Background/Aims: Irritable bowel syndrome with diarrhoea (IBS-D) is a chronic, functional bowel disorder characterized by abdominal pain or diarrhoea and altered bowel habits, which correlate with intestinal hyperpermeability. MicroRNAs (miRNAs) are involved in regulating intestinal permeability in IBS-D. However, the role of miRNAs in regulating intestinal permeability and protecting the epithelial barrier remains unclear. Our goals were to (i) identify differential expression of miRNAs and their targets in the distal colon of IBS-D rats; (ii) verify in vitro whether occludin (OCLN) and zonula occludens 1 (ZO1/TJP1) were direct targets of miR-144 and were down-regulated in IBS-D rats; and (iii) determine whether down-regulation of miR-144 in vitro could reverse the pathological hallmarks of intestinal hyperpermeability via targeting OCLN and ZO1. Methods: The IBS-D rat model was established using 4% acetic acid and evaluated by haematoxylin-eosin (HE) staining. The distal colon was obtained in order to perform miRNA microarray analysis and to isolate and culture colonic epithelial cells. When differential expression of miRNA was found, the results were verified by qRT-PCR, and the target genes were further explored by bioinformatics analysis. Correlation analyses were carried out to compare the expression of miRNA and target genes. Then, mutants, miRNA mimics and inhibitors of the target genes were constructed and transfected to colonic epithelial cells. qRT-PCR, western blotting, enzyme-linked immunosorbent assays (ELISAs) and dual-luciferase assays were used to investigate the expression of miR-144 and OCLN, ZO1 in IBS-D rats. Results: There were 8 up-regulated and 18 down-regulated miRNAs identified in the IBS-D rat model. Of these, miR-144 was markedly up-regulated and resulted in the down-regulation of OCLN and ZO1 expression. Overexpression of miR-144 by transfection of miR-144 precursor markedly inhibited the expression of OCLN and ZO1. Further studies confirmed that OCLN and ZO1 were direct targets of miR-144. Additionally, intestinal hyperpermeability was enhanced by miR-144 up-regulation and attenuated by miR-144 down-regulation in IBS-D rat colonic epithelial cells. Moreover, rescue experiments showed that overexpression of OCLN and ZO1 significantly eliminated the inhibitory effect of miR-144, which showed a stronger effect on the attenuation of intestinal hyperpermeability. Conclusion: Up-regulation of miR-144 could promote intestinal hyperpermeability and impair the protective effect of the epithelial barrier by directly targeting OCLN and ZO1. miR-144 is likely a key regulator of intestinal hyperpermeability and could be a potential therapeutic target for IBS-D.

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Background/AimsMicroRNAs (miRNAs) were reported to be responsible for intestinal permeability in diarrhea-predominant irritable bowel syndrome (IBS-D) rats in our previous study. However, whether and how miRNAs regulate visceral hypersensitivity in IBS-D remains largely unknown.MethodsWe established the IBS-D rat model and evaluated it using the nociceptive visceral hypersensitivity test, myeloperoxidase activity assay, restraint stress-induced defecation, and electromyographic (EMG) activity. The distal colon was subjected to miRNA microarray analysis followed by isolation and culture of colonic epithelial cells (CECs). Bioinformatic analysis and further experiments, including dual luciferase assays, quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay, were used to detect the expression of miRNAs and how it regulates visceral hypersensitivity in IBS-D rats.ResultsThe IBS-D rat model was successfully established. A total of 24 miRNAs were differentially expressed in the distal colon of IBS-D rats; 9 were upregulated and 15 were downregulated. Among them, the most significant upregulation was miR-200a, accompanied by downregulation of cannabinoid receptor 1 (CNR1) and serotonin transporter (SERT). MiR-200a mimic markedly inhibited the expression of CNR1/SERT. Bioinformatic analysis and luciferase assay confirmed that CNR1/SERT are direct targets of miR-200a. Rescue experiments that overexpressed CNR1/SERT significantly abolished the inhibitory effect of miR-200a on the IBS-D rats CECs.ConclusionsThis study suggests that miR-200a could induce visceral hyperalgesia by targeting the downregulation of CNR1 and SERT, aggravating or leading to the development and progression of IBS-D. MiR-200a may be a regulator of visceral hypersensitivity, which provides potential targets for the treatment of IBS-D.

