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Back to Journal »Journal of Inflammation Research» Volume 14

PLIN2 mediates neuroinflammation and oxidative/nitrosative stress by down-regulating the phosphatidylethanolamine in the ventrolateral medulla of stress-induced hypertension rats

Authors: Zhang S, Hu L, Han C, Huang R, Ooi K, Qian X, Ren X, Chu D, Zhang H, Du D, Xia C 

Published on November 30, 2021, the 2021 volume: 14 pages 6331-6348

DOI https://doi.org/10.2147/JIR.S329230

Single anonymous peer review

Reviewing editor: Professor Quan Ning

Shutian Zhang,1,* Li Hu,2,* Chengzhi Han,1 Renhui Huang,1 Kokwin Ooi,1 Xinyi Qian,1 Xiaorong Ren,1 Dechang Chu,3 Haili Zhang,3 Dongshu Du,4 Chunmei Xia1 1Physiology and Fudan Pathophysiology, School of Basic Medicine, Shanghai 200032; 2 Department of Cardiology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200240; 3 School of Agriculture and Bioengineering, Heze University, Heze 274000; 4 School of Life Sciences, Shanghai University, Shanghai, 200444 *above The author has equal contributions to this article. Corresponding author: Xia Chunmei, School of Basic Medicine, Fudan University, Shanghai 200032, China Phone +86 21 54237612-805 Email [Email Protection] Du Dongshu, School of Life Sciences, Shanghai University 200444 People’s Republic of China Email [Electronics Mail protection] Purpose: Oxidative/nitrosative stress, neuroinflammation and their intimate interaction mediate the excessive activation of sympathetic hypertension. Excessive inflammation is characterized not only by increased pro-inflammatory cytokines (PIC), but also by increased mitochondrial dysfunction, reactive oxygen species (ROS) and nitric oxide (NO). Recent data indicate that both phospholipids and lipid droplets (LD) are effective regulators of microglia physiology. Method: Stressed rats were subjected to complex stressors for 15 days, and PLIN2-siRNA or scrambled-siRNA (SC-siRNA) was administered to the ventrolateral medulla oblongata (RVLM). Analyze lipids by mass spectrometry-based quantitative lipidomics. Detect the phenotype and proliferation of microglia LDs in RVLM in rats; evaluate blood pressure (BP) and myocardial damage in rats. The antioxidant/nitrosative stress effects of phosphatidylethanolamine (PE) were explored in cultured primary microglia. Results: Lipidomic analysis showed that 75 individual lipids in RVLM were significantly unbalanced due to stress [PE is the most common], indicating that the lipid composition changes with stress. In vitro, prorenin stress induces the accumulation of LDs and increases PICs, which can be blocked by siRNA-PLIN2 in microglia. PLIN2 knockdown up-regulated PE synthesis in microglia. The antioxidant/nitrosative stress effect delivered by PE was confirmed by the reduction of ROS and the reduction of 3-NT and MDA in microglia treated with prorenin. PLIN2 knockdown in RVLM blocked the number of iNOS+ and PCNA+ microglia, lowered blood pressure, and reduced cardiac fibrosis and hypertrophy in stressed rats. Conclusion: PLIN2 mediates microglial polarization/proliferation by down-regulating PE in RVLM of stressed rats. The delivery of PE is a promising strategy to combat neuroinflammation and oxidative/nitrosative stress in stress-induced hypertension. Keywords: lipid droplets, PLIN2, phosphatidylethanolamine, lipidomics, microglia expansion, neuroinflammation, stress, RVLM, oxidative/nitrosative stress

There is evidence that chronic stress is one of the important pathogenic factors of hypertension and target organ damage. 1,2 Stress leads to activation of the HPA axis and increased drive of the cardiovascular central sympathetic nerves, which ultimately leads to an increase in blood pressure. The activation of microglia is one of the most significant brain changes caused by chronic stress. 3,4 There is increasing evidence that stress can cause gliosis and inflammation in rodent RVLM. 5-7 It is worth noting that the excessive inflammatory response is not only manifested as an increase in inflammatory cytokines, but also an increase in mitochondrial dysfunction, reactive oxygen species (ROS) and nitric oxide (NO). Overactive brain regions of the renin-angiotensin system (RAS), oxidative/nitrosative stress, and neuroinflammation play a key role in triggering the center to enhance sympathetic nerve activity. 8-12 We have previously reported that stress causes mitochondrial damage in RVLM microglia. 13,14 Microglial mitochondrial autophagy-lysosomal dysfunction mediates the activation of NLRP3 inflammasomes in the cells of stress-induced hypertension mice, thereby promoting the release of pro-inflammatory factors. 13,14 In vitro, we found that stressed microglia showed increased mitochondrial ROS (mitoROS). 15 However, the molecular mechanisms of stress-induced microglia M1 polarization and oxidative/nitrosative stress and their effects on hypertensive heart damage are not fully understood.

A variety of blood-derived and peripheral-derived stimuli activate microglia and mediate the formation of inflammation in the heart sympathetic center. 16,17 In addition to the M1 pro-inflammatory activation and M2 anti-inflammatory activation, which have received much attention, neuroinflammation is also accompanied by microglia proliferation, leading to microglia proliferation. 18,19 proved that chronic stress and various metabolic stresses can induce the proliferation and activation of microglia in the heart sympathetic center such as the paraventricular nucleus (PVN). 20-23

Recent data indicate that both lipid droplets and phospholipids are effective regulators of microglia physiology. 24-26 On the one hand, phospholipids provide a metabolic basis for the proliferation and activation of stress-related inflammatory cells; on the other hand, it also acts as a pro-inflammatory mediator, mediated through oxidative stress or Toll-like receptor (TLR)-dependent pathways Neuroinflammation. 27-31 Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are the two most abundant phospholipids in eukaryotic cells. The changes of PE and PC content are sensitively regulated by chronic stress and exercise, and the abnormal conversion rate/ratio is also involved in the pathogenesis of metabolic syndrome [32-35]. It has been found that the decrease or increase of PE and the conversion of PE to PC are related to the occurrence of non-alcoholic fatty liver disease and obesity. 36-38 It has been reported that chronic stress leads to a decrease in the synthesis of PE in the brain. In vitro injection of PE can protect the neurotoxicity caused by the stress hormone corticosterone. 32,39 The imbalance of phospholipid synthesis and transformation is also involved in the inflammatory activation and promotion of macrophages. The release of inflammatory factors such as IL-1β indicates that the phospholipid metabolism disorder represented by the down-regulation of PE may be related to the neuroinflammation caused by stress. 40,41 The potential anti-inflammatory and antioxidant effects of PE delivery need to be further explored. She verified.

