT3/IL-17 A signaling pathway was activated in LPS-induced liver injury in chickens,and DHM had an inhibitory effect on STAT3/IL-17 A signaling pathway,reducing liver inflammatory response and alleviating liver injury.(2)Protective effect of DHM on LPS-induced hepatocyte injury in chicken: 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.Liver injury index,inflammatory factors and IL-17 A content were detected.ROS content of liver cells was detected by immunofluorescence method.Inflammatory cytokines,STAT3,IL-17 A and TRAF6 m RNA expression,STAT3 activation and TRAF6 protein expression were detected.Chicken primary hepatocytes were isolated and cultured,and the chicken hepatocyte injury model was successfully replicated after 12 h of LPS treatment.Hepatocyte injury indicators,ROS levels,inflammatory factors,STAT3,IL-17 A and TRAF6 m RNA expression,inflammatory factors and IL-17 A content,and STAT3 activation and TRAF6 protein expression were highly significant higher in the LPS group compared with the control group(p < 0.01).Hepatocyte injury indicators,ROS levels,inflammatory factors,STAT3,IL-17 A and TRAF6 m RNA expression,inflammatory factors and IL-17 A content,and STAT3 activation and TRAF6 protein expression were all highly significantly reduced in the DHM+LPS group compared with the LPS group(p < 0.01).The above results indicated that STAT3/IL-17 A signaling pathway was activated in LPS-induced hepatocyte injury,and DHM could reduce the content of ROS and have an inhibitory effect on STAT3/IL-17 A signaling pathway,reduce the inflammatory response of hepatocytes and alleviate hepatocyte injury.(3)DHM interferes with chicken hepatocyte injury by regulating STAT3/IL-17 A signaling pathway: IL-17 A can recruit the downstream gene TRAF6 to induce the production of pro-inflammatory factors,while activated STAT3 can directly promote the secretion of IL-17 A.Therefore,the STAT3 inhibitor C188-9 was used in this experiment,The optimal concentration was determined to be 3 μM by detecting cell viability and liver injury index.Control group,LPS group,DHM+LPS group,DHM group,C188-9+LPS group,C188-9 group,C188-9+DHM+LPS group and C188-9+DHM group were established.STAT3 m RNA expression and activation,IL-17 A m RNA expression and content,TRAF6 m RNA and protein expression,inflammatory factor m RNA expression and content were detected.Compared with the LPS group,STAT3 m RNA expression and STAT3 activation were both significantly reduced in the C188-9+LPS group(p < 0.01),indicating that C188-9 could inhibit STAT3 transcription and reduce STAT3 activation;IL-17 A m RNA expression and content were significantly reduced(p < 0.01),indicating that C188-9inhibited STAT3/IL-17 A signaling pathway;TRAF6 m RNA and protein expression and downstream inflammatory factor m RNA expression and content were significantly reduced(p <0.01),indicating that inhibition of STAT3/IL-17 A signaling pathway could further reduce TRAF6 expression and the expression and release of downstream inflammatory factors.Compared with the LPS group,STAT3 m RNA expression and STAT3 activation were highly significantly reduced in both the C188-9+DHM+LPS and DHM+LPS groups(p < 0.01);IL-17 A m RNA expression and content were highly significantly reduced(p < 0.01),and there was no significant difference between the C188-9+LPS,DHM+LPS and C188-9+DHM+ LPS group,indicating that DHM could inhibit the activation of STAT3/IL-17 A signaling pathway by suppressing the transcription and activation of STAT3;TRAF6 m RNA and protein expression and inflammatory factor m RNA expression and content were highly significantly reduced(p < 0.01),indicating that DHM can reduce TRAF6 and its downstream inflammatory factor expression and release by inhibiting the activation of STAT3/IL-17 A signaling pathway and alleviate the inflammatory response of hepatocytes.In summary,the STAT3/IL-17 A signaling pathway is activated in LPS-induced liver injury,and DHM can reduce the expression of downstream proteins and pro-inflammatory cytokines by inhibiting the activation of STAT3/IL-17 A signaling pathway to alleviate the inflammatory response and mitigate LPS-induced liver injury in chickens.