It has been previously reported that psoralen, one of the active ingredients in Psoralea corylifolia, could induce osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), suggesting its potential to treat osteoporosis. Additionally, runt-related transcription factor 2 (Runx2) is a transcription factor that plays vital roles in BMSC osteogenic differentiation. However, whether and how microRNAs (miRNAs/miRs) modulate osteogenic differentiation induced by psoralen have not yet been examined, to the best of the authors' knowledge. The present study aimed to identify the miRNA target genes that regulate osteogenic differentiation of BMSCs induced by psoralen. A Cell Counting Kit-8 assay and alizarin red staining were used to detect the viability and osteogenic differentiation of BMSCs, respectively, under treatment with psoralen. miRNA microarray analysis was performed to identify the differentially expressed miRNAs under treatment with psoralen. A bioinformatics analysis and a luciferase reporter assay were conducted to identify the targets of miR-488. Finally, the mechanisms of miR-488 in psoralen-induced BMSC osteogenic differentiation were investigated using overexpression or inhibition methods in vitro. Cell viability was elevated and osteogenic differentiation of BMSCs was improved under treatment with psoralen. miRNA microarray analysis and further validation by reverse transcription-quantitative PCR revealed that miR-488 was downregulated during psoralen-induced BMSC osteogenic differentiation. Bioinformatics analysis and experimental validation by a luciferase reporter assay identified Runx2 as a potential target of miR-488. Overexpression of miR-488 by transfection with miR-488 mimics markedly inhibited the expression of Runx2, Osterix and alkaline phosphatase, whereas, the inhibition of miR-488 expression by the miR-488 inhibitor promoted their expression compared with the control. Rescue assays demonstrated that Runx2 overexpression partially rescued the inhibitory effect of miR-488 on BMSC osteogenic differentiation. The present results suggested that miR-488 is a negative regulator of psoralen-induced BMSC osteogenic differentiation by targeting Runx2, providing a possible therapeutic target for osteoporosis.

BackgroundSerum exosomal microRNAs (miRNAs) have been suggested as novel biomarkers for various diseases, especially gastric cancer (GC). But circulating biomarkers for Chronic atrophic gastritis (CAG) which is defined as precancrerous lesions of GC remain largely elusive. To investigate serum exosomal miRNAs that are differently expressed in CAG patients and Chronic nonatrophic gastritis (CNAG) may be helpful for its diagnosis and therapy.MethodsPatients were recruited according to the diagnosis and exclusioncriteria. RNA was extracted from serum exosomes of 30 CAG and 30 CNAG patients. The miRNA expression profiles were analyzed by next generation sequencing and were validated by qRT-PCR. Receiver operating characteristic (ROC) analysis has been used to evaluate the diagnostic value.Results30 CAG patients and 30 CNAG patients were recruited in our study. sRNA-seq results showed that hsa-miR-3591-3p, − 122-3p, and − 122-5p of the top 10 miRNAs (hsa-miR-148a-3p, − 122-3p, − 486-3p, −451a, − 122-5p, − 3591-3p, − 486-5p, −151a-3p, −92a-3p, −320a) were significantly upregulated in exosomes from CAG patients versus those from CNAG patients, but hsa-miR-451a, −151a-3p, and -92a-3p were significantly downregulated. Furthermore, qRT-PCR analysis confirmed that hsa-miR-122-5p and hsa-miR-122-3p were significantly upregulated in CAG samples, but hsa-miR-122-3p hadnot a steable expression. ROC curves showed that the AUC for hsa-miR-122-5p was 0.67 (95% CI 0.52–0.82, SE 62%, SP 86%). A sum of the four miRNAs (panel 1, hsa-miR-122-5p, −451a, −151a-3p, and -92a-3p) did not significantly improve the diagnostic potential (AUC 0.63, 95% CI 0.47 to 0.78). Correlation analysis showed that the expression of hsa-miR-122-5p differed significantly between patients based on atrophic (Moderate atrophic vs. Absent, P value was 0.036.) and IM (compare moderate-severe, absent and mild P values were 0.001 and 0.014, respectively). However, there were no differences between groups based on age, gender, dysplasia, or chronic or active inflammation.ConclusionThese results suggested that hsa-miR-122-5p in serum exosomes might serve as a potential biomarker for CAG diagnosis.Trial registrationChinese Clinical Trial Registy (ChiCTR-IOR-16008027, Date of Registration:2016-03-01).Electronic supplementary materialThe online version of this article (10.1186/s12885-019-5328-7) contains supplementary material, which is available to authorized users.