Lipid droplets (LDs) are the most important organelles that regulate lipid metabolism. On the one hand, lipid droplets act as storage organelles for neutral fatty acids and provide substrates for phospholipid synthesis. On the other hand, the lipid droplet membrane serves as a functional docking site for enzymes involved in phospholipid synthesis. 42 The lipid droplet membrane protein perilipins performs the function of recruiting enzymes involved in phospholipid synthesis. Highly proliferating cells, including cancer cells and inflammatory cells, continue to contain a large number of LDs, which provides a solid foundation for cell proliferation and the production of inflammatory signaling molecules. 43-45 Inhibition of LDs accumulation regulates the metabolic pattern of proliferating cells and partially blocks cell proliferation signals. The 46-48 week lipoprotein is an important structural protein of the LDs membrane. It separates the hydrophobic lipid core from the cytoplasm and participates in the maintenance of LDs homeostasis. PLIN2 is an inherent perilipin protein whose expression and phosphorylation are involved in lipolysis, fat phagocytosis, lipid droplet fusion and signal transduction. 49-52 PLIN2 activation-mediated accumulation of LDs has been observed in microglia activated by various inflammatory stimuli, which is closely related to the production of ROS and inflammatory cytokines. 24,53,54 Our preliminary data indicate that LDs accumulate in stressed microglia. In addition, lipidomics analysis shows that PE is the most down-regulated lipid in RVLM of stressed rats, indicating the lipid composition Changes with pressure.

We have previously reported that prorenin (a component of the RAS system) and HMGB1 (a stress response molecule) mediate mitochondrial damage and block autophagy in SIH's RVLM microglia. 13,14 The discovery of mitochondrial damage and mitochondrial autophagy prompted us to further explore the molecular mechanism of stress-induced microglia activation from the perspective of metabolic remodeling. In our current study, we confirmed that PLIN2-induced PE deficiency is related to microglia polarization/proliferation and oxidative/nitrosative stress in RVLM. Exogenous PE supplements can effectively block the oxidative/nitrosative stress induced by prorenin in cultured microglia. Central PLIN2 siRNA administration can block neuroinflammation and then reverse the increase in blood pressure and myocardial damage caused by stress.

Nitrotyrosine ELISA kit (ab113848) and phosphatidylethanolamine detection kit (ab241005) were purchased from Abcam (Cambridge, Massachusetts). L-α-phosphatidylethanolamine (PE) (L130310) was purchased from Aladdin (Shanghai, China). Mito-SOX (M36008), BODIPY probe (D3835) and WGA (W11261) were purchased from Thermo Fisher Scientific (Waltham, USA). GSH and GSSG detection kit (S0053), lipid peroxidation MDA detection kit (S0131M), hematoxylin and eosin staining kit (C0105M) were purchased from Beyotime (Shanghai, China). Masson Tricolor Staining Kit (DC0032) was purchased from Leagene (Beijing, China). NE ELISA kit (E-EL-0047c) was purchased from Elabscience (Wuhan, China). SC-siRNA (sc-37007); PLIN2-siRNA (sc-270227) was purchased from Santa Cruz Biotechnology (California, USA). Antibodies: PLIN2 (ab108323), iNOS (ab178945), Iba-1 (ab153696), Iba-1 (ab283342), PGP 9.5 (ab72911), TNF-α (ab205587), PCNA (ab26-tro13939) and IL-1β ( ab254360) from Abcam (Cambridge, MA). c-fos (#2250, CST) was purchased from Cell Signaling Technology (Beverly, MA). 4-hydroxynonenal (MA5-27570) was purchased from Thermo Fisher Scientific (Waltham, USA).

Male adult Sprague-Dawley rats (8 weeks old, 250-300 g) were from the Animal Experiment Center of Fudan University. All experimental procedures are in compliance with the guidelines of the Institutional Ethics Committee of Fudan University and approved by the Animal Care Committee of Fudan University. All efforts are made to minimize the number of mice used and reduce their pain. The rats were kept in a 24°C temperature-controlled animal laboratory, under a 12-hour light/dark cycle, and fed with standard food and tap water ad libitum. The experimental design is shown in Figure 1. SC-siRNA or PLIN2-siRNA was microinjected into the RVLM of rats to concentrate the interference genes. Figure 1 The experimental flow chart of the research.

Figure 1 The experimental flow chart of the research.

RVLM stereotactic surgery and microinjection were performed as described in our previous study. 5 In brief, rats were anesthetized intraperitoneally with a mixed anesthetic composed of 140 g urethane, 7 g chloralose and 7 g borax at a dose of 7 mL/kg and dissolved in 1 L of normal saline. Place the rat's head on a stereotaxic device (Neurostar Tubingen, Germany) and bend it to an angle of 45°. The animal was intubated with a polyethylene tube and breathed indoor air spontaneously. The occipital bone was removed and the fourth ventricle of the rat was exposed. According to the regulations in the Paxinos map, make a glass micropipette and insert the RVLM (1.5-1.9 mm in front of obex, 1.5-2.0 mm on the right side of the midline, and 6.6-7.0 mm deep on the back of the cerebellum) and Watson. 55 To knock out PLIN2 in RVLM, rats were anesthetized and received bilateral RVLM microinjection of PLIN2 siRNA (0.3 μg in 0.7 μL 10 mM JetSITM). The promiscuous siRNA was used as a control. After the injection, the skull is sealed with bone wax and the surface of the skull is disinfected. Then the skin suture is interrupted and the incision is disinfected. A heating pad was used to maintain the body temperature of the rats at 37°C until they recovered from the anesthesia. The rats were injected with procaine penicillin (1000 IU, intramuscular injection) after the operation.

As described in our previous publication, compound stress was applied to rats. In short, the rat is placed in a 22 cm × 22 cm × 28 cm cage, and computer-controlled intermittent electric shocks (35-75 V, 0.5 millisecond duration) are given every 2-30 seconds. At the same time, the noise (range, 88-98 decibels) produced by the buzzer was given to the rat as a conditioned stimulus. 6 Rats are under pressure twice a day for 2 hours for 15 consecutive days. The sham control rats were subjected to sham stress; they were placed in cages of the same size and kept for the same time, but they were not subject to foot vibration or noise pressure.

