miR-146a调节TLR4信号通路对慢性炎性疼痛的影响
发布时间：2018-12-17 20:08
【摘要】：[研究背景及目的]炎性疼痛普遍存在于临床情况中,也是困扰患者的主要慢性疼痛类型。机制研究发现,小胶质细胞及其分泌的促炎症因子参与炎性疼痛的发生与长期维持。而近年来的研究揭示,miR-146a对炎症反应有负向调节作用,且很可能也参与了疼痛机制。本研究的目的是希望通过人为干预,在体外和体内实验中,高表达miR-146a,验证它能否减少小胶质细胞激活后及大鼠炎性疼痛模型中脊髓的促炎症因子表达,并能否缓解大鼠炎性疼痛。[研究方法]体外——PCR法检测细菌脂多糖(LPS)刺激小胶质细胞株(BV2细胞)后的miR-146a表达水平；在miR-146a模拟物转染BV2细胞后,采用ELISA法检测细胞上清液中的TNFa和IL-6表达情况；同时分别采用PCR和western-blot检测细胞中IRAK1和TRAF6的基因和蛋白表达情况。体内——采用完全氟氏佐剂(CFA)制备大鼠慢性炎性疼痛模型,鞘内注射miR-146a模拟物进行中枢神经系统内转染；采用机械和热痛刺激进行疼痛行为学检测；提取腰段脊髓RNA,采用实时荧光定量PCR检测中枢神经系统内miR-146a、TLR4、IRAK1、TRAF6、TNFa和IL-6表达变化。[研究结果]体外——与正常组或阴性对照组比较,采用10nM、50nM、100nM三个转染浓度对BV2细胞进行miR-146a模拟物转染,均可明显增加细胞内miR-146a含量；其中以50nM转染浓度效率最佳；LPS刺激导致BV2细胞激活,可以明显增加细胞内IRAK1和TRAF6表达,也可以明显诱发细胞分泌更多的TNFa和IL-6;用miR-146a模拟物转染可以明显降低TNFa和IL-6的表达水平,同时明显降低细胞内TRAF6,而非IRAK1的表达。体内——在CFA诱发的大鼠炎性疼痛中,miR-146a出现异常表达,在CFA注射后第1天有所降低,在第3天显著升高,第7天有所回落,但仍然显著高于对照组；而中枢神经系统内的TLR4、IRAK1、 TRAF6、TNFa和IL-6随着时间逐渐升高；鞘内注射miR-146a模拟物可以明显缓解大鼠的疼痛,其机械缩足阈值和热缩足潜伏期均有所回升；PCR分析证实miR-146a模拟物鞘内注射可以显著增加脊髓内miR-146a含量,同时可以降低促炎症因子TNFa口IL-6的表达水平,并且TLR4信号通路上的IRAK1和TRAF6分子的表达亦有明显降低。[研究结论]1) miR-146a模拟物的体外和体内转染,都可以有效提高细胞或组织内的miR-146a含量；2) miR-146a可以减少LPS导致的BV2细胞促炎症因子分泌,并且该作用可能是通过负反馈调节TLR4信号通路上的TRAF6分子而进行；3)miR-146a可以减轻CFA诱导的大鼠炎性疼痛,很有可能参与了慢性疼痛的发展、维持及调节。
[Abstract]:Background and objective: inflammatory pain is common in clinical conditions and is the main chronic pain type in patients. It is found that microglia and its proinflammatory cytokines are involved in the occurrence and long-term maintenance of inflammatory pain. Recent studies have revealed that miR-146a plays a negative role in inflammatory response and may also be involved in the pain mechanism. The aim of this study is to investigate whether the overexpression of miR-146a, can reduce the expression of pro-inflammatory factors in spinal cord after activation of microglia and in rat inflammatory pain model by human intervention in vitro and in vivo. And can alleviate the inflammatory pain in rats. [methods] the expression of miR-146a in microglia (BV2 cells) stimulated by lipopolysaccharide (LPS) was detected by PCR method in vitro. After transfection of miR-146a mimics into BV2 cells, the expression of TNFa and IL-6 in supernatant was detected by ELISA assay, and the gene and protein expression of IRAK1 and TRAF6 were detected by PCR and western-blot, respectively. In vivo-chronic inflammatory pain model of rats was established by complete fluorine adjuvant (CFA), intrathecal injection of miR-146a mimics for central nervous system transfection, mechanical and thermal pain stimulation for pain behavior detection; The expression of miR-146a,TLR4,IRAK1,TRAF6,TNFa and IL-6 in central nervous system (CNS) was detected by real-time fluorescence quantitative PCR (PCR). [results] compared with the normal group or the negative control group in vitro, the miR-146a content in BV2 cells was significantly increased by transfection of miR-146a mimics with 10 nm M ~ (50) nM ~ (10) nM ~ (100) nm. The concentration efficiency of 50nM transfection was the best. LPS stimulation led to activation of BV2 cells, which significantly increased the expression of IRAK1 and TRAF6, and induced more TNFa and IL-6; secretion. Transfection with miR-146a mimics could significantly reduce the expression of TNFa and IL-6, and TRAF6, instead of IRAK1 in cells. In vivo-in CFA induced inflammatory pain in rats, the expression of miR-146a was abnormal, decreased on the first day after CFA injection, increased significantly on the third day, and decreased on the 7th day, but still significantly higher than that of the control group. However, TLR4,IRAK1, TRAF6,TNFa and IL-6 in the central nervous system increased gradually with time, and intrathecal injection of miR-146a mimics significantly alleviated the pain in rats, and the mechanical foot contraction threshold and the latent period of thermal contraction increased. PCR analysis showed that intrathecal injection of miR-146a mimics could significantly increase the content of miR-146a in the spinal cord and decrease the expression of IL-6 in the mouth of TNFa, and the expression of IRAK1 and TRAF6 in TLR4 signaling pathway. [conclusion] 1) transfection of miR-146a mimics in vitro and in vivo can effectively increase the content of miR-146a in cells or tissues; 2) miR-146a can reduce the secretion of inflammatory cytokines induced by LPS in BV2 cells, and this effect may be mediated by negative feedback regulation of TRAF6 molecules in TLR4 signaling pathway. 3) miR-146a can alleviate the inflammatory pain induced by CFA in rats, which may be involved in the development, maintenance and regulation of chronic pain.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R402
