Brain-Derived Small Extracellular Vesicles from Dahl Salt-Sensitive Rats with High Salt Diet Induce Inflammation and Oxidative Stress
Authors: Chen Xinqian , Yan Xin , Gingerich Leah , Chen Qinghui , Bi Lanrong , Shan Zhiying 
Published: May 2023

It has been reported that small extracellular vesicles (sEVs ≤ 200 nm) are implicated in the pathogenesis of multiple diseases including hypertension. However, the role of brain-derived sEVs in the development of salt sensitive hypertension (SSHTN) remains unclear. We hypothesize that brain-derived sEVs from high salt diet-treated rats can induce inflammation and oxidative stress in the central nervous system (CNS). To test this hypothesis, brain-derived sEVs of Dahl salt-sensitive rats with high salt (HS) diet (Dahl-HS-sEV) were used to treat primary brain neuronal cultures and microinjected into brain lateral ventricles, respectively, proinflammatory cytokines, chemokines, and oxidative stress markers were measured through real-time PCR or fluorescent probes. sEVs isolated from Sprague Dawley (SD) rats with normal salt (NS) diet (SD-NS-sEV) were used as a control. Briefly, we isolated sEVs from brain tissues using ultracentrifugation and identified sEVs with scanning electron microscopy, dynamic light scattering and western blots. Primary neurons derived from neonatal SD rats were incubated with either Dahl-HS-sEV (4μg/mL), or SD-NS-sEV (4μg/mL) for 24 h. The mRNA levels of inflammatory factors, neuronal activity indicator (c-Fos) and NADPH oxidase subunits (CYBA and CYBB) were tested by real-time PCR. Results showed that Dahl-HS-sEV incubation increased mRNA levels of inflammatory cytokines including TNFα (2.3-fold) and IL1β (3.7-fold) with statistical significance (P<0.05). It also significantly increased (P<0.05) mRNA levels of chemokines including CCL2 (2.4-fold), CCL5 (2.1-fold), and CCL12 (4.2-fold). In addition, Dahl-HS-sEV treatment increased mRNA levels of transcription regulator, NF-κB (1.4-fold), and neuronal activation marker, c-FOS (1.3-fold), as well as CYBA (1.7-fold), in primary neurons, compared to SD-NS-sEV-treated cells (P<0.05). We further tested mitochondrial reactive oxygen species (ROS) levels using fluorescent probes in primary neurons. Confocal images showed that Dahl-HS sEV significantly increased mitochondrial ROS levels, with total fluorescence intensity increased 1.6-fold relative to SD-NS-sEV treatment (P<0.01). Subsequently, we tested the effect of sEVs on the inflammatory cytokine marker expression in the brain. SD-NS rats received intracerebroventricular injection of either Dahl-HS-sEV (5.5 μg /rat, n=4) or SD-NS-sEV (5.5 μg/rat, n=4) and euthanized 6 h after injection. Their brains were removed and paraventricular nucleus (PVN) were punched out. RNAs were isolated from PVN tissues and used for real-time PCR assessment. Results showed that Dahl-HS-sEV significantly increased (P<0.05) PVN mRNA levels of IL1β (4.3-fold), CCL5 (2.6-fold), IL-6 (3.4-fold) and NOS2 (5.2-fold) in SD-NS rats 6 h after injection. These results suggested that in SSHTN, brain-derived sEVs may induce central inflammation and oxidative stress, which in turn results in an elevation of arterial blood pressure. This work was supported by NIH grant R01HL 163159 (Shan) and R15HL 150703 (Shan), and Portage Health Foundation Mid-Career Award (Shan). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.