RVLM from 8 stressed rats and 8 sham rats was used for lipidome analysis. Lipidomic testing and analysis were performed by OE biotech (Shanghai, China), see the work of Wu et al. and Chen et al. for details. 56,57 To briefly explain, the solution of dichloromethane/methanol (2/1, v/v) homogenized with pure water is used to extract lipids. Lysophosphatidylcholine (17:0, 0.01 mg/mL) and L-2-chlorophenylalanine (0.3 mg/mL) were used as internal standards. Isopropanol/methanol (1/1, v/v) is used as the solvent. Lipidomic analysis was performed on the Ultimate-3000 UPLC system combined with the Q-Exactive hybrid quadrupole-Orbitrap MS system (Thermo Scientific), which used a 100×2.1-mm hypersil GOLD 1.9-μm C18 column (Thermo Scientific) Scientific). All raw data were obtained using Xcalibur software (version 3.0, Thermo Scientific). LipidSearch (version 4.0, Thermo Scientific) is used for lipid identification and quantification.

The culture of primary microglia is the same as our previous publication. In short, the medulla oblongata containing RVLM was taken from 1-2 days old Sprague-Dawley rats and then sacrificed by decapitation. 13 The location of RVLM was determined based on the map of Watson and Paxinos. 55 Both sides of the RVLM (as described above) are collected using micropores, and then the minced tissues are incubated in a dissection medium containing glucose, bovine serum albumin (BSA) and HEPES and 0.025% trypsin at 37°C 20 minutes. Then, the cells were inoculated in Dulbecco's modified Eagle medium (DMEM) containing GlutaMax and high glucose (4.5 g/L) at 3 × 105 cells/cm2, supplemented with 10% fetal bovine serum, 0.1 mg/mL chain And 100 U/mL of penicillin in a 75 cm2 polylysine-coated culture flask. On day 9, resuspend the cells after centrifugation (150 × g for 10 minutes). The cell viability was evaluated by the trypan blue exclusion method. When identified by staining with specific microglia markers, the purity of the cultured microglia is >95%. The in vitro experimental design is shown in Figure 1. siRNA was purchased from Santa Cruz Biotechnology and transfected into microglia to knock out PLIN2.

Rats were anesthetized intraperitoneally (ip) with sodium pentobarbital (50 mg/kg). Before the rats were sacrificed, the cardiac diastolic and systolic functions of the rats were evaluated by transthoracic echocardiography. The image was processed at 21 MHz using an MS-250 transducer operated by a Vevo 2100 color Doppler ultrasound scanner (FUJIFILM VisualSonics Inc., Toronto, ON, Canada). The blood pressure was measured for 5 minutes through the femoral artery cannula using a pressure sensor and a polygraph (Powerlab, AD Instruments, Australia). Make limb leads to record ECG. The analysis of heart rate variability (HRV) also comes from the analysis module in POWER LAB.

Alexa Fluor 488 conjugate with Masson trichrome stain, hematoxylin-eosin (HE) stain and wheat germ agglutinin stain (WGA, cell membrane stain) was used for 5 µm thin myocardial sections. Interstitial fibrosis (excluding vascular spaces) was evaluated on Masson's trichrome stained sections. In short, ImageJ software (NIH, Bethesda, MD, USA) uses two independent observers to threshold the red-green-blue superimposed images of Masson's three-color slices to determine the fibrotic tissue area and the total myocardial tissue area Ratio. As mentioned earlier, HE and/or WGA stained sections are used to assess cardiomyocyte hypertrophy by measuring the cross-sectional area. 58

Perform immunofluorescence staining as previously described. After the rats were deeply anesthetized, the left ventricle was perfused with 300 mL of 0.01 M PBS (pH 7.4), followed by 300 mL of 4% paraformaldehyde in 0.1 M PBS. RVLM slices were fixed for 4 hours, then placed in 20% and 30% sucrose gradients, and dehydrated overnight at 4°C. Subsequently, 30 μm thick free-floating coronal sections containing RVLM were washed in PBS, and then permeabilized with 0.3% Triton X-100 for 30 minutes, and then incubated with 5% goat serum at 37°C for 1 hour to block non- Specific protein. The section and the primary antibody [antibody: PLIN2, iNOS, TNF-α, IL-1β, PCNA, 3-nitrotyrosine, 4-hydroxynonenal, c-fos, Iba-1 (microglia marker [The Donkey Anti-Goat IgG H&L (Alexa Fluor 488), Donkey Anti-Goat IgG H&L (Cy5), Donkey Anti-Rabbit) overnight at 4°C. IgG H&L (Alexa Fluor 647) and donkey anti-rabbit IgG H&L (Alexa Fluor 488)] were kept at room temperature for 2 hours. The sections were observed under the FluoView FV300 laser scanning confocal microscope (Zeiss, Carl Zeiss, Germany). Use ImageJ software to process immunofluorescence images.

BODIPY+ staining is used to evaluate the LD (the largest projection of the z-stack in the entire slice) from six randomly selected fields of view. Two independent observers manually calculate the number of LDs in RVLM. In order to measure the average size of lipid droplets, the BODIPY+ signal was randomly analyzed six times using the "Analyze Particles" function of ImageJ 1.45 s (ImageJ website: http://imagej.nih.gov.laneproxy.stanford.edu/ij/) The selected field of view.

The total protein 13-15 was extracted from RVLM tissue according to our previously described western blotting procedure. Homogenize RVLM tissue in a lysis buffer containing 1% NP40 and 1 mmol/L PMSF. Extract the same amount of protein sample from each rat, extract protein from RVLM homogenate, and analyze protein expression by Western blot. In short, the protein samples (20 μg each) were subjected to SDS/PAGE in an 8-12% gradient gel (Invitrogen, Carlsbad, CA) and transferred to a PVDF membrane. Incubate the primary antibody and then incubate with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG. ECL detection reagent (WBKLS0050; Millipore) was used to evaluate the amount of protein, and the immunostaining band was visualized and quantified by an automated chemiluminescence image analysis system (Tanon-5200; Tanon Science & Technology, Shanghai, China). The data was normalized by developing β-actin as a loading control. Except for β-actin (1:5000), the concentration of all antibodies is 1:1000.

This procedure has been reported in our previous publications. 14 In short, RVLM tissue is fixed, infiltrated, washed with phosphate buffer, dehydrated and embedded in epoxy resin, and allowed to polymerize at 70°C for 24 hours. The block containing RVLM was sliced ​​at 70-80 nm using an ultramicrotome (Ultracut; Leica), collected on a grid and stained with uranyl acetate and lead citrate. Check the grid under an 80 kV transmission electron microscope (H-700; Hitachi, Tokyo, Japan).

Measure the mitochondrial superoxide anion to reflect the mitochondrial ROS level according to the manufacturer's instructions of the MitoSOX Red kit (Thermo Fisher Scientific). In short, prepare 5 μM MitoSOX reagent working solution and incubate cells at 37°C in the dark for 10 minutes. Cells incubated without MitoSOX reagent are used for background subtraction. Determine the average fluorescence intensity and normalize all samples to control samples.

