褪黑素对布比卡因脊髓神经毒性的神经保护的作用机制

Neuroprotective mechanism of melatonin on spinal cord neurotoxicity of bupivacaine

目的:探讨褪黑素(MT)通过抑制小胶质细胞增殖、激活对布比卡因脊髓神经毒性大鼠模型的神经保护的作用机制。方法:选择24只SPF级雄性SD大鼠随机分为4组:假手术组(S组)、生理盐水组(N组)、布比卡因组(B组)、褪黑素与布比卡因共处理组(M组)。4组大鼠行鞘内置管后分别做如下处理:B组及M组各鞘内注射5%布比卡因0.12 mL/kg,N组鞘内注射生理盐水0.12 mL/kg,每组各给药3次,每次间隔90 min;M组鞘内注射后立即腹腔注射MT 12.5 mg/kg,B组及N组鞘内注射后立即腹腔注射与M组等量MT溶媒,各组均连续3 d给药,每天1次;S组鞘内置管后,不行鞘内注射,腹腔注射MT溶媒同B组。各组于给药后3 d取材,取腰膨大处脊髓组织,HE染色光镜下观察各组脊髓组织损伤情况,Western blotting法检测各组脊髓组织Bcl-2、Bax、Caspase-3、CD86、IL-1β的蛋白表达,免疫荧光法检测各组小胶质细胞的增殖情况。结果:与S组比较,N组脊髓组织损伤程度,Bcl-2、Bax、Caspase-3、CD86、IL-1β蛋白表达及IBA1免疫阳性细胞数无明显差异(均P>0.05)。与N组相比,B组和M组脊髓组织损伤程度较重,脊髓组织空泡化及损伤神经细胞增多,Bax、Caspase-3、CD86、IL-1β蛋白表达升高,Bcl-2蛋白表达降低,小胶质细胞增多(均P<0.05)。与B组比较,M组脊髓组织损伤较轻,神经细胞尼氏体溶解及噬神经现象较少,Bax、Caspase-3、CD86、IL-1β蛋白表达降低,Bcl-2蛋白表达升高,小胶质细胞增殖减少(均P<0.05)。结论:MT可以减轻大鼠鞘内注射布比卡因引起的脊神经毒性,减少脊髓组织细胞的凋亡,其机制可能与其抑制小胶质细胞增殖激活相关。

Objective:To explore the neuroprotective mechanism of melatonin(MT)on rats spinal cord neurotoxicity model of bupivacaine by inhibiting the proliferation and activation of microglial.Methods:24 SPF male SD rats were randomly divided into four groups:sham-operation group(S group),normal saline group(N group),bupivacaine group(B group),melatonin and bupivacaine co-treatment group(M group).After intrathecal catheterization,rats in B group received intrathecal injection of 5%bupivacaine(0.12 mL/kg)and group N received an equal volume of normal saline,3 times with an interval of 90 min.M group rats were intrathecally injected with 5%bupivacaine(0.12 mL/kg)and thereafter intraperitoneally injected with MT(12.5 mg/kg).B and N groups rats were also injected with a similar volume of MT vehicle,once a day for 3 consecutive days.S group rats were injected with MT vehicle only after intrathecal catheterization.Three days after drug administration,the spinal cord tissues at lumbar enlargement were collected from each group.HE staining was used to observe the injury of spinal cord tissues.The protein expressions of Bcl-2,Bax,Caspase-3,CD86 and IL-1βin spinal cord tissues were detected by western blotting.The proliferation of microglia was detected by immunofluorescence.Results:There were no significant differences with regard to above indexes between S and N groups(P>0.05).Compared with N group,the degree of spinal cord injury in group B and M was more severe,the spinal cord tissue vacuolation,the number of injured nerve cells and microglia,and the protein expression of Bax,Caspase-3,CD86 and IL-1βincreased in B and M groups,while the protein expression of Bcl-2 was decreased(P<0.05).Compared with B group,Nissl body dissolution,phagocytosis in nerve cells,the number of microglia,and the protein expressions of Bax,Caspase-3,CD86 and IL-1βwere decreased,whereas the protein expression of Bcl-2 was increased in M group(P<0.05).Histopathological study revealed severe spinal cord injury in B group but mild in M group.Conclusion:MT can reduce spinal neurotoxicity and spinal cord tissue cells apoptosis induced by intrathecal injection of bupivacaine,and the mechanism may be related to the inhibition of microglia proliferation.