脊髓背角神经元凋亡在炎性痛大鼠吗啡耐受中作用研究

STUDY ON THE EFFECT OF NEURONAL APOPTOSIS OF SPINAL DORSAL HORN ON ARTHRITIS RATS WITH CHRONIC MORPHINE TOLERANCE

目的:探讨NMDAR及谷氨酸转运体(GT)在阿片耐受机制中的作用,以及脊髓神经元凋亡在慢性阿片耐受形成机制中的意义。方法:将60只健康雄性SD大鼠行鞘内置管后随机分为6组(n=10),空白对照组(A)于左后足踝关节腔注射生理盐水50μl,其他5组注射CFA50μl。3d后分别经鞘内给予生理盐水10μl(A和B)、吗啡10μg(C组)、吗啡20μg(D组)、吗啡10μg+NM-DA受体抑制剂MK-80110nM(E组)、吗啡10μg+GT抑制剂PDC10μg(F组),1日2次,连续给药7d。检测大鼠左后肢机械缩爪阈值评价其痛行为学,于给药后第7天取出左侧腰膨大处脊髓进行TUNEL染色和Westernblotting检测。结果:C、D两组关节炎大鼠在鞘内给予吗啡第7天形成较稳定的吗啡耐受,MK-801和PDC分别对吗啡耐受的机械痛敏具有抑制和促进作用。B组大鼠脊髓背角凋亡细胞数及凋亡相关蛋白表达与A组比较,差异无统计学意义。与B组比较,C、D组大鼠脊髓背角的凋亡细胞数显著增加(P<0.01),Bax和caspase-3蛋白表达上调(P<0.01),Bcl-2表达下调(P<0.01)。与C组比较,E组大鼠脊髓背角凋亡细胞显著减少(P<0.05),Bax和caspase-3蛋白表达下调(P<0.01),Bcl-2表达上调(P<0.05);F组凋亡细胞增多(P<0.01),Bax和caspase-3蛋白表达上调(P<0.01),Bcl-2表达下调(P<0.01)。结论:脊髓神经元凋亡可能是慢性阿片耐受的神经基础,NMDAR及GT在阿片耐受的神经细胞凋亡过程中发挥作用。

Objective：To investigate the effect of NMDAR and glutamate transporter on the mechanism of morphine tolerance development,and to elucidate the implication of neuronal apoptosis of spinal dorsal horn in chronic morphine tolerance.Methods：60 healthy male SD rats implanted intrathecal catheters were randomized into 6 groups.The blank control group （A） was injected 50μl saline at left hind ankle while other five groups were injected 50μl CFA instead.All groups received respectively intrathecal injections of 10 μl saline （A and B）,10 μg morphine （C）,20 μg morphine（D）,10 μg morphine ＋10nM MK-801（E）,10 μg morphine ＋10 μg PDC（F）,twice daily for 7 days.Mechanical withdrawal threshold （MWT） of rats was examined to evaluate their behaviors.After MWT examination on day 7,the left lumbar intumescentias of rats were isolated for TUNEL stain and western blotting.Results：Morphine tolerance formed stably in group C and group D on day 7.MK-801 and PDC inhibited and enhanced mechanical hyperalgesia of morphine tolerance,respectively.There was no significant difference of spinal dorsal horn apoptotic cells and apoptosis related protein expression between group A and B.Compared with group B,apoptotic cells in group C and D increased （P0.01）;the expression of Bax and caspase-3 was upregulated （P0.01）,and Bcl-2 downregulated（P0.01）.In comparison with group C,apoptotic cells decreased in group E （P0.05） but increased in group F （P0.01）;the expression of Bax and caspase-3 in group E was downregulated but in group F was upregulated （P0.01）;the expression of Bcl-2 in group E and F was upregulated （P0.05） and downregulated （P0.01）,respectively.Conclusion：Spinal neural apoptosis may be the basis of chronic opioid tolerance developing.NMDAR and GT contribute to the neural apoptosis in opioid tolerance.