MicroRNA-181a Contributes To Gastric Hypersensitivity By Regulating Toll-like Receptor 4 In Diabetic Rats

Aims:This study aims to investigate the mechanism and interaction of microRNA 181a(miR-181 a)and toll-like receptor 4(TLR4)in gastric hypersensitivity of diabetic rats.Methods:1.Diabetes was induced by a single intraperitoneal(i.p.)injection of streptozotocin(STZ;65mg/kg)in adult female SD rats.2.Behavioral test:Measurement of the gastric balloon distention was classified on normal and diabetic rats.3.Analyzed the expression of TLR4 and microRNA(T7-T10 DRGs)by western blotting(WB)and real-time quantitative PCR(RT-PCR),respectively.4.Using dual-luciferase reporter gene assay to detect the targeting regulation of microRNA and TLR4.5.Fluorescent-immunohistochemistry and Flourescence in situ hybridization(FISH)were uesd to detect the expression of TLR4 and co-location of TLR4 and miR-181a.Results:1.Diabetic rats began to be more sensitive to the graded gastric balloon distention 2 weeks after injection of STZ and lasted for at least 4 weeks.Intrathecal injection of TLR4 inhibitor CLI-095 significantly alleviated gastric hypersensitivity in diabetic rats.2.The expression of TLR4 had a remarkable increase in T7-10 DRGs of diabetic rats.3.MiR-181a,miR-23a,miR-218 and miR-7a were predicted by bioinformatics technology to affect the expression of TLR4.RT-PCR results showed that the expression of miRNA-181a,miR-23a,miR-218 and miRNA-7a in T7-T10 DRGs of diabetic rats were significantly decreased.Dual-luciferase reporter gene assay showed that microRNA-181a,microRNA-23a,microRNA-218 had targeting regulation roles on TLR4,while microRNA-7a had no regulation effect on TLR4.4.Intrathecal injection of microRNA-181a agomir significantly alleviated gastric hypersensitivity and down-regulated the expression of TLR4 in T7-T10 DRGs of diabetic rats.5.TLR4 and miR-181a were distributed in NeuN positive neurons of T7-T10 DRGs in control rats,and TLR4 and miR-181a were co-localized in DRG neurons.Conclusion:MicroRNA-181a and TLR4 in gastric-specific DRGs were involved in diabetic gastric hypersensitivity.The decreased microRNA-181a might contribute to diabetic gastric hypersensitivity by upregulating TLR4 expression.These findings might provide potential targets for clinical treatment.