| Good growth and development of piglets are the foundation for healthy breeding of pigs.However,early weaning can easily cause weaning stress in piglets,induce intestinal microbiota disruption,intestinal barrier dysfunction and occurrence of intestinal inflammation,leading to diarrhea in piglets,even high mortality,which seriously harm the health of pig breeding.In recent decades,weaning stress,intestinal inflammation and diarrhea have been difficult problems for pig farmers and have become an important topic for animal husbandry researchers.The extracts of traditional Chinese herbal plants,which have multiple biological effects,have become a research hotspot for ensuring the health of piglets.Eucommia ulmoides Oliver is a unique relic plant in China.Extract Eucommia ulmoides Oliver leaf(EEUO)had been included in the variety catalogue of feed additives of China.Chlorogenic acid(CGA)is one of the main active components.This project included EEUO feeding experiment in piglets,intestinal inflammation experiments in piglets,mice and cell experiments,focusing on the mechanism of EEUO and CGA in alleviating intestinal inflammation of piglets.The following research was conducted:Experiment 1: A total of 36 piglets(21 d of age)were selected for a 28-day feeding experiment.The results showed that the body weight and average daily gain of piglets in the EEUO group were significantly higher than that in the CON group(P < 0.05),and FCR was significantly better than that in the CON group(P < 0.05).In addition,EEUO supplementation improved the anti-inflammatory and antioxidative capacities of piglets and enhanced the integrity of intestinal barrier.The results indicated that 1000mg/kg EEUO supplementation could promote growth,and possibly through its antioxidative and anti-inflammatory functions.Experiment 2: A total of 54 piglets(21 d of age)were selected.An intestinal inflammation model in piglets was constructed by intraperitoneal injection of 80 μg/kg LPS.The results showed that LPS treatment significantly increased the m RNA expressions of(IL-6,IL-1β,TNF-α)(P < 0.05),which was significantly decreased by EEUO supplementation(P < 0.05).Moreover,the m RNA expression of IL-10 was enhanced by EEUO(P < 0.05).In addition,EEUO supplementation could increase the protein expression of autophagy related genes ATG5(P < 0.01)and ATG7(P < 0.01),and also decrease the expression of P62 and NF-κB p65(P < 0.05).The results of this chapter showed that EEUO could improve LPS-induced intestinal inflammation in piglets,and the potential mechanism included inhibition of the NF-κB signaling pathway,downregulation of NLRP3 and its downstream related gene expression,as well as activation of autophagy.Experiment 3: In the intestinal inflammation model experiment,the colonic microbial composition,short-chain fatty acid(SCFAs)and bile acids levels were conducted.The results showed that the relative abundance of pathogenic bacteria Rikenellaceae＿RC9＿gut＿group(P = 0.09)was increased and the relative abundance of beneficial bacteria Prevotellaceae＿NK3B31＿group(P = 0.088)was decreased in the LPS group compared to that of CON group.However,addition of EEUO reversed the negative effects of LPS treatment on the intestinal microbiota,and also increased the relative abundance of beneficial genus Clostridia＿UCG-014(P = 0.077).The results of SCFAs showed that the levels of acetic acid and butyric acid were significantly decreased in the LPS group compared to the CON group(P < 0.05),whereas the level of butyric acid was significantly increased after EEUO supplementation(P < 0.05).The results of LC-MS/MS showed that the levels of lithocholic acid(LCA)and secondary bile acids(SBA)in the colonic digesta of piglets were significantly decreased in the LPS group(P < 0.05),whereas the levels of LCA and SBA were improved after EEUO supplementation.It was suggested that EEUO supplementation improved gut microbiota and its-derived metabolites butyrate and SBA in piglets.Experiment 4: The research was conducted in dextran sulfate sodium(DSS)-induced colitis model of mice.The increased DAI score,decreased body weight and shortened colon length of mice indicated that the colitis model was successfully constructed.CGA supplementation significantly reduced DAI score,decreased body weight and shortened colon length.The m RNA expression of pro-inflammatory cytokine(IL-1β,IL-6 and TNF-α)were significantly increased compared to the CON group(P < 0.01),which were decreased by the CGA supplementation.Furthermore,CGA treatment enhanced the m RNA expression of anti-inflammatory cytokine IL-10 and anti-oxidative stress indexes(CAT,GPX1,GPX2 and SOD1)in the colonic tissue(P < 0.01).The results indicated that CGA could play its own anti-inflammatory and antioxidative roles to alleviate DSS-induced UC in mice.Experiment 5: The research was conducted on the microbial composition of colonic chyme in colitis model of mice.The results showed that the relative abundance of Turicibacter(P < 0.01)and Romboutsia(P < 0.05)was significantly increased,while the relative abundance of Alistipes(P < 0.05)bacteria was significantly decreased in the DSS group compared with the CON group at the genus level.However,CGA supplementation reversed the negative effects of DSS treatment on the intestinal microbiota.SCFAs analysis revealed that the level of butyrate was significantly increased after CGA supplementation compared to the LPS group.The results indicated that CGA exerted anti-inflammatory effects by improving the levels of intestinal microbiota and butyrate.Experiment 6: Exploring the mechanism of CGA in alleviating inflammatory response of IPEC-J2 cells.The results showed that the m RNA expression of P62(P <0.05)was significantly increased in the LPS group compared to the CON group,whereas the expression of P62 was significantly decreased and the expression of ATG5,ATG7 and LC3 B were significantly enhanced after CGA supplementation(P < 0.05).In addition,the m RNA expression of ZO-1 and Occludin were increased by CGA supplementation(P < 0.05).After inhibiting autophagy with the autophagy inhibitor 3-MA,the results showed that the m RNA expression of IL-1β,IL-6,TLR-4 and NF-κB were significantly decreased in the CGA+LPS group compared to the LPS group(P <0.05),and the CGA+3-MA+LPS group also decreased the m RNA expression of the above genes,but there was no statistical difference compared with LPS group(P > 0.05).The results revealed that CGA could alleviate inflammatory response in LPS-induced IPEC-J2 cells by activating autophagy and inhibiting NF-κB signal pathway.In summary,this study demonstrates that feed additives EEUO and CGA alleviate inflammatory responses in piglets by inhibiting the NF-κB signaling pathway,activating autophagy,regulating gut microbiota and metabolites butyric acid and SBA.The findings offer guidance for the development and application of novel feed additives in the future livestock and poultry field. |
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