静脉注射咪达唑仑对蛛网膜下腔出血大鼠脑神经炎症和损伤的改善作用及其机制

Effects and mechanism of intravenous injection of midazolam on neuroinflammation and brain injury in rats with subarachnoid hemorrhage

目的 探讨咪达唑仑(MDZ)对蛛网膜下腔出血(SAH)大鼠脑神经炎症和损伤的改善作用,及其对环腺苷酸(cAMP)/蛋白激酶A(PKA)/环腺苷酸应答元件结合蛋白(CREB)信号通路的调节作用。方法 取Wistar大鼠65只,随机分为假手术组10只及造模组55只,造模组通过向枕大池二次注入血液的方法建立SAH模型,假手术组向枕大池注入等体积生理盐水。将造模成功的50只SAH大鼠随机分为SAH组、MDZ低剂量组、MDZ中剂量组、MDZ高剂量组、MDZ高剂量+H-89组,每组10只。MDZ高、中、低剂量组分别经颈静脉注射0.30、0.15、0.05 mg/kg MDZ,同时腹腔注射等体积生理盐水;MDZ高剂量+H-89组经颈静脉注射0.3 mg/kg MDZ,同时腹腔注射5 mg/kg PKA抑制剂H-89;SAH组、假手术组颈静脉及腹腔注射等体积生理盐水;每天1次,连续3 d。干预结束后,对大鼠进行神经功能评分;采集静脉血,ELISA法检测炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β、IL-6水平;每组随机选取3只大鼠,干/湿法评估脑水含量;剩余大鼠进行SAH等级评分,HE染色观察脑组织形态学变化;Western blotting法检测cAMP/PKA/CREB通路相关蛋白表达。结果 与假手术组相比,SAH组海马区神经元细胞核碎裂、坏死现象,神经功能评分、cAMP、p-PKA/PKA、p-CREB/CREB表达降低,IL-1β、IL-6、TNF-α、脑含水量、SAH等级评分增加(P均<0.05);与SAH组相比,MDZ高、中、低剂量组神经元损伤有不同程度缓解,神经功能评分、cAMP、p-PKA/PKA、p-CREB/CREB表达增加,IL-1β、IL-6、TNF-α、脑含水量、SAH等级评分降低(P均<0.05);与MDZ高剂量组相比,MDZ高剂量+H-89组神经元损伤加剧,神经功能评分、cAMP、p-PKA/PKA、p-CREB/CREB表达降低,IL-1β、IL-6、TNF-α、脑含水量、SAH等级评分增加(P均<0.05)。结论 MDZ可减轻SAH大鼠脑神经炎症,抑制早期脑损伤;其机制可能与cAMP/PKA/CREB通路激活有关。

Objective To investigate the effects of midazolam(MDZ)on neuroinflammation and brain injury in rats with subarachnoid hemorrhage(SAH)and its regulatory effect on cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cAMP-response element binding protein(CREB)signaling pathway.Methods Sixty-five Wistar rats were randomly grouped into the sham operation group(10 rats)and model group(55 rats).In the model group,we established the SAH rat models by injecting blood into the occipital cistern twice,while rats in the sham surgery group were injected with equal volume of normal saline into the occipital cistern.The 50 successfully modeled SAH rats were randomly grouped into the SAH group,low-dose MDZ group,medium-dose MDZ group,high-dose MDZ group,and high-dose MDZ+H-89 group,with 10 rats in each group.The rats in the high-,medium-,and low-dose MDZ groups were injected with 0.30,0.15,and 0.05 mg/kg MDZ via jugular vein,respectively,while the same volume of normal saline was injected intraperitoneally;the rats in the high-dose MDZ+H-89 group received intravenous injection of 0.3 mg/kg MDZ and in⁃traperitoneal injection of 5 mg/kg PKA inhibitor H-89;the rats in the SAH group and sham operation group were injected with equal volume of normal saline into the jugular vein and abdominal cavity,once a day,for 3 consecutive days.After the intervention,the rats were scored for neurological function;venous blood was collected,and the levels of inflammatory factors tumor necrosis factor(TNF)-α,interleukin-1β(IL-1β)and interleukin-6(IL-6)were detected by ELISA.Three rats in each group were randomly selected,and the brain water content was evaluated by dry/wet method.The remaining rats were scored by SAH,and HE staining was applied to observe the morphological changes of brain tissues;Western blot⁃ting was applied to detect the expression of cAMP/PKA/CREB pathway-related proteins.Results Compared with the sham operation group,the neurons in the SAH group showed nuclear fragmentation and necrosis,the neurological function score,the expression levels of cAMP,p-PKA/PKA,and p-CREB/CREB decreased,and the IL-1β,IL-6,TNF-α,brain water content and SAH score increased(all P<0.05).Compared with the SAH group,the neuron injury in the high-dose,medium-dose,and low-dose MDZ groups was alleviated to varying degrees,the neurological function score,the expression levels of cAMP,p-PKA/PKA,and p-CREB/CREB increased,and the IL-1β,IL-6,TNF-α,brain water content and SAH score decreased(all P<0.05).Compared with the high-dose MDZ group,the neuron injury in the high-dose MDZ+H-89 group was aggravated,the neurological function score,the expression levels of cAMP,p-PKA/PKA,and p-CREB/CREB decreased,and the IL-1β,IL-6,TNF-α,brain water content and SAH score increased(all P<0.05).Conclusion MDZ can alleviate neuroinflammation and inhibit early brain injury in SAH rats,which may be related to the activation of cAMP/PKA/CREB pathway.