血红素加氧酶1对糖尿病大鼠脊髓背角神经元凋亡及痛敏的影响

Effect of Heme Oxygenase-1 on Apoptosis in Spinal Dorsal Horn Neurons and Neuropathic Pain in Diabetic Rats

目的探讨血红素加氧酶1(heme oxygenase-1,HO-1)对链脲菌素(streptozotocin,STZ)诱导的糖尿病大鼠脊髓背角神经元凋亡和神经病理性疼痛的影响以及两者之间可能存在的关系。方法成年雄性SPF级Wistar大鼠24只,体重180～220g,随机分为3组(n=8/组):正常对照组(C组),糖尿病组(B组),Hemin干预组(A组)。A组、B组单次腹腔注射链脲菌素65mg/kg建立糖尿病模型。建模成功后,A组Hemin25μmol/kg隔天腹腔注射1次,连续6周,B、C组同一时间腹腔注射等体积生理盐水。于注射STZ前以及注射STZ后每周测定大鼠后肢机械性缩足反应阈值(mechanical withdrawal threshold,MWT),6周后麻醉下取坐骨神经电镜观察病理学变化,处死大鼠取L4-6脊髓,尼氏体染色法光镜下观察脊髓背角组织的形态学变化,免疫组化法测定脊髓背角HO-1蛋白的表达,原位末端标记法(TdT-mediated dUTP nick end labeling,TUNEL法)检测脊髓背角凋亡神经元并计算凋亡指数。结果 A组大鼠MWT于注射STZ后28d时较B组明显增加(P<0.05),35d时显著增加(P<0.01),42d达峰值;A组大鼠于注射STZ后42d时脊髓背角神经元中HO-1的表达较B组增强,而B组较C组也有所增强;A组脊髓背角病理改变程度、坐骨神经髓鞘病变程度及脊髓背角神经元细胞凋亡程度均较B组轻微。结论糖尿病神经病理性疼痛大鼠中存在着脊髓背角神经元的凋亡,HO-1表达上调可对其有一定的抑制作用,并能够减轻神经病理性疼痛,糖尿病大鼠痛敏的形成与脊髓背角神经元凋亡之间可能存在着一定的关系。

Objective To investigate the effect of heme oxygenase-1（HO-1） on apoptosis in spinal dorsal horn neurons and neuropathic pain in diabetic rats,and to research the possible relationship between neuropathic pain and the neurons apoptosis.Methods Twenty-four male SPF Wistar rats,weighing 180-220g,were randomly divided into 3 groups（n=8/group）：group C（normal group）,group B（rats with diabetes mellitus） and group A（rats with diabetes mellitus accepting Hemin treatment）.In groups A and B,65mg/kg streptozocin（STZ） was injected intraperitoneally（IP） to induce diabets mellitus.Group C received equal volume of buffered saline instead.Three days later blood glucose concentration was measured.In groups A and B,rats with blood glucose 16.7mmol/L were excluded from the study.Group A were injected Hemin 25μmol/kg q.2d for 6 weeks.The mechanical withdrawal threshold（MWT） of the paw was measured with von Frey filaments before STZ injection and at 1,2,3,4,5 and 6 weeks after STZ injection.Rats were then sacrificed,and the lumbar segments（L4-6） of the spinal cord were removed.Pathologic changes were observed by Nissal＇s staining under electronic microscope.HO-1 expression was detected by immuno-histochemistry.Apoptosis in the dorsal horn neurons was detected by TdT-mediated dUTP nick end labeling（TUNEL）.Results The MWT of group A was increased significantly than group B at 4 weeks after STZ injection（P〈0.05） and at 5 and 6 weeks after STZ injection（P〈0.01）.The HO-1 expression in group A was significantly higher than that in group B which was higher than in group C.Compared with group B,the histopathological damage to the neurons and the destruction of sciatic nerve myelin sheath was lighter and the apoptotic index was lower in group A.Conclusion HO-1 can reduce neuronal apoptosis in spinal dorsal horn and attenuate neuropathic pain in diabetic rats.Neuropathic pain and the neurons apoptosis have a certain relationship.