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目的观察鞘内注射高选择性蛋白激酶A抑制剂H-89对慢性神经病理痛大鼠脊髓背角磷酸化环磷酸腺苷反应元件结合蛋白(pCREB)表达的影响。方法成年雌性SD大鼠58只,体重230-270g。采用右侧慢性坐骨神经结扎的方法建立慢性神经病理痛模型。第一部分,取28只模型大鼠随机分为4组(n=7),H1、H2、H4组分别单次鞘内注射1、2、4nmolH-89[用二甲亚砜(DMSO,10mmol/L)溶解成10μl],Con组单次鞘内注射10mmol/L DMSO10μl。给药前和给药后15、30、60min分别测定右侧50%缩足反射阈值(MWT)和热缩足反射潜伏期(TWL)。第二部分,取24只模型大鼠随机分成4组(n=6),Con组:单次鞘内注射10mmol/L DMSO10μl,H1、H2、H4组分别单次鞘内注射H-891、2、4nmol(10μl);另取6只大鼠实施假手术后,单次鞘内注射10mmol/LDMSO10μl(sham组)。第二部分大鼠给药后30min处死,取L4,5脊髓,免疫组织化学染色观察脊髓背角pCREB的表达。结果与给药前比较,H2组MWT给药后15min增加,H4组给药后15、30minMWT增加,TWL延长(P<0.05或0.01);与Con组比较,H2组MWT给药后15min增加,H4组给药后15、30minMWT增加,TWL延长(P<0.05或0.01)。与Con组比较,Sham、H1、H2和H4组脊髓背角pCREB免疫反应阳性神经元数量和表达降低(P<0.05或0.01)。结论鞘内注射H-89抑制了神经病理痛大鼠脊髓背角pCREB表达,PKA/CREB信号通路的激活参与了慢性神经病理痛的维持。
Objective To observe the effects of intrathecal injection of high selective protein kinase A inhibitor H-89 on the expression of phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB) in spinal dorsal horn of chronic neuropathic pain rats. Methods 58 adult female Sprague-Dawley rats weighing 230-270g. A chronic neuropathic pain model was established by ligating the right chronic sciatic nerve. In the first part, 28 rats were randomly divided into 4 groups (n = 7). The rats in H1, H2 and H4 groups were given intrathecal injection of 1, 2 and 4 nmol of H-89 [dimethylsulfoxide (DMSO, 10 mmol / L) dissolved into 10μl], Con group intrathecal injection of 10mmol / L DMSO10μl. The right 50% reduction threshold (MWT) and the thermal contraction foot reflex latency (TWL) were measured before administration and at 15, 30, 60 minutes after administration. In the second part, 24 rats were randomly divided into 4 groups (n = 6). Con group: intrathecal injection of 10mmol / L DMSO 10μl, H-891,2 , 4nmol (10μl); another 6 rats were sham operated, a single intrathecal injection of 10mmol / LDMSO10μl (sham group). The second part of the rats were killed 30min after administration, L4,5 spinal cord was taken and the expression of pCREB in spinal dorsal horn was observed by immunohistochemical staining. Results Compared with preconditioning group, MWT of H2 group increased 15 min after administration of MWT, MWT increased and TWL prolonged 15 min and 30 min after H4 administration (P <0.05 or 0.01) The MWT increased and TWL prolonged at 15 and 30 minutes after H4 administration (P <0.05 or 0.01). Compared with Con group, the number and expression of pCREB immunoreactive neurons in spinal dorsal horn of Sham, H1, H2 and H4 groups decreased (P <0.05 or 0.01). Conclusion Intrathecal injection of H-89 inhibits the expression of pCREB in the spinal dorsal horn of neuropathic pain rats and the activation of PKA / CREB signaling pathway is involved in the maintenance of chronic neuropathic pain.