| Astragalus Polysaccharide(APS)is the main bioactive substance of Astragalus in inhibiting inflammation,In veterinary clinics,it is often used as a feed additive to enhance immunity.It s anti-inflammatory mechanism in inflammatory diseases is still not the field of veterinary,especially poultry.In order to further reveal the molecular mechanism of APS's anti-inflammatory effect in poultry and provide more practical and reliable theoretical basis for veterinary clinical application.In this study,the chicken macrophage cell line HD11(chicken macrophage cell line)and the chicken embryonic fibroblast cell line DF-1(DF-1)were used as research objects to establish an in vitro inflammation model and the effects of the expression of inflammatory cytokines IL-1? and TNF-?,as well as the inhibitors of cytokine signaling 3(SOCS3)on the regulation mechanism of inflammatory signaling pathways NF-?Bp65 and p38 MAPK.Immune cells HD11 and tissue cells DF-1 were divided into control group(C),Lipopolysaccharide group(LPS group,L),Astragalus Polysaccharide group(APS group,A)and Astragalus Polysaccharide inhibited Lipopolysaccharide group(APS+LPS group,A+L),detect IL-1? of each group at different time points after LPS stimulation(HD11: 2,6,12,24 and 48 h,DF-1 cells: 6,12,24,48 and 72 h),TNF-?,NF-?Bp65,p38 MAPK and SOCS3 m RNA and protein levels.At the same time,in order to explore the anti-inflammatory effect and mechanism of APS in clinical application,an SPF chicks inflammatory response model was constructed and divided into control group(C),LPS group(L),APS group(A),and Astragalus Polysaccharide inhibited Lipopolysaccharide group(A+L),and tested Changes of immune organ index,proinflammatory cytokines IL-1? and TNF-? protein content and jejunum morphology in each treatment group.The main findings are as follows:(1)By examining the effects of different concentrations of LPS on the viability of two types of cells and the expression of IL-1?,it can be seen that in HD11 cells,when the final concentration of LPS is 0.5 ?g/m L,the expression of proinflammatory factors can be induced significantly(P<0.05)elevated,there is a strong inflammatory response;in DF-1 cells,when the final concentration of LPS is 2 ?g/m L,it can induce a significant(P<0.05)increase in the expression of pro-inflammatory cytokines,and there is a significant inflammatory response;and SPF chicks induced a strong inflammatory response by oral administration of 1 mg/kg BW LPS.(2)In HD11 cells,the expression and protein content of pro-inflammatory cytokines IL-1? and TNF-? in group L and group A cells were significantly(P<0.05)higher than those in group C at 2-48 h,but The levels of APS group were lower than those of group L;the m RNA expression and protein content of IL-1? and TNF-? in cells of group A+L were significantly(P<0.05)lower than those of group L from 12 to 48 h.(3)In HD11 cells,the expression of NF-?Bp65,p38 MAPK and SOCS3 m RNA in inflammation-related signaling pathways in group L and group A were significantly(P<0.05)higher at 2-48 h,2-24 h and 2-48 h,respectively.In group C,but the increase in group A was lower than that in group L;the expression of NF-?Bp65 m RNA in cells of group A+L was significantly(P<0.05)lower than that in group L at 2-48 h,and the expression level of SOCS3 m RNA was 2-48 h was significantly(P<0.05)higher than group L.The gray ratio of P-NF-?Bp65/NF-?Bp65 protein content in group L and group A was significantly(P<0.05)higher than that in group C;SOCS3 protein content in group A was significantly higher than group C;A+L group P-NF-?Bp65/NF-?Bp65 and P-p38MAPK/p38 MAPK protein content gray ratio were significantly(P<0.05)lower than group L,SOCS3 protein content was significantly(P<0.05)higher than group L;SOCS3 protein content significantly(P<0.05)higher than L group.It is suggested that APS treatment alone can promote the release of cytokines such as IL-1? and TNF-? in chicken macrophages to play an immune enhancing role;while in the LPS-induced HD11 cell inflammation model,APS pretreatment can be suppressed by high expression of SOCS3 over-activation of NF-?Bp65 and p38 MAPK reduces the release of inflammatory cytokines IL-1? and TNF-?,thereby playing a role in inhibiting inflammation.