Effect of Electroacupuncture on Intestinal Mucosal Barrier in IBS-D Rats: Analysis Based on RNA-seq.

Abstract

Objective: Currently, transcriptome-level investigations into the therapeutic mechanisms of electroacupuncture (EA) on intestinal mucosal barrier dysfunction in diarrhoea-predominant irritable bowel syndrome (IBS-D) models remain scarce. This study was designed to establish a comprehensive competing endogenous RNA (ceRNA) network through integrated RNA sequencing (RNA-seq) and bioinformatics analyses while elucidating the underlying mechanisms through which EA restores intestinal barrier integrity in IBS-D rats via modulation of the long non-coding RNA (lncRNA)-microRNA (miRNA) -messenger RNA (mRNA) regulatory network.

Methods: The IBS-D model was established by neonatal maternal separation (NMS), 4% acetic acid enema, and restrain stress (RS). The rats were randomly divided into three groups: control, model and EA groups. After 2 weeks of EA, the morphological changes of the rat colon were observed by hematoxylin-eosin staining (HE) and Transmission electron microscope (TEM), and the expression of substances related to the damage of the intestinal mucosal barrier was detected by Enzyme-linked Immunosorbent Assay (ELISA) and Western blot (WB) to verify the protective effect of EA on the intestinal mucosal barrier of IBS-D rats. Then RNA-seq was used to analyse rat colon differentially expressed RNAs (DE RNAs) and construct relevant ceRNA networks. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the differentially expressed mRNAs (DE mRNAs) altered by EA to elucidate the mechanism of EA in improving the damage of the intestinal mucosal barrier. Finally, Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) was used to verify the RNA-seq results, and WB and immunofluorescence (IF) were used to verify the involvement of mast cells (MCs) in the relevant signalling pathways regulated by EA.

Results: Firstly, EA had an alleviating effect on the intestinal mucosal barrier damage in IBS-D rats. Then, RNAseq results showed that 426 DE mRNAs, 342 differentially expressed lncRNAs (DE lncRNAs) and 10 differentially expressed miRNAs (DE miRNAs) were up-regulated and 429 DE mRNAs, 362 DE lncRNAs and 48 DE miRNAs were down-regulated by EA. Meanwhile, the ceRNA networks of 7 DE lncRNAs-miR-139-3p-Bid and 7 DE lncRNAs-miR-378b-Slc4a5 were successfully constructed. GO indicated that EA protected the intestinal mucosal barrier of IBS-D rats mainly by regulating a series of defense responses (e.g., against viruses and bacteria), participating in regulating the secretion and transport of hormones, and affecting the function of cytokines. KEGG indicated that there were key signal pathways such as antigen processing and presentation, neuroactive ligand-receptor interaction, PPAR signaling pathway and glutathione metabolism, which were related to the participation of MC in immune inflammation after degranulation. RT-qPCR results were consistent with RNA-seq. Further experiments confirmed that EA ameliorated the damage to the intestinal mucosal barrier in IBS-D rats by inhibiting MC activation.

Conclusion: This study explored the multi-system, multi-level and multi-target mechanism of EA in the treatment of IBS-D through RNA-seq and found that EA could improve the intestinal mucosal barrier damage in IBSD rats. The overall regulatory effect of EA was related to the regulation of the ceRNA networks composed of 7 DE lncRNAs-miR-139-3p-Bid and 7 DE lncRNAs-miR-378b-Slc4a5 by affecting multiple genes in IBS-D rats.