| The purpose of this experiment was to study the effects of methionine and lysine deletion and dietary protein levels on the autophagy of gastrointestinal tract and liver tissues of Xiangdong black goatss,and to explore the mechanism of action.The method of reducing dietary protein levels was used to study the effects of low-protein diets on the expression of autophagy-related proteins and genes in the gastrointestinal tract and liver tissues of Xiangdong black goats.In addition,the mechanism of autophagy in the liver tissue of Xiangdong black goats was studied by transcriptomics.This test includes four parts,the research results are as follows:Experiment 1: Effect of dietary methionine and lysine deletion on autophagy of gastrointestinal tract and liver tissues of Xiangdong black goatsThis experiment first selected 30 Xiangdong black goats with an average body weight of 18 ± 0.5 kg.The single-factor completely randomized trial design was randomly divided into 3 groups,namely the normal diet group(control group Con),the methionine-deficient group(MD),and lysine-deficient group(LD).Effects of different treatments on autophagy in rumen epithelium,jejunum mucosa,ileum mucosa and liver tissues of Xiangdong black goats were studied through determine the expression of autophagy-related proteins and genes of rumen epithelium,jejunum mucosa,ileum mucosa and liver tissues of Xiangdong black goats.The expression levels of autophagy marker proteins LC3 and p62 in the rumen epithelium,jejunum mucosa,ileum mucosa and liver tissue of Xiangdong black goats under different treatment conditions were analyzed by immunohistochemistry.The results showed that there was no significant difference in LC3 optical density between rumen epithelium,jejunum and ileum mucosa and liver tissues of Xiangdong Black Goats(P > 0.05).Except for jejunum,there was no significant difference in p62 optical density among other tissues(P > 0.05),the p62 optical density in jejunal mucosal tissue in lysine-deficient group(LD)was significantly higher than that in the normal group(control group Con)and the methionine-deficient group(MD)(P < 0.05).The mRNA expression levels of autophagy-related genes in the rumen epithelium,jejunum mucosa,and ileum mucosa of the Xiangdong black goats in different treatment groups were analyzed.The results showed that the expression level of ATF4 mRNA in rumen tissue of LD group was significantly higher than that of control group and MD group(P < 0.01),FOXO3 mRNA expression level was significantly higher than that of the control group(P <0.01);in jejunal mucosa,ATF4 mRNA expression level was highest in LD group(P <0.01),while Beclin-1 mRNA expression level was lowest(P <0.01);In the ileal mucosa,the ATF mRNA expression level in the LD group was the highest,which was significantly higher than that in the control group and the MD group(P <0.01).Dietary methionine and lysine deletions had no significant effect on the mRNA expression levels of Atg13 and eIF2 a in all tissues(P > 0.05).Experiment 2: Effect of dietary methionine and lysine deficiency on the metabolomics of jejunum tissues of Xiangdong black goatsAccording to the results of the first experiment,the deletion of dietary lysine reduced the level of autophagy in jejunum.Therefore,this part of the study conducted a preliminary study on the mechanism of autophagy by analyzing the metabolomics of jejunal mucosa tissues in different treatment groups.The test results showed that compared with the Con group,the MD group jejunum tissue sorbitol and N-acetylglucosamine levels were significantly increased(P <0.05),Valeric acid,phenethylamine,resveratrol,Met-Ala,Ile-His,Leu-Ala and other 12 metabolites were significantly lower than the control group(P < 0.05).The levels of dimethyluric acid,Leu-Ala,thiazide,5-methylcytosine,homocysteine,methyluridine,inosine,uridine and isopentenyl pyrophosphate in jejunal tissue of LD group were significantly higher than Control group(P <0.05);5-aminovalerate hydrochloride,benzazoline,o-toluic acid,L-cystine,p-fluorophenylalanine and other 7 metabolites were significantly lower than the control(P < 0.05).Analysis of the metabolic pathways of the differential metabolites showed that compared with the Con group,the metabolic pathways involved in the differential metabolites of MD group were mainly D-arginine and D-ornithine metabolism,phenylalanine metabolism,pentose phosphate pathway,amino sugar and nucleotide sugar metabolic pathway.Compared LD with the Con group,the metabolic pathways involved in differential metabolites are mainly cysteine and methionine metabolism and purine metabolism.Experiment 3: Effect of low protein diet on autophagy of liver and gastrointestinal tract of Xiangdong black goatsTwenty-four female Xiangdong black goats of similar growth weight(19.55 ± 1.01 kg)were selected.They were randomly divided into 2 groups using a randomized block test design.Each group had 12 heads,which were the control group(CP content 10.50%,dry matter basis)And low protein group(CP content 5.50%,dry matter basis).The results showed that compared with the control group,the optical density of autophagy marker proteins LC3 and p62 in the liver tissue,jejunal mucosa and ileum mucosa of Xiangdong black goatss in the low protein group were significantly higher than that in the control group(P < 0.05).There was no significant difference in the optical density of LC3 and p62 in rumen epithelium between the two treatments(P> 0.05).According to the analysis of mRNA expression levels of autophagy-related genes in tissues of Xiangdong black goatss above different treatments,the expression level of autophagy gene p62 mRNA in the ileal mucosa of the low protein group was significantly increased(P <0.05),and apoptosis-related gene P53 and autophagy-related gene ATF4 mRNA expression levels were significantly increased in liver tissue(P <0.05).In this experiment,except for the above-mentioned tissues and genes,there was no significant difference in mRNA expression levels of other tissues and autophagy-related genes(P> 0.05).Experiment 4: Effect of low-protein diets on liver transcriptomics of Xiangdong black goatsOn the basis of experiment three,transcriptomics analysis was performed on the liver tissue of Xiangdong back goats under low protein diet.The results showed that compared with the normal group,a total of 198 differential genes were selected in the low protein group,of which 112 were up-regulated(meet the condition FDR <0.05,logFC> 1),and 86 were down-regulated(FDR <0.05,logFC <-1).A total of 198 differentially expressed genes were enriched into 177 KEGG pathways,of which 39 differentially expressed genes enriched pathways reached a significant level(Q value <0.05).KEGG analysis of differentially expressed genes involves amino acid metabolism,lipid metabolism,cell growth and death,protein digestion and absorption,etc.Among them,10 genes related to amino acid metabolism,7 genes involved in cell growth and death,12 genes involved in lipid metabolic genes,29 genes involved in digestive system,7 differentially expressed genes involved in energy metabolism.There are 25 KEGG pathways involved in amino acid metabolism.In addition to the significantly enriched KEGG pathway,compared with the normal group,the differentially expressed genes in the liver transcriptome of Xiangdong black goats are also involved in apoptosis and autophagy pathways and related pathways in the process of autophagy.However,the enrichment of the KEGG pathway has not reached a significant level.The above experiments show that dietary lysine deletion can induce autophagy in goats jejunum tissue through mTORC1 pathway.At the same time,in the absence of dietary lysine,the body up-regulates ATF4 gene expression levels to maintain the metabolic balance of amino acids in rumen,jejunum,and ileum tissues..Metabolomics analysis showed that the increase in cell autophagy caused by the lack of dietary lysine is likely to be achieved by upregulating homocysteine to regulate the mTOR signaling pathway.The low protein diet induced the occurrence of autophagy in the liver tissue,jejunal mucosa and ileum mucosa of Xiangdong black goats,which significantly changed the gene expression pattern in the liver tissue.The body's immune system induces various diseases of the body,which adversely affects the growth and development of the body. |
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