依托咪酯对脊髓损伤大鼠的脑保护作用及其机制研究

Cerebral protective effect of etomidate in rats with spinal cord injury and its mechanism

目的探究依托咪酯对脊髓损伤(SCI)大鼠的脑保护作用,并分析相关作用机制。方法62只SPF级SD雄性大鼠,取50只复制SCI大鼠(成功48只),将模型复制成功大鼠随机分为模型组及依托咪酯低、中、高剂量组,每组12只;剩余12只大鼠为假手术组(只去除T_(9)~T_(11)锥板,不进行打击锤锥击,其余操作同模型组)。依托咪酯低、中、高剂量组大鼠分别尾静脉注射0.3 mg/kg、0.9 mg/kg、2.7 mg/kg依托咪酯脂肪乳注射液1次/d,连续4周;假手术组和模型组大鼠同时间尾静脉注射等量生理盐水。用药4周后评估各组大鼠神经行为学;采用酶联免疫吸附试验(ELISA)试剂盒测定各组大鼠脑脊液白细胞介素1β(IL-1β)、白细胞介素18(IL-18)水平;苏木精-伊红(HE)染色观察各组大鼠脊髓组织病理学变化;实时荧光定量聚合酶链反应检测各组大鼠脊髓组织核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)和半胱氨酸天门冬氨酸蛋白水解酶-1(Caspase-1)mRNA的表达;Western blotting检测各组大鼠脊髓组织NLRP3和Caspase-1蛋白的表达。结果与假手术组比较,模型组及依托咪酯低、中、高剂量组大鼠BBB评分降低(P<0.05),脑脊液IL-1β、IL-18水平升高(P<0.05),脊髓组织NLRP3、Caspase-1 mRNA和蛋白相对表达量升高(P<0.05);但依托咪酯低、中、高剂量组大鼠BBB评分高于模型组(P<0.05),脑脊液IL-1β、IL-18水平低于模型组(P<0.05),脊髓组织NLRP3、Caspase-1 mRNA和蛋白相对表达量低于模型组(P<0.05),均具有剂量依赖性。结论依托咪酯可发挥SCI脑保护作用,其对中枢神经系统损伤的保护作用可能是通过抑制NLRP3/Caspase-1通路活化,抑制炎症反应实现的。

Objective To investigate the cerebral protective effect of etomidate in rats with spinal cord injury(SCI),and to analyze the underlying mechanism.Methods Fifty of the 62 SPF male SD rats were used to establish the SCI models,and the successfully-established rat models(n=48)were randomly divided into model group(n=12),low-dose etomidate group(n=12),medium-dose etomidate group(n=12)and high-dose etomidate group(n=12).The rest 12 rats were set as sham-operation group where only the T_(9) to T_(11) vertebral plates were removed without injuring the spinal cord.The low-,medium-and high-dose etomidate groups were injected 0.3 mg/kg,0.9 mg/kg,and 2.7 mg/kg of etomidate fat emulsion injection,respectively through the tail veins once a day for 4 weeks.The rats in sham-operation group and model group were injected with the same amount of normal saline via tail veins at the same time.The neurobehavioral scores were evaluated after 4 weeks,and the levels of interleukin-1(IL 1β)and IL-18 in the cerebral spinal fluid(CSF)were measured via enzyme-linked immunosorbent assay(ELISA).Hematoxylin and eosin(HE)staining was used to observe the histopathological changes of the spinal cord in each group.The mRNA levels of NLRP3 and Caspase-1 in spinal cord tissues of rats were detected by quantitative real time polymerase chain reaction(qRT-PCR).The protein levels of NLRP3 and Caspase-1 were measured by Western blotting.Results Compared with the sham-operation group,the BBB scores were decreased,the levels of IL-1βand IL-18 in CSF were higher,and the mRNA and protein levels of NLRP3 and Caspase-1 in spinal cord tissues were increased in the model group,and low-,medium-and high-dose etomidate groups(P<0.05).However,the BBB scores were higher,and the levels of IL-1βand IL-18 in CSF as well as the mRNA and protein levels of NLRP3 and Caspase-1 in spinal cord tissues were lower in the low-,medium-and high-dose etomidate groups than those in the model group(P<0.05).Conclusions Etomidate plays a cerebral protective role in SCI,potentially by inhibiting the activation of NLRP3/Caspase-1 pathway and attenuating the inflammatory response.