【作者】 潘韫丹；

【导师】 黄晓元；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 目的1.观察鞘内注射核转录因子-κB(nuclear factor-kappaB,NF-κB)抑制剂吡咯烷二硫氨基甲酸(PDTC)对坐骨神经慢性挤压伤(chronic constriction injury,CCI)大鼠痛阈和脊髓NF-κBp-p65、小胶质细胞活性、CX3CR1、p-p38MAPK、TNFα表达的影响。2.观察NF-κB抑制剂吡咯烷二硫氨基甲酸(PDTC)对体外TNFα诱导的BV-2细胞CX3CR1基因和蛋白表达的影响。3.观察鞘内注射白藜芦醇(Resveratrol,Res)对坐骨神经慢性挤压伤(CCI)大鼠痛阈和脊髓NF-κB-p65、小胶质细胞活性、CX3CR1、TNFα表达的调节,并探讨其对临床疼痛治疗的机制。方法1.288只成年雄性SD大鼠,体质量250～300 g,鞘内置管成功后,随机分8组(n=36):正常对照组:实验大鼠不做任何处置;假手术组:仅暴露左侧坐骨神经不结扎;假手术+PDTC组:假手术处理大鼠并经鞘内置管注入PDTC1000pmol/d;CCI组:大鼠CCI手术处理;CCI+PDTC组1:CCI处理大鼠分为2个亚组,分别在CCI术前1天和CCI第3天开始经鞘内置管注入PDTC100pmol/d;CCI+PDTC组2:CCI处理大鼠分为2个亚组,分别在术前1天和CCI第3天开始鞘内注入PDTC1000pmol/d;假手术+盐水组:假手术处理大鼠并鞘内注入生理盐水;CCI+盐水组:CCI术处理大鼠并经鞘内注入生理盐水。鞘内置管5 d后按每组处理不同,开始鞘内输注生理盐水和不同剂量的PDTC,鞘内注射1 d后分别建立大鼠CCI神经病理性疼痛模型和假手术模型,测定大鼠的痛敏值,并在鞘内注射4d后取脊髓腰膨大部进行免疫组化测定NF-κBp-p65、OX42、TNFα表达、Western Blot检测CX3CR1、p-p38MAPK蛋白表达、RT-PCR检测CX3CR1mRNA的表达、并且进行脊髓切片HE染色。2.采用体外培养的永生态小胶质细胞BV-2细胞,设立对照组和实验组,实验组经TNFα(20ng/ml)、TNFα+PDTC、PDTC孵育,于10min,20min,40min,60min,2h,3h,4h,6h,8h采用MTT方法和倒置显微镜分别测定小胶质细胞的生长率及形态改变,免疫细胞化学测定NF-κBp-p65表达,Western blot和RT-PCR检测CX3CR1蛋白和mRNA表达情况。3.252只成年雄性SD大鼠,体质量250～300 g,鞘内置管成功后,随机分7组(n=36):正常对照组:实验大鼠不做任何处置;假手术组:仅暴露左侧坐骨神经不结扎;假手术+Res组:假手术处理大鼠经鞘内置管注入Res;CCI组;CCI+Res组:大鼠分为2个亚组,分别在CCI术前1天和CCI第3天开始经鞘内置管注入Res500ug/d;假手术+盐水组:假手术处理大鼠并鞘内注入生理盐水;CCI+盐水组:CCI术处理大鼠并经鞘内注入生理盐水。按各组处理,鞘内置管5d后开始分别鞘内输注生理盐水和Res,鞘内注射1d后分别建立大鼠CCI神经病理性疼痛模型和假手术模型,测定大鼠的痛敏值,并在鞘内注射4 d后取脊髓腰膨大部进行免疫组化测定NF-κBp-p65、OX42、TNFα表达、Western Blot检测CX3CR1蛋白表达、并进行脊髓切片HE染色。结果1.CCI组与sham组比较大鼠术后各时点痛敏阈值明显下降,并在术后第3日降至最低点(P＜0.05),脊髓CX3CR1、p-p38MAPK、p-p65、OX42、YNFα的表达明显升高(P＜0.05)。PDTC+CCI组与CCI组比较,术后各时点大鼠痛敏阈值增高,脊髓CX3CR1、p-p38MAPK、p-p65、OX42、TNFα的表达下降(P＜0.05)。HE染色假手术+PDTC组与假手术+盐水组比较,未见明显脊髓组织病理损伤。2.TNFα刺激后BV-2细胞形态突起增粗;刺激20 min内NF-κBp-p65核表达细胞显著增多(P＜0.05),1 h达高峰,TNFα诱导的CX3CR1蛋白和mRNA表达分别于4h和2h达到高峰,PDTC(100μmol/l)可抑制TNFα诱导的NF-κBp-p65核表达,明显降低了TNFα诱导的CX3CR1蛋白和mRNA表达。3.CCI组与sham组比较大鼠术后各时点痛敏阈值明显下降,并在术后第3日降至最低点(P＜0.05),脊髓CX3CR1、p-p65、OX42、TNFα的表达明显升高(P＜0.05)。Res+CCI组与CCI组比较,术后各时点大鼠痛敏阈值增高,脊髓CX3CR1、p-p65、OX42、TNFα的表达下降(P＜0.05)。HE染色假手术+Res组与假手术+盐水组比较,未见明显脊髓组织病理损伤。结论1.鞘内给予PDTC可剂量依赖性地减轻CCI大鼠的病理性疼痛,并抑制脊髓CX3CR1、p-p38MAPK、p-p65、TNFα的表达和小胶质细胞活性。活化NF-κB通路可能通过上调脊髓CX3CR1、p-p38MAPK、TNFα的表达和小胶质细胞活性而参与了神经病理性疼痛的发生。2.TNFα可激活BV-2细胞NF-κB通路,改变细胞形态,使细胞体积增大突起增粗,增加细胞CX3CR1蛋白和mRNA表达,其激活的细胞内机制可能与核因子-κB的活化有关。3.鞘内给予Res可减轻CCI大鼠的病理性疼痛,并抑制脊髓CX3CR1、p-p65、TNFα的表达和小胶质细胞活性,这可能成为Res止痛作用的机制之一。更多还原

【Abstract】 Objectives:1.To examine the effects of intrathecal nuclear factor-kappaB(NF-κB) inhibitor,pyrrolidine dithiocarbamate(PDTC),on the development of neuropathic pain,spinal microglial activation and spinal NF-κBp-p65、CX3CR1、p-p38MAPK、tumor necrosis factor(TNF)αexpression induced by sciatic chronic constriction injury(CCI) in rats. 2.To examine the effects of NF-κB inhibitor PDTC on TNFα-induced CX3CR1 expression in BV-2 microglial cells.3.To examine the effects of intrathecal resveratrol(Res) on the development of neuropathic pain, spinal microglial activation and spinal NF-κBp-p65、CX3CR1、TNFαexpression induced by sciatic CCI in rats,and investigate the therapeutic potential