脊髓损伤小鼠通过激活小胶质细胞MyD88通路促进神经元铁死亡  

Activating MyD88 signal pathway in the microglia promotes neuronal ferroptosis after spinal cord injury

作　　者：黄乔 李涛 赵莉莉[1,2] 陈志阳 高小川 刘芳芳 武胜昔 邝芳 HUANG Qiao;LI Tao;ZHAO Lili;CHEN Zhiyang;GAO Xiaochuan;LIU Fangfang;WU Shengxi;KUANG Fang(Department of Neurobiology,School of Basic Medicine,Air Force Medical University,Xi'an 710032;Department of Neurology,the Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004;Department of Anesthesiology,the Second Affiliated Hospital of Air Force Medical University,Xi'an 710038,China)

机构地区：[1]空军军大学基础医学院神经生物学教研室,西安710032 [2]西安交通大学第二附属医院神经内科,西安710004 [3]空军军医大学第二附属医院麻醉科,西安710038

出　　处：《神经解剖学杂志》2023年第1期71-78,共8页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81671217);陕西省重点产业链项目(2022ZDLSF02-05)。

摘　　要：目的:探索脊髓损伤(SCI)后小胶质细胞(MG)炎性活化对神经元铁死亡的作用及其关键信号通路,为修复脊髓损伤寻找新的治疗靶点。方法:小鼠胸段脊髓夹伤后3 d,通过免疫荧光检测钙接头蛋白(Iba-1)与4-羟基壬烯醛(4HNE)的表达。以脂多糖(LPS)及髓系分化初级反应蛋白88(MyD88)抑制剂ST2825处理小鼠原代小胶质细胞,另以糖氧剥夺(OGD)处理SH-SY5Y细胞,再将两种细胞用Transwell共培养24 h,CCK-8法检测SH-SY5Y细胞活力,碘化丙啶(PI)染色结合流式细胞仪分析SH-SY5Y细胞死亡情况,用Western Blot和细胞免疫荧光染色检测小胶质细胞的炎性活化情况和SH-SY5Y细胞铁死亡分子表达情况。结果:小鼠脊髓夹伤后3 d可见损伤局部神经元铁死亡与激活的小胶质细胞密切相关。与对照组相比,LPS刺激上调小胶质细胞中MyD88、核因子-κB(NF-κB)、诱导型一氧化氮合酶(iNOS)和白细胞介素1β(IL-1β)等分子表达。激活的小胶质细胞与SH-SY5Y细胞共培养,可促进OGD诱导的SH-SY5Y细胞死亡率进一步增加,同时使胱氨酸/谷氨酸逆向转运蛋白(xCT)、谷胱甘肽过氧化物酶4(GPX4)、血红素加氧酶1(HO-1)、铁蛋白重链(FTH1)等分子的表达进一步降低。而MyD88抑制剂ST2825能抑制小胶质细胞的促炎性激活,明显减轻SH-SY5Y细胞铁死亡发生。结论:SCI局部神经元铁死亡与激活的小胶质细胞密切相关,小胶质细胞促炎性激活可促进神经元的铁死亡。Objective:To explore the effect and key signaling of microglia(MG)inflammatory response on neuronal ferroptosis after spinal cord injury(SCI),so as to find new therapeutic target for spinal cord injury.Methods:Three days after spinal cord crush injury,the expressions of ionized calcium binding adaptor molecule-1(Iba-1)and 4-hydroxynonenal(4HNE)were detected with immunofluorescence.MG were treated with lipopolysaccharide(LPS)and myeloid differentiation primary response protein 88(MyD88)inhibitor ST2825,which led to activation or inhibition of MyD88 signaling.SH-SY5Y cells were treated with oxygen and glucose deprivation(OGD),and then the two cells were co-cultured with transwell for 24 h.The cell viability of SH-SY5Y cells was detected by CCK-8 assay.The death of SH-SY5Y cells was analyzed by propidium iodide(PI)staining and flow cytometer.The activation of inflammation in MG and molecules related to ferroptosis in SH-SY5Y cells were detected by Western Blot and immunofluorescent staining.Results:Three days after SCI surgery,neuronal ferroptosis was adjacent to the activated MG in the spinal cord.Compared with the control group,LPS stimulated up-regulated the expression of MyD88,nuclear factor-κB(NF-κB),inducible nitric oxide synthase(iNOS)and interleukin-1β(IL-1β)in microglia cells.The co-culture of activated microglia with SH-SY5Y cells can promote the further increase of OGD-induced SH-SY5Y cell mortality.At the same time,the expression of cystine/glutamate reverse transporter(xCT),glutathione peroxidase 4(GPX4),heme oxygenase 1(HO-1),ferritin heavy chain(FTH1)and other molecules was further reduced.MyD88 inhibitor ST2825 inhibited the proinflammatory activation of MG and significantly reduced the occurrence of ferroptosis in the SH-SY5Y cells.Conclusion:Neuronal ferroptosis in SCI is strongly linked to the activated MG.Proinflammatory activation of MG can promote neuronal ferroptosis.Inhibition of MyD88 signaling can alleviate MG inflammatory response and neuronal ferroptosis.

关 键 词：脊髓损伤 神经元铁死亡 小胶质细胞 髓系分化初级反应蛋白88 细胞共培养 小鼠 

分 类 号：R651.2[医药卫生—外科学]