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. 2023 Feb 14;43(6):2871–2882. doi: 10.1007/s10571-023-01325-9

Macrophage/Microglia Sirt3 Contributes to the Anti-inflammatory Effects of Resveratrol Against Experimental Intracerebral Hemorrhage in Mice

Jidong Sun 1,#, Chen Pu 3,#, ErWan Yang 1,#, Hongchen Zhang 1, Yuan Feng 1, Peng Luo 1, Yuefan Yang 1, Lei Zhang 1, Xia Li 1, Xiaofan Jiang 1,✉,#, Shuhui Dai 1,2,✉,#
PMCID: PMC11410121  PMID: 36786945

Abstract

Intracerebral hemorrhage (ICH) is a devastating stroke type with high mortality and disability. Inflammatory response induced by macrophages/microglia (M/Ms) activation is one of the leading causes of brain damage after ICH. The anti-inflammatory effects of resveratrol (RSV) have already been evaluated in several models of central nervous system disease. Therefore, we designed the current study to assess the role of RSV in ICH and explore its downstream mechanism related to Sirt3. The autologous artery blood injection was administrated to create an ICH mouse model. M/Ms-specific Sirt3 knockout Sirt3f/f; CX3CR1-Cre (Sirt3 cKO) mouse was used to evaluate the role of Sirt3 on RSV treatment. Neuronal function and hematoma volume were assessed to indicate brain damage. The pro-inflammatory marker (CD16) and cytokine (TNF) were measured to evaluate the inflammatory effects. Our results showed that RSV treatment alleviates neurological deficits, reduces cell death, and increases hematoma clearance on day 7 after ICH. In addition, RSV effectively suppressed CD16+ M/Ms activation and decreased TNF release. In Sirt3 cKO mice, the protective effects of RSV were abolished, indicating the potential mechanism of RSV was partially due to Sirt3 signaling activation. Therefore, RSV could be a promising candidate and therapeutic agent for ICH and Sirt3 could be a potential target to inhibit inflammation.

Graphical Abstract

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Supplementary Information

The online version contains supplementary material available at 10.1007/s10571-023-01325-9.

Keywords: Intracerebral hemorrhage, Sirt3, Resveratrol, Microglia, Neuroinflammation

Introduction

Intracerebral hemorrhage (ICH) is the most lethal type of stroke characterized by spontaneous bleeding in the brain parenchyma, which is associated with early mortality rates over 40% (Revilla-Leon et al. 2022). The extensive inflammatory response is a crucial factor participating in the pathological process of brain injury and long-term disability after ICH (Hirsch et al. 2022). Macrophages/microglia (M/Ms) are the first responders to brain injury and the main drivers of inflammation in multiple central nervous system (CNS) diseases (Sarkar 2022). M/Ms-derived inflammation is a dynamic process at different stages after ICH which has comprehensive roles in brain damage and recovery. The polarization state of M/Ms varies in different microenvironmental statuses and is defined by released cytokines and chemokines (Li et al. 2022b). It is evident that M1-like M/Ms are pro-inflammatory while M2-like M/Ms are involved in tissue remodeling (Martinez-Rojas et al. 2022). Emerging preclinical research has proved that suppression of inflammatory activity exerts improved functional outcomes after ICH onset (Magid-Bernstein et al. 2022). It has been shown that anti-inflammatory molecules and medicines represent a potential therapeutic strategy for ICH.

Resveratrol (RSV) is a well-known natural compound that widely exists in plant species and belongs to the flavonoid polyphenol family (Kong et al. 2022). Resveratrol displays anti-inflammatory and neuroprotective properties against ischemic stroke, Alzheimer's Disease, postoperative cognitive dysfunction, and other CNS disorders (Koronowski et al. 2017; Yan et al. 2020; Li et al. 2021). However, its role and the underlying mechanism after ICH remains to be determined. A recent study on pulmonary arterial hypertension indicated that RSV activated Sirtuins, a family of deacetylases, to protect mitochondrial integrity and energetics (Bernal-Ramirez et al. 2021). Sirtuins signaling is essential in regulating numerous biological processes in many cell types, including redox homeostasis, energetic metabolism, and cytogenesis (Jablonska et al. 2022; Martino et al. 2022). Among the Sirtuins family, Sirt3 primarily exists in mitochondria and is the main mitochondrial deacetylase. In several disease models, restoration of Sirt3 has recently been found to suppress inflammatory activity by regulating NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation (Martino et al. 2022). In our previous work, we found that Sirt3 in M/Ms is responsible for M/Ms polarization and M/Ms-specific knockout of Sirt3 enhanced inflammatory response after ICH (Dai et al. 2022). Therefore, we wondered whether RSV could regulate M/Ms-induced inflammation through activating Sirt3 after ICH.

