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-κB 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.

Neuroinflammation plays hypertensive roles in the uninjured autonomic nuclei of the central nervous system, while its mechanisms remain unclear. The present study is to investigate the effect of neuroinflammation on autophagy in the neurons of the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone reside. Stress-induced hypertension (SIH) was induced by electric foot-shock stressors with noise interventions in rats. Systolic blood pressure (SBP) and the power density of the low frequency (LF) component of the SAP spectrum were measured to reflect sympathetic vasomotor activity. Microglia activation and pro-inflammatory cytokines (PICs (IL-1β, TNF-α)) expression in the RVLM were measured by immunoblotting and immunostaining. Autophagy and autophagic vacuoles (AVs) were examined by autophagic marker (LC3 and p62) expression and transmission electron microscopy (TEM) image, respectively. Autophagy flux was evaluated by RFP-GFP-tandem fluorescent LC3 (tf-LC3) vectors transfected into the RVLM. Tissue levels of glutamate, gamma aminobutyric acid (GABA), and plasma levels of norepinephrine (NE) were measured by using high-performance liquid chromatography (HPLC) with electrochemical detection. The effects of the cisterna magna infused minocycline, a microglia activation inhibitor, on the abovementioned parameters were analyzed. SIH rats showed increased SBP, plasma NE accompanied by an increase in LF component of the SBP spectrum. Microglia activation and PICs expression was increased in SIH rats. TEM demonstrated that stress led to the accumulation of AVs in the RVLM of SIH rats. In addition to the Tf-LC3 assay, the concurrent increased level of LC3-II and p62 suggested the impairment of autophagic flux in SIH rats. To the contrary, minocycline facilitated autophagic flux and induced a hypotensive effect with attenuated microglia activation and decreased PICs in the RVLM of SIH rats. Furthermore, SIH rats showed higher levels of glutamate and lower level of GABA in the RVLM, while minocycline attenuated the decrease in GABA and the increase in glutamate of SIH rats. Collectively, we concluded that the neuroinflammation might impair autophagic flux and induced neural excitotoxicity in the RVLM neurons following SIH, which is involved in the development of SIH.

Purpose: Endoplasmic reticulum stress (ERS) plays an important role in the pathogenesis of lung ischemia/reperfusion (I/R) injury. Cyclic GMP-AMP synthase (cGAS) is a cytosol dsDNA sensor, coupling with downstream stimulator of interferon genes (STING) located in the ER, which involves innate immune responses. The aim of our present study was to investigate the effects of cGAS on lung I/R injury via regulating ERS. Methods: We used Sprague-Dawley rats to make the lung I/R model by performing left hilum occlusion-reperfusion surgery. cGAS-specific inhibitor RU.521, STING agonist SR-717, and 4-phenylbutyric acid (4-PBA), the ERS inhibitor, were intraperitoneally administered in rats. Double immunofluorescent staining was applied to detect the colocalization of cGAS or BiP, an ERS protein, with alveolar epithelial type II cells (AECIIs) marker. We used transmission electron microscopy to examine the ultrastructure of ER and mitochondria. Apoptosis and oxidative stress in the lungs were assessed, respectively. The profiles of pulmonary edema and lung tissue injury were evaluated. And the pulmonary ventilation function was measured using a spirometer system. Results: In lung I/R rats, the cGAS-STING pathway was upregulated, which implied they were activated. After cGAS-STING pathway was inhibited or activated in lung I/R rats, the ERS was alleviated after cGAS was inhibited, while when STING was activated after lung I/R, ERS was aggravated in the AECIIs, these results suggested that cGAS-STING pathway might trigger ERS responses. Furthermore, activation of cGAS-STING pathway induced increased apoptosis, inflammation, and oxidative stress via regulating ERS and therefore resulted in pulmonary edema and pathological injury in the lungs of I/R rats. Inhibition of cGAS-STING pathway attenuated ERS, therefore attenuated lung injury and promoted pulmonary ventilation function in I/R rats. Conclusion: Inhibition of the cGAS-STING pathway attenuates lung ischemia/reperfusion injury via alleviating endoplasmic reticulum stress in alveolar epithelial type II cells of rats. Graphical Abstract:

Accumulating evidence suggests that neuroinflammation and oxidative stress in cardiovascular center contribute to the pathological processes underlying hypertension. Microglia activation triggers the inflammation and oxidative stress. Melatonin is a documented potent anti-inflammatory regent and antioxidant, the underlying roles of melatonin in regulating microglia activation via mitochondria remain unclear. In present study, we investigated the protective role of melatonin in decreasing M1 phenotype switching via attenuating mitochondrial oxidative damage in dependence on uncoupling protein 2 (UCP2) pathway in microglia. Prorenin (20 nmol/L; 24 hr) was used to induce inflammation in cultured microglia. Mitochondrial morphology was detected by transmission electron microscope. The reactive oxygen species (ROS) production by using DCFH-DA fluorescence imaging and mitochondrial membrane potential (MMP, ΔΨm) was evaluated by JC-1 staining. The indicator of the redox status as the ratio of the amount of total NADP⁺ to total NADPH, and the expression of 6 subunits of NADPH oxidase is measured. The pro-inflammatory cytokines releasing was measured by qPCR. UCP2 and activated AMPKα (p-AMPKα) expression were examined by immunoblot. Melatonin (100 μM) markedly alleviated the M1 microglia phenotype shifting and abnormal mitochondria morphology. Melatonin attenuated prorenin-induced ΔΨm increasing and ROS overproduction. Melatonin decreased the redox ratio (NADP⁺/NADPH) and the p47phox and gp91phox subunits of NADPH oxidase expression in prorenin-treated microglia. These effects were reversed in the presence of UCP2 siRNA. Our results suggested that the protective effect of melatonin against prorenin-induced M1 phenotype switching via attenuating mitochondrial oxidative damage depending on UCP2 upregulation in prorenin-treated microglia.

Increased microglial activation and neuroinflammation within autonomic brain regions such as the rostral ventrolateral medulla (RVLM) have been implicated in stress-induced hypertension (SIH). Prorenin, a member of the brain renin-angiotensin system (RAS), can directly activate microglia. The present study aimed to investigate the effects of prorenin on microglial activation in the RVLM of SIH rats. Rats were subjected to intermittent electric foot-shocks plus noise, this stress was administered for 2 h twice daily for 15 consecutive days, and mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) were monitored. The results showed that MAP and RSNA were augmented, and this paralleled increased pro-inflammatory phenotype (M1) switching. Prorenin and its receptor (PRR) expression and the NLR family pyrin domain containing 3 (NLRP3) activation were increased in RVLM of SIH rats. In addition, PLX5622 (a microglial depletion agent), MCC950 (a NLRP3 inhibitor), and/or PRO20 (a (Pro)renin receptor antagonist) had antihypertensive effects in the rats. The NLRP3 expression in the RVLM was decreased in SIH rats treated with PLX5622. Mito-tracker staining showed translocation of NLRP3 from mitochondria to the cytoplasm in prorenin-stimulated microglia. Prorenin increased the ROS-triggering M1 phenotype-switching and NLRP3 activation, while MCC950 decreased the M1 polarization. In conclusion, upregulated prorenin in the RVLM may be involved in the pathogenesis of SIH, mediated by activation of the microglia-derived NLRP3 inflammasome. The link between prorenin and NLRP3 in microglia provides insights for the treatment of stress-related hypertension.