脊髓Pellino1敲减对长春新碱诱导大鼠神经病理性痛的影响

Effect of Pellino1 knockdown in rat spinal cords on neuropathic pain induced by vincristine

目的探讨敲减脊髓Pellino1(Peli1)对长春新碱诱导大鼠神经病理性痛的影响及其机制。方法将正常大鼠行鞘内置管手术,术后随机分为干扰短发夹RNA(shscr)组和Peli1短发夹RNA(sh Peli1)组,分别于鞘内置管术后第2天(D2)至D4每天1次鞘内注射表达shscr或sh Peli1的慢病毒。将正常大鼠行鞘内置管手术,术后随机分为正常对照组、化疗诱导神经病理性痛(CINP)组、CINP+shscr组和CINP+sh Peli1组,除正常对照组外,其余各组从D5开始隔日ip给予长春新碱125 mg·kg^(-1)(共4次)建立CINP模型,CINP+shscr组和CINP+sh Peli1组大鼠分别于D2~D4每天1次鞘内注射shscr和sh Peli1。分别采用机械缩足反射阈值(MWT)和热缩足反射潜伏期(TWL)评价大鼠机械痛觉过敏和热痛觉过敏;Western印迹法检测大鼠脊髓Peli1、小胶质细胞标志物离子钙接头蛋白1(Iba1)和星形胶质细胞标志物胶质细胞原纤维酸性蛋白(GFAP)表达水平及p38丝裂原活化蛋白激酶(p38 MAPK)、c-Jun氨基末端激酶(JNK)和细胞外信号调节激酶1/2(ERK1/2)磷酸化水平;免疫荧光化学检测脊髓组织切片Iba1蛋白表达水平;RT-PCR法检测脊髓Peli1和Iba1 mRNA表达水平;ELISA检测脊髓组织中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)和IL-1β含量。结果与shscr组相比,在D12,sh Peli组MWT和TWL无明显差异,而脊髓Peli1蛋白和mRNA表达均明显下调(P<0.05)。与正常对照组比较,CINP模型组大鼠在D8,D10和D12 MWT和TWL均明显降低(P<0.01);在D12,CINP模型组大鼠脊髓组织中Peli1和Iba1蛋白及mRNA表达水平、p38 MAPK,JNK和ERK1/2蛋白磷酸化水平及脊髓匀浆中TNF-α,IL-6和IL-1β含量均明显上调(P<0.01)。与CINP+shscr组比较,CINP+sh Peli1组在D8,D10和D12,MWT和TWL明显增加(P<0.05,P<0.01);在D12,脊髓Peli1和Iba1蛋白和mRNA表达水平、p38 MAPK,JNK和ERK1/2蛋白磷酸化水平及脊髓组织中TNF-α,IL-6和IL-1β含量均明显下调(P<0.05,P<0.01)。而在D12,各组大鼠脊髓GFAP蛋白和mRNA表达均无明显差异。结论敲减脊髓Peli1明显抑制长春新碱诱导的大鼠神经病理性痛,其机制可能与抑制脊髓小胶质细胞活化和MAPK信号通路介导的炎症反应有关。

OBJECTIVE To investigate the effect of Pellino1(Peli1)knockdown in spinal cords on neuropathic pain induced by vincristine and the mechanism.METHODS Normal rats were subjected to intrathecal catheterization and randomly divided into the interfering short hairpin RNA(shscr)and the Peli1 short hairpin RNA(sh Peli1)groups after surgery.Rats were injected intrathecally with lentivirus expressing shscr and sh Peli1 once daily from the 2 ndday after intrathecal catheterization(D2)to D4,respectively.Normal rats were subjected to intrathecal catheterization and randomly divided into normal control,CINP,CINP+shscr and CINP+sh Peli1 groups.An animal model of chemotherapy-induced neuropathic pain(CINP)was established via intraperitoneal injection of vincristine 125 mg·kg^(-1) on four alternate days beginning from D5 except in the normal control group.Rats were injected intrathecally with shscr and sh Peli1 once daily from D2-D4,respectively.Mechanical allodynia and heat hyperalgesia were evaluated by the mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL),respectively.The expression levels of astrocyte marker-glial fibrillary acidic protein(GFAP)and microglial marker-ionized calcium binding adapter molecule1(Iba1),as well as the phosphorylation levels of p38 mitogen-activated protein kinase(p38 MAPK),c-Jun N-terminal kinases(JNK)and extracellular signal-regulated kinases1/2(ERK1/2)in spinal cords of rats were detected by Western blotting.The expression level of Iba1 was detected by immunofluorescence chemistry in spinal cord tissue section.The mRNA relative expressions of Peli1,GFAP and Iba1 in spinal cords of rats were measured by RT-PCR.The contents of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in spinal cord homogenates of rats were determined by ELISA.RESULTS Compared with the shscr group,the protein and mRNA expressions of Peli1 in the spinal cord were obviously downregulated(P<0.05),but there was no significant difference in MWT or TWL in the sh Peli1 group on D12.Compared with the normal control group,MWT and TWL in the CINP group were significantly decreased on D8,D10 and D12(P<0.01).The protein and mRNA expression levels of Peli1 and Iba1,the phosphorylation levels of p38 MAPK,JNK and ERK1/2,and the contents of TNF-α,IL-6 and IL-1βin the spinal cord of rats were evidently upregulated in the CINP group on D12(P<0.01).Compared with the CINP+shscr group,MWT and TWL in the CINP+sh Peli1 group were significantly increased on D8,D10 and D12(P<0.05,P<0.01).The protein and mRNA expression levels of Peli1 and Iba1,phosphorylation levels of p38 MAPK,JNK and ERK1/2,as well as the contents of TNF-α,IL-6 and IL-1βin the spinal cord in the CINP+sh Peli1 group were evidently downregulated on D12(P<0.05,P<0.01).There was no significant difference in the protein and mRNA expressions of GFAP between these groups on D12.CONCLUSION Spinal Peli1 knockdown obviously inhibits neuropathic pain induced by vincristine,and the mechanism may be related to the inhibition of microglia activation and MAPK signaling pathway mediated inflammatory reaction in the spinal cord of rats.