According to the manufacturer's instructions, use a PE detection kit (ab241005, Abcam) to determine the concentration of PE in rat RVLM and/or in vitro microglia.

ELISA analysis was performed according to the manufacturer's instructions. The plasma NE concentration of rats was measured by using NE ELISA kit (E-EL-0047c, Elabscience). The 3-NT concentration in the supernatant of microglia culture medium was measured using the nitrotyrosine ELISA kit (ab113848, Abcam).

Use SPSS 11.0 software to analyze the obtained data. Data are expressed as mean ± standard error of mean (SEM). Through analysis of variance, then use GraphPad Prism software to perform Tukey multiple comparison post-hoc test (if the variances are equal) or Dunnett T3 (if the variances are not equal) to evaluate the treatment group mean. A P value of <0.05 is considered significant for all tests.

In order to discover the ultrastructural differences between rat sham operation and stress RVLM, we analyzed their cytoplasmic content by transmission electron microscopy. We have observed that representative lipid droplets (LDs) are present in stress, but rarely in sham RVLM in rats. In addition, LDs are mostly adjacent to or contained in lysosomes (Figure 2A). In order to study whether the dynamics of LDs in RVLM neurons/microglia changes in stressed rats, BODIPY is a dye that specifically labels neutral lipids and is commonly used to detect LDs, using PGP9.5 (neuron Marker)/Iba for double immunofluorescence staining. 1 (Microglia labeling) is executed (Figure 2B and C). The results show that there are many and greater LDs in the RVLM of stressed rats (Figure 2D and E). Compared with sham rats, the percentage of BODIPY+ neurons/microglia in RVLM is 3 times higher under stress (Figure 2F and G). LDs also react with their lipid droplet surface protein Perilipin 2 (PLIN2). Then we detected PLIN2 expression in the RVLM of rat brain slices (Figure 2H and I), and we found that more PLIN2+ neurons/microglia were counted under pressure than sham rats (Figure 2J and K). Taken together, these data indicate that stress induces the accumulation of LDs in RVLM, accompanied by an increase in PLIN2 expression. Figure 2 Stress promotes the accumulation of LDs in RVLM. (A) Representative transmission electron micrographs of RVLM in stressed and sham rats. The yellow arrow indicates LD. Scale bar = 2 μm. (B) Double immunofluorescence staining of BODIPY (a specific probe for LD) using PGP9.5 (a neuronal marker) in the RVLM of rats. (C) Double immunofluorescence staining of BODIPY and Iba-1 (a microglia marker) in rat RVLM. (D-G) Quantification of the number of BODIPY+ lipid droplets in RVLM (D), the average size of BODIPY+ lipid droplets in RVLM (E), BODIPY+/PGP9.5+ cell percentage (F) and BODIPY+/Iba-1+ percentage in RVLM Cell (G). (H) Double immunofluorescence staining of PLIN2 and PGP9.5 in rat RVLM. (I) Double immunofluorescence staining of PLIN2 and Iba-1 in rat RVLM. (J and K) PLIN2+/PGP9.5+ cell percentage (J) and PLIN2+/Iba-1+ cell percentage (K) in RVLM. The scale bar in (B), (C), (H) and (I) = 20 μm. Data are expressed as mean ± SD. n = 6–8, *p <0.05, t test.

Figure 2 Stress promotes the accumulation of LDs in RVLM. (A) Representative transmission electron micrographs of RVLM in stressed and sham rats. The yellow arrow indicates LD. Scale bar = 2 μm. (B) Double immunofluorescence staining of BODIPY (a specific probe for LD) using PGP9.5 (a neuronal marker) in the RVLM of rats. (C) Double immunofluorescence staining of BODIPY and Iba-1 (a microglia marker) in rat RVLM. (D-G) Quantification of the number of BODIPY+ lipid droplets in RVLM (D), the average size of BODIPY+ lipid droplets in RVLM (E), BODIPY+/PGP9.5+ cell percentage (F) and BODIPY+/Iba-1+ percentage in RVLM Cell (G). (H) Double immunofluorescence staining of PLIN2 and PGP9.5 in rat RVLM. (I) Double immunofluorescence staining of PLIN2 and Iba-1 in rat RVLM. (J and K) PLIN2+/PGP9.5+ cell percentage (J) and PLIN2+/Iba-1+ cell percentage (K) in RVLM. The scale bar in (B), (C), (H) and (I) = 20 μm. Data are expressed as mean ± SD. n = 6–8, *p <0.05, t test.

Lipid droplets are organelles that regulate cell lipid metabolism. The accumulation of lipid droplets in microglia indicates that lipid metabolism disorders may be involved in RVLM neuroinflammation. Non-targeted lipidomics analysis was performed to study the effect of stress on the lipidome of RVLM and its association with neuroinflammation. Unsupervised PCA showed a clear separation between the sham group and the pressure group, where R2X[1] was 0.285 and R2X[2] was 0.194 (Figure 3A). A significant difference in metabolic phenotype was further observed from the OPLS-DA model, Q2 was 0.75 (Figure 3B). In addition, the PLS-DA model verified a significant separation between the sham operation group and the stress group, with R2[Y] of 0.998 (Figure 3C). A total of 1,341 lipids were manually identified and quantified based on their MS/MS spectra (Figure 3D). Among them, 75 lipids were significantly unbalanced due to pressure. The selection criteria were VIP> 1.0 and p-value <0.05. The relative expression levels between the sham operation group and the pressure group were visualized in a heat map based on cluster analysis (Figure 3E). It is worth noting that the reduction in PE (20:0e/22:4) is the largest among the detected lipids. According to KEGG pathway enrichment analysis (https://www.kegg.jp/), the identified lipid types are divided into 17 metabolic pathways (Figure 3F). Among them, glycerophospholipid metabolism showed the highest diversity, followed by choline and retrograde endocannabinoid metabolism. Figure 3 Stress causes a deficiency of phosphatidylethanolamine in RVLM. (A) PCA scoring chart, (B) OPLS-DA scoring chart and (C) PLS-DA scoring chart for fake and stressed rats. (D) The volcano map is represented by the pressure plotted against -log10 and the false log2 (multiple change). (E) The heat map of 75 different metabolites determined from the comparison of sham rats and stressed rats. The color from green to red in the heat map indicates the increase in metabolites from low to high. (F) Pathway enrichment analysis based on altered metabolites. Data are expressed as mean ± SD. n = 8, p <0.05, t test.