[Abstract]:Background and objective: inflammatory pain is common in clinical conditions and is the main chronic pain type in patients. It is found that microglia and its proinflammatory cytokines are involved in the occurrence and long-term maintenance of inflammatory pain. Recent studies have revealed that miR-146a plays a negative role in inflammatory response and may also be involved in the pain mechanism. The aim of this study is to investigate whether the overexpression of miR-146a, can reduce the expression of pro-inflammatory factors in spinal cord after activation of microglia and in rat inflammatory pain model by human intervention in vitro and in vivo. And can alleviate the inflammatory pain in rats. [methods] the expression of miR-146a in microglia (BV2 cells) stimulated by lipopolysaccharide (LPS) was detected by PCR method in vitro. After transfection of miR-146a mimics into BV2 cells, the expression of TNFa and IL-6 in supernatant was detected by ELISA assay, and the gene and protein expression of IRAK1 and TRAF6 were detected by PCR and western-blot, respectively. In vivo-chronic inflammatory pain model of rats was established by complete fluorine adjuvant (CFA), intrathecal injection of miR-146a mimics for central nervous system transfection, mechanical and thermal pain stimulation for pain behavior detection; The expression of miR-146a,TLR4,IRAK1,TRAF6,TNFa and IL-6 in central nervous system (CNS) was detected by real-time fluorescence quantitative PCR (PCR). [results] compared with the normal group or the negative control group in vitro, the miR-146a content in BV2 cells was significantly increased by transfection of miR-146a mimics with 10 nm M ~ (50) nM ~ (10) nM ~ (100) nm. The concentration efficiency of 50nM transfection was the best. LPS stimulation led to activation of BV2 cells, which significantly increased the expression of IRAK1 and TRAF6, and induced more TNFa and IL-6; secretion. Transfection with miR-146a mimics could significantly reduce the expression of TNFa and IL-6, and TRAF6, instead of IRAK1 in cells. In vivo-in CFA induced inflammatory pain in rats, the expression of miR-146a was abnormal, decreased on the first day after CFA injection, increased significantly on the third day, and decreased on the 7th day, but still significantly higher than that of the control group. However, TLR4,IRAK1, TRAF6,TNFa and IL-6 in the central nervous system increased gradually with time, and intrathecal injection of miR-146a mimics significantly alleviated the pain in rats, and the mechanical foot contraction threshold and the latent period of thermal contraction increased. PCR analysis showed that intrathecal injection of miR-146a mimics could significantly increase the content of miR-146a in the spinal cord and decrease the expression of IL-6 in the mouth of TNFa, and the expression of IRAK1 and TRAF6 in TLR4 signaling pathway. [conclusion] 1) transfection of miR-146a mimics in vitro and in vivo can effectively increase the content of miR-146a in cells or tissues; 2) miR-146a can reduce the secretion of inflammatory cytokines induced by LPS in BV2 cells, and this effect may be mediated by negative feedback regulation of TRAF6 molecules in TLR4 signaling pathway. 3) miR-146a can alleviate the inflammatory pain induced by CFA in rats, which may be involved in the development, maintenance and regulation of chronic pain.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R402
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相关期刊论文 前3条
1 陈烨;陈彦青;李德龙;;大鼠鞘内注射方法的改进与验证[J];福建医科大学学报;2009年02期
2 王海燕;李扬;陈梅红;张p，
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