脊髓哺乳动物雷帕霉素靶蛋白信号通路参与大鼠外周神经损伤诱发的痛觉过敏
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摘要
目的:探讨哺乳动物雷帕霉素(rapamycin,RAPA)靶蛋白(mammalian target of RAPA,m TOR)信号通路是否通过激活脊髓背角星形胶质细胞参与外周神经损伤诱发的大鼠痛觉过敏。方法:取健康雄性Sprague-Dawley(SD)大鼠30只,随机分为6组(n=5):1 d组(D1组)、4 d组(D4组)、7 d组(D7组)、14 d组(D14组)、正常组、假手术组。其中D1,D4,D7,D14组建立坐骨神经慢性压榨损伤(chronic constriction injury,CCI)模型,Normal组不做处理,Sham组仅暴露坐骨神经。于CCI术后第1,4,7,14天分别测定各组大鼠左后肢机械缩足阈值(paw withdrawal mechanical threshold,PWMT)和热缩足潜伏期(paw withdrawal thermal latency,PWTL)。D1,D4,D7,D14组分别于CCI术后第1,4,7,14天,假手术组和正常组于相应第14天采集腰段脊髓。采用免疫组织化学法观察m TOR在大鼠脊髓的分布,采用real-time PCR和蛋白质印迹法检测CCI大鼠腰段脊髓m TOR mRNA和蛋白的表达。另取雄性SD大鼠30只,完成鞘内置管后,随机分为6组(n=5):空白组、CCI组、早给药组(CCI+early RAPA组)、早溶剂组[CCI+early二甲基亚砜(dimethylsulfoxide,DMSO)组]、晚给药组(CCI+later RAPA组)、晚溶剂组(CCI+later DMSO组)。空白组不建立CCI模型也不给药;CCI组建立左后肢CCI模型;CCI+early RAPA组于CCI术后4 h开始鞘内注射1%RAPA 10μL,连续给药3 d;CCI+early DMSO组于CCI术后4 h开始鞘内注射同浓度等体积溶剂4%DMSO 10μL作为对照;CCI+later RAPA组于CCI术后第7天开始鞘内注射1%RAPA 10μL,连续给药3 d;CCI+later DMSO组于CCI术后第7天开始鞘内注射同浓度等体积溶剂DMSO10μL作为对照。各组于鞘内置管前后以及CCI术后隔天测定痛阈。于CCI术后第14天取腰段脊髓,免疫组织化学检测脊髓背角胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达。结果:m TOR免疫组织化学阳性颗粒广泛分布于正常脊髓神经元胞浆中;D14组大鼠术后第1,4,7,14天的PWMT与其基础值比较明显降低,术后第4,7,14天的PWTL与其基础值比较明显降低(P<0.05或P<0.01);与正常组比较,CCI组(D1,D4,D7和D14组)大鼠腰段脊髓m TOR的mRNA和蛋白表达显著增加(P<0.05或P<0.01);与CCI+early DMSO组相同时间点比较,CCI+early RAPA组CCI术后第4,6,8,10,12,14天的PWMT和PWTL均升高(P<0.05或P<0.01);与CCI+later DMSO组相同时间点比较,CCI+later RAPA组CCI术后第8,10,12,14天的PWMT和PWTL均升高(P<0.05或P<0.01);CCI+early RAPA组与CCI+early DMSO组、CCI+later RAPA组与CCI+later DMSO组相比较,CCI大鼠术侧腰段脊髓背角GFAP免疫组织化学阳性面积与吸光度值均下降(均P<0.05或P<0.01)。结论:脊髓m TOR信号通路可能通过脊髓背角星形胶质细胞活化参与外周神经损伤诱发的痛觉过敏。
Objective:To investigate whether mammalian target of rapamycin(mTOR) signaling pathway is involved in peripheral nerve injury-induced hyperalgesia through activation of spinal dorsal astrocytes in rats.Methods:A total of 30 male Sprague-Dawley(SD) rats were randomly divided into 6 groups(n=5):the 1 day group(D1 group),the 4 days group(D4 group),the 7 days group(D7 group),the 14 days group(D14 group),the normal group and the sham group.The sciatic nerve chronic constriction injury(CCI) model was established in the D1,D4,D7 and D14 group.The normal group received no treatment while the sham group was only exposed the sciatic nerve.Paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency(PWTL) were measured at the 1 st,4 th,7 th,and 14 th day after CCI in the different groups.Lumbar spinal cord were harvested on the 1 st,4 th,7 th and 14 th day in the D1,D4,D7,D14 group correspondingly,which were harvested on the 14 th day in the normal group and the sham