| Methionine(Met),as an essential amino acid for livestock and poultry,has extensive and important biological functions in the body.The metabolites of Met,S-adenosyl methionine(SAM)and S-adenosyl-homocysteine(SAH),can affect the methylation level of the body and thus affect the function of the body.Commonly used feed additives that provide Met include DL-Met and DL-methionine hydroxy analogue(DL-HMTBA).Studies have shown that dietary Met restriction can reduce intestinal permeability by changing the expression of intestinal tight proteins,improving intestinal epithelial barrier function,and HMTBA has also been shown to have a protective effect on intestinal barrier function.In the previous laboratory,it was found in intestinal porcine epithelial cell line that DL-HMTBA can significantly increase the mRNA level of the tight junction protein Claudin compared with L-Met.And it was found that this effect may be related to the increased mRNA stability caused by the decrease of RNA m6A modification level.However,at Met-restricted levels,the effect of limiting Met intake with HMTBA as a Met source,or adding HMTBA on top of Met-restriction,on intestinal epithelial barrier function is unclear.Therefore,in this experiment,by using Salmonella typhimurium-induced intestinal barrier function damage model in C57BL/6 mice,under the conditions of Met restriction and HMTBA addition,we explored the effects of intestinal Met metabolism,intestinal barrier function,and DNA and RNA methylation levels.Variety.The main research contents and results are as follows:In this experiment,60 male C57BL/6 mice of 6-8 weeks,12 mice in each group,were randomly divided into NI(Non-Infected)group,NM(Normal Met)group,MR(Met Restriction)group and HR(HMTBA Restriction)group and MR+H(Met Restriction+HMTBA)group were fed 0.86%Met,0.86%Met,0.17%Met,0.17%HMTBA,0.17%Met+0.69%HMTBA diets for 46 days,respectively.On the 42nd day of the experiment,the NI group was treated with PBS by gavage,and the NM,MR,HR,and MR+H groups were infected with Salmonella typhimurium,and the mice were slaughtered and sampled.During the experiment,the mice were guaranteed to drink water freely,and the food intake was recorded every day.Changes in mouse body weight were recorded every five days.Four days after infection,the mice were slaughtered and sampled to detect bacterial load,tissue inflammatory factors and expression levels of tight junction proteins,and LC-MS to detect Met metabolites and m~6A methylation levels.The main results are as follows:1.Mice body weight and feed intake:Before ST infection,compared with the NM group,the daily feed intake and daily gain of the HR group were significantly lower(P<0.01),however the daily feed intake and daily gain of the MR+H group were significantly increased(P<0.01).After ST infection,compared with the NI group,the body weight of the four infected groups was significantly decreased on the third day after infection(P<0.05);compared with the NM group,the body weight and feed intake of the MR+H group were significantly decreased(P<0.05).The above results indicated that under normal Met levels,HMTBA as a partial Met source could improve growth performance,but this effect could not be maintained under ST infection.2.Mice organ index and bacterial load:Compared with the NI group,the liver and spleen indexes of the four infection groups were significantly increased(P<0.05),and there were a large number of bacteria in the viscera and feces;compared with the NM group,the liver and spleen indices were significantly decreased in the MR group(P<0.05),and the bacterial load in the viscera was significantly decreased(P<0.05),and the liver and spleen indices were significantly increased in the MR+H group(P<0.05).The bacterial load in the viscera and feces of the mice in the NM group and the MR+H group was significantly higher than that in the MR group and the HR group(P<0.05).The above results indicate that restricting Met intake with Met or HMTBA as Met source can alleviate organ enlargement and bacterial translocation caused by ST infection.3.Intestinal morphological structure and tight junction protein expression in mice:Compared with the NI group,the colon length of the mice in the infection group was significantly shortened(P<0.05).There were more inflammatory cell infiltration in the lamina propria,and the epithelial cells in the mucosal layer were