Chlorogenic Acid And Curcumin Regulate Ammonia Nitrogen-induced Inflammatory Response Of Yellow Catfish Macrophages

A study was carried out to investigated the anti-infalmmatory mechanisms of chlorogenic acid（CGA）and curcumin（CUR）on ammonia nitrogen-induced inflammation in head kidney macrophages of Pelteobagrus fulvidraco.Macrophages from head kidney of P.fulvidraco were isolated and cultured by discontinuous density gradient method.The value of 24 h inhibitory concentration(IC₅₀)of ammonia nitrogen,and the cell survival of CGA and CUR treatment were measured by MTT assay.The m RNA levels of inflammatory factors,anti-oxidant enzymes,anti-apoptotic gene and macrophage polarization were detected by RT-q PCR.The level of ROS was detected by Determination of DCF,and the apoptosis of macrophages was detected by TUNEL staining.The main results are as follows:（1）Isolation,culture and identification of macrophages from head kidney of P.fulvidraco:Discontinuous density gradient method was used to isolate macrophages from head kidney of P.fulvidraco.The purification rate of macrophages from head kidney of P.fulvidraco was over 98%.Giemsa staining and ultrastructural observation were used for identifing macrophages.The nucleus of macrophages were round,oval or irregular in shape.The nuclear lamina and organelles remained intact and clear.The melanin granules and phagocytic vesicles were distributed in the cell cytosol,and many pseudopodium were observed in the cell.（2）Anti-inflammatory effects of chlorogenic acid on ammonia nitrogen-induced inflammation in head kidney macrophages of P.fulvidraco:The cells were divided into six groups,as follows:blank control group（CON）,ammonia nitrogen group（0.23 mg/L,AM）,chlorogenic acid（125μmol/L,CGA）,and ammonia nitrogen+chlorogenic acid（5,25,125μmol/L,5A,25A,125A）.The cells were pretreated with three different concentrations（5,25,125μmol/L）of CGA for 1 h and then incubated with ammonia nitrogen for 24 h.There were six replicates in each group.The half-maximal inhibitory concentration(IC₅₀)of macrophages was 0.23mg/l under ammonia nitrogen stress.The relative survival rate of macrophages of AM group were lowest（P<0.05）,in contrast,CON group were highest,and were significantly higher than that of 5A,25A and 125A groups（P<0.05）.The m RNA expression levels of IL-1,IL-6,TNF-α,COX-2,NF-κB p65 in AM group were higher than that in CON group（P<0.05）,and CGA significantly downregulated the m RNA expression levels of above genes（P<0.05）.The m RNA expression of SOD in 25A group was significantly higher than that in CON group（P<0.05）.Expression of Bcl-2 and Arg-1 genes were remarkably downregulated in cells of AM group（P<0.05）.The relative level of ROS in AM group was significantly higher than that in other groups（P<0.05）.The relative level of ROS in AM group was significantly higher than that in other groups（P<0.05）.There was no significant difference between CGA group and con group（P>0.05）,but it was significantly lower than that in 5A,25A and 125A groups（P<0.05）.Cell apoptosis was analysed by TUNEL staining（red for apoptotic cells）.A large number of apoptotic cells were observed in AM group,but only very few apoptotic cell in other groups.（3）Anti-inflammatory effects of curcumin on ammonia nitrogen-induced inflammation in head kidney macrophages of P.fulvidraco:The cells were divided into six groups,as follows:blank control group（CON）,ammonia nitrogen group（0.23 mg/L,AM）,curcumin（45μmol/L,CUR）,and ammonia nitrogen+chlorogenic acid（5,25,45μmol/L,5A,25A,45A）.The cells were pretreated with three different concentrations（5,25,45μmol/L）of CUR for 1 h and then incubated with ammonia nitrogen for 24 h.There were six replicates in each group.The half-maximal inhibitory concentration(IC₅₀)of macrophages was 0.23mg/l under ammonia nitrogen stress.The relative survival rate of macrophages of AM group were significantly lower than that of CON,CUR,25A and 45A groups（P<0.05）.The m RNA expression levels of IL-1,IL-6,TNF-α,COX-2,NF-κB p65,SOD,GPx were highest in AM group（P<0.05）,and CUR significantly downregulated the m RNA expression levels of above genes（P<0.05）.In contrast,the m RNA expression of Bcl-2 and Arg-1 genes were lowest among groups（P<0.05）,and CUR remarkably upregulated the m RNA expression of above genes（P<0.05）.The relative level of ROS in AM group was the highest,which was significantly higher than other groups（P<0.05）.There was no significant difference between 5A and 45A groups（P>0.05）,but they were significantly higher than con,cur and 25A groups（P<0.05）.The relative level of ROS in cur group was the lowest,which was significantly lower than con and 25A groups（P<0.05）.A large number of apoptotic cells were observed in AM group,but only very few apoptotic cell in other groups（red for apoptotic cells）.Conclusion:Ammonia induced inflammation and oxidative stress.Pro-inflammatory effects of ammonia were related to the NF-κB/COX-2 pathway.Ammonia caused macrophage apoptosis;CGA and CUR could alleviate the inflammatory response,oxidative stress and apoptosis of macrophages induced by ammonia nitrogen stress,and induce M2 polarization of macrophages.CGA and CUR may exert their antioxidant activities by directly neutralizing free radicals or other anti-inflammatory pathways.