of Res in neuropathic pain.Methods 1.288 male Sprague-Dawley(SD) rats(250～300 g) fitted with intrathecal(i.t.) catheters,underwent surgery.Randomly,rats were divided into 8 groups (n=36):normal control group:rats didn’t undergo any surgery;sham group:rats underwent surgery of exposing sciatic nerve without ligation; sham+PDTC group:rats undergoing sham surgery were intrathecally administrated PDTC(1000pmol/d);CCI group:rats underwent sciatic CCI surgery;CCI+PDTC group 1:Rats were divided into 2 subgroups, and intrathecal PDTC(100pmol/d) was infused 1 day before and 3 days after sciatic CCI respectively;CCI+PDTC group 2:Rats were divided into 2 subgroups,and intrathecal PDTC(1000pmol/d) was infused 1 day before and 3 days after sciatic CCI respectively;sham+saline group: Intrathecal normal saline(NS) was infused 1 day before and 3 days after sham surgery respectively;CCI+saline group:Intrathecal NS was infused 1 day before and 3 days after sciatic CCI,respectively.5 days after intrathecal catheters implantation,rats were administrated various intrathecal medicine.And 1 day after administration,rats experiented CCI and sham surgery,respectively.The rat hind paw withdrawal threshold(WT) to mechanical stimuli and withdrawal latency(WL) to radiant heat were determined before surgery and from day 1 to 7 following CCI.4 days after administration,the spinal cord around lumbar enlargement was removed.Spinal microglial activation was evaluated with OX-42 immunoreactivity and spinal NF-κBp-p65、TNFαexpression were determined by immunohistochemisty,spinal CX3CR1、p-p38MAPK expression were assessed by western blotting,spinal CX3CR1 mRNA expression was assessed by reverse transcriptase polymerase chain reation(RT-PCR),and spinal cord sections were also stained with hematoxylin and eosin(HE).2.BV-2 microglial cells were cultured in vitro.For experiments,cells were treated with TNFα(20ng/ml) as the experimental group,TNFα(20ng/ml) and PDTC(100μmol/l) as TNFα+PDTC group,PDTC(100μmol/l) as PDTC group,and serum-free medium as the control group.At the time point of 10min,20min,40min, 60min,2h,3h,4h,6h,8h after treatment,we used MTT,the inverted microscope,RT-PCR,immunocytochemistry and western blotting to assess cell viability,morphology changes,NF-κBp-p65 protein expression、CX3CR1 protein and mRNA expression.3.252 male Sprague-Dawley(SD) rats(250～300 g) fitted with intrathecal catheters, underwent surgery.Randomly,rats were divided into 7 groups(n=36): normal control group:rats didn’t undergo any surgery;sham group:rats underwent surgery of exposing sciatic nerve without ligation;sham+Res group:rats with sham surgery were intrathecally administrated Res (500ug/d);CCI group:rats underwent sciatic CCI surgery;CCI+Res group:Rats were divided into 2 subgroups and intrathecal Res(500ug/d) was infused 1 day before and 3 days after sciatic CCI respectively; sham+saline group:Intrathecal normal saline(NS) was infused 1 day before and 3 days after sham surgery,respectively;CCI+saline group: Rats were divided into 2 subgroups and intrathecal