肥大细胞稳定剂对脓毒症小鼠脑损伤的保护作用

Effect of mast cells stabilizer cromolyn on sepsis induced brain injury

目的探讨肥大细胞(MCs)稳定剂色甘酸钠(Cro)在脓毒症小鼠脑损伤中的保护作用。方法采用盲肠结扎穿刺法(CLP)诱导小鼠SAE模型并用Cro进行治疗。甲苯胺蓝及组化染色检测MCs激活;神经反射评分(NRS)和Y迷宫评估小鼠神经功能和认知功能;免疫印迹法(Western blot)和ELISA分别用于海马紧密连接(TJ)蛋白和炎性因子的检测。多组间比较用方差分析。结果CLP组小鼠生存率、体重、NRS、活动和探索学习能力均低于Sham组;而CLP小鼠海马MCs激活数量、脑含水量(BWC)、荧光素钠(FS)渗出量和炎性因子含量均高于Sham组;此外,CLP组小鼠海马TJ蛋白表达低于Sham组。经过Cro治疗后CLP小鼠海马区MCs激活数量(TB:4.53±1.69比2.93±1.03,F=10.430,P<0.05;Tryptase:5.40±1.99比4.00±1.46,F=9.753,P<0.05)、炎性因子释放[TNF-α:(790.00±84.35)pg/ml比(610.50±69.54)pg/ml,F=28.560,P<0.01;IL-1β(357.30±50.68)pg/ml比(269.30±15.44)pg/ml,F=22.560,P<0.01]、BWC(F=17.290,P<0.05]和FS渗出量均显著低于Sham组(F=27.970,P<0.01),而TJ蛋白ZO-1(F=57.230,P<0.05)和Occludin表达高于Sham组(F=71.410,P<0.01)。同时,CLP小鼠生存率(56.7%比73.3%,P<0.05)、NRS(2.50±1.05比7.30±1.51,t=6.452,P<0.01)、C区活动距离及时间显著高于Sham组[(3161±2303)cm比(7659±3150)cm,t=2.824,P<0.05;(26.66±18.83)%比(57.49±22.41)%,t=2.579,P<0.05]。结论Cro通过减少MCs激活和减轻炎性反应,从而保护CLP小鼠血脑屏障,改善神经功能和认知功能。

Objective To investigate the protective effect of cromolyn(Cro),a mast cells(MCs)stabilizer,in brain injury of septic mice.Methods We used cecal ligation puncture(CLP)to induce sepsis associated encephalopathy(SAE)and treated with Cro.MCs was detected by toluidine blue(TB)and tryptase staining.Neural function and cognitive function were evaluated by neural reflex score(NRS)and Y-maze,respectively.Hippocampal tight junction(TJ)protein and inflammatory factors detected by Western blotting and ELISA,respectively.Using SPSS 23.0 statistical software to analyze data,measurement data were displayed as mean±standard deviation(mean±SD),using the single factor analysis of variance is compared between groups.Results The survival rate,body weight,NRS,activity and exploratory learning ability of CLP mice were lower than those of Sham mice.And the number of activated MCs,brain water content(BWC),FSextravasation and inflammatory cytokines in hippocampus of CLP mice were higher than those of Sham mice.Additionally,TJ proteins expression in hippocampus of CLP mice waslower than those of Sham mice.After Cro treatment,the number of activated MCs(TB:4.53±1.69 vs.2.93±1.03,F=10.430,P<0.05;Tryptase:5.40±1.99 vs.4.00±1.46,F=9.753,P<0.05),inflammatory cytokines[TNF-α:(790.00±84.35)pg/ml vs.(610.50±69.54)pg/ml,F=28.560,P<0.01;IL-1β:(357.30±50.68)pg/ml vs.(269.30±15.44)pg/ml,F=22.560,P<0.01]BWC(F=17.290,P<0.01)and FSextravasation(F=27.970,P<0.01)in hippocampus of CLP mice were decreased significantly.However,TJproteins ZO-1(F=57.230,P<0.05)and Occludin(F=71.410,P<0.01)were up-regulated.Meanwhile,the survival rate(56.7%vs.73.3%,P<0.05),NRS(2.50±1.05 vs.7.30±1.51,t=6.452,P<0.01),and the movement distance and time in C zone[(3161±2303)cm vs.(7659±3150)cm,t=2.824,P<0.05;(26.66±18.83)%vs.(57.49±22.41)%,t=2.579,P<0.05]in CLP mice was significantly increased.Conclusion Cro protects the blood-brain barrier and improves neurological,and cognitive function in CLP mice by reducing MCs activation and alleviating inflammatory responses.