摘要
背景:研究发现,腰椎间盘突出症患者的疼痛机制与炎症相关,自噬与椎间盘疾病及炎症反应密切相关,miR-206表达异常可促进骨骼疾病的发生。目的:探讨miR-206对腰椎间盘突出大鼠髓核炎症、镇痛及自噬相关蛋白的作用机制。方法:SPF级雄性SD大鼠60只按照随机数字方法分为对照组、模型组、miR-206 mimics-NC组、miR-206 mimics组、miR-206 inhibitor-NC组及miR-206 inhibitor组。除对照组外均建立腰椎间盘突出症大鼠模型,建模后第10天miR-206 mimics-NC组、miR-206 mimics组、miR-206 inhibitor-NC组及miR-206 inhibitor组分别在损伤处注射miR-206 mimics-NC、miR-206 mimics(miR-206模拟物)、miR-206 inhibitor-NC及miR-206 inhibitor(miR-206抑制物),均20μmol/L,10μL,1次/d,连续注射4 d;对照组及模型组均注射等剂量生理盐水。Von Frey纤维丝测定大鼠双侧后足机械性缩足反射阈值,热痛测试仪检测大鼠双侧后足热刺激缩足反射潜伏期;苏木精-伊红染色观察背根神经节组织形态;qPCR检测各组大鼠髓核组织炎症因子磷脂酶A2、环氧化酶2、前列腺素E2、肿瘤坏死因子α、白细胞介素1β表达;免疫印迹检测自噬相关蛋白LC3Ⅰ、Beclin-1表达。结果与结论:①造模后3,7及14 d时,与对照组相比,模型组大鼠机械性缩足反射阈值、热刺激缩足反射潜伏期降低,磷脂酶A2、环氧化酶2、前列腺素E2、肿瘤坏死因子α、白细胞介素1β、LC3Ⅰ、Beclin-1表达升高(P<0.05);miR-206 inhibitor-NC组、miR-206 mimics-NC组上述指标与模型组相比差异无显著性意义(P>0.05);②与miR-206 mimics-NC组相比,miR-206 mimics组大鼠机械性缩足反射阈值、热刺激缩足反射潜伏期降低,磷脂酶A2、环氧化酶2、前列腺素E2、肿瘤坏死因子α、白细胞介素1β、LC3Ⅰ、Beclin-1水平升高(P<0.05);与miR-206 inhibitor-NC组相比,miR-206 inhibitor组大鼠上述指标呈相反变化,差异均有显著性意义(P<0.05);③结果说明,抑制miR-206可以显著改善腰椎间盘突出症大鼠髓核炎症因子水平,提高疼痛阈值,降低细胞自噬,其作用机制与抑制LC3Ⅰ、Beclin-1表达相关。
BACKGROUND:Pain mechanisms in patients with lumbar disc herniation are associated with inflammation,autophagy is closely related to intervertebral disc diseases and inflammatory response,and aberrant miR-206 expression can trigger skeletal diseases.OBJECTIVE:To investigate the mechanism of miR-206 on inflammation,analgesia and autophagy related proteins in nucleus pulposus in rats with lumbar disc herniation.METHODS:Sixty SPF male Sprague-Dawley rats were randomly divided into control group,model group,miR-206 mimics-NC group,miR-206 mimics group,miR-206 inhibitor-NC group and miR-206 inhibitor group.Animal models of lumbar disc herniation were established except for the control group.Ten days after modeling,miR-206 mimics-NC group,miR-206 mimics group,miR-206 inhibitor-NC group and miR-206 inhibitor group were injected with miR-206 mimics-NC(20μmol/L,10μL),miR-206 mimics(20μmol/L,10μL),miR-206 inhibitor-NC(20μmol/L,10μL)and miR-206 inhibitor(20μmol/L,10μL),respectively.Administration was given once a day for 4 continuous days.The control group and model group were injected with the same dose of normal saline.The paw withdrawal mechanical threshold of bilateral hind feet was measured by Von Frey filaments,and the paw withdrawal thermal latency of bilateral hind feet was measured by heat pain tester.The morphology of dorsal root ganglia was observed by hematoxylin-eosin staining.The expressions of inflammatory factors phospholipase A2,cyclooxygenase 2,prostaglandin E2,tumor necrosis factorα,and interleukin 1βin nucleus pulposus were detected by qPCR.The expressions of autophagy-related proteins LC3I and Beclin-1 were detected by western blot assay.RESULTS AND CONCLUSION:At 3,7,and 14 days after modeling,the paw withdrawal mechanical threshold and paw withdrawal thermal latency were both decreased in the model group compared with the control group,while the levels of phospholipase A2,cyclooxygenase 2,prostaglandin E2,tumor necrosis factorα,interleukin 1β,LC3I and Beclin-1 increased(P<0.05).The above indexes showed no significant changes in the miR-206 inhibitor-NC group and miR-206 mimics-NC group compared with the model group(P>0.05).Compared with the miR-206 mimics-NC group,the miR-206 mimics group had lower paw withdrawal mechanical threshold and paw withdrawal thermal latency and higher levels of phospholipase A2,cyclooxygenase 2,prostaglandin E2,tumor necrosis factorα,interleukin 1β,LC3I,and Beclin-1 levels(P<0.05).Compared with the miR-206 inhibitor-NC group,the rats in the miR-206 inhibitor group showed opposite changes in the above indicators,and there were significant differences between the two groups(P<0.05).To conclude,inhibition of miR-206 can significantly improve the level of inflammatory factors in nucleus pulposus of rats with lumbar disc herniation,increase pain threshold,and reduce autophagy.The mechanism is related to the inhibition of LC3I and Beclin-1 expression.
作者
王美
索娜
于欢
杨建博
Wang Mei;Suo Na;Yu Huan;Yang Jianbo(Harrison International Peace Hospital,Hengshui 053000,Hebei Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2024年第11期1712-1718,共7页
Chinese Journal of Tissue Engineering Research
基金
河北省卫生健康委员会医学科学研究项目(20211299)。
