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. 2018 Oct 19;24(1):55.
doi: 10.1186/s10020-018-0053-2.

Protective role of down-regulated microRNA-31 on intestinal barrier dysfunction through inhibition of NF-κB/HIF-1α pathway by binding to HMOX1 in rats with sepsis

Cheng-Ye Zhan  1 , Di Chen  2 , Jin-Long Luo  1 , Ying-Hua Shi  1 , You-Ping Zhang  1
Affiliations

Protective role of down-regulated microRNA-31 on intestinal barrier dysfunction through inhibition of NF-κB/HIF-1α pathway by binding to HMOX1 in rats with sepsis

Cheng-Ye Zhan et al. Mol Med. .

Abstract

Background: Intestinal barrier dysfunction is a significant clinical problem, commonly developing in a variety of acute or chronic pathological conditions. Herein, we evaluate the effect of microRNA-31 (miR-31) on intestinal barrier dysfunction through NF-κB/HIF-1α pathway by targeting HMOX1 in rats with sepsis.

Methods: Male Sprague-Dawley rats were collected and divided into the sham group, and the cecum ligation and perforation group which was subdivided after CACO-2 cell transfection of different mimic, inhibitor, or siRNA. Levels of serum D-lactic acid, diamine oxidase and fluorescence isothiocyanate dextran, FITC-DX concentration, and bacterial translocation were detected. Superoxidedismutase (SOD) activity and malondialdehyde (MDA) content were evaluated using the colorimetric method and an automatic microplate reader, respectively. Additionally, the levels of tumor necrosis factor, interleukin (IL)-6, and IL-10 were tested using enzyme-linked immunosorbent assay. The expression of miR-31, HMOX1, NF-κB, HIF-1α, IκB, ZO-1 and Occludin were assessed by reverse transcription quantitative polymerase chain reaction and Western blot analysis.

Results: Inhibition of miR-31 decreased intestinal mucosal permeability and intestinal barrier function. The increased levels of miR-31 could cause oxidative damage and affect the expression of inflammatory factors in intestinal tissue of rats. HMOX1 was confirmed as a target gene of miR-31. MiR-31 affected intestinal mucosal permeability and intestinal barrier function, as well as oxidative damage and inflammation level by regulating HMOX1. Down-regulation of miR-31 inhibited NF-κB/HIF-1α pathway related genes by regulating HMOX1 expression. Furthermore, inhibition of miR-31 increased survival rates of rats.

Conclusion: Overall, the current study found that inhibition of miR-31 protects against intestinal barrier dysfunction through suppression of the NF-κB/HIF-1α pathway by targeting HMOX1 in rats with sepsis.

Keywords: HMOX1; Intestinal barrier dysfunction; NF-κB/HIF-1α pathway; Sepsis; microRNA-31.

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Conflict of interest statement

Ethics approval and consent to participate

The experimental procedures and the use of animals were approved by the ethics committee on animal experimentation of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology.

Consent for publication

Consent for publication was obtained from the participants.

Competing interest

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Intestinal mucosa permeability was affected by miR-31 levels. a levels of D-lactic acid in serum of rats increased after the treatment of miR-31 mimic; (b) levels of DAO in serum of rats elevated after treating with miR-31 mimic; (c) levels of FD-40 in serum of rats upregulated by miR-31 mimic; *, p < 0.05, vs. the sham group; #, p < 0.05, vs. the sepsis group; NC negative control, DAO Diamine oxidase
Fig. 2
Fig. 2
MiR-31 caused oxidative damage and affected the levels of inflammatory factors in intestinal tissue of rats. a content of MDA increased after treating with miR-31 mimic; (b) SOD activity was inhibited by miR-31 mimic; (c) elevated levels of TNF-α after the treatment of miR-31 mimic; (d) increased levels of IL-6 following the treatment of miR-31 mimic; (e), increased levels of IL-10 by miR-31 mimic; *, p < 0.05, vs. the sham group; #, p < 0.05, vs. the sepsis group; MDA malondiadehyde, SOD superoxidedismutase, NC negative control, TNF tumor necrosis factor, IL interleukin
Fig. 3
Fig. 3
HMOX1 was the target gene of miR-31. a HMOX1 was confirmed as a target gene of miR-31 by the biology predicted website microRNA.org; (b) dual-luciferase reporter assay showed HMOX1 was a target gene of miR-31; (c) Western blot analysis detected that protein levels of HMOX1 were significantly increased after treating miR-31 inhibitor; (d) inhibition of miR-31 could increase protein levels of HMOX1; *, p < 0.05, vs. the corresponding control group
Fig. 4
Fig. 4
MiR-31 can influence intestinal mucosa permeability through regulation of HMOX1. a increased levels of D-lactic acid in serum of rats after the treatment of siRNA-HMOX1; (b) siRNA-HMOX1 could increase levels of DAO in serum of rats; (c) increased levels of FD-40 in serum of rats by siRNA-HMOX1; *, p < 0.05, vs. the NC group; NC negative control, DAO Diamine oxidase
Fig. 5
Fig. 5
MiR-31 affects oxidative damage and the levels of inflammatory factors in intestinal tissue of rats by regulating HMOX1. a siRNA-HMOX1 increased content of MDA; (b), SOD activity was down-regulated by siRNA-HMOX1; (c) increased levels of TNF-α after the treatment of siRNA-HMOX1; (d) increased levels of IL-6 following the treatment of siRNA-HMOX1; (e) increased levels of IL-10 by siRNA-HMOX1; *, p < 0.05, vs. the NC group; MDA, malondiadehyde; SOD, superoxidedismutase; NC negative control, TNF tumor necrosis factor, IL interleukin
Fig. 6
Fig. 6
MiR-31 resulted in histopathological changes in intestine tissues of rats. a histopathological changes of the intestine tissues of the rats could be caused by miR-31 (HE staining 200 ×); (b) pathological injury of small intestine tissue in rats was abated by inhibiting miR-31; *, p < 0.05, vs. the sham group; #, p < 0.05, vs. the sepsis group; NC negative control, HE hematoxylin and eosin
Fig. 7
Fig. 7
MiR-31 regulated expression of the NF-κB/HIF-1α pathway related genes by inhibiting HMOX1. a-b RT-qPCR detected that the mRNA levels of NF-κB, HIF-1α, HMOX1, IκB, ZO-1, and Occludin in the ileal tissue of the rats were affected by miR-31 through HMOX1; (c-d), Western blot analysis detected that protein levels of IκB, p-IκB, ZO-1, HMOX1, HIF-1α, NF-κB, and Occludin in the ileal tissue of the rats were regulated by miR-31 via HMOX1; *, p < 0.05, vs. the sham group; #, p < 0.05, vs. the sepsis group; NC negative control
Fig. 8
Fig. 8
MiR-31 reduced the survival rates of rats. n = 15
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