髓样分化蛋白2介导脓毒症相关性脑病的机制研究  

Mechanism of myeloid differentiation factor 2 on mediating sepsis-associated encephalopathy

作　　者：贾琪 顾婷婷 李仪 王嘉炜 李谨 方宗平 张西京 Jia Qi;Gu Tingting;Li Yi;Wang Jiawei;Li Jin;Fang Zongping;Zhang Xijing(Department of Anaesthesiology and Perioperative Medicine,Xijing Hospital,the Fourth Military Medical University,Xi'an 710032,Shaanxi,China;Department of Intensive Care Unit,Xijing Hospital,the Fourth Military Medical University,Xi'an 710032,Shaanxi,China)

机构地区：[1]空军军医大学第一附属医院重症医学科,陕西西安710032 [2]空军军医大学第一附属医院麻醉与围术期医学科,陕西西安710032

出　　处：《中华危重病急救医学》2023年第11期1200-1206,共7页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81871603,82272190)。

摘　　要：目的通过脂多糖(LPS)建立脓毒症相关性脑病(SAE)体外模型,探讨人髓样分化蛋白2(MD2)在LPS导致神经元死亡过程中的作用及内在机制。方法选择健康C57BL/6J孕鼠,孕期14~18 d,取胎鼠脑皮质组织,用0(空白对照组)、1、5和10 g/L的LPS刺激神经元后24 h,检测乳酸脱氢酶(LDH)释放量,观察神经元死亡情况,并用酶联免疫吸附试验(ELISA)检测炎症因子白细胞介素(IL-6、IL-1β)水平,以确定建立SAE体外神经炎症模型的最佳LPS剂量。将细胞分为空白对照组和LPS组,用原位末端缺刻标记法(TUNEL)染色检测细胞凋亡情况,用蛋白质免疫印迹试验(Western blotting)检测相关蛋白标志物活化天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)表达、坏死性凋亡相关蛋白神经元受体相互作用丝氨酸/苏氨酸蛋白激酶3(RIPK3)及磷酸化RIPK3(p-RIPK3)水平,用免疫荧光化学染色检测p-RIPK3和微管相关蛋白2(MAP2)的表达,以评估细胞死亡类型及神经元死亡程度;用Western blotting法检测MD2表达,免疫荧光化学染色观察p-RIPK3及MD2在神经元中的表达和分布,以评估MD2是否参与炎症反应促进神经元死亡。此外,将细胞分为空白对照组、LPS组和MD2干扰肽组(LPS+TC组),检测IL-6、IL-1β、LDH水平,评估干扰MD2能否减轻LPS诱导的神经炎症。结果10 g/L LPS可诱导明显的神经元死亡,该浓度刺激神经元24 h后LDH释放量较空白对照组明显增加(相对释放量:1.45±0.04比1.00±0.00,P<0.01),神经元发生凋亡及坏死性凋亡,炎症因子IL-6、IL-1β水平显著增加〔IL-6(相对水平):1.94±0.04比1.00±0.00,IL-1β(相对水平):1.53±0.09比1.00±0.00,均P<0.01〕。与空白对照组比较,LPS组TUNEL检测细胞凋亡明显增多,活化caspase-3表达明显增加(活化caspase-3/GAPDH:1.55±0.10比1.00±0.00,P<0.01),p-RIPK3/RIPK3比值明显升高(相对值:1.54±0.06比1.00±0.00,P<0.05),神经元周围p-RIPK3表达显著增多,提示脓毒症环境下神经元发生显�ObjectiveTo investigate the role and underlying mechanism of human myeloid differentiation protein 2(MD2)in the process of neuronal death induced by lipopolysaccharide(LPS)by establishing an in vitro model of sepsis-associated encephalopathy(SAE)by LPS.MethodsHealthy C57BL/6J mice at 14-18 days of gestation were selected,and brain cortical tissue was taken from fetal mice.Neurons were stimulated with 0(control),1,5 and 10 g/L of LPS for 24 hours.The release of lactate dehydrogenase(LDH)was detected and the death of neurons was observed.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors interleukins(IL-6,IL-1β),in order to determine the optimal dose of LPS for establishing an in vitro neuroinflammation model of SAE.The cells were divided into blank control group and LPS group.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling(TUNEL)was used to discover apoptosis.Western blotting was used to detect the expression of the relevant protein markers activated caspase-3,necroptosis-associated protein neuronal receptor-interacting serine/threonine-protein kinase 3(RIPK3)and phosphorylated RIPK3(p-RIPK3).Immunofluorescence chemical staining was used to detect the expressions of p-RIPK3 and microtubule-associated protein 2(MAP2)to evaluate the type of cell death and the degree of neuronal death.Western blotting was used to detect MD2 expression.Immunofluorescence chemical staining was performed to observe the expression and distribution of p-RIPK3 and MD2 in neurons to assess whether MD2 was involved in the inflammatory response promoting neuronal death.In addition,the cells were divided into blank control group,LPS group,and MD2 interfering peptide group(LPS+TC group),and the levels of IL-6,IL-1βand LDH were detected to evaluate whether interfering with MD2 can alleviate LPS induced neuroinflammation.Results10 g/L LPS induced notable neuronal death,and the release of LDH in neurons stimulated with this concentration for 24 hours was significantly higher than t

关 键 词：脓毒症相关性脑病 神经炎症 凋亡 坏死性凋亡 髓样分化蛋白2 

分 类 号：R459.7[医药卫生—急诊医学] R747.9[医药卫生—治疗学]