肾上腺髓质素受体信号活动参与吗啡耐受的细胞学机制—对胶质细胞的激活

发布时间：2018-08-31 13:04

【摘要】：肾上腺髓质素(adrenomedullin, AM)是降钙素基因相关肽(calcitonin gene related peptide, CGRP)家族的一员,与CGRP具有相似的功能。AM在脊髓背角浅层和背根神经节感觉神经元都有表达,是一种疼痛相关肽。最近的研究发现,慢性应用吗啡,通过μ-阿片受体和蛋白激酶C信号通路导致AM表达上调,引起吗啡耐受,鞘内注射AM受体拮抗剂AM22-52可翻转吗啡耐受。这一结果提示,AM参与了吗啡耐受的形成。但是其促进吗啡耐受的细胞学机制尚不清楚。为揭示AM参与吗啡耐受的细胞学机制。本研究通过在体和离体实验,分别采用AM受体拮抗剂和AMsiRNA抑制AM受体,观察阻断AM受体功能后,慢性应用吗啡诱导的胞内痛介导质兴奋性氨基酸和促炎性细胞因子的变化。并采用行为学、实时荧光定量PCR、蛋白印迹、免疫荧光组化等技术研究AM调节促炎性细胞因子的细胞学机制,揭示AM参与吗啡耐受的机制。结果显示：(1)鞘内给予AM受体拮抗剂AM22-52(10nmol),能抑制吗啡耐受大鼠脊髓背角和血浆中兴奋性氨基酸(谷氨酸和天门冬氨酸)释放增加,还能抑制脊髓背角中促炎性细胞因子IL-1β、IL-6mRNA表达增加。(2) AM siRNA (50nM)干扰离体培养的DRG外植体,能抑制慢性应用吗啡(3.3μM)诱导的DRG外植体中促炎性细胞因子IL-1β、IL-6mRNA表达增加。(3)单独连续7天鞘内注射AM受体激动剂AM1-50(10μg),能引起大鼠脊髓背角中IL-1β、IL-6和TNF-α mRNA表达增加,却引起DRG中IL-1β、IL-6和TNFα mRNA表达下降。(4)单独连续7天鞘内注射AM1-50,能引起脊髓背角星型胶质细胞和小胶质细胞特异性标志物GFAP和OX-42表达增加；而鞘内注射AM22-52,能抑制慢性应用吗啡诱导的大鼠脊髓背角GFAP和OX-42表达增加。(5)免疫荧光双重染色显示在大鼠脊髓背角中AM受体的结合部件CRLR和RAMP2与小胶质细胞标志物OX-42共表达。(6)连续7天鞘内注射AM1-50,可引起脊髓背角磷酸化p38蛋白表达增加；鞘内联合注射小胶质细胞选择性抑制剂米诺环素可抑制慢性应用AM1-s0诱发的痛觉过敏,抑制p38磷酸化水平增加,抑制促炎性细胞因子IL-1β、IL-6、TNF-α mRNA表达增加。以上结果表明：AM受体通过调节兴奋性氨基酸(谷氨酸和天门冬氨酸)和促炎性因子IL-1β、IL-6和TNF-α合成参与吗啡耐受。AM受体通过直接方式引起星型胶质细胞和小胶质激活,促使其增加IL-1β、IL-6和TNF-α合成,引起吗啡耐受。其作用机制之一是通过小胶质细胞p38MAPK信号通路介导。
[Abstract]:Adrenomedullin (adrenomedullin, AM) is a member of calcitonin gene-related peptide (calcitonin gene related peptide, CGRP) family. It has similar function with CGRP. AM is expressed in the sensory neurons of dorsal horn and dorsal root ganglion. It is a kind of pain related peptide. Recent studies have found that chronic morphine administration through 渭 -opioid receptor and protein kinase C signaling pathway leads to upregulation of AM expression, leading to morphine tolerance, and intrathecal injection of AM receptor antagonist AM22-52 can reverse morphine tolerance. These results suggest that AM is involved in the development of morphine tolerance. However, the cytological mechanism for promoting morphine tolerance is unclear. To elucidate the cytological mechanism of AM involved in morphine tolerance. In this study, in vivo and in vitro, AM receptor antagonists and AMsiRNA were used to inhibit AM receptor, respectively. After blocking the function of AM receptor, the changes of intracellular excitatory amino acids and pro-inflammatory cytokines induced by morphine were observed. The cytological mechanism of AM regulating pro-inflammatory cytokines was studied by means of behavioral, real-time fluorescent quantitative PCR, Western blotting and immunofluorescence histochemistry, and the mechanism of AM involved in morphine tolerance was revealed. The results showed that: (1) intrathecal administration of AM receptor antagonist AM22-52 (10nmol) inhibited the increased release of excitatory amino acids (glutamate and aspartate) in spinal dorsal horn and plasma of morphine tolerant rats. It also inhibited the expression of pro-inflammatory cytokine IL-1 尾 -mil 6 mRNA in spinal dorsal horn. (2) AM siRNA (50nM) interfered with DRG explants cultured in vitro. The expression of pro-inflammatory cytokine (IL-1 尾) and IL-6 mRNA in DRG explants induced by chronic morphine administration (3.3 渭 M) was inhibited. (3) Intrathecal injection of AM receptor agonist AM1-50 (10 渭 g),) alone for 7 days could increase the expression of IL-1 尾 IL-6 and TNF- 伪 mRNA in the dorsal horn of spinal cord of rats. However, the expression of IL-1 尾 -IL-6 and TNF 伪 mRNA decreased in DRG. (4) Intrathecal injection of AM1-50, alone for 7 days increased the expression of GFAP and OX-42, the specific markers of astrocyte and microglia in the dorsal horn of spinal cord. Intrathecal injection of AM22-52, inhibited the increase of GFAP and OX-42 expression in the spinal dorsal horn of rats induced by chronic morphine. (5) Immunofluorescence double staining showed that CRLR and RAMP2, the binding components of AM receptor, and microglia markers in the dorsal horn of spinal cord of rats. (6) Intrathecal injection of AM1-50, for 7 days increased the expression of phosphorylated p38 protein in the dorsal horn of spinal cord. Intrathecal injection of minocycline, a selective microglia inhibitor, could inhibit hyperalgesia induced by chronic AM1-s0, increase the level of p38 phosphorylation, and inhibit the expression of pro-inflammatory cytokine IL-1 尾, IL-6, TNF- 伪 mRNA. These results suggest that the receptor of 1: AM participates in the activation of astrocytes and microglia by regulating the synthesis of excitatory amino acids (glutamic acid and aspartic acid) and the pro-inflammatory factors IL-1 尾, IL-6 and TNF- 伪. It increased the synthesis of IL-1 尾 -IL-6 and TNF- 伪 and induced morphine tolerance. One of its mechanisms is mediated by microglial p38MAPK signaling pathway.
【学位授予单位】：福建师范大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96

【共引文献】