NS was infused 1 day before and 3 days after sciatic CCI respectively.5 days after intrathecal catheters implantation,rats were administrated various intrathecal medicine.And 1 day after administration,rats experiented sciatic CCI and sham surgery according to groups arrangement.The rat hind paw withdrawal threshold to mechanical stimuli and withdrawal latency to radiant heat were determined before surgery and from day 1 to 7 following CCI.4 days after administration,the spinal cord around lumbar enlargement was removed.Spinal microglial activation was evaluated with OX-42 immunoreactivity and spinal NF-κBp-p65、TNFαexpression were determined by immunohistochemisty,spinal CX3CR1 expression was assessed by western blotting and spinal cord sections were also stained with hematoxylin and eosin.Results 1.Compared with sham group,a significant decrease in WT and WL was observed after surgery in CCI group,and the lowest point was 3 days after surgery(P＜0.05), spinal CX3CR1、p-p38MAPK、p-p65、OX42、TNFαexpression were significantly increased(P＜0.05).The WTs and WLs of PDTC+CCI group were significantly increased compared with CCI group,spinal CX3CR1、p-p38MAPK、p-p65、OX42,TNFαexpression were significantly decreased(P＜0.05).HE staining didn’t show obvious spinal histopathologic change in sham+Res group compard with sham+NS group.2.TNFα-stimulated BV-2 cells showed a thicker process,and a significant increase of NF-κBp-p65 expression in cell nuclear within 20 min(P＜0.05),peaked at 1 h.TNFα-induced CX3CR1 protein and mRNA expression increased significantly,peaked at 4h and 2h after treatment, and PDTC(100μmol/l) suppressed TNFα-induced NF-κBp-p65 expression,significantly downregulated TNFα-induced CX3CR1 protein and mRNA expression.3.Compared with sham group,a significant decrease in WT and WL was observed after surgery in CCI group,and the lowest point was 3 days after surgery(P＜0.05),spinal CX3CR1、p-p65、OX42、TNFαexpression were significantly increased(P＜0.05).The WTs and WLs of Res+CCI group were significantly increased compared with CCI group,spinal CX3CR1、p-p65、OX42、TNFαexpression were significantly decreased(P＜0.05).HE staining didn’t show obvious spinal histopathologic change in sham+Res group compard with sham+NS group.Conclusions:1.Intrathecal PDTC attenuated the CCI-induced neuropathic pain in rats,and suppressed spinal CX3CR1、p-p38MAPK、p-p65、TNFαexpression and microglial activation.And the activation of NF-κB pathway may contribute to spinal CX3CR1、p-p38MAPK、TNFαupregulation and microglial activation in neuropathic pain.2.TNFαrapidly activated the NF-κB pathway,changed the morphology of cells and upregulated CX3CR1 protein and mRNA expression in BV-2 microglial cells.And the morphological changes and upregulation of CX3CR1 expression may be mediated by NF-κB pathway.3.Inthathecal Res attenuated the CCI-induced neuropathic pain in rats,and suppressed spinal CX3CR1、p-p65、TNFαexpression and microglial activation.The regulation of spinal neuroinflammation may contribute to therapeutic potential of Res in neuropathic pain.更多还原