嘌呤能离子通道型受体7对小鼠创伤性脑损伤后神经炎症及脑水肿的影响

Effect of P2X7R on neuroinflammation and brain edema after traumatic brain injury in mice

背景创伤性脑损伤(traumatic brain injury,TBI)后的炎症反应及脑水肿是造成颅内压升高等严重并发症的重要因素。嘌呤能离子通道型受体7(purinergic ligand-gated ion channel 7 receptor,P2X7R)具有介导先天免疫反应的重要作用,P2X7R的活化可触发炎症反应的激活,与神经系统疾病的炎症反应密切相关。目的观察P2X7R对小鼠创伤性脑损伤后神经炎症及脑水肿的影响。方法使用经典的控制性皮质撞击模型构建小鼠创伤性脑损伤模型。免疫荧光观察小胶质细胞形态和P2X7R含量变化,干湿重法测小鼠脑组织含水量。使用脂多糖(lipopolysaccharide,LPS)作用于小胶质细胞系(BV2细胞系)模拟创伤性脑损伤后小胶质细胞的激活,Western blot测定BV2细胞系P2X7R蛋白含量,ELISA测定BV2细胞系肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、IL-6和IL-12的表达水平;采用条件培养基共培养法探索活化的小胶质细胞对内皮细胞(bEnd3细胞系)的影响,Western blot测定bEnd3细胞系的紧密连接蛋白Claudin-5的蛋白含量。结果动物实验结果表明,急性创伤性脑损伤后小胶质细胞增多且显著活化,P2X7R主要表达于小胶质细胞,抑制P2X7R可降低创伤性脑损伤小鼠的脑组织含水量,显著缓解脑水肿(P<0.05)。细胞实验结果显示,激活的小胶质细胞P2X7R含量升高,TNF-α、IL-1β、IL-6和IL-12表达量显著增加,而抑制P2X7R可以减少上述炎症因子的分泌(P<0.05)。共培养实验提示抑制P2X7R可增加创伤性脑损伤后内皮细胞紧密连接蛋白的表达量。结论抑制P2X7R有助于降低小鼠急性创伤性脑损伤继发的神经炎症和脑水肿,其作用机制有待进一步研究。

Background Inflammation and brain edema after traumatic brain injury(TBI)are important factors causing severe complications such as increased intracranial pressure.Purinergic ligand-gated ion channel 7 receptor(P2X7R)plays an important role in mediating innate immune response.Its activation can trigger the activation of inflammatory response,which is closely related to the inflammatory response of nervous system diseases.Objective To observe the effect of P2X7R on neuroinflammation and brain edema after traumatic brain injury in mice.Methods The classic controlled cortical impaction model was utilized to establish a mouse model of traumatic brain injury.The morphology of microglia and the content of P2X7R were observed by immunofluorescence.The water content of mouse brain tissue was measured by wet-dry weight method.Lipopolysaccharide(LPS)was applied to microglia cell line(BV2 cell line)to simulate the activation of microglia after traumatic brain injury.The protein content of P2X7R in BV2 cell line was determined by Western blot.The expression levels of TNF-α,IL-1β,IL-6 and IL-12 in BV2 cell line were determined by ELISA.Conditioned medium co-culture was utilized to explore the effect of activated microglia on endothelial cells,and the protein content of claudin-5 in bEnd3 cell line was detected by Western blot.Results The results of animal experiments showed that after acute traumatic brain injury,microglia were increased and significantly activated and P2X7R was mainly colocalized to the microglial cell.And inhibition of P2X7R could reduce the water content of brain tissue in mice with traumatic brain injury,and significantly alleviate brain edema.The results of cell experiments showed that the content of P2X7R,TNF-α,IL-1β,IL-6 and IL-12 in activated microglia was increased,while inhibition of P2X7R could reduce the secretion of the above inflammatory factors(P<0.05).Co-culture experiment suggested that inhibition of P2X7R could increase the expression of tight junction protein in endothelial cells after traumatic brain injury.Conclusion Inhibition of P2X7R can reduce neuroinflammation and brain edema secondary to acute traumatic brain injury in mice,but its mechanism needs further study.