In this study, we evaluated the effects of resveratrol on the inflammatory response induced by M/Ms activation after ICH with an experimental ICH mouse model. Furthermore, the transgenic macrophages/microglia specific Sirt3 knockout mice were used to verify the mechanism of resveratrol.

Materials and Methods

Animals

Thirty 6–8 week-old healthy C57BL/6 J male mice (weighted 20–25 g) were provided by the Experimental Animal Center of Fourth Military Medical University. 12 8 week-old Sirt3f/f; CX3CR1-Cre (Sirt3 cKO) male mice and 12 Sirt3f/f mice (weighted 20–25 g) were constructed and propagated by Model Organisms Co. (Shanghai, China). All the procedures in this study were approved by the Animal Ethics Committee of Fourth Military Medical University (No. 2014-81371447, Xi’an, China). The mice were housed (5 mice in a standard cage, 10 mice in a large flat cage) under a 12 h light/dark cycle in a temperature-(25 ± 2 °C) and humidity-(52 ± 5%) controlled room with free access to food and water. The required minimum sample size was determined as 6 per group using the G*Power 3.1 software according to the previously published article (Faul et al. 2009). Behavioral tests were performed to exclude the animals with abnormal performance. The C57BL/6 J animals were assigned to Sham, ICH + Vehicle (ICH + Veh) and ICH + Resveratrol (ICH + RSV) evenly and randomly using a computer-generated random allocation sequence according to the previous published article (Karp et al. 2022). All the Sirt3 cKO and Sirt3f/f mice received ICH onset and resveratrol treatment. The experiments were performed by researchers who were blinded to the study design and animal assignment.

Experimental Intracerebral Hemorrhage Model

ICH was induced by intrastriatal injection of 30 μl autologous whole artery blood following previously published protocols (Wang et al. 2021). Mice were anesthetized with an intraperitoneal injection of 0.3% pentobarbital sodium (P3761, Sigma-Aldrich, US) at 40 mg/kg dosage and placed on the stereotactic frame (68,046, RWD Life Science, China). A burr hole was drilled near the right coronal suture. Then, the right femoral artery of the mice was carefully exposed, and catheterization was performed. 30 μl of whole blood was withdrawn from the femoral artery and injected into the right basal ganglia according to coordinates as follows: 3.5 mm ventral, 0.2 mm anterior, and 2.5 mm lateral to the bregma at the rate of 3 μl/min. The needle was slowly withdrawn 10 min after the injection. The heating pad was used to maintain mice body temperature at 37 °C during the surgery and recovery and 5 mg/kg carprofen (33,975, Sigma-Aldrich, US) was subcutaneously injected once after surgery to release pain.

Neurobehavioral Function Evaluation

A cylinder test of forelimb asymmetry and a corner turn test were conducted to detect the neurological function of mice as previously reported (Dai et al. 2022). Briefly, for the cylinder test, mice were placed in a transparent polyester cylinder (10 cm diameter) and allowed to place their paws on the wall freely. The numbers of wall touch with left, right, or both forelimb paws were counted during 3 min. The forelimb asymmetry score was calculated as [(right-left)/(right + left + both)] × 100%.

For the corner turn test, mice were placed with the head to a corner of 30 degrees angle. The mice could turn either to the right or left to exit the corner. The test was repeated 20 times, and the percentage of right turns was calculated as the corner turn score. The mice were allowed rest 30 s after each trail and not to be touched right after each turn.

Drug Administration

Resveratrol (R5010, Sigma-Aldrich, US) was dissolved in corn oil. The mice in the treatment group received daily intraperitoneal injection of resveratrol at the concentration of 30 mg/kg from 3 days before surgery and lasted until 7 days after ICH onset according to the previously published studies (Li et al. 2022a). The mice in the vehicle group were injected with corn oil for the same time course.

Hematoxylin–eosin (H&E) Staining and Hematoma Volume Evaluation

The 4 μm-thick brain sections were deparaffized in xylene, and then washed with distilled water and alcohol at different concentrations. For H&E staining, the sections were firstly stained with hematoxylin (G1120, Solarbio, China) for 5 min and washed with tap water. Next, the sections were counterstained with eosin (G1120, Solarbio, China) for 15–30 s and dehydrated with alcohol. Xylene was then used to clear the color, and neutral gum was used to seal the sections. The section number with hematoma was counted as N. The section with the most extensive clot was chosen, and the clot size was calculated as Smax mm2 with Image Pro Plus 6.0 software. The hematoma volume was calculated as follows: V = 0.002 × N × Smax mm3.

