| The intestine is an important digestive organ as well as the the body’s largest immune organ,the integrity of the intestinal barrier and the immune function are the guarantee of the body’s health.Bacillus subtilis is a kind of beneficial bacteria,current research mainly focused on its growth promotion,although there are reports on its ability to maintain the intestinal barrier and inhibit inflammation,the mechanism by which Bacillus subtilis affects the intestinal barrier and immunity is still unknown.It has not been reported whether the dosage of Bacillus subtilis and the age of broilers will affect the effect of Bacillus subtilis.In this study,different doses of Bacillus subtilis were added to the diet and samples were collected at different times to study the effects of Bacillus subtilis doses and ages of white feather broilers on the intestinal barrier and inflammation as well as the mechanisms of it.In this study,white feather broilers were selected as experimental animals with a breeding scale of 60,000,and they were randomly divided into 3 groups,they were fed with a basic diet(Basic Diet Group,BD)and basic diet with 1‰(Low Dose Group,LD)and 1.5‰(High Dose Group,HD)Bacillus subtilis supplement as experimental groups,each with 8 replicates.A chicken was randomly selected from each repetition on 20 d and 40 d,blood samples were collected for routine blood testing;intestinal and spleen tissues were taken to make paraffin sections to observe the morphological structure;the intestinal tissues was made into electron microscope samples to observe the ultrastructure of the surface of the villi;intestinal tissues were collected to detect the expression of intestinal tight junction protein,key genes of Tolllike receptors(TLRs)pathway and bile acid pathway by q PCR;the protein expression of Nuclear factor-kappa B(NF-κB)and Farnesoid X receptor(FXR)were detected by Western Blot;Chromatin immunoprecipitation(CHIP)technology were adapted to detect histone modifications in the FXR promoter region.The results are as follows:1.The morphology of intestinal villi in the control group and the test group was complete.Compared with the control group,the length of villi in the duodenal LD group at 20 d and 40 d increased(P <0.05),and the depth of crypts in the duodenal and jejunum HD group at 40 d increased(P <0.05);under the scanning electron microscope,the goblet cells and columnar epithelial cells on the surface of the intestinal villi of the test group and the control group were normal,and no difference was found.The q PCR results showed that the expression of tight junction protein genes in the experimental group were significantly reduced(P < 0.05),and there were more tight junction proteins changed on 40 d than on the 20 d when comparing the experimental group with the control group,but the changes in the tight junction protein genes were non-dose-dependent.2.No difference was found in the number of inflammatory cells showed by blood test results;No significant difference was revealed in the area of white pulp through the results of HE staining of the spleen.The q PCR results showed that at 20 d,the m RNA level of TLRs in the duodenal HD group were significantly lower than that in the LD group(P < 0.05),and the Myeloid differentiation primary response protein MYD88(MYD88)in the experimental group was significantly higher than that in BD;at 40 d,Tlr4 and Tlr5 of duodenal,jejunum and ileum experimental group were significantly(P < 0.05)or extremely significantly reduced(P < 0.01),but the change showed non-dose-dependence,the NF-κB of the jejunum LD group was significantly higher than that of the BD,and there was no difference in NF-κB,interleukin-1β(IL-1β)and tumor necrosis factor α(TNF-α)in other intestines(P > 0.05);Western Blot results showed that there was no difference in ileal NF-κB protein expression at20 d(P > 0.05),and significantly decreased at 40 d(P < 0.05),but there was no difference in p-NF-κB expression between experimental groups the BD group at 20 d and 40 d(P > 0.05).3.Compared with the control group,nuclear receptor subfamily 1 group H member 4(Nr1h4),the gene encoding FXR,in HD group of jejunum at 20 d was significantly increased(P <0.05);The Nr1h4 in HD group of the duodenum,jejunum and ileum was significantly reduced at 40 d(P <0.05),and the Nr1h4 of the ileum LD group was extremely significantly lower than that of the BD group(P <0.01);at 20 d,nuclear receptor subfamily 2 group B member 1(Nr2b1),the gene encoding Retinoid X receptor α(RXRA),in duodenum and jejunum extremely significantly reduced(P < 0.01),Nr2b1 was significantly reduced in the duodenum at 40 d(P < 0.05);at 40 d,solute carrier family 10 member 2(Slc10a2),the gene encoding Apical sodium-dependent bile acid transporter(ASBT),in ileal HD was significantly reduced(P < 0.05);The expression of FXR protein in the ileal LD group was significantly reduced at 40 days(P < 0.05);in FXR promoter,acetylation of histone H3 protein subunit(H3Ac)level of HD group was significantly reduced(P < 0.05),Trimethylation of lysine 4(H3K4me3)was significantly reduced in the experimental group(P < 0.05),the monomethylation of lysine 9 on histone H3 protein subunit(H3K9me)in the LD group was significantly reduced(P < 0.05),the level of trimethylation of lysine 27 on histone H3 protein subunit(H3K27me3)in the experimental group was significantly lower than that in the control group(P < 0.05).The above results indicated that the length of villi and crypt depth were increased by the addition of Bacillus subtilis to the fee,the expression of intestinal tight junction protein was reduced,and the effect of Bacillus subtilis increased with the age of broilers.The blood test results show that the test group and the control group are both No inflammation occurred.The q PCR results showed that the Tlr of the experimental group was reduced,and the Western Blot results showed that the expression of NF-κB protein was reduced,suggesting that the TLR pathway was inhibited.The bile acid pathway genes Slc10a2 and Nr1h4 decreased,and FXR protein expression decreased at 40 d.histone modifications in the FXR promoter region was detected by CHIP,and the results showed that the level of H3K4me3 decreased,therefore,it is speculated that the histone modification of the FXR promoter was affected by Bacillus subtilis,the expression of ASBT and FXR genes was reduced,as well as the expression of FXR protein,and the bile acid pathway was inhibited,which leads to the reduction of intestinal tight junction proteins,the enhancement of absorption function and the inhibition of the TLR pathway;There was no correlation between the effect and dose of Bacillus subtilis,but the effect was time-dependent. |
PDF Full Text Request