group.Distribution of mTOR in rat spinal cord was assessed by immunohistochemistry.The expressions of mTOR mRNA and protein in the spinal cord in different groups were determined by real-time PCR and Western blotting,respectively.Another 30 male intrathecal catheterized SD rats were randomly divided into 6 groups(n=5):a blank group,a CCI group,a CCI+early rapamycin(RAPA) group,a CCI+early dimethylsulfoxide(DMSO) group,a CCI+ later RAPA group,and a CCI+later DMSO group.The blank group didn't received any treatment;The CCI group was carried out the treatment of CCI model in the left hind limbs.10 μL of 1% RAPA was given to the CCI+early RAPA group intrathecally at 4 hours after CCI for 3 days;the CCI+later RAPA group were treated with the same dose of RAPA on the 7 th days after CCI for 3 days;the CCI+early DMSO group and the CCI+later DMSO group were injected with the same volume of 4% DMSO at the corresponding time as controls.The PWTL and PWMT were measured before and after intrathecal catheterization,and every other day after CCI.The lumbar spinal cords were selected and the expression of glial fibrillary acidic protein(GFAP) in spinal dorsal horn were examined by immunohistochemistry in the 14 th day after CCI.Results:The immunohistochemistry positive particles of mTOR were widely distributed in the cytoplasm of the normal spinal neurons.Compared with the base line,the PWMT in the D14 group on the 1 st,4 th,7 th and 14 th day after CCI were significantly lower,and the PWTL on the 4 th,7 th and 14 th day after CCI were also significantly lower(P<0.05 or P<0.01).The expressions of mTOR mRNA and protein in the CCI groups(D1,D4,D7 and D14 group) were significantly increased than those in the normal group(P<0.05 or P<0.01).Compared with the CCI+early DMSO group,the PWMT and PWTL in the CCI+early RAPA group were obviously increased on 4 th,6 th,8 th,10 th,12 th or 14 th day after CCI(P<0.05 or P<0.01);compared with the CCI+later DMSO group,the PWMT and PWTL in the CCI+later RAPA group were also significantly increased at the 8 th,10 th or 14 th day after CCI(P<0.01 or P<0.05).The GFAP immunohistochemistry positive area and absorbance value in the dorsal horn of the lumbar spinal cord in the CCI rats were decreased in the CCI+early RAPA group compared with the CCI+early DMSO group(P<0.05 or P<0.01),and which were also decreased in the CCI+later RAPA group compared with the CCI+later DMSO group(P<0.05 or P<0.01).Conclusion:mTOR signaling pathway may be involved in hyperalgesia induced by peripheral nerve injury via spinal astrocyte activation in the dorsal horn of the spinal cord.
