Study Of Quercetin Protect From Salmonella Enterica Serotype Typhimurium Lipopolysaccharide-induced The Duodena And Hearts Injury In Chicken Embryos

The protective mechanism of quercetin alleviates lipopolysaccharide(LPS,from Salmonella enterica serotype Typhimurium)-induced duodenal and cardiac inflammation,autophagy,and programmed cell death in chicken embryos model remain elusive.This was a basic study of antibiotics alternatives in poultry farming and breeding.In order to protect LPS exposure from environment or intestinal microbiotas,specific pathogen free chicken embryos were selected for eatablishing the duodenal and cardiac inflammation model by LPS.Chiken embryos were allocated as the control group,PBS group,PBS with ethanol group,LPS groups(125 ng/egg,500 ng/egg or 1000 ng/egg),quercetin groups(40nmo L/egg),quercetin treatment group Ⅰ(40 nmo L quercetin/egg + 125 ng LPS/egg),quercetin treatment group Ⅱ(40 nmo L quercetin/egg + 500 ng LPS/egg).Embryos were injected with abovementioned solutions via the allantoic cavity at embryonic day 15;the duodena and hearts of the embryos were collected at embryonic day 19 for histopathological examination,real-time quantitative polymerase chain reaction,immunohistochemical investigations,and Western blotting.The results demonstrated that quercetin improved the inflammatory cell infiltration in the peyer’s patch of the intestinal mucosa and inflammation reactions in the hearts after LPS induction.Quercetin decreased the m RNA expressions of inflammatory-related factors(TLR4,TNFα,NF-κB1,IFNγ,IL-1β,IL-6,IL-8,p38,MMP3,and MMP9)after LPS induction in the duodena and hearts.The immunopositivities to TLR4,MMP3,and MMP9 in the villi,crypts,and lamina propria of the intestinal mucosa were increased after LPS induction when compared with that of the PBS group,whereas quercetin could significantly decrease the duodenal immunopositivities to TLR4,MMP3,and MMP9 in the treatment group when compared with that of the LPS group.Quercetin also decreased the protein expression of TLR4,IL-1β,MMP3,and MMP9 after LPS induction in the duodena.The immunopositivities to TLR4 and MMP9 in the cytoplasma of myocardiocytes after LPS induction significantly increased when compared with that of the PBS group,whereas quercetin significantly decreased the immunopositivities to TLR4 and MMP9 in myocardiocytes of the treatment group when compared with that of the LPS group.Quercetin decreased the protein expression of TLR4,IFNγ,MMP3,and MMP9 after LPS induction in the hearts.LPS reduced the duodenal m RNA expression of mucin 2,but upregulated the m RNA and protein expression of Claudin 1,Occludin,and ZO-1.However,quercetin could reverse the m RNA and protein exrepssions of mucin 2,Claudin 1,Occludin,and ZO-1.The immunopositivities to Claudin 1 and ZO-1 in the duodenal villi,crypts,lamina propria,and tunica muscularis were increased after LPS induction when compared with that of the PBS group.Nevertheless,quercetin could decrease the immunopositivity to Claudin 1 in the cytoplasma of myocardiocytes after LPS induction when compared with that of the LPS group.Quercetin treatment significantly decreased the m RNA expression of Claudin 1 and ZO-1 LPS-induced increase in the hearts.Quercetin could decrease the immunopositivity to Claudin 1 in the cytoplasma of myocardiocytes after LPS induction as compared with that of the LPS group.Therefore,quercetin ameliorates the TLR4/NF-κB1/p38 siganaling pathway after LPS induction in the duodena and hearts,atteanuates the inflammatory cytokine storm,decreases the decomposition of extracellular matrix,improves the permeability of the duodena and hearts.Quercetin could downregulate autophagy-associated gene m RNA expression(LKB1,PEPT1,PPARα,SGLT1,AMPKα1,AMPKα2,Beclin-1,ATG5,ATG7,LC3 A,and LC3B)in the duodena after LPS induction.Quercetin decreased the protein expressions to AMPKα2,ATG5,LC3-Ⅰ,and LC3-Ⅱ in the villi,crypts,lamina propria,and tunica muscularis of the intestinal mucosa after LPS induction when compared with that of the LPS group.Quercetin could significantly downregulate cardiac autophagy-related gene m RNA expression(APOA4,PPARα,SGLT1,AMPKα1,AMPKα2,Beclin-1,ATG5,ATG7,and LC3B).The immunopositivities to AMPKα2,LC3-Ⅰ,and LC3-Ⅱ in the cytoplasm of myocardiocytes in LPS group were significantly increased when compared with that of the PBS group.Nevertheless,quercetin could decrease the immunopositivity to AMPKα2,LC3-Ⅰ,and LC3-Ⅱ in the myocardiocytes of the treatment group.Quercetin markedly decreased the cardiac protein expression of AMPKα1,LC3-Ⅰ,and LC3-Ⅱ after LPS induction.Thus,quercetin improves the energy deficiency of the duodena and hearts after LPS induction,and alleviates the autophagy signaling pathway induced by LPS.Quercetin decreased the duodenal and cardiac gene m RNA expressions of programmed cell death factors(Fas,CASP1,CASP12,CASP3,Drp1,and RIPK1)after LPS induction;meanwhile,quercetin decreased the duodenal and cardiac protein expression of CASP1 and CASP3 after LPS induction.The immunopositivities to CASP1 and CASP3 in the villi,crypts,lamina propria,myenteric plexuses,peyer’s patches,and tunica muscularis were increased after LPS induction when compared with that of the PBS group.Nevertheless,quercetin reduced the duodenal immunopositivity to CASP1 and CASP3 in the treatment group.Hence,quercetin ameliorates the Pyroptosis and apoptosis siganaling pathways after LPS induction in the duodena and hearts.In conclusion,the evidence suggests quercetin can alleviate duodenal and cardiac inflammation induced by LPS through modulating autophagy and programmed cell death.