Figure 3 Stress causes a deficiency of phosphatidylethanolamine in RVLM. (A) PCA scoring chart, (B) OPLS-DA scoring chart and (C) PLS-DA scoring chart for fake and stressed rats. (D) The volcano map is represented by the pressure plotted against -log10 and the false log2 (multiple change). (E) The heat map of 75 different metabolites determined from the comparison of sham rats and stressed rats. The color from green to red in the heat map indicates the increase in metabolites from low to high. (F) Pathway enrichment analysis based on changed metabolites. Data are expressed as mean ± SD. n = 8, p <0.05, t test.

To determine the effect of LDs on PE synthesis, we used siRNA to knock down PLIN2 to reduce the accumulation of LDs in RVLM/in vitro microglia, and then quantified the PE concentration. The PE detection kit is used to quantify PE in RVLM/microglia. We found that compared with the vehicle group, intra-RVLM microinjection of PLIN2-siRNA significantly reduced the protein level of PLIN2 in the RVLM of stressed rats (Figure 4A and B). Immunofluorescence staining of PLIN2 was used to verify the knockdown efficiency of PLIN2-siRNA in RVLM (Figure 4C and D). The test results of the PE detection kit showed that stress induced PE deficiency in RVLM, while PLIN2-siRNA inhibited its decline (Figure 4E). Figure 4 Knockdown of T PLIN2 alleviates PE deficiency in RVLM under stress of rat and/or prorenin-treated microglia. (A and B) Western blot results show that the PLIN2 protein in the RVLM of PLIN2-siRNA microinjected rats is deleted. (C and D) Immunofluorescence staining also showed that the PLIN2 protein in RVLM was deleted in PLIN2-siRNA microinjected rats. Scale bar = 100 μm. (E) The results of the PE assay show that knocking down PLIN2 alleviates stress-induced PE deficiency in RVLM in rats. (F and G) Immunofluorescence staining results showed that PLIN2 deletion was achieved in cultured microglia. Scale bar = 4 μm. (H and I) BODIPY staining showed that PLIN2 knockdown attenuated the accumulation of LDs in cultured microglia stimulated by renin. Scale bar = 4 μm. (J) The results of the PE assay further confirmed that knockdown of PLIN2 alleviated PE deficiency in microglia treated with prorenin. Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test.

Figure 4 Knockdown of T PLIN2 alleviates PE deficiency in RVLM under stress of rat and/or prorenin-treated microglia. (A and B) Western blot results show that the PLIN2 protein in the RVLM of PLIN2-siRNA microinjected rats is deleted. (C and D) Immunofluorescence staining also showed that the PLIN2 protein in RVLM was deleted in PLIN2-siRNA microinjected rats. Scale bar = 100 μm. (E) The results of the PE assay show that knocking down PLIN2 alleviates stress-induced PE deficiency in RVLM in rats. (F and G) Immunofluorescence staining results showed that PLIN2 deletion was achieved in cultured microglia. Scale bar = 4 μm. (H and I) BODIPY staining showed that PLIN2 knockdown attenuated the accumulation of LDs in cultured microglia stimulated by renin. Scale bar = 4 μm. (J) The results of the PE assay further confirmed that knockdown of PLIN2 alleviated PE deficiency in microglia treated with prorenin. Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test.

Our previous studies have confirmed that prorenin is an important pro-inflammatory intermediate molecule for the activation of RVLM microglia induced by pro-hypertensive stress. Therefore, in all in vitro experiments in this study, prorenin (20 nmol/L, 24 hours) was used to stimulate microglia. Transfection of cells with PLIN2-siRNA significantly reduced PLIN2 expression (Figure 4F and G). It is worth noting that after knocking out PLIN2, the number of LD in microglia decreased (Figure 4H and I), confirming that PLIN2 is a key determinant of LD accumulation in microglia. In addition, through the PE detection kit test, we found that prorenin significantly reduced PE in microglia, and knockdown of PLIN2 inhibited these effects (Figure 4J). Taken together, these results indicate that knockdown of PLIN2 alleviates PE deficiency in compressed RVLM/prorenin-treated microglia.

In view of the reduced PE of compressed microglia, we speculate that PE may play a potential role in the pathophysiology of microglia. Exogenous PE was delivered in prorenin-treated microglia to investigate whether PE deficiency is related to microglia oxidation/nitrosation stress in vitro. We first used the Mito-SOX probe to detect the level of ROS represented by mitochondrial superoxide anion, and found that a large amount of ROS was produced in microglia by stimulation of renin, and PE supplementation inhibited the production of ROS (Figure 5A and B) . The ratio of GSSG/GSH is measured to reflect the consumption of the antioxidant system. Our results showed that PE supplementation reduced the ratio of GSSG/GSH in prorenin-treated microglia (Figure 5C), indicating that PE supplementation increased the antioxidant capacity of microglia under stress. Use 3-nitrotyrosine (3-nitrotyrosine, 3NT) competitive ELISA to assess the level of nitrosative stress to determine the level of nitrotyrosine modified protein. We observed that prorenin induces nitrosative stress in compressed microglia, which can be attenuated by PE supplementation (Figure 5D). Finally, we tested the level of lipid peroxidation with the MDA kit and found that PE supplementation inhibited the lipid peroxidation induced by prorenin in microglia (Figure 5E). Figure 5 Delivery of exogenous PE can ameliorate the oxidative/nitrosative stress induced by prorenin in microglia. (A and B) Representative images show the staining and intensity quantification of mitoROS fluorescent probes. Scale bar = 4 μm. (C) Use GSH and GSSG detection kits to detect the GSSG/GSH ratio. The results indicate that exogenous PE delivery attenuates the increase in the prorenin-induced GSSG/GSH ratio in cultured microglia. (D) The level of nitrosation indicator 3-NT quantified using a nitrotyrosine ELISA kit. The results indicate that the delivery of PE can ameliorate the nitrosative stress induced by prorenin in cultured microglia. (E) Use MDA kit to measure lipid peroxidation. The data suggests that PE supplementation can reduce lipid peroxidation in microglia stimulated by renin. Data are expressed as mean ± SD. n = 3–5, *p <0.05, #p <0.05, t test.

Figure 5 Delivery of exogenous PE can ameliorate the oxidative/nitrosative stress induced by prorenin in microglia. (A and B) Representative images show the staining and intensity quantification of mitoROS fluorescent probes. Scale bar = 4 μm. (C) Use GSH and GSSG detection kits to detect the GSSG/GSH ratio. The results indicate that exogenous PE delivery attenuates the increase in the prorenin-induced GSSG/GSH ratio in cultured microglia. (D) The level of nitrosation indicator 3-NT quantified using a nitrotyrosine ELISA kit. The results indicate that the delivery of PE can ameliorate the nitrosative stress induced by prorenin in cultured microglia. (E) Use MDA kit to measure lipid peroxidation. The data suggests that PE supplementation can reduce lipid peroxidation in microglia stimulated by renin. Data are expressed as mean ± SD. n = 3–5, *p <0.05, #p <0.05, t test.