Objective:To investigate whether mammalian target of rapamycin(mTOR) signaling pathway is involved in peripheral nerve injury-induced hyperalgesia through activation of spinal dorsal astrocytes in rats.Methods:A total of 30 male Sprague-Dawley(SD) rats were randomly divided into 6 groups(n=5):the 1 day group(D1 group),the 4 days group(D4 group),the 7 days group(D7 group),the 14 days group(D14 group),the normal group and the sham group.The sciatic nerve chronic constriction injury(CCI) model was established in the D1,D4,D7 and D14 group.The normal group received no treatment while the sham group was only exposed the sciatic nerve.Paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency(PWTL) were measured at the 1 st,4 th,7 th,and 14 th day after CCI in the different groups.Lumbar spinal cord were harvested on the 1 st,4 th,7 th and 14 th day in the D1,D4,D7,D14 group correspondingly,which were harvested on the 14 th day in the normal group and the sham group.Distribution of mTOR in rat spinal cord was assessed by immunohistochemistry.The expressions of mTOR mRNA and protein in the spinal cord in different groups were determined by real-time PCR and Western blotting,respectively.Another 30 male intrathecal catheterized SD rats were randomly divided into 6 groups(n=5):a blank group,a CCI group,a CCI+early rapamycin(RAPA) group,a CCI+early dimethylsulfoxide(DMSO) group,a CCI+ later RAPA group,and a CCI+later DMSO group.The blank group didn't received any treatment;The CCI group was carried out the treatment of CCI model in the left hind limbs.10 μL of 1% RAPA was given to the CCI+early RAPA group intrathecally at 4 hours after CCI for 3 days;the CCI+later RAPA group were treated with the same dose of RAPA on the 7 th days after CCI for 3 days;the CCI+early DMSO group and the CCI+later DMSO group were injected with the same volume of 4% DMSO at the corresponding time as controls.The PWTL and PWMT were measured before and after intrathecal catheterization,and every other day after CCI.The lumbar spinal cords were selected and the expression of glial fibrillary acidic protein(GFAP) in spinal dorsal horn were examined by immunohistochemistry in the 14 th day after CCI.Results:The immunohistochemistry positive particles of mTOR were widely distributed in the cytoplasm of the normal spinal neurons.Compared with the base line,the PWMT in the D14 group on the 1 st,4 th,7 th and 14 th day after CCI were significantly lower,and the PWTL on the 4 th,7 th and 14 th day after CCI were also significantly lower(P<0.05 or P<0.01).The expressions of mTOR mRNA and protein in the CCI groups(D1,D4,D7 and D14 group) were significantly increased than those in the normal group(P<0.05 or P<0.01).Compared with the CCI+early DMSO group,the PWMT and PWTL in the CCI+early RAPA group were obviously increased on 4 th,6 th,8 th,10 th,12 th or 14 th day after CCI(P<0.05 or P<0.01);compared with the CCI+later DMSO group,the PWMT and PWTL in the CCI+later RAPA group were also significantly increased at the 8 th,10 th or 14 th day after CCI(P<0.01 or P<0.05).The GFAP immunohistochemistry positive area and absorbance value in the dorsal horn of the lumbar spinal cord in the CCI rats were decreased in the CCI+early RAPA group compared with the CCI+early DMSO group(P<0.05 or P<0.01),and which were also decreased in the CCI+later RAPA group compared with the CCI+later DMSO group(P<0.05 or P<0.01).Conclusion:mTOR signaling pathway may be involved in hyperalgesia induced by peripheral nerve injury via spinal astrocyte activation in the dorsal horn of the spinal cord.
引文
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[18]Takuma K,Matsuda T,Hashimoto H,et al.Role of Na+-Ca2+exchanger in agonist-induced Ca2+signaling in cultured rat astrocytes[J].J Neurochem,1996,67(5):1840-1845.
[19]Takeda K,Sawamura S,Tamai H,et al.Role for cyclooxygenase 2 in the development and maintenance of neuropathic pain and spinal glial activation[J].Anesthesiology,2005,103(4):837-844.
[20]DubovyP,KlusákováI,Hradilová-Sví?enskáI,et al.Activation of astrocytes and microglial cells and CCL2/CCR2 upregulation in the dorsolateral and ventrolateral nuclei of periaqueductal gray and rostral ventromedial medulla following different types of sciatic nerve injury[J].Front Cell Neurosci,2018,12:40.