BackgroundTong-Xie-Yao-Fang (TXYF) has been shown to be effective in diarrhoea-predominant irritable bowel syndrome (IBS-D) patients. However, the underlying mechanism remains to be clarified. The aim of this study was to investigate the efficacy and related mechanisms of TXYF in an IBS-D rat model.MethodsThe IBS-D rat model was established with 4% acetic acid and evaluated by haematoxylin-eosin (HE) staining. Then, IBS-D rats were divided into control, TXYF and rifaximin groups and treated intragastrically with normal saline, TXYF and rifaximin, respectively, for 14 days. The following indicators were measured before and after treatment: defecation frequency, faecal water content (FWC) and colorectal distension (CRD). Histopathological changes in the distal colon were observed after treatment. The expression of OCLN and ZO1 in the distal colon of IBS-D rats reflected the intestinal mucosal permeability, as measured by qRT-PCR, western blot, and enzyme-linked immunosorbent assays (ELISAs). The NF-κB and Notch signalling pathways and inflammation-related factors were investigated.ResultsAfter treatment with TXYF, the defecation frequency, FWC and CRD were significantly lower than those in the model group (P < 0.05). HE staining showed that colonic epithelial cells (CECs) in the IBS-D rats displayed significant oedema, impaired intestinal mucosal integrity and an increased influx of inflammatory cells. A significant reduction in granulocyte and CEC oedema was observed after the administration of TXYF and rifaximin compared to that of the model group and blank group (P < 0.05). TXYF significantly upregulated the expression of OCLN and ZO-1 and downregulated inflammation-related factors (IL-6, IL-1β, and TNF-α and the chemokine KC) in IBS-D rats compared to those in the model group rats (P < 0.05). In terms of the NF-κB and Notch signalling pathways, the expression of NICD, p-ERK, Hes-1 and p-P65 decreased significantly in the TXYF and rifaximin groups, while the expression of ATOH1 increased significantly compared to that in the model group (P < 0.05).ConclusionTXYF can effectively improve intestinal permeability and enhance intestinal mucosal barrier function, which may be related to inhibition of the inflammatory cascade and the NF-κB and Notch signalling pathways.

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Psoralen, one of the active ingredients in Psoralea corylifolia, has been previously reported to regulate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). A previous study revealed that psoralen can regulate the expression levels of microRNA-488 and runt-related transcription factor 2 (Runx2) to promote the osteogenic differentiation of BMSCs. However, the underlying signalling pathway in this process remains to be fully elucidated. BMSCs have also been confirmed to play a key role in the occurrence and development of osteoporosis, and are expected to be potential seed cells in the treatment of osteoporosis. In order to explore the potential signalling pathways of psoralen acting on BMSCs, in the present study, human BMSCs (hBMSCs) were treated with different concentrations of psoralen (0.1, 1, 10 and 100 µmol/l) and the TGF-β receptor I (RI) inhibitor SB431542 (5 µmol/l) in vitro for 3, 7 or 14 days. Cell Counting Kit-8 and MTT assays were used to measure cell proliferation and cell viability of hBMSCs following psoralen administration. Alkaline phosphatase (ALP) activity and alizarin red S staining were used to assess the osteogenic differentiation ability of hBMSCs. Reverse transcription-quantitative PCR and western blotting were used to measure the expression of osteogenic differentiation-related genes [bone morphogenetic protein 4 (BMP4), osteopontin (OPN), Runx2 and Osterix] and proteins associated with the TGF-β/Smad3 pathway [TGF-β1, TGF-β RI, phosphorylated (p-)Smad and Smad3]. Psoralen was found to increase the proliferation and viability of hBMSCs. Although different concentrations of psoralen enhanced ALP activity and the calcified nodule content in hBMSCs, the enhancement effects were more potent at lower concentrations (0.1, 1 and 10 µmol/l). The expression of BMP4, OPN, Osterix, Runx2, TGF-β1, TGF-β RI and p-Smad3 was also promoted by psoralen at lower concentrations (0.1, 1 and 10 µmol/l). In addition, whilst SB431542 could inhibit calcium deposition and osteogenic differentiation-related gene expression in hBMSCs, psoralen effectively reversed the inhibitory effects of SB431542. In conclusion, psoralen accelerates the osteogenic differentiation of hBMSCs by activating the TGF-β/Smad3 pathway, which may be valuable for the future clinical treatment of osteoporosis.