4-Hydroxynonenal (4-HNE) is a biomarker of α, β-unsaturated hydroxyenal and oxidative/nitrosative stress. Double immunofluorescence staining of 4-HNE and microglia marker Iba-1 showed that stress induced an increase in 4-HNE levels, which was inhibited by PLIN2-siRNA (Figure 6A and B). In addition, we also observed that PLIN2-siRNA reduced the proportion of 4-HNE+ microglia (Figure 6C), indicating that PLIN2-siRNA reduced the stress-induced oxidative stress in RVLM. In addition, immunofluorescence results showed that stress-induced nitrosative stress product 3-NT in RVLM increased, which was inhibited by PLIN2-siRNA (Figure 6D and E). Under stress, 3-NT+microglia in rat RVLM The proportion of cells is reduced (Figure 6F). In addition, Western blot analysis showed that compared with the sham operation group, the expression of Iba-1, 4-HNE and 3-NT in the RVLM of stressed rats increased, while the administration of PLIN2 siRNA significantly inhibited Iba-1, 4 -HNE and 3-NT (P <0.05, Figure S1). These results indicate that knockdown of PLIN2 inhibits the oxidative/nitrosative stress of RVLM in rats. Figure 6 PLIN2 knockdown reduced RVLM oxidative/nitrosation stress in stressed rats. Representative double immunofluorescence staining shows the colocalization image of 4-HNE (oxidation/nitrosation indicator) and Iba-1 (A), the quantification of 4-HNE immunopositive intensity (B), and 4-HNE+/Iba -Percentage of 1+ cells in rat RVLM (C). Representative double immunofluorescence staining shows the colocalization image of 3-NT (nitrosation indicator) and Iba-1 (D), the quantification of 3-NT immunopositive intensity (E), and 3-NT+/Iba-1 + Percentage of cells in rat RVLM (F). Scale bar = 50 μm. Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test.

Figure 6 PLIN2 knockdown reduced RVLM oxidative/nitrosation stress in stressed rats. Representative double immunofluorescence staining shows the colocalization image of 4-HNE (oxidation/nitrosation indicator) and Iba-1 (A), the quantification of 4-HNE immunopositive intensity (B), and 4-HNE+/Iba -Percentage of 1+ cells in rat RVLM (C). Representative double immunofluorescence staining shows the colocalization image of 3-NT (nitrosation indicator) and Iba-1 (D), the quantification of 3-NT immunopositive intensity (E), and 3-NT+/Iba-1 + Percentage of cells in rat RVLM (F). Scale bar = 50 μm. Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test.

Next, we investigated whether microglia polarization and proliferation are regulated by PLIN2. We found that microinjection of PLIN2-siRNA reduced the proportion of iNOS+ microglia (M1 polarization) in the RVLM of stressed rats (Figure 7A and B). Consistently, PLIN2-siRNA reduced the number of PCNA+ (proliferation marker) microglia in the RVLM of stressed rats (Figure 7C and D). It is worth noting that PLIN2-siRNA reduced the expression of pro-inflammatory cytokines, IL-1β and TNF-α (Figure 7E-G). These data indicate that PLIN2 knockdown significantly reduces the inflammatory polarization and proliferation of RVLM microglia in stressed rats. Figure 7 PLIN2 knockdown attenuates the polarization and proliferation of RVLM microglia in stressed rats. Representative dual immunofluorescence image of iNOS (M1 polarization marker) co-localized with Iba-1 (A) and percentage of iNOS+/Iba-1+ cells in rat RVLM (B). Scale bar = 50 μm. Representative dual immunofluorescence image of PCNA (proliferation marker) co-localized with Iba-1 (C) and PCNA+/Iba-1+ cell percentage (D) in rat RVLM. Scale bar = 50 μm. The representative immunofluorescence image of IL-1β (upper image)/TNF-α (lower image) (E) and the quantification of the immunopositive intensity of IL-1β(F)/TNF-α(G) in RVLM. Data are expressed as mean ± SD. Scale bar = 50 μm. n = 6–8, *p <0.05, #p <0.05, t test.

Figure 7 PLIN2 knockdown attenuates the polarization and proliferation of RVLM microglia in stressed rats. Representative dual immunofluorescence image of iNOS (M1 polarization marker) co-localized with Iba-1 (A) and percentage of iNOS+/Iba-1+ cells in rat RVLM (B). Scale bar = 50 μm. Representative dual immunofluorescence image of PCNA (proliferation marker) co-localized with Iba-1 (C) and PCNA+/Iba-1+ cell percentage (D) in rat RVLM. Scale bar = 50 μm. The representative immunofluorescence image of IL-1β (upper image)/TNF-α (lower image) (E) and the quantification of the immunopositive intensity of IL-1β(F)/TNF-α(G) in RVLM. Data are expressed as mean ± SD. Scale bar = 50 μm. n = 6–8, *p <0.05, #p <0.05, t test.

Use PGP9.5 in RVLM to double immunofluorescence staining of neuron activation marker c-fos to study sympathetic preneuron activation. The results showed that PLIN2-siRNA reduced the proportion of c-fos+ neurons in the RVLM of stressed rats (Figure 8A and B). In addition, PLIN2 knockdown in RVLM reduced serum norepinephrine (NE) levels, which reflected the decreased peripheral sympathetic nerve activity in stressed rats (Figure 8C). Heart rate variability analysis showed that the LF/HF ratio was reduced in stressed rats, and central PLIN2 knockdown suppressed this decrease (Figure 8D). BP measurement showed that the mean arterial blood pressure (MAP) of stressed rats was significantly increased, while PLIN2-siRNA attenuated the increase of MAP in stressed rats (Figure 8E). These data indicate that PLIN2 knockdown in RVLM improves cardiovascular sympathetic overactivation and hypertension in stressed rats. Figure 8 PLIN2 knockdown reduced the sympathetic overactivation of stressed rats and lowered blood pressure. Representative dual immunofluorescence image of c-fos (neuron activation marker) co-localized with PGP9.5 (A) and c-fos+/PGP9.5+ cell percentage (B) in RVLM. Scale bar = 100 μm. (C) ELISA assay showed that PLIN2 knockdown in RVLM attenuated the increase in plasma NE induced by stress in rats. (D) The results of heart rate variability (HRV) analysis showed that the knockdown of PLIN2 in RVLM improved the stress-induced increase in LF/HF ratio in rats, which means cardiac sympathetic/parasympathetic tone imbalance and sympathetic overactivation . (E) The mean arterial blood pressure (mmHg) was recorded and analyzed. The data showed that PLIN2-siRNA reduced pressure-induced hypertension in rats. Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test.

