| Weaning stress diarrhea in piglets has caused enormous economic losses to the pig industry,and the sensitization of soybean antigen proteins is considered to be the main cause of diarrhea in weaned piglets.Meanwhile,soybean 7S globulin is the most important and most allergenic antigen protein in soybean.Therefore,to explore the mechanism and prevention of intestinal injury caused by soybean 7S globulin in weaned piglets is of great significance to solve the problem of weaned piglets diarrhea.Micro RNA（mi RNA）is a kind of non-coding RNA involved in the post-transcriptional regulation of genes.It plays an important role in the proliferation,differentiation and apoptosis of intestinal cells,the formation of intestinal tight junction and intestinal inflammation,and is closely related to the homeostasis and integrity of intestinal epithelium.However,the changes and functions of mi RNA in IPEC-J2 cells induced by soybean 7S globulin have not been reported.Therefore,in this study,the effect of soybean 7S globulin on mi RNA expression profile of IPEC-J2 cells was detected by small RNA sequencing（s RNA-seq）,and the most down-regulated mi RNA（ssc-mi R-221-5p）was screened.CCK-8,Ed U,q RT-PCR,Western Blot,ELISA and flow cytometry were used to explore the function of ssc-mi R-221-5p in intestinal porcine epithelial cells（IPEC-J2）induced by soybean 7S globulin.The main results are as follows:1.Establishment and analysis of mi RNA expression profile in soybean 7S globulin-induced IPEC-J2 cells injurys RNA-seq technique was used to detect the changes of mi RNA expression profile in soybean 7S globulin-induced IPEC-J2 cells injury.Under the condition of︱log2FC︱>1and P.adj<0.05.15 differentially expressed mi RNA were screened,of which 9 were down-regulated（ssc-mi R-221-5p was the most down-regulated）.6 expressions were up-regulated（sscmi R-129a-5p was the most significantly up-regulated）.A total of 1470 candidate target genes were predicted by 15 differentially expressed mi RNA.The functions of candidate target genes GO are mainly concentrated in biological processes such as hormone secretion regulation,signal release,cell connection and transcriptional inhibitory activity,while candidate target genes are mainly enriched in Hedgehog signal pathway,thiamine metabolism,propionic acid metabolism and fatty acid synthesis metabolism.These GO functions and pathways are closely related to cell proliferation and differentiation,immune regulation,inflammatory response and so on.Fifteen differentially expressed mi RNA were verified by q RT-PCR,and the expression trend was consistent with that of s RNA-seq.2.The effect of ssc-mi R-221-5p on the proliferation and apoptosis of IPEC-J2 cells induced by soybean 7S globulinIn this experiment,we knocked down and over-expressed ssc-mi R-221-5p in IPEC-J2cells.Through morphological observation,CCK-8 detection and Ed U staining,it was found that the morphology and structure of IPEC-J2 cells induced by soybean 7S globulin were significantly improved after overexpression of ssc-mi R-221-5p.The cell activity increased significantly,the number of Ed U positive cells increased,and cell proliferation was significantly promoted,but it was reversed after knocking down ssc-mi R-221-5p（P<0.05）.Through the detection of apoptosis,ROS,m RNA protein and protein level of Annexin V-FITC/PI cells,it was found that overexpression of ssc-mi R-221-5p could significantly reduce the apoptosis rate of IPEC-J2 cells induced by soybean 7S globulin,decrease the positive rate of ROS,down-regulate the m RNA and protein expression of Bax,and simultaneously up-regulate the m RNA and protein expression of Bcl2 and Bcl-x L,but reverse after knocking down ssc-mi R-221-5p（P<0.05）.Therefore,ssc-mi R-221-5p has the effect of promoting proliferation and anti-apoptosis of IPEC-J2 cells induced by soybean 7S globulin.3.Effects of ssc-mi R-221-5p on mechanical barrier damage and inflammatory response of IPEC-J2 cells induced by soybean 7S globulinThe effect of ssc-mi R-221-5p on mechanical barrier damage of IPEC-J2 cells induced by soybean 7S globulin was evaluated by TEER value,sodium fluorescein permeability,cell damage related indexes（LDH,ALP,DAO and IFABP1）and tight junction protein expression.The results showed that ssc-mi R221-5p was overexpressed.Compared with the negative control group,the TEER value of IPEC-J2 monolayer cells increased significantly,the permeability of sodium fluorescein decreased significantly,the contents of LDH,ALP,DAO and IFABP1 in the culture supernatant decreased significantly,and the m RNA and protein expression levels of ZO-1,Claudin-1 and Occludin were significantly up-regulated,while knocking down ssc-mi R221-5p was the opposite（P<0.05）.This suggests that ssc-mi R-221-5p can alleviate the barrier damage of IPEC-J2 cells induced by soybean 7S globulin by reducing cell permeability,protecting cell integrity and promoting the expression of tight junction protein.By detecting the secretion and m RNA expression of pro-inflammatory cytokines（IL-6,IL-8,IL-1β,TNF-α）and anti-inflammatory cytokines（IL4,IL-10）,it was found that overexpression of ssc-mi R-221-5p could significantly inhibit the secretion and m RNA expression of IL-6,IL-8,IL-1β,TNF-αand significantly promote the secretion of IL-4,IL-10 and m RNA expression,while knocking down ssc-mi R-221-5p was the opposite（P<0.05）.It is suggested that ssc-mi R-221-5p can reduce the inflammatory damage of IPEC-J2 cells induced by soybean 7S globulin.Conclusion:During the injury of IPEC-J2 cells induced by soybean 7S globulin,there are 15 differentially expressed mi RNA and corresponding to 1470 mi RNA-m RNA relationship pairs,among which ssc-mi R-221-5p is the most down-regulated mi RNA.Through the functional study of ssc-mi R-221-5p,it is found that ssc-mi R-221-5p can promote cell proliferation,inhibit apoptosis,reduce cell permeability,protect cellular mechanical barriers and inhibit inflammation,and can effectively alleviate the damage of soybean 7S globulin to IPEC-J2 cells.The results of this study provide a theoretical basis for further revealing the mechanism of diarrhea in weaned piglets caused by soybean 7S globulin,screening targeted drugs that promote the expression of ssc-mi R-221-5p and the genetic improvement and breeding of diarrhea resistant pigs. |
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