对miR-206在大鼠下肢缺血再灌注损伤过程中炎症反应的影响分析

Influence of miR-206 on inflammatory response during lower limb ischemia-reperfusion injury in rats

目的下肢缺血再灌注损伤(IRI)机制与炎症反应密切相关,而miR-206在血管炎症反应中起着关键作用,本文旨在探讨miR-206对大鼠下肢IRI过程中炎症反应的影响。方法将48只SD雄性大鼠采用随机数字表法分为4组:Sham组(游离出股动脉,不做夹闭股动脉处理)、IRI组(制备大鼠下肢IRI模型,尾部静脉注射0.9%氯化钠溶液)、IRI+miR-206 agomir组(模型建立后尾部静脉miR-206激动剂miR-206 agomir)和IRI+miR-206 antagomir组(模型建立后尾部静脉注射miR-206拮抗剂miR-206 antagomir),每组12只。麻醉后处死取大鼠股动脉,记录苏木精-伊红(HE)染色和病理分析观察股动脉壁形态变化;TUNEL法检测大鼠股动脉细胞凋亡情况;大鼠股动脉组织中氧化应激产物超氧化物歧化酶(SOD)和丙二醛(MDA)检测;蛋白质印迹法检测大鼠股动脉中炎症因子迁移率族蛋白B1(HMGB1)、肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)表达。对数据行单因素方差分析、LSD检验。结果IRI+miR-206 agomir组miR-206 mRNA表达水平较IRI组和IRI+miR-206 antagomir组显著升高(t=7.81、9.39,P<0.05),IRI+miR-206 antagomir组miR-206 mRNA表达水平较IRI组显著降低(t=5.39,P<0.05)。HE染色光镜下观察IRI+miR-206 agomir组股动脉损伤减轻,IRI+miR-206 antagomir组股动脉损伤加重。IRI+miR-206 agomir组与IRI组和IRI+miR-206 antagomir组相比较SOD活性显著增加(t=4.54、8.25,P<0.05),而MDA水平显著降低(t=4.09、6.27,P<0.05)。IRI+miR-206 antagomir与IRI组相比较SOD活性显著降低(t=4.52,P<0.05),而MDA水平显著增加(t=3.40,P<0.05),HMGB1、TNF-a和IL-6表达水平组间总体比较,差异均有统计学意义(F=124.90、20.05、94.73,P<0.05);IRI+miR-206 agomir组HMGB1、TNF-a和IL-6蛋白表达水平与IRI组和IRI+miR-206 antagomir组相比较,显著降低(t=7.68、17.7,t=2.59、4.63,t=9.21、10.32;P<0.05)。IRI+miR-206 antagomir组HMGB1和IL-6蛋白表达水平较IRI组显著升高(t=4.26、2.56,P<0.05)。结论miR-206高表达可抑制大鼠下肢IRI过程中炎症反应,为临床治疗下肢IRI提供了思路。

Objective The mechanism of lower limb ischemia-reperfusion injury(IRI)is closely related to inflammatory reaction,and miR-206 plays a key role in vascular inflammation.This study aims to investigate the effect of miR-206 on inflammatory response during lower limb IRI in rats.Methods Forty-eight male SD rats were divided into 4 groups according to the random number table:Sham group(free femoral artery,no clipping femoral artery treatment),IRI group(the rat model of lower limb IRI was established,and 0.9%sodium chloride solution was injected into tail vein),IRI+miR-206 agomir group(miR-206 agonist miR-206 agomir group after model establishment),IRI+miR-206 antagomir group(miR-206 antagonist miR-206 antagomir group after model establishment),with 12 rats in each group.Rats were killed after anesthesia,and the femoral artery was taken,and HE staining and pathological analysis were recorded to observe the morphological changes of femoral artery wall.Apoptosis of rat femoral artery was detected by TUNEL.Detection of oxidative stress products superoxide dismutase(SOD)and malondialdehyde(MDA)in rat femoral artery;western blotting was used to detect the expression of high mobility group B1(HMGB1),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in rat femoral artery.Data were statistically analyzed with one-way analysis of variance and least significant difference test.Results The expression level of miR-206 mRNA in IRI+miR-206 agomir group was significantly higher than those in IRI group and IRI+miR-206 antagomir group(t=7.81,9.39;P<0.05),while the expression level of miR-206 mRNA in IRI+miR-206 antagomir group was significantly lower than that in IRI group(t=5.39,P<0.05);Observation under HE staining light microscope,the ischemia-reperfusion femoral artery injury of the injured lower limbs in IRI+miR-206 agomir group was alleviated,while the ischemia-reperfusion femoral artery injury of the injured lower limbs in IRI+miR-206 antagomir group was aggravated.Compared with IRI group and IRI+miR-206 antagomir group,SOD activity in IRI+miR-206 agomir group was significantly increased(t=4.54,8.25;P<0.05),while MDA level was significantly decreased in IRI+miR-206 agomir group(t=4.09,6.27;P<0.05).Compared with IRI group,SOD activity in IRI+miR-206 antagomir group was significantly decreased(t=4.52,P<0.05),while MDA level in IRI+miR-206 antagomir group was significantly increased(t=3.40,P<0.05).The expression levels of HMGB1,TNF-a and IL-6 were compared among the groups,and the differences were statistically significant(F=124.90,20.05,94.73;P<0.05);The expression levels of HMGB1、TNF-a and IL-6 of IRI+miR-206 agomir group were significantly lower than those of IRI group and IRI+miR-206 antagomir group(t=7.68,17.7;t=2.59,4.63;t=9.21,10.32;P<0.05).The expression levels of HMGB1,IL-6 of IRI+miR-206 antagomir group were significantly higher than that of the IRI group(t=4.26,2.56;P<0.05).Conclusion The high expression of miR-206 can inhibit the inflammatory level during lower limb IRI in rats,which provides a way for clinical treatment of lower limb IRI.