| As one of the most harmful stress pathogenic factors in southern China,heat stress(HS)can cause intestinal mucosal disappearance,villi shortening,crypt shallowing,upregulated expression of inflammatory factors and diarrhea in pigs,which shows typical characteristics of inflammatory bowel disease(IBD)and does great harm to pig industry.Little is known about the mechanism of HS-induced IBD,which seriously hinders the further development of effective preventive measures.It is of great significance to systematically reveal the molecular mechanism of heat stress induced mucosal injury in pigs for the development of prevention and control strategies based on target intervention.In this study,30 2-month-old(16 ± 1kg)healthy Landrace boars(Leizhou black male pig × Duroc female pig)were randomly divided into control group and HS group.The environment temperature of the control group was 21 ± 3 °C,and the pigs in the HS group were raised in the artificial climate greenhouse with the environment temperature of 35 ± 1 °C,and the relative humidity was 75% ～ 85%for each group.Pigs were slaughtered on the 1st,7th,14 th and 21 th day after HS treatment,and the peripheral blood,intestinal tissue and contents were collected.16 S r RNA sequencing,transcriptomics and metabonomics were used to detect the structure of intestinal flora,metabolites of intestinal flora and differentially expressed genes in intestinal tissue under HS.The expression of TLR4,STAT6,MYLK and tight junction protein(TJs)in intestinal tract were detected by Western blot and RT-q PCR,etc.;the heat shock model of IPEC-J2 cells and their monolayer fusion epithelium,3D intestinal organ in vitro was established to analyze the expression changes of HSP70,TLR4,STAT6 and MYLK;the TLR4 overexpression / silencing model was established to study the regulatory mechanism of TLR4 / STAT6 and MYLK;the HS model of mice was established to study the effect of chitosan-gentamicin conjugate(CS-GT)on intestinal mucosal injury.The results showed that:(1)the liver function,renal function and blood biochemical indexes of HS-pigs were significantly changed;the levels of COR,ET,DAO,D-lactic acid,IL-4 and IL-13 in peripheral blood were significantly increased;the integrity of intestinal tissue was damaged,bleeding,lymphatic infiltration and villus height were observed.The expression of IL-4 and IL-13 m RNA in intestinal tissue was increased and the level of LPS in colonic contents was increased,but the concentration of s Ig A was decreased.(2)In the early stage of HS treatment,the contents of acetic acid and butyric acid of short chain fatty acids in pig colon decreased significantly(P <0.05),and then gradually returned;the diversity and structure of colonic microorganisms changed significantly,which was associated with the changes of metabolites;on the first day of HS,92 up-regulated genes and 60 down regulated genes were identified comparing with control group,and the difference was significant with the extension of heat stress groups.It’s showed that the expression of TLR4 / STAT6 /MYLK was significantly increased;the differential genes were clustered into multiple immune regulatory signaling pathways,such as TLR4 / STAT6 / MYLK,and were associated with the changes of metabolites;the expression of heat shock protein and key proteins of TLR4 / STAT6 / MYLK signaling pathway in duodenum,cecum and colon of HS-pigs were significantly increased,while the expression of TJs was down regulated;(3)after heat shock treatment of IPEC-J2 cells,the expression of key proteins of TLR4 / STAT6 / MYLK signaling pathway were significantly up-regulated,while the expression of TJs was down regulated,and recovered with the recovery of heat shock;gene silencing / overexpression further confirmed that HSP70 significantly activated TLR4 / STAT6 / MYLK signaling pathway,which led to the phosphorylation of MLC and down regulated the expression of TJs.(4)Heat stress significantly activated TLR4 / STAT6 / MYLK tight junction protein signaling pathway,which was significantly inhibited after administration of 150 mg / kg CS-GT;(5)In vitro,CS-GT significantly improved the decrease of transmembrane resistance and the increase of FITC dextran permeability of heat shock treated IPEC-J2 monolayer fusion epithelium,and the optimal concentration was 15 μg / ml.CS-GT significantly inhibited the activation of TLR4 / STAT6 / MYLK signaling pathway and up-regulated the expression of TJs in heat shock IPEC-J2 cells and 3D intestinal organs of mice(P <0.01),the molecular docking analysis shows CS-GT can target to inhibit TLR4 activation..The results showed that:(1)HS could cause abnormal physiological and biochemical indexes and intestinal mucosal damage in pigs.(2)The structure,diversity and metabolite levels of intestinal microorganisms in HS-pigs changed significantly,which activated a variety of immune and inflammatory signal transduction pathways,especially TLR4 / STAT6 / MYLK signaling pathway.(3)Under heat stress,HSP70 or LPS activated TLR4 / STAT6 / MYLK signaling pathway and down regulated TJs expression.(4)CS-GT can significantly improve HS-induced intestinal mucosal injury and protect intestinal mucosal barrier.(5)CS-GT protects the integrity of intestinal mucosa by inhibiting the activation of TLR4 / STAT6 / MYLK signaling pathway. |
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