Effect of remimazolam on a rat model of cerebral ischaemia/reperfusion injury by inhibiting the NOD-like receptor pyrindomaincontaining three inflammasome
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目的 探讨瑞马唑仑通过抑制核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体介导的焦亡改善大鼠脑缺血再灌注损伤的机制.方法 6～8周龄清洁健康SD雄性大鼠48只,随机数字分为4组,假手术组(N组)、脑缺血再灌注(MCAO模型组,C组)、MCAO+瑞马唑仑组(R组)、MCAO+MCC950组(M组),每组12只.再灌注24 h后进行神经功能评分,收集血清及脑组织样本,酶联免疫吸附试验(ELISA)检测血清白细胞介素-6(IL-6)和肿瘤坏死因子(TNF-α)的含量.测定脑梗死面积、苏木精-伊红(HE)染色检测脑组织病理情况,蛋白质印迹法(Western blot)检测并分析脑组织的NLRP3、凋亡相关斑点样蛋白(ASC)、天冬氨酸特异性半胱氨酸蛋白酶-1(Caspase-1)、白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)的蛋白水平,多组间比较采用单因素方差分析,不符合正态分布的计量资料采用M(P25～P75)表示.多组间比较采用Kruskal-Wallis H检验.结果 R组和M组神经功能评分均低于C组,差异有统计学意义(1.00±0.00比3.13±0.35、1.38±0.74 比 3.13±0.35,F=32.147,P＜0.05);R 组与 M 组脑梗死率低于C 组(0.227±0.085 比0.307±0.061、0.184±0.046 比 0.307±0.061,F=5.255,P＜0.05);ELISA 检测 IL-6 含量表达中,R 组和 M 组低于 C 组[(54.394±28.684)pg/ml 比(103.238±16.718)pg/ml、(44.010±17.066)pg/ml 比(103.238±16.718)pg/ml,F=3.604,P＜0.05];ELISA 检测 TNF-α 含量表达中,R 组和 M 组低于C 组[(6.242±2.110)pg/ml 比(25.523±6.828)pg/ml、(5.645±1.266)pg/ml 比(25.523±6.828)pg/ml,F=6.609,P＜0.05];R组与M组两组神经元细胞比C组排列整齐、细胞数目增多,胞周空泡减少.NLRP3蛋白表达中,R组和M组低于C组(0.390±0.039比0.489±0.050、0.338±0.023 比 0.489±0.050,F=3.824,P＜0.05);ASC 蛋白表达中,M 组低于 C 组(0.316±0.035 比 0.498±0.083,F=4.949,P＜0.05);Caspase-1 蛋白表达中,M 组低于 C 组(0.312±0.031比 0.429±0.010,F=8.798,P＜0.05);IL-1β 蛋白表达中,M 组低于 C 组(0.229±0.012 比 0.303±0.067,F=2.663,P＜0.05);IL-18 蛋白表达中,R 组和 M 组低于 C 组(0.426±0.017 比 0.556±0.084、0.365±0.026 比 0.556±0.084,F=3.716,P＜0.05).结论 瑞马唑仑在一定程度上可以抑制NLRP3介导的焦亡,减少炎性因子的激活和释放,最终减轻脑缺血再灌注损伤,为围手术期脑保护提供参考.
Objective To study the mechanism responsible for the therapeutic effect of remimazo-lam in a rat model of cerebral ischaemia/reperfusion injury(CI/RI)via NOD-like receptor pyrindomain-containing three-mediated pyroptosis.Methods A total of 48 clean and male Sprague-Dawley rats,aged 6-8 weeks,were randomly divided into 4 groups(n=12 each):Sham(N)group,middle cerebral artery occlusion(MCAO)model(C)group,MCAO+remimazolam(R)group and MCAO+MCC950(M)group.The score for neurological assessment was obtained 24 h after reperfusion,while serum and brain tissue samples were collected.Enzyme-linked immunosorbent assay was conducted to measure serum levels of interleukin-6(IL-6)and tumour necrosis factor-alpha(TNF-α),and the cerebral infarction area was measured.Haematoxylin and eosin staining was conducted to analyze brain tissue pathology,Western blot-ting was performed to detect and analyze the protein levels of NLRP3,ASC,Caspase-1,IL-1β and IL-18 in brain tissue.The measurement data conforming to the normal distribution were expressed,one-way anal-ysis of variance was used for the comparison between multiple groups,the measurement data not conforming to the normal distribution were expressed by M(P25-P75),and Kruskal-Wallis H test was used for the com-parison between multiple groups.Results The neurological function scores in R group and M group were lower than those in C group,and the difference was statistically significant(1.00±0.00 vs.3.13±0.35,1.38±0.74 vs.3.13±0.35,F=32.147,P＜0.05).The infarction rates in R and M groups were lower than those in C group(0.227±0.085 vs.0.307±0.061,0.184±0.046 vs.0.307±0.061,F=5.255,P＜0.05).The expression of IL-6 in R and M groups was lower than that in C group[(54.394±28.684)vs.(103.238±16.718)pg/ml,(44.010±17.066)vs.(103.238±16.718)pg/ml,F=3.604,P＜0.05].The expression of TNF-α in R and M groups was lower than that in C group[(6.242±2.110)vs.(25.523±6.828)pg/ml,(5.645±1.266)vs.(25.523±6.828)pg/ml,F=6.609,P＜0.05].Compared with C group,neurons in the R and M groups displayed a more orderly arrangement,increased neuron count,reduced pericellular vacuolation.The protein expression levels of NLRP3 was lower in R and M groups than in C group(0.390±0.039 vs.0.489±0.050,0.338±0.023 vs.0.489±0.050,F=3.824,P＜0.05).The protein expression levels of ASC in M group was lower than in C group(0.316±0.035 vs.0.498±0.083,F=4.949,P＜0.05).The protein expression levels of Caspase-1 in M group was lower than in C group(0.312±0.031 vs.0.429±0.010,F=8.798,P＜0.05).The protein expres-sion levels of IL-1 β in M group were lower than in C group(0.229±0.012 vs.0.303±0.067,F=2.663,P＜0.05).The protein expression levels of IL-18 in R and M groups were lower than in C group(0.426±0.017 vs.0.556±0.084,0.365±0.026 vs.0.556±0.084,F=3.716,P＜0.05).Conclusion Remimazolam can prevent NLRP3-mediated pyroptosis and reduce the activation and release of inflammatory factors,ultimately relieving CI/RI.This work provides a reference for perioperative brain protection.
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