| Subacute rumen acidosis(SARA)is a common nutritional metabolic disease in intensive ruminant production,which has caused widespread concern in the word.Previous studies in this field were limited to the effect of SARA on rumen epithelium in ruminants,however,there are few reports about the effect of intestinal epithelial barrier function.Therefore,this paper will systematically study the damage of SARA on intestinal epithelial barrier function and its possible regulatory mechanism in dairy goats from the tissue,molecular and histological levels.1.Effects of SARA on the morphological structure of intestinal epithelium in dairy goatsTwelve lactating goats with good body condition and similar body weight(62.13±4.76 kg)and permanent ruminal fistula were randomly divided into two groups:control group(n=6)and SARA group(n=6).The dairy goats in control group were fed a basic diet with NFC/NDF of 1.15,while those in SARA group were fed four experimental diets with NFC/NDF of 1.15,1.49,2.12 and 2.66 in turn to induced SARA.Each diet lasted for 15 days.Experimental period was 60 days.The rumen pH value was continuously monitored by pH acidimeter 3 days before the end of each diet fed,as the basis for determining the occurrence of SARA,when the rumen pH value at 5.2～5.5 lasts more than 3 hours in one day.The above is regardedas the successful establishment of SARA model.On this basis,three dairy goats in control group and SARA group were respectively selected for 12 hours,and then slaughtered,and epithelial tissues of duodenum,jejunum,cecum and intestine were collected.The morphological structure and intercellular ultrastructure of intestinal epithelium were observed by optical microscopy and transmission electron microscopy.The results showed that:(1)SARA significantly increased the villus height and crypt depth of duodenum and jejunum in dairy goats(P<0.05),and the mucosal thickness increased significantly in jejunum(P<0.05),but not in duodenum(P>0.05),decreased the ratio of villus height to crypt depth of duodenum(P>0.05)and jejunum(P<0.05),and caused serious cell depression and crypt necrosis on the surface of cecum and colon epithelium.(2)SARA reduced the electron density of epithelial tight junctions in duodenum,jejunum and colon,increased the intercellular space,swelled mitochondria,and ruptured or disappeared partial mitochondrial ridges in dairy soats.These results indicated that SARA damages intestinal epithelium morphology and integrity.2.Effect of SARA on intestinal epithelial permeability in dairy goatsThe experimental design,experimental animals and diets were the same as experiment 1.The electrophysiological parameters of intestinal epithelial tissue and the flow rate of fluorescein isothiocyanate-dextran 4(FD4)were measured by Ussing chamber system.The contents of LPS,D-lactic acid contents and activity of DAO in plasma were measured to reflect the effect of SARA on intestinal epithelial penneability in dairy goats.The results showed that:(1)Isc,Gt and FD4 flow rates in duodenum,jejunum and colon epithelium of dairy goats in SARA group were significantly higher than those in control group(P<0.05),and their PD was significantly lower than that in control group(P<0.05).(2)Compared with the control group,the plasma LPS contents,D-lactic acid contents and DAO activity of dairy goats in SARA group were significantly increased(P<0.05).These results indicated that SARA occurs in dairy goats,resulting in a significant increase in intestinal epithelial permeability.3.Effect of SARA on the expression of intercellular tight junction protein in intestinal epithelial cells of dairy goatsThe experimental design,experimental animals and diets were the same as experiment1.In this study,real-time fluorescence quantitative PCR(qRT-PCR),immunohistochemical SP method and Western-Blot technique were used to detect the mRNA expression and protein expression levels of intestinal epithelial Claudin-1,Claudin-4,Claudin-7,Occludin and ZO-1 genes,in order to reveal the damage mechanism of SARA on intestinal epithelial barrier function in dairy goats at molecular level.The results showed that SARA significantly up-regulated mRNA expression and protein expression levels of Claudin-1,Claudin-4,Claudin-7 and ZO-1 in duodenal epithelium(P<0.05),and up-regulated mRNA expression(P<0.05)and protein expression(P>0.05)levels of Occludin.and also significantly up-regulated the expression levels of Claudin-4,Claudin-7,Occludin and ZO-1 in jejunal epithelium(P<0.05),down-regulated gene expression levels of Claudin-1,but no significant difference(P>0.05);also up-regulated mRNA expression and protein expression levels of Claudin-1(P<0.05),Claudin-4(P>0.05)and Occludin(P<0.05)in colon epithelium,while the expression levels of Claudin-7(P>0.05)and ZO-1(P>0.05)were down-regulated.These results indicated that SARA altered the expression of tight junction genes in intestinal epithelium,and the expression levels were different in different intestinal segments.4.Transcriptionomic analysis of duodenal tissue in SARA dairy goatsIn this study,RNA-seq technique was used to sequence the transcriptome of duodenal tissue in dairy goats.The results showed that a total of 30 GB Clean reads were obtained by filtration from control group and SARA group.The total number of genes matched to the genome was 26 724,of which 273 were only expressed in the control group,473 were only expressed in the SARA group,and 12 989 were expressed in both groups.Then,the differentially expressed genes(DEGs)were screened using edgeR software at a significant level of P<0.05 and |log2FC|≥1 A total of 495 DEGs were obtained.Of which 177 genes were up-regulated and 318 genes were down-regulated.The GO function annotation and KEGG pathway enrichment analysis of DEGs showed that these up-regulated genes were mainly related to apoptosis,inflammation,leukocyte migration and other functions.The down-regulated genes were mainly related to ion transport,fatty acid metabolism,lipid transport(metabolism),organic acid transport(metabolism),and these DEGs were mainly enriched in tight junction signaling pathway,chemokine signaling pathway and calcium ion signaling pathway.The above results showed that SARA destroyed the morphological structure of intestinal epithelium,resulting in impaired barrier function and increased epithelial permeability.. |
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