(4)In DF-1 cells,the m RNA expression and protein content of pro-inflammatory cytokines IL-1? and TNF-? in group L cells were significantly(P<0.05)higher than those in group C at 6-72 h;group A The m RNA expression and protein content of IL-1? and TNF-? in the cells were significantly(P<0.05)higher than those in group C at 6-72 h,but were lower than those in group L to varying degrees;the m RNA expression and protein content of IL-1? and TNF-? in cells in group A+L were significantly(P<0.05)lower than that in group L from 12 to 72 h.(5)In DF-1 cells,the expression of NF-?Bp65 m RNA in inflammation-related signaling pathways in groups L and A was significantly higher than that in group C at 6-72 h(P<0.05),but the increase in group A is lower than that in group L;SOCS3m RNA expression in group A was significantly(P<0.05)higher than that in group C at 6-72 h;NF-?Bp65 m RNA expression in cells in group A+L was significantly(P<0.05)lower than that in group L at 6-72 h,the expression of SOCS3 m RNA was significantly(P<0.05)higher at 6-72 h than in group L;there was no statistically significantly(P>0.05)difference in the expression of p38 MAPK m RNA in each treatment group.The gray ratio of P-NF-?Bp65/NF-?Bp65 protein content in group L and group A was significantly(P<0.05)higher than that in group C;SOCS3 protein content in group A was significantly(P<0.05)higher than group C;A+L group P-NF-?Bp65/NF-?Bp65 protein content gray ratio was significantly(P<0.05)lower than group L,SOCS3 protein content was significantly(P<0.05)higher than group L;P-p38MAPK/p38 MAPK protein between treatment groups There was no significantly(P>0.05)difference in the change of the content gray ratio.It shows that in fibroblasts,APS treatment alone can promote the release of cytokines such as IL-1? and TNF-? to play an immune enhancement role;while in the LPS-induced DF-1 cell inflammation model,APS pretreatment can pass SOCS3 High expression inhibits the excessive activation of NF-?Bp65,reduces the release of inflammatory cytokines IL-1? and TNF-?,and thus plays a role in inhibiting inflammation.(6)In the SPF chicks inflammation model,the thymus and bursa organ indexes of group L were significantly(P<0.05)lower than that of group C;the organ index of thymus and bursa of A+ L treatment group were significantly(P<0.05)higher than that of L group;there was no statistically significant difference in spleen organ index among the treatment groups(P>0.05).The height of jejunum villus,the depth of crypts and the surface area of villus were significantly(P<0.05)higher in group L than in group C,and the width of villus and the ratio of villus glands were not statistically(P>0.05)different from the C group;the height of jejunum villus,the depth of crypts,the width of villus and the surface area of villi were significantly(P<0.05)higher in group A+L than in group L.Serum levels of proinflammatory cytokines IL-1? and TNF-? in group L and group A were significantly(P<0.05)higher than group C,and group A was lower than group L in varying degrees;The protein content of IL-1? and TNF-? in serum of group A+L were significantly(P<0.05)lower than that of group L.It shows that APS pretreatment can enhance immunity by increasing chick immune organ index and intestinal structure and shape,thereby reducing the secretion of pro-inflammatory cytokines and inhibiting the occurrence of inflammatory reactions.By combining the results of in vivo and in vitro studies,a systematic analysis of the molecular mechanism of APS in veterinary medicine,especially poultry anti-inflammatory effect,provides a new theoretical basis for elucidating the mechanism of poultry inflammatory diseases and the opening and application of veterinary clinical drugs. |
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