Figure 8 PLIN2 knockdown reduced the sympathetic overactivation of stressed rats and lowered blood pressure. Representative dual immunofluorescence image of c-fos (neuron activation marker) co-localized with PGP9.5 (A) and c-fos+/PGP9.5+ cell percentage (B) in RVLM. Scale bar = 100 μm. (C) ELISA assay showed that PLIN2 knockdown in RVLM attenuated the increase in plasma NE induced by stress in rats. (D) The results of heart rate variability (HRV) analysis showed that the knockdown of PLIN2 in RVLM improved the stress-induced increase in LF/HF ratio in rats, which means cardiac sympathetic/parasympathetic tone imbalance and sympathetic overactivation . (E) The mean arterial blood pressure (mmHg) was recorded and analyzed. The data showed that PLIN2-siRNA reduced pressure-induced hypertension in rats. Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test.

Myocardial remodeling, including cardiomyocyte hypertrophy and interstitial fibrosis, is a process that develops in response to pathological stimuli such as pressure overload and neuroendocrine activation. In order to study the myocardial remodeling in SIH rats, HE, Masson and WGA staining were used to evaluate the morphological changes of the left ventricle of rats (Figure 9A). Compared with the sham operation group, the increased area of ​​myocardial fibrosis (Figure 9B) and the cross-sectional area of ​​cardiomyocytes (Figure 9C) in the stressed rats were larger, while the central PLIN2 knockdown improved myocardial fibrosis and myocardial fibrosis in the stressed rats. Hypertrophy. In addition, stressed rats showed increased heart weight (HW), heart weight/tibia length ratio (HW/TL), left ventricular weight (LW), and left ventricular weight/tibia length ratio (LW/TL), and these The increase was attenuated by microinjecting PLIN2-siRNA into RVLM (Figure 10A-D). Corresponding to the histopathological changes, we measured several key cardiac parameters in rats by echocardiography. A representative image of echocardiographic M-mode imaging is shown in Figure 10E. Heart rate (HRs), left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) did not change significantly (Figure 10F-H), and the end-diastolic posterior wall thickness (LVPWd) of stressed rats increased significantly) and false magnification Compared with mice, PLIN2 knockdown in RVLM inhibited the above changes (Figure 10I). All these laboratory evidences indicate that PLIN2 knockdown can inhibit stress-induced myocardial damage. Figure 9 PLIN2 knockdown improved myocardial histopathological changes in stressed rats. Representative images of hematoxylin-eosin (HE) staining (upper image), Masson-trichrome staining (middle image), and WGA staining (lower image) of left ventricular tissue of the heart. (A) Scale bar = 50 μm. Quantitative analysis of fibrosis area (B) and cross-sectional area (C) showed that PLIN-siRNA can reduce myocardial fibrosis and hypertrophy in stressed rats. Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test. Figure 10 PLIN2 knockdown improves cardiac dysfunction in stressed rats. (AD) Measure the heart weight (HW), heart weight/tibia length ratio (HW/TL), left ventricular weight (LVW) and left ventricular weight/tibia length ratio (LVW/TL) groups of different rats. Representative M-mode images of echocardiography show (E) and heart rate (HRs), left ventricular ejection fraction (LVEF), fraction shortening (LVFS), and end-diastolic posterior wall (LVPWd) thickness are quantified (F)-I ). Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test.

Figure 9 PLIN2 knockdown improved myocardial histopathological changes in stressed rats. Representative images of hematoxylin-eosin (HE) staining (upper image), Masson-trichrome staining (middle image), and WGA staining (lower image) of left ventricular tissue of the heart. (A) Scale bar = 50 μm. Quantitative analysis of fibrosis area (B) and cross-sectional area (C) showed that PLIN-siRNA can reduce myocardial fibrosis and hypertrophy in stressed rats. Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test.

Figure 10 PLIN2 knockdown improves cardiac dysfunction in stressed rats. (AD) Measure the heart weight (HW), heart weight/tibia length ratio (HW/TL), left ventricular weight (LVW) and left ventricular weight/tibia length ratio (LVW/TL) groups of different rats. Representative M-mode images of echocardiography show (E) and heart rate (HRs), left ventricular ejection fraction (LVEF), fraction shortening (LVFS), and end-diastolic posterior wall (LVPWd) thickness are quantified (F)-I ). Data are expressed as mean ± SD. n = 6–8, *p <0.05, #p <0.05, t test.

The microglia phenotype highlights the strong changes in lipid and lipoprotein metabolism to meet their functions, and the situation in which they encounter chronic stress is unclear. In our current study, first of all, we found significant M1 polarization and microglia proliferation in RVLM of stressed rats, accompanied by an increase in the accumulation of LDs. Secondly, the results of non-targeted lipid metabolomics showed that the content of phosphatidylethanolamine (PE) in the RVLM area of ​​stressed rats was significantly reduced. Third, we observed that the activation of PLIN2, one of the LDs proteins, leads to the accumulation of LDs, which leads to the lack of PE. Fourth, exogenous PE supplements can reduce the oxidative/nitrosative stress induced by prorenin stress in microglia. Finally, we found that central knockdown of PLIN2 reduced stress-induced RVLM neuroinflammation, hypertension, and myocardial damage. Our research shows that the pressure including the accumulation of LDs is mediated by PLIN2, which interferes with the PE metabolism and proliferation in microglia.

It is noted that PE is one of the most abundant phospholipids in eukaryotes. It is involved in the formation, fusion and fluidity of the phospholipid bilayer. It is closely related to redox homeostasis, mitochondrial function and endoplasmic reticulum stress (ERS). .59 The reduction of PE has been reported in various stress and metabolic shock models, which are closely related to inflammation, oxidative stress, mitochondrial dysfunction, and autophagy. 60-62 In addition to PE, another phosphatidylcholine (PC), with the largest content, also changed in stressed rats. We found that stress may cause the PC/PE ratio to increase. Previous literature reports that obesity and various stresses lead to an increase in the PC/PE ratio, which is accompanied by the initiation of inflammation and metabolic remodeling. 63-66 It is important to note that CCTα, a rate-phospholipid synthesis restriction enzyme, is not expressed in the rat brain, and CCTβ expression is only detected in Purkinje cells. 67 This indicates that the de novo synthesis of phospholipids is not the main pathway for most brain cells. Another thing that cannot be ignored is the conversion of PE/PC by phosphatidylethanolamine N-methyltransferase (PEMT), which leads to a decrease in PE and an increase in PC. 68 It is well known that the catalytic activity of PEMT is involved in Alzheimer's disease, obesity and non-alcoholic fatty liver disease. 38,69,70 PEMT knockout mice on a high-fat diet seem to have a greater increase in systolic blood pressure. 71 All of these indicate a potential relationship between PE deficiency and autonomic disorders.