Immunohistochemical (IHC) and Immunofluorescent (IF) Staining

Immunohistochemical staining was performed with a commercial Metal Enhanced DAB Substrate Kit (DA1016, Solarbio, China). Briefly, polyformaldehyde (PFA)-fixed, paraffin-embed tissue samples were cut into 2 μm-thick slices. The deparaffinized sections were submerged in EDTA at 95 °C for 20 min to retrieve antigens. Afterward, sections were permeabilized in a blocking solution containing 1% bovine serum albumin (BSA, A1933, Sigma-Aldrich, US) and 0.5% Triton X-100 at RT for 2 h. The sections were incubated with rabbit anti-Iba-1 (17,198, Cell Signaling Technology, US, 1:800) antibody overnight at 4 °C, followed by incubation with anti-rabbit IgG-peroxidase conjugate (DC03L, Sigma-Aldrich, US) for 2 h at RT, and DAB working solution for 30 min at RT in the darkness. The nucleus was counterstained with eosin.

For immunofluorescent staining, Sirt3 or CD16 was double-labeled with Iba-1. The sections were incubated with primary antibodies against Sirt3 (PA5-115903, Invitrogen, US, 1:200) or CD16 (80,366, Cell Signaling Technology, 1:50) with Iba-1 (MABN92, Sigma-Aldrich, US, 1:800) overnight at 4 °C. Alexa Fluor™ 488-labled goat anti-mouse (A-11001, Invitrogen, US, 1:800), Alexa Fluor™ 594-labled goat anti-rabbit (A-11012, Invitrogen, US, 1:400) and Alexa Fluor™ 594-labled goat anti-rat (A-11007, Invitrogen, US, 1:100) secondary antibodies were then used to detect these primary antibodies. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was used to counterstain the nucleus.

For analysis, six non-overlapping views in the peri-hematoma area of each section were captured, and three discontinuous sections of the same layers were chosen for each mouse. The total amount of Iba-1-positive (Iba-1+), Sirt3+Iba-1+, and CD16+Iba-1+ cells were calculated and analyzed.

Cellular Cytokine Release Measurement

The level of inflammatory cytokine tumor necrosis factor (TNF) in peri-hematoma brain tissues was measured by ELISA kit (430,907, BioLegend, US) according to the manufactory's instruction.

Western Blot Analysis

The expression of Sirt3 was determined by Western blotting, and β-actin was used as a loading control. Briefly, total protein samples in peri-hematoma brain tissue were extracted, and the protein concentration was measured with the BCA protein assay reagent (71,285, Thermo Fisher Scientific, US). 30 μg of proteins were loaded in a 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. The electrophoresis and transfer conditions were 90 V, 2.5 h, room temperature (RT), and 100 V, 1.5 h, 4 °C, respectively. Afterward, the membranes were blocked with 10% no-fat milk fat at RT for 1 h before incubating with primary antibodies (Sirt3 PA5-115,903, Invitrogen, US 1:800, β-actin ab6276, Abcam, US, 1:5000) at 4 °C overnight. After being washed three times with PBS for 10 min, the membranes were incubated with HPR-conjugated secondary antibodies for 2 h at RT. The membranes were then rinsed, and bands were detected by a SuperSignal chemiluminescence detection kit (S0500, Sigma-Aldrich, US). Images were obtained using ChemiDoc Imaging System (Bio-Rad, US). Band intensities were quantified using Image Pro Plus 6.0 software and normalized to β-actin.

Statistical Analysis

All the results were expressed as mean ± SD. The analysis was performed using GraphPad Prism 8 software. After the normality and variance homogeneity test was performed using Shapiro–Wilk test (details for each experiment were available in supplementary materials Table S3) and Levene's test, the Student t test was used to compare the difference between two groups passed Levene's test while Welch's t test was used between two groups not passed, and one-way ANOVA with Dunnett’s post-hoc test was used for multiple group comparisons. For behavioral test analysis, two-way ANOVA followed by Bonferroni’s test was performed. P-value < 0.05 was considered statistically significant. The sample size was determined based on PASS 15 software.

Results

Resveratrol Attenuates Brain Damage After Intracerebral Hemorrhage in Mice

To investigate the effects of resveratrol (RSV) on ICH-induced brain damage in mice, Foreelimb use asymmetry and corner turn test were used to evaluate the behavioral performance after ICH-induced damage to the brain. Interestingly, resveratrol could only attenuate the behavioral deficits on day 7 after ICH but had limited effects on days 1 and 3 (Fig. 1A, B, Table S1). H&E staining was further performed to measure the hematoma volume after 7 days of ICH onset. As it was shown in Fig. 1C, D, the hematoma volume was significantly decreased when treated with resveratrol compared with its ICH counterparts (4.8 ± 1.1 vs 2.8 ± 1.3 mm3). TUNEL staining indicated that after resveratrol treatment, the number of apoptotic cells decreased significantly compared ICH group (Fig. 1E, F, 15.4% ± 2.1% vs 8.2% ± 2.9%). Taken together, resveratrol had beneficial effects against ICH-induced brain damage at 7 days in mice.

Fig. 1.

