水通道蛋白4对一氧化碳中毒后迟发性脑病大鼠神经损伤的影响

目的 探讨水通道蛋白4(Aquaporin 4,AQP4)对一氧化碳(Carbon monoxide,CO)中毒后迟发性脑病(Delayed encephalopathy,DEACMP)大鼠神经损伤的影响。方法 将210只雄性SD大鼠随机分为空白对照(Blank control,BC)组、CO中毒(CO)组、钠-钾-氯共转运体(Na⁺-K⁺-Cl^- cotransporter,NKCC)抑制剂处理(布美他尼)组、p38-丝裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)抑制剂处理(MAPK)组和AQP4特异性抑制剂处理(AQP4抑制剂)组,每组各42只; 根据造模后不同时间点将每组大鼠进一步分为染毒3、6、12、24、48、72 h和7 d后共7个亚组,每亚组各6只; 取大鼠脑前额叶皮质组织,计算脑皮质含水量,采用HE法观察脑皮质形态; 采用免疫组化链霉菌抗生物素蛋白-过氧化物酶(Streptavidin-perosidase,SP)染色法测定大鼠脑皮质AQP4,p38 MAPK,NKCC1、胶质纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)和S100钙结合蛋白B(S100 calcium binding protein B,S100B)蛋白表达水平。结果 与BC组比较,CO组大鼠在染毒3、6、12、24、48、72 h后尾静脉COHB水平和脑皮质含水量显著升高,脑皮质AQP4,p38 MAPK,NKCC1,GFAP和S100B蛋白表达水平显著升高,染毒7 d后恢复正常(P<0.05); 与CO组比较,布美他尼组、MAPK组和AQP4抑制剂组大鼠脑皮质含水量显著降低,脑皮质AQP4,p38 MAPK,NKCC1,GFAP和S100B蛋白表达水平显著降低,且AQP4抑制剂组变化更明显(P<0.05)。结论 p38-MAPK/NKCC信号通路可能参与调控CO中毒DEACMP大鼠脑皮质AQP4表达,抑制AQP4表达可有效减轻大鼠脑水肿并改善预后,有望成为预防和治疗DEACMP的新靶点。

Effect of AQP4 on nerve damage in rats with delayed encephalopathy after acute carbon monoxide poisoning

ObjectiveTo investigate the effect of aquaporin 4(AQP4)on nerve damage in rats with delayed encephalopathy after acute carbon monoxide poisoning(DEACMP).Methods 210 male SD rats were randomly divided into blank control group(BC group), carbon monoxide poisoning group(CO group), NKCC inhibitor treatment group(Bumetanide group), p38-MAPK inhibitor treatment group(MAPK group), and AQP4 specific inhibitor treatment group(AQP4 inhibitor group)of 42 rats each. Then, the rats in each group were further divided into 7 subgroups at 3, 6, 12, 24, 48, 72 h and 7 d after modeling, with 6 rats in each subgroup. The prefrontal cortex tissues of rats were taken, the water content of the cortex was calculated, and the morphology of the cortex was observed by HE method. Immunohistochemical SP staining was used to determine the protein expression of AQP4, P38 MAPK, NKCC1, GFAP and S100B in rat cerebral cortex.Results Compared with BC group, COHB concentration and cerebral cortex water content in CO group were significantly increased at 3, 6, 12, 24, 48 and 72 h after modeling. Meanwhile, the protein expression levels of AQP4, P38 MAPK, NKCC1, GFAP and S100B in cerebral cortex were significantly increased, and returned to normal at 7d after modeling(P<0.05). When compared with CO group, the cerebral cortex water content of rats in Bumetanide group, MAPK group and AQP4 inhibitor group was significantly reduced, and the protein expression levels of AQP4, P38 MAPK, NKCC1, GFAP and S100B in cerebral cortex were significantly decreased, and all those changes were more significant in AQP4 inhibitor group(P<0.05).Conclusion The p38-MAPK/NKCC signaling pathway might be involved in regulating the expression of AQP4 in cerebral cortex of DEACMP rats. Moreover, inhibiting the expression of AQP4 could effectively reduce the cerebral edema and improve the prognosis, which is expected to be a new strategy for the prevention and treatment of DEACMP.