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中华损伤与修复杂志(电子版) ›› 2023, Vol. 18 ›› Issue (03) : 249 -255. doi: 10.3877/cma.j.issn.1673-9450.2023.03.012

论著

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
史孟杰, 贺仕才, 刘斐, 闫燕, 代毅, 王辉()   
  1. 710068 西安,陕西省人民医院血管外科
  • 收稿日期:2023-02-12 出版日期:2023-06-01
  • 通信作者: 王辉
  • 基金资助:
    陕西省科技攻关资助项目(2022SF-079)

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

Mengjie Shi, Shicai He, Fei Liu, Yan Yan, Yi Dai, Hui Wang()   

  1. Department of Vascular Surgery, Shaanxi Provincial People′s Hospital, Xi′an 710068, China
  • Received:2023-02-12 Published:2023-06-01
  • Corresponding author: Hui Wang
引用本文:

史孟杰, 贺仕才, 刘斐, 闫燕, 代毅, 王辉. 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[J/OL]. 中华损伤与修复杂志(电子版), 2023, 18(03): 249-255.

Mengjie Shi, Shicai He, Fei Liu, Yan Yan, Yi Dai, Hui Wang. Influence of miR-206 on inflammatory response during lower limb ischemia-reperfusion injury in rats[J/OL]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2023, 18(03): 249-255.

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.

图1 各组大鼠股动脉组织形态学分析(苏木精-伊红 ×400)。A示Sham组股动脉三层膜结构清晰、完整、正常;B示IRI组股动脉三层膜组织明显水肿,边界模糊,红细胞大量聚集伴炎性细胞浸润,外膜纤维结缔组织水肿增厚,局部断裂缺损;C示IRI+miR-206 agomir组内皮细胞水肿、壁增厚、中性粒细胞浸润、弹性纤维层损伤程度较IRI组减轻;D示IRI+miR-206 antagomir组内皮细胞水肿、壁增厚、中性粒细胞浸润、弹性纤维层损伤程度较IRI组加重
图2 光镜下各组大鼠股动脉壁细胞凋亡情况(苏木精-伊红 ×400)。A示Sham组光镜下个别细胞出现细胞凋亡现象;B示IRI组光镜下可见细胞出现凋亡现象;C示IRI+miR-206 agomir组细胞凋亡现象较IRI组减少;D示IRI+miR-206 antagomir组细胞凋亡现象较IRI组明显
图3 蛋白质印迹法检测各组大鼠股动脉组织HMGB1、IL-6和TNF-a蛋白表达水平。HMGB1为迁移率族蛋白B1,TNF-α为肿瘤坏死因子-α,IL-6为白细胞介素6,GAPDH为3-磷酸甘油醛脱氢酶;条带上方1、2、3、4分别指示Sham组、IRI组、IRI+miR-206 agomir组、IRI+miR-206 antagomir组
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