Experimental Research On The Effect Of MicroRNA-21 Inhibitor On A Rat Model Of Intervertebral Disc Degeneration

Objective To validate the role and underlying mechanisms of miR-21 inhibitor in a rat model of intervertebral disc degeneration.Methods(1)A total of 60 1-year-old female Sprague-Dawley rats(150-200 g)were used in strict accordance with the guidelines for the Care and Use of Laboratory Animals.Rats were randomly divided into four groups of 15.The normal control group received a skin incision,which were subsequently sutured.The rats in the three other groups underwent a previously described surgical procedure to induce the development of lumbar intervertebral disc degeneration.(2)method:The rats were anesthetized by an intraperitoneal injection of 100 mg/kg 10%chloral hydrate.The sacrospinal muscles,spinous processes,supraspinous ligaments,interspinous ligaments and posterolateral halves of the bilateral zygapophysial joints of the lumbar spine were removed.(3)rats in the model(control)group received a tail vein injection of normal saline;rats in the scramble group received a tail vein injection of 80 mg/kg/day control oligonucleotides;rats in the antagomiR-21 group received a tail vein injection of 80 mg/kg/day of antagomiR-21.Injections were administered for 8 weeks.Following this,Lumbar spines,were removed en bloc;the paravertebral muscles and the posterior columns were fully removed.The vertebral pulp and annulus fibrosus were isolated for immunohistochemical analysis of HIF-1? and VEGF expression.The proteoglycan content in the lumbar spines was detected using the phloroglucinol method.The apoptosis of lumbar vertebrae was observed by TUNEL staining.Results(1)AntagomiR-21 treatment decreased the expression of HIF-1? in the vertebral pulp and annulus fibrosus.In the model and scramble groups,positive staining for HIF-la(brown and yellow)was observed in the nucleus and cytoplasm.The model and scramble groups exhibited significantly increased expression of HIF-la in the vertebral pulp and annulus fibrosus compared with the control group.Compared with the scramble group,antagomiR-21 treatment significantly decreased HIF-la expression in the vertebral pulp and annulus fibrosus.(2)AntagomiR-21 treatment decreased VEGF expression in the vertebral pulp and annulus fibrosus.Positive staining for the expression of VEGF(brown and yellow)was predominantly observed in the cytoplasm.Similar to the expression pattern of HIF-1?,VEGF expression in the model and scramble groups was significantly increased compared with the control group and VEGF expression was significantly decreased in the antagomiR-21 treatment group compared with the scramble group.(3)AntagomiR-21 treatment increased the lumbar spine proteoglycan content.The results revealed that the proteoglycan content of the lumbar spine was significantly decreased in the model group compared with the control group.AntagomiR-21 treatment significantly increased the proteoglycan content in lumbar spines compared with the scramble group.(4)AntagomiR-21 treatment inhibited cell apoptosis in lumbar spines.The number of TUNEL-positive cells(brown)in the model group was significantly increased compared with the control group.As expected,antagomiR-21 treatment significantly decreased the number of TUNEL-positive cells compared with the scramble group.ConclusionsAntagomiR-21 treatment exerted a protective role in the rat model of intervertebral disc degeneration by increasing the proteoglycan content and inhibiting cell apoptosis,at least in part through HIF-la and VEGF expression regulation.The findings may demonstrate that antagomiR-21 may be a novel treatment for intervertebral disc degeneration.