摘要
目的探讨创伤性脑损伤后大鼠脑内巨噬细胞分化的亚群种类和创伤后神经元过度自噬和凋亡的关系。方法选择45只雄性SD大鼠建立创伤性脑损伤(TBI)模型,然后根据随机数字表法分为Sham组(假手术大鼠,右心室注射5μL氯化钠溶液)、TBI组(TBI模型大鼠,右心室注射5μL氯化钠溶液)和3-MA组(TBI模型大鼠,右心室注射5μL 600 nmol的3-MA),每组各15只。造模后1~14 d测定Sham组和TBI组大鼠脑水肿程度;流式细胞术检测Sham组、TBI组和3-MA组大鼠脑损伤处巨噬细胞分化的亚群种类,采用蛋白质印迹(Western blotting)法测定了3组大鼠脑损伤处自噬相关通路蛋白、凋亡相关通路蛋白的表达水平以及自噬效应性信号通路PI3K/AKT通路的磷酸化水平。结果TBI造模手术后第1~14天时,TBI组大鼠脑水肿程度显著高于Sham组(P<0.05);与Sham组相比,TBI组和3-MA组M1亚群比例和M1/M2比值均显著升高,3-MA组M1亚群比例则较TBI组显著降低(P<0.05);与Sham组相比,TBI组和3-MA组大鼠脑损伤组织中凋亡相关蛋白cleaved-Caspase-3和Bax的相对表达水平显著升高,Bcl-2的相对表达水平显著降低,3-MA组cleaved-Caspase-3和Bax的相对表达水平则较TBI组显著升高,Bcl-2水平显著降低(P<0.05);与Sham组相比,TBI组和3-MA组的p-PI3K和p-AKT的磷酸化水平显著升高,而3-MA处理后,p-PI3K和p-AKT的磷酸化水平较TBI组显著降低(P<0.05)。结论TBI能够诱导大鼠脑内M1巨噬细胞亚群激增并导致脑损伤处神经元发生过度自噬和凋亡,同时,当自噬被抑制后,脑损伤处的凋亡被进一步激活;TBI大鼠神经元的过度自噬引发PI3K/AKT信号通路被激活,抑制自噬能够明显下调被激活的PI3K/AKT磷酸化水平。
Objective To investigate the relationship between the subgroups of macrophages and the over-reactive autophagy and apoptosis of post-traumatic neurons in the rat brain after traumatic brain injury.Methods Forty-five male SD rats were chosen to establish traumatic brain injury(TBI)models,then according random number table,they were divided into Sham group(sham operated rats,injection of 5μL of saline into the right ventricle),TBI group(TBI model rats,injection of 5μL of saline into the right ventricle)and 3-MA group(TBI model rats,injection of 5μL of 3-MA into the right ventricle),15 rats in each group,and the degree of cerebral edema in the two groups of rats was measured 1 to 14 days after modeling.Flow cytometry was used to determine the subgroups of macrophages in the Sham group,TBI group and 3-MA group after TBI surgery.Western blotting was used to determine the expression levels of autophagy-related pathway proteins and apoptosis-related pathway proteins,as well as the phosphorylation level of autophagy effective signal pathway,PI3K/AKT pathway of the injury brain in the Sham group,TBI group,and 3-MA treatment group.Results On the 1st to 14th days after TBI model surgery,the degree of cerebral edema in the TBI group was significantly higher than that in the Sham group(P<0.05);Compared with the Sham group,the M1 subgroup ratio and M1/M2 ratio of the TBI group and the 3-MA group were significantly increased,and the 3-MA group was significantly lower than that of the TBI group(P<0.05);Compared with the Sham group,the relative expression levels of apoptosis-related proteins cleaved-Caspase-3 and Bax in the brain injury tissues of the TBI and 3-MA groups were significantly increased,and the relative expression levels of Bcl-2 were significantly reduced,while the relative expression levels of cleaved-Caspase-3 and Bax in the 3-MA group were significantly higher than those in the TBI group,and the Bcl-2 level was significantly lower(P<0.05);Compared with the Sham group,the phosphorylation levels of p-PI3K and p-AKT in the TBI group and 3-MA group were significantly increased,and after 3-MA treatment,the phosphorylation levels of p-PI3K and p-AKT were significantly lower than those in the TBI group(P<0.05).Conclusion TBI can induce a sharp increase of M1 macrophage subpopulations in the TBI rat brain and lead to over-reactive autophagy and apoptosis of the neurons in the brain injury area.Besides,when autophagy is inhibited,the apoptosis of the neurons is further activated.Over-reactive autophagy of TBI rat neurons triggers activation of the PI3K/AKT signaling pathway,and inhibition of autophagy can significantly down-regulate the activated PI3K/AKT phosphorylation level.
作者
杨波
郭海波
王磊
YANG Bo;GUO Hai-bo;WANG Lei(Department of Neurosurgery,Chaoyang Central Hospital,Liaoning Province,Chaoyang Liaoning 122000,China.)
出处
《临床和实验医学杂志》
2021年第3期233-237,共5页
Journal of Clinical and Experimental Medicine
基金
国家自然科学基金青年项目(编号:GN-2018R0002)。
