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HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia
Journal of Neuroinflammation ( IF 9.3 ) Pub Date : 2020-01-10 , DOI: 10.1186/s12974-019-1673-3
Shutian Zhang , Li Hu , Jialun Jiang , Hongji Li , Qin Wu , Kokwin Ooi , Jijiang Wang , Yi Feng , Danian Zhu , Chunmei Xia

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. 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. 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/RAGEfl/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-κB activation and reduced PIC release in exogenous disulfide HMGB1 (ds-HMGB1)-stimulated microglia. 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.

中文翻译:

HMGB1 / RAGE轴通过削弱小胶质细胞的吞噬通量来介导小鼠应激性RVLM神经炎症

延髓腹侧延髓(RVLM)中的小胶质细胞介导的神经炎症在应激性高血压(SIH)的病因中起作用。据报道自噬通过小胶质细胞的免疫表型转换影响炎症。高迁移率族盒1(HMGB1)充当自噬的调节剂并启动促炎细胞因子(PIC)的产生,但其潜在机制仍不清楚。对受压的小鼠进行连续两次连续15天每天两次的断续性电脚电击加噪音,持续2小时。在小鼠中,通过无创尾套法和分别放置的铂铱电极监测血压(BP)和肾交感神经活性(RSNA)。将siRNA-HMGB1(siHMGB1)显微注射到小鼠的RVLM中,以研究HMGB1对小胶质M1激活的影响。将mRFP-GFP串联荧光LC3(tf-LC3)载体转染到RVLM中,以评估自溶酶体形成/自噬通量的过程。RAB7,溶酶体相关膜蛋白1(LAMP1)的表达和溶酶体pH值变化用于评估小胶质细胞中的溶酶体功能。通过透射电子显微镜观察或通过在共聚焦显微镜下检查LC3和MitoTracker共定位来鉴定线粒体。我们显示慢性应激增加了HMGB1的细胞质易位及其在小胶质细胞中受体RAGE表达的上调。在应激的Cre-CX3CR1 / RAGEfl / fl小鼠的RVLM中线粒体损伤,氧化应激和M1极化减弱。HMGB1 / RAGE轴在应激诱导的线粒体通量的早期增加,而在小胶质细胞中线粒体通量的后期受到损害,这是由于GFP-RFP-LC3-II点的GFP荧光猝灭减少以及溶酶体与线粒体的共定位降低。在受压的小胶质细胞中,RAB7和LAMP1的表达降低,而敲除RAGE可逆转这些作用,并导致溶酶体的酸性增加。RVLM中的siHMGB1导致SIH小鼠的BP降低和RSNA降低。当使用自噬诱导剂雷帕霉素促进线粒体通量时,这种处理导致外源二硫化物HMGB1(ds-HMGB1)刺激的小胶质细胞中NF-κB激活减弱,PIC释放减少。总的来说,
更新日期:2020-01-11
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