Fig. 1

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Behavioral performance and hematoma volume after resveratrol (RSV) treatment of ICH in mice. A Forelimb use asymmetry and B Corner turn tests were performed to measure the behavioral performance before ICH and at 1, 3, and 7 days after ICH with or without RSV treatment. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s test, F(df1,df2) = 1,10, *P = 0.03 (A) and *P = 0.03 (B) compared with the ICH + Veh group. C The representative images of H&E staining indicated the hematoma volume (circled with yellow lines), scale bar = 500 μm. D The analysis of hematoma volume with continuous slices, data were presented as mean ± SD and passed Levene's test with F = 0.02, P = 0.89. Student t-test was performed, t(df) = 10, *P = 0.02 compared with the ICH + Veh group. E The representative images of TUNEL staining in the peri-hematoma area, scale bar = 20 μm. F The analysis of the TUNEL-positive cell number percentage of total DAPI positive cell number. Data were presented as mean ± SD and passed Levene's test with F = 0.60, P = 0.46. Student t-test was performed, t(df) = 10, *P < 0.001 compared with the ICH + Veh group

Resveratrol Suppresses Neuroinflammation Induced by Macrophage/Microglia Activation

Since RSV had anti-inflammation effects in multiple CNS disease (Singh and Bodakhe 2022), we further verified whether it could regulate microglia-related inflammation after ICH. The results of IHC staining indicated that in the peri-hematoma area, the number of Iba-1 positive M/Ms decreased when mice were treated with RSV compared with Veh group (Fig. 2A and B, 513 ± 86 vs 321 ± 75). The production of TNF was also reduced by RSV treatment compared with Veh (Fig. 2C, 18.2 ± 4.7 vs 9.6 ± 3.3 pg/ml). The expression of pro-inflammation marker CD16 in M/Ms was detected with double-inflorescent staining. In the ICH + RSV group, the number of cells expressing both Iba-1 and CD16 around hematoma decreased significantly compared with ICH + Veh group (Fig. 2D and E, 109 ± 27 vs 52 ± 25). These results demonstrated that the treatment of RSV exhibited an anti-inflammatory effect against ICH.

Fig. 2.

Fig. 2

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The activation of pro-inflammatory macrophage/microglia and inflammatory response after RSV treatment. A The representative images of IHC staining for detecting Iba-1 positive M/Ms in the peri-hematoma area on day 7 after ICH with or without RSV treatment, scale bar = 20 μm. B The number of Iba-1 positive cells was calculated. Data were presented as mean ± SD, and passed Levene's test with F = 0.03, P = 0.86. Student t-test was performed, t(df) = 10, *P = 0.002 compared with the ICH + Veh group. C TNF concentration in the brain tissue around the hematoma was measured with ELISA. Data were presented as mean ± SD, and passed Levene's test with F = 0.16, P = 0.70. Student t-test was performed, t(df) = 10, *P = 0.005 compared with the ICH + Veh group. D The representative images of IF staining of Iba-1 and pro-inflammation marker CD16 in the peri-hematoma area, scale bar = 20 μm. E The Iba-1+CD16+ cell number was calculated, data were presented as mean ± SD, and passed Levene's test with F = 0.02, P = 0.90. Student t-test was performed, t(df) = 10, *P = 0.003 compared with the ICH + Veh group

Resveratrol Enhances Expression of Sirt3 in Macrophage/Microglia After Intracerebral Hemorrhage

We have reported that Sirt3 is critical in regulating M/Ms polarization and neuroinflammation after ICH (Dai et al. 2022). Therefore, we were considering whether Sirt3 was engaged in the anti-inflammatory effects of RSV. Western blot analysis was used to detect the expression of Sirt3 after treatment with RSV. Sirt3 expression increased significantly compared with the ICH + Veh group (Fig. 3A and B, 0.12 ± 0.03 vs 0.27 ± 0.09 vs 0.78 ± 0.14). Double-immunofluorescent staining was performed to directly observe the expression of Sirt3 in M/Ms around hematoma, and it was shown that RSV significantly enhanced Sirt3 expression in Iba-1 positive M/Ms (Fig. 3C and D, 96 ± 22 vs 287 ± 77). The above results indicated that Sirt3 expression in M/Ms could be increased by RSV treatment, which might be the potential mechanism of anti-inflammatory effects of RSV against M/Ms activation.

Fig. 3.

Fig. 3

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The expression of Sirt3 in macrophage/microglia after RSV treatment. A Western blot was used to detect the expression of Sirt3 in peri-hematoma tissue after RSV treatment. B The relative gray value of bands in each group was calculated and analyzed, data were presented as mean ± SD and passed Levene's test with P = 0.02. One-way ANOVA with Dunnett’s post-hoc test for multiple group comparisons. #P < 0.001 compared with the Sham group, *P < 0.001 compared with the ICH + Veh group. C The representative images of double-immunