MicroRNA Files in the Prevention of Intestinal Ischemia/Reperfusion Injury by Hydrogen Rich Saline

Abstract:

Background Hydrogen rich saline (HRS) has been proven effective against ischemia reperfusion(I/R) injury. However, knowledge on the underlying signaling events remain poor. Having recent highlight of microRNAs(miRNAs) in mediating intestinal I/R injury, we hypothesized that HRS may protect intestine against I/R injury by regulating miRNAs. Method Mice were given intraperitoneal injection of saline or HRS once daily for 5 consecutive days before undergoing intestinal I/R which was induced by 60-min ischemia followed by 180-min reperfusion of superior mesenteric artery. The intestine was collected for histopathological assay, miRNA microarray profiling, Real-Time PCR, and Western blotting. Next, miR-199a-3p mimics or inhibitor were transfected into IEC-6 cells to explore the relationship between HRS treatment and miR-199a-3p. Results I/R-induced mucosal injury and epithelial cells apoptosis were attenuated by HRS pretreatment. A total of 64 intestinal I/R-responsive miRNAs were altered significantly by HRS pretreatment, in which we validated 4 novel miRNAs with top significance by Real-Time PCR, namely miR-199a-3p, miR-296-5p, miR-5126, and miR-6538. Particularly, miR-199a-3p was drastically increased by I/R but reduced by HRS. Computational analysis predicts insulin-like growth factor(IGF)-1, mammalian target of rapamycin(mTOR), and phosphoinositide-3-kinase(PI3K) regulatory subunit 1 as targets of miR-199a-3p, suggesting involvement of the pro-survival pathway, IGF-1/PI3K/Akt/mTOR. In in vitro experiment, HRS treatment reduced miR-199a-3p level, increase IGF-1, PI3K and mTOR mRNA expression, restore IEC-6 cells viability, and this protective effects were reversed under miR-199a-3p mimics treatment. Conclusion Collectively, miR-199a-3p may serve a key role in the anti-apoptotic mechanism of HRS which contributes to its protection of the intestine against I/R injury.