HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia
HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia
复制标题
HMGB1 RAGE 轴通过损害小胶质细胞的线粒体自噬通量介导小鼠应激诱导的 RVLM 神经炎症
DOI：
10.1186/s12974-019-1673-3
复制
发表时间：
2020-01-10
影响因子：
9.3
通讯作者：
Xia, Chunmei
中科院分区：
文献类型：
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作者：
Zhang, Shutian;Hu, Li;Xia, Chunmei
背景：延髓头端腹外侧区小胶质细胞介导的神经炎症在应激性高血压(SIH)的发病机制中起重要作用。据报道，自噬通过小胶质细胞的免疫表型转换影响炎症反应。高迁移率族蛋白1(HMGB1)作为自噬的调节因子，启动促炎细胞因子(PIC)的产生，但其潜在机制尚不清楚。方法应激小鼠给予间歇性电击加噪声，连续15d，每日2次，持续2 h。采用无创尾套法和铂-铱电极分别监测血压(BP)和肾交感神经活动(RSNA)。将siRNA-HMGB1(SiHMGB1)注射到小鼠RVLM内，观察HMGB1对小胶质细胞M1活化的影响。将MRFP-GFP-串联荧光LC3(Tf-LC3)载体导入RVLM，观察自溶酶体形成/自噬通量的变化。通过RAB7、溶酶体相关膜蛋白1(LAMP1)的表达和溶酶体pH的变化来评价小胶质细胞的溶酶体功能。通过透射电子显微镜观察或在共聚焦显微镜下检查Lc3和MitoTracker的共定位来鉴定有丝分裂。结果慢性应激使小胶质细胞HMGB1胞浆易位增加，其受体RAGE表达上调。Cre-CX3CR1/RAGE(fl/fl)小鼠的RVLm线粒体损伤、氧化应激和M1极化明显减轻。HMGB1/RAGE轴在应激诱导的丝裂原吞噬通量的早期阶段增加，而在小胶质细胞的晚期吞噬通量减弱，表现为GFP-RFP-LC3-II斑点的GFP荧光猝灭减少和溶酶体与线粒体的共存减少。RAB7和LAMP1在应激的小胶质细胞中的表达降低，而RAGE的敲除逆转了这些作用，并导致溶酶体酸性增加。RVLM中的siHMGB1可降低SIH小鼠的血压，降低RSNA。当使用自噬诱导剂雷帕霉素促进有丝分裂吞噬通量时，这种处理导致外源性二硫化物HMGB1(DS-HMGB1)刺激的小胶质细胞中核因子-kappaB的激活和PIC释放减少。结论抑制HMGB1/RAGE轴的激活可导致应激诱导的吞噬细胞流量增加，从而减少小胶质细胞介导的神经炎症活动，从而降低RVLM的交感血管收缩驱动力。
Background Microglial mediated neuroinflammation in the rostral ventrolateral medulla (RVLM) plays roles in the etiology of stress-induced hypertension (SIH). It was reported that autophagy influenced inflammation via immunophenotypic switching of microglia. High-mobility group box 1 (HMGB1) acts as a regulator of autophagy and initiates the production of proinflammatory cytokines (PICs), but the underlying mechanisms remain unclear. Methods The stressed mice were subjected to intermittent electric foot shocks plus noises administered for 2 h twice daily for 15 consecutive days. In mice, blood pressure (BP) and renal sympathetic nerve activity (RSNA) were monitored by noninvasive tail-cuff method and platinum-iridium electrodes placed respectively. Microinjection of siRNA-HMGB1 (siHMGB1) into the RVLM of mice to study the effect of HMGB1 on microglia M1 activation was done. mRFP-GFP-tandem fluorescent LC3 (tf-LC3) vectors were transfected into the RVLM to evaluate the process of autolysosome formation/autophagy flux. The expression of RAB7, lysosomal-associated membrane protein 1 (LAMP1), and lysosomal pH change were used to evaluate lysosomal function in microglia. Mitophagy was identified by transmission electron microscopic observation or by checking LC3 and MitoTracker colocalization under a confocal microscope. Results We showed chronic stress increased cytoplasmic translocations of HMGB1 and upregulation of its receptor RAGE expression in microglia. The mitochondria injury, oxidative stress, and M1 polarization were attenuated in the RVLM of stressed Cre-CX3CR1/RAGE(fl/fl) mice. The HMGB1/RAGE axis increased at the early stage of stress-induced mitophagy flux while impairing the late stages of mitophagy flux in microglia, as revealed by decreased GFP fluorescence quenching of GFP-RFP-LC3-II puncta and decreased colocalization of lysosomes with mitochondria. The expressions of RAB7 and LAMP1 were decreased in the stressed microglia, while knockout of RAGE reversed these effects and caused an increase in acidity of lysosomes. siHMGB1 in the RVLM resulted in BP lowering and RSNA decreasing in SIH mice. When the autophagy inducer, rapamycin, is used to facilitate the mitophagy flux, this treatment results in attenuated NF-kappa B activation and reduced PIC release in exogenous disulfide HMGB1 (ds-HMGB1)-stimulated microglia. Conclusions Collectively, we demonstrated that inhibition of the HMGB1/RAGE axis activation led to increased stress-induced mitophagy flux, hence reducing the activity of microglia-mediated neuroinflammation and consequently reduced the sympathetic vasoconstriction drive in the RVLM.