Our research shows that knocking down PLIN2 can partially restore PE content by reducing LDs in microglia stimulated by renin. As lipid storage organelles, LDs are also an integration platform for lipid metabolism networks. In particular, the perilipin protein represented by PLIN2 is involved in the degradation of LDs and the utilization of neutral fatty acids, as well as the regulation of the interaction between LDs and other organelles. 72-74 Our current study shows that in activated microglia, increased PLIN2 mediates LDs accumulation, and PLIN2 knockdown inhibits LDs accumulation, which is consistent with other studies. In addition, previous studies have also reported that changes in PEMT activity are closely related to the fusion of LDs. The intervention of PE degradation and the increase of PC synthesis mediated by PEMT can promote the fusion of LDs, which indicates that there is a potential link between the degradation of LDs and the increase of PE synthesis. 75 In addition, it should not be overlooked that research reports claim that LDs act as the attachment interface of enzymes in phospholipid synthesis and metabolism and regulate phospholipid production. 76-78 Therefore, follow-up studies need to further explore the molecular mechanism of phospholipid biosynthesis and metabolic disorders caused by the accumulation of LDs in microglia.

In our current study, we demonstrated that supplementation with exogenous PE can prevent the inflammatory activation of microglia. Previous studies reported that increased mitochondrial PE can stimulate the function of respiratory chain proteins (including cytochrome c oxidase and succinate reductase), release mitochondrial aerobic respiration potential, increase ATP synthesis and improve cell metabolism. 79 Lack of PE leads to mitochondrial fragmentation and excessive fusion, accompanied by mitochondrial structural disorder observed under a transmission electron microscope. 80 Lack of PE can impair the formation of respiratory supercomplexes and/or membrane integration, which can be alleviated by supplementing PE. 81 In addition, the role of PE in autophagy has been noted. PE-modified LC3 mediates the formation and transport of autophagosomes, and plays a key role in the initiation of selective autophagy and autophagosome-lysosome fusion. 82 PE deficiency directly leads to the destruction of autophagy, and the disorder of selective autophagy is closely related to the development of mitochondria and endoplasmic reticulum stress. Our previous publications indicated that oxidative stress is an important event that triggers mitochondrial dysfunction and autophagy blockade in stressed microglia. This study further confirmed that PE supplementation can reduce the production of mitoROS and the accumulation of oxidative/nitrosative stress products in microglia stimulated by renin.

The oxidative stress/nitrosative stress of the sympathetic nerve center is a key mechanism for the excessive activation of sympathetic nerves in hypertension and other cardiovascular events (including myocardial infarction). RAS (Renin-Angiotensin System) is the most important trigger of oxidative/nitrosative stress in the cardiovascular center. 13,83 Microglia are the main cells that produce ROS/NO in the brain under pathological stimulation. 84,85 Studies have confirmed that various pro-hypertensive stresses induce the activation of local RAS system in the sympathetic center and cause neuroinflammation and oxidative stress. RAS components in the circulation also stimulate sympathetic nerve activity or directly activate through subfornix organs-paraventricular nucleus-RVLM The sympathetic center leaks through the blood-brain barrier. 86,87 Reducing the RAS effect in brain areas such as RVLM can significantly inhibit the sympathetic nerve output and blood pressure increase caused by various pressures. However, the molecular mechanism of oxidative/nitrosative stress induced by the RAS system has not been fully explored. AT1 receptor activation induces NADPH oxidase (NOX) activation and iNOS expression are common mechanisms observed in various hypertension models and myocardial infarction stress models. 88,89 events.

Our previous studies have confirmed that prorenin is an important part of the RAS system, which induces oxidative stress and activation of NOXs through prorenin receptors, leading to M1 polarization of microglia in RVLM, which is related to stress-induced hypertension. The increase in rat sympathetic nerve output is related. .15 In our current study, we further found that PLIN2-mediated increase in LD leads to oxidative/nitrosative stress in RVLM of stressed rats, and PE supplementation prevents ROS/nitrosative stress induced by prorenin Increase in product. These results indicate that the lack of PE in the sympathetic center mediated by PLIN2 is involved in the development of stress-induced hypertension. This study provides a new treatment strategy for interference with stress hypertension and hypertensive heart damage.

In summary, we demonstrated that inhibiting PLIN2 in RVLM can reduce oxidative/nitrosative stress, inhibit sympathetic overdrive and protect the heart from hypertrophy and fibrosis in stressed rats.

3-NT, 3-nitrotyrosine; 4-HNE, 4-hydroxynonenal; CSA, cross-sectional area; CTP, phosphocholine cytidine transferase; electrocardiogram, echocardiogram; GSH, glutathione Peptides; H & E staining, hematoxylin and eosin staining; HPA, hypothalamic-pituitary-adrenal axis; HR, heart rate; HRV, heart rate variability; HW, heart weight; HW/TL, heart weight/tibia length ratio ; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; LDs, lipid droplets; LF/HF, low frequency/high frequency; LVEF, left ventricular ejection fraction; LVFS, left ventricular shortening fraction ; LVPWd, left ventricular end-diastolic posterior wall; LW, left ventricular weight; LW/TL, left ventricular weight/tibia length ratio; MAP, mean arterial blood pressure; MDA, malondialdehyde; mitoROS, mitochondrial reactive oxygen species; NE, de Norepinephrine; NLRP3, the pyrin domain of the NLR family, containing 3; NO, nitric oxide; PC, phosphatidylcholine; PCNA, proliferating cell nuclear antigen; PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine N-methyl Base transferase; PIC, pro-inflammatory cytokines; PLIN2, Perilipin 2; PVN, paraventricular nucleus; RAS, renin-angiotensin system; RVLM, rostral ventrolateral medulla; SEM, mean ± standard error of mean ; SIH, stress hypertension; TLRs, Toll-like receptors.

This work was supported by the National Natural Science Foundation of China [81770423, 32071111, 31871151, 31571171] and the Science and Technology Program of Shaoxing Science and Technology Bureau (2020B33004).

The authors report no conflicts of interest in this work.

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