[21]Cao H,Zhang YQ.Spinal glial activation contributes to pathological pain states[J].Neurosci Biobehav Rev,2008,32(5):972-983.
[2]Campbell JN,Meyer RA.Mechanisms of neuropathic pain[J]Neuron,2006,52(1):77-92.
[3]Laplante M,Sabatini DM.mTOR signaling in growth control and disease[J].Cell,2012,149(2):274-293.
[4]Jaworski J,Sheng M.The growing role of m TOR in neuronal development and plasticity[J].Mol Neurobiol,2006,34(3):205-219.
[5]Bennett GJ,Xie YK.A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man[J].Pain1988,33(1):87-107.
[6]Hou Y,Wang L,Gao J,et al.A modified procedure for lumbar intrathecal catheterization in rats[J].Neurol Res,2016,38(8):725-732.
[7]Yaksh TL,Rudy TA.Chronic catheterization of the spinal subarachnoid space[J].Physiol Behav,1976,17(6):1031-1036.
[8]Magnuson B,Ekim B,Fingar DC.Regulation and function of ribosomal protein S6 kinase(S6K)within m TOR signalling networks[J].Biochem J,2012,441(1):1-21.
[9]Loewith R,Jacinto E,Wullschleger S,et al.Two TOR complexes only one of which is rapamycin sensitive,have distinct roles in cell growth control[J].Mol Cell,2002,10(3):457-468.
[10]Yip CK,Murata K,Walz T,et al.Structure of the human mTORcomplex I and its implications for rapamycin inhibition[J].Mol Cell2010,38(5):768-774.
[11]Banko JL,Poulin F,Hou L,et al.The translation repressor 4E-BP2 is critical for eIF4F complex formation,synaptic plasticity,and memory in the hippocampus[J].J Neurosci,2005,25(42):9581-9590.
[12]Melemedjian OK,Khoutorsky A.Translational control of chronic pain[J].Prog Mol Biol Transl Sci,2015,131:185-213.
[13]Duan Z,Li J,Pang X,et al.Blocking mammalian target of rapamycin(m TOR)alleviates neuropathic pain induced by chemotherapeutic bortezomib[J].Cell Physiol Biochem,2018,48(1):54-62.
[14]Asante CO,Wallace VC,Dickenson AH.Formalin-induced behavioural hypersensitivity and neuronal hyperexcitability are mediated by rapid protein synthesis at the spinal level[J].Mol Pain2009,5(1):27.
[15]Géranton SM,Jiménez-Díaz L,Torsney C,et al.A rapamycinsensitive signaling pathway is essential for the full expression of persistent pain states[J].J Neurosci,2009,29(47):15017-15027.
[16]Obara I,Medrano MC,Signoret-Genest J,et al.Inhibition of the mammalian target of rapamycin complex 1 signaling pathway reduces itch behaviour in mice[J].Pain,2015,156(8):1519-1529.
[17]Wieseler-Frank J,Maier SF,Watkins LR.Glial activation and pathological pain[J].Neurochem Int,2004,45(2/3):389-395.
[18]Takuma K,Matsuda T,Hashimoto H,et al.Role of Na+-Ca2+exchanger in agonist-induced Ca2+signaling in cultured rat astrocytes[J].J Neurochem,1996,67(5):1840-1845.
[19]Takeda K,Sawamura S,Tamai H,et al.Role for cyclooxygenase 2 in the development and maintenance of neuropathic pain and spinal glial activation[J].Anesthesiology,2005,103(4):837-844.
[20]DubovyP,KlusákováI,Hradilová-Sví?enskáI,et al.Activation of astrocytes and microglial cells and CCL2/CCR2 upregulation in the dorsolateral and ventrolateral nuclei of periaqueductal gray and rostral ventromedial medulla following different types of sciatic nerve injury[J].Front Cell Neurosci,2018,12:40.
[21]Cao H,Zhang YQ.Spinal glial activation contributes to pathological pain states[J].Neurosci Biobehav Rev,2008,32(5):972-983.