亚甲蓝对机械性脑损伤小鼠的神经保护作用及其机制研究

Neuroprotective effect and mechanism of methylene blue on mice with stab wound injury

目的探讨亚甲蓝对小鼠机械性脑损伤的影响及其作用机制。方法将50只SPF级C56BL/6雄性小鼠,体质量范围为20~25 g,采用随机数字表法分为假手术组(Sham组,n=10)、机械性脑损伤组(SWI组,n=10)、亚甲蓝组(MB组,n=10)、核转录因子κB(NF-κB)抑制剂组(PDTC组,n=10)、亚甲蓝联合NF-κB抑制剂组(MB+PDTC组,n=10)。利用针刺伤建立小鼠机械性脑损伤模型,MB组腹腔注射亚甲蓝1 mg/kg治疗。时间损伤严重程度(NSS)评分评价小鼠神经功能变化;原位末端转移酶标记(TUNEL)检测脑内神经细胞凋亡情况;苏木精-伊红(HE)染色观察病理损伤情况;酶联免疫吸附测定(ELISA)检测小鼠脑组织中白细胞介素(IL)-1β,IL-6,肿瘤坏死因子α(TNF-α)水平;蛋白质印迹法(Western blotting)检测小鼠脑组织中小胶质细胞(Iba-1)、星形胶质细胞(GFAP)表达;Western blotting检测各组大鼠脑内NF-κB p65,Iκ-α及p-IκB-α蛋白表达水平。结果与SWI组相比,MB组小鼠神经功能明显改善;MB能够明显减少神经细胞凋亡;MB能减轻病理损伤;MB组脑组织中IL-1β、IL-6和TNF-α水平均降低;小鼠脑组织Iba-1、GFAP蛋白明显减少;同时,NF-κB p65、p-IκB-α蛋白表达降低,IκB-α蛋白表达升高;但是给予NF-κB抑制剂后,MB的治疗作用被抑制。结论亚甲蓝能有效改善针刺伤导致的神经功能障碍,抑制神经细胞凋亡及神经炎症,其作用机制可能与抑制NF-κB信号通路有关。

Objective To investigate the effects of methylene blue(MB)on stab wound injury(SWI)and its possible mechanism in mice model.Methods Fifty SPF grade C57BL/6 mice,weighing 20-25 g,were randomly divided into sham-operated group(Sham group,n=10),stab wound injury group(SWI group,n=10),MB-treated group(MB group,n=10),NF-κB inhibitor group(PDTC group,n=10)and MB combined with inhibitor group(MB+PDTC,n=10)group.The mice underwent the stab wound injury by the needle,mice in MB groups were intraperitoneally injected with 1 mg/kg MB after the model formation.The NSS score was used to analyze neural func‐tion in mice;Neural cells apoptosis was analysis by TUNEL;HE staining was used to observe pathological damage;The levels of L-1β,IL-6,TNF-αin brain tissue were tested by ELISA;The expression of Iba-1,GFAP was tested by Western blotting;NF-κB p65,Iκ-α,pIκB-αprotein expression was assessed by Western blotting.Results Compared to SWI group,neurological function was significantly improved in MB group;The number of cell apoptosis was significant decreased;MB alleviated pathological damage;The levels of IL-1β,IL-6 and TNF-αwere significantly decreased;The expression of Iba-1,GFAP was significantly decreased.In addition,MB also inhibit‐ed the expression of NF-κB p65 and p-IκB-αprotein,while enhanced the expression of IκB-αprotein,however,NF-κB signaling path‐way was inhibited,the anti-inflammatory effect of MB was abolished.Conclusion Methylene blue could effectively improve neurologi‐cal dysfunction caused by stab wound injury,inhibit neuronal apoptosis and neuroinflammation,and its mechanism may be related to inhibition of NF-κB signaling pathway.