Effect Of Berberine Hydrochloride On Chicken Macrophages Infected By APEC

Chicken E.coli disease is a systemic bacterial infection caused by avian pathogenic Escherichia coli(APEC),which is a serious threat to the development of the poultry industry.It can cause respiratory diseases such as pneumonia,ballooning,pulmonary congestion and pulmonary hemorrhage and other systemic infections such as sepsis,pericarditis,perihepatitis,tubulitis,peritonitis and enteritis.Pulmonary macrophages,as resident immune cells in the lung,play an important role in coordinating pulmonary immunity,fighting APEC infections,and clearing invading pathogens.When the body suffers from disease or environmental factors that cause the body’s"Wei Qi"to be insufficient,the body’s intrinsic immune macrophage function is reduced,and APEC takes advantage of the"deficiency"to enter and cause the occurrence of chicken E.coli disease.Therefore,regulation of macrophage activity to suppress lung inflammation in APEC infections has become an important treatment tool.In order to investigate the role of berberine hydrochloride in regulating macrophages against APEC infection,we established an in vitro model of APEC-infected chicken macrophages and investigated the infection effect of APEC on chicken macrophages at different temperatures,different infection complexes and different infection times;secondly,we analyzed the ability and mechanism of berberine hydrochloride in regulating chicken macrophages in response to APEC infection.Finally,we investigated the regulatory effect and mechanism of berberine hydrochloride on the generation of extracellular trap network by APEC-induced chicken lung macrophages through pretreatment of chicken lung macrophages with berberine hydrochloride,in order to reveal the role of berberine hydrochloride in regulating macrophage resistance to APEC infection and its mechanism,and to provide a research basis for the prevention and treatment of chicken E.coli disease and the development of anti-infective drugs.Firstly,to establish an in vitro model of APEC-infected chicken macrophages,we detected the changes of relative expression of i NOS,IL-1β,IL-6,TNF-αand IL-10 genes after infection of HD11 cells by avian pathogenic E.coli at different infection replicates,different infection times and different infection temperatures by fluorescence quantitative PCR;and detected the changes of relative expression of i NOS,IL-1β,IL-6,TNF-αand IL-10 genes after infection of HD11 cells at 41℃using Gress method and DCFH-DA method,respectively.The changes of NO and ROS production in HD11 cells at different infection times and different infection replicates at 41℃were detected by Gress method and DCFH-DA method,respectively.The results showed that the relative gene expressions of i NOS,IL-1β,IL-6 and TNF-αwere time-dependent from 0.5 to 4 h and reached the peak at 4 h(P<0.01);at 4 h,the relative gene expressions of i NOS,IL-1β,IL-6 and TNF-αshowed a decreasing trend with the increase of infection replication.When the incubation temperature was 41°C,the expression level of IL-10 gene increased with the increase of infection level,and the infection time was 4 h.The expression level of IL-10 gene reached the maximum when MOI=5~3;NO and ROS levels increased significantly(P<0.05)at the early stage of infection and decreased with the increase of infection level;the relative expression of four pro-inflammatory genes was significantly higher than 37°C(P<0.05),and their relative gene expressions reached the maximum when MOI=5~1(P<0.01).Therefore,the infection plural of MOI=5~1,the infection time of 4 h and the infection temperature of 41℃were selected as the infection conditions for the chicken macrophage model of in vitro infection with avian pathogenic E.coli.Then,to investigate the anti-inflammatory mechanism of berberine hydrochloride in regulating chicken macrophages in response to APEC infection,we determined the concentrations of berberine hydrochloride that had no effect on APEC growth and no cytotoxicity to HD11 from 0 to 30μM by detecting the cytotoxicity and minimum inhibitory concentration(MIC)of the drug;then,we examined the effects of berberine hydrochloride 7.5μM,15μM and 30μM on the inflammation and inflammation caused by APEC-infected chicken macrophages.The effects of berberine hydrochloride 7.5μM,15μM and 30μM on the inflammation and MAPK and NF-κB signaling pathways induced by APEC-infected chicken macrophages were then examined.The results showed that 7.5μM,15μM and 30μM berberine hydrochloride significantly reduced the relative expression of pro-inflammatory genes IL-1β,IL-6 and TNF-αand inhibited the phosphorylation of MAPK(JNK,ERK1/2,p38)and NF-κB(p65,IκB)signaling pathways.These results suggest that berberine hydrochloride can reduce the level of inflammatory gene expression after APEC infection of chicken macrophages with HD11 through MAPK as well as NF-κB by regulating M1 polarization induced by APEC infection.Finally,to investigate the effect of berberine hydrochloride on APEC-induced extracellular traps production in chicken lung macrophages,we obtained chicken lung macrophages by lung lavage and then induced extracellular trap network production in chicken lung macrophages with APEC,by detecting the ability of extracellular traps production and MAPK expression in chicken lung macrophages after berberine treatment.The results showed that APEC induced extracellular traps structure in chicken lung macrophages,and berberine hydrochloride attenuated the production of extracellular traps in lung macrophages through ERK1/2 and p38MAPK signaling pathways.In conclusion,MOI=5~1,infection time of 4 h and infection temperature of 41℃were the infection conditions for the in vitro infection of chicken macrophages by APEC;berberine hydrochloride could inhibit the inflammatory response induced by APEC infection by altering M1-type macrophages without affecting the growth of APEC,and regulate the generation of APEC-induced extracellular traps in chicken lung macrophages to exert anti-infective effect.