severely damaged.The mRNA levels of ZO-1,Occludin and Claudin in the colon were significantly down-regulated in the NM group(P<0.05).Compared with the NM group,the mRNA levels of ZO-1 and Occludin in the ileum and colon were significantly up-regulated in the MR and HR groups(P<0.05).The detection of LPS and D-LC related to intestinal permeability found that the content of LPS and D-LC was significantly increased in the NM group compared with the NI group(P<0.05).Compared with the NM group,the LPS content of the MR group was significantly lower(P<0.05).The above results indicated that limiting Met intake with either Met or HMTBA as Met source could increase the expression of tight junction proteins and reduce intestinal permeability in the inflammatory state.4.Intestinal inflammatory factors:Compared with the NI group,the mRNA levels of IL-6,IL-1βand TNF-αin the ileum and colon were significantly up-regulated in the NM group(P<0.05),while IL-10 was significantly higher in the ileum and colon.Levels were significantly down-regulated in the ileum and not significantly different in the colon(P>0.05).Compared with the NM group,the mRNA levels of IL-6,IL-1βand TNF-αin the ileum and colon were significantly down-regulated in both the MR and HR groups(P<0.05).The mRNA levels of IL-10 in the colon in the MR groups and HR groups were significantly higher than those in the NM group and the MR+H group(P<0.05),and the mRNA levels of IL-10 and IL-6 in the MR+H group were significantly down-regulated(P<0.05).The above results suggest that restriction of Met intake with Met or HMTBA as Met source can alleviate intestinal damage caused by ST infection.5.Met metabolites:Compared with the NI group,the Met content in the tissue and plasma of the NM group was not significantly different,and the contents of MTA,SAH and SAM were significantly decreased(P<0.05).The colon and plasma contents of Met,SAM,SAH and SAM/SAH were significantly decreased in the MR and HR groups compared with the NM group(P<0.05),but there was no significant difference in Met,SAH and SAM/SAH in the MR+H group.The contents of MTA in the colon and ileum were significantly increased in the MR and HR groups(P<0.05).The SAH content in the ileum was significantly decreased in the MR and HR groups(P<0.05).The content of SAM in the ileum of the NM group and the MR group was significantly higher than that of the HR group and the MR+H group(P<0.05).and the SAM/SAH value of the MR+H group was significantly higher than that of the other three infection groups(P<0.05).The above results suggest that limiting Met intake with Met or HMTBA as Met source can affect SAH and SAM levels in vivo by reducing Met content.6.Methylation levels:Compared with the NI group,the m~6A and m~5C levels of the NM group in the colon were significantly decreased(P<0.05),and the m~6A and m~5C levels of the NM group in the ileum weren’t significantly differents.Compared with the NM group,m~6A levels in the colon and ileum were significantly lower in the MR group(P<0.05).Compared with the NI group,the mRNA levels of METTL3,YTHDF1 and WTAP in the NM group were significantly up-regulated in the colon(P<0.05).Compared with the NM group,the mRNA levels of METTL3,YTHDF1 and WTAP in the MR group were significantly down-regulated in the colon and ileum(P<0.05),and the expressions of METTL3 and YTHDF1 in the HR group and MR+H group were significantly down-regulated in the colon and ileum(P<0.05).And the expression of DNA methylation-related proteases also showed a similar trend.These results suggest that limiting Met intake with Met or HMTBA as Met sources may improve intestinal barrier function by affecting the level of RNA m~6A or DNA m~5C.In summary,the main conclusions of this study are:1.Restriction of Met intake with Met or HMTBA as the sole Met source can significantly improve intestinal barrier impairment and intestinal inflammation caused by Salmonella typhimurium.The mechanism may be that low levels of HMTBA and Met affect the intestinal barrier function by changing the body’s Met metabolism,affecting the level of RNA m~6A or DNA m~5C.2.Supplementing HMTBA to the normal level of Met on the basis of Met restriction can improve the growth performance of mice,but it can’t improve the impaired intestinal physical barrier function caused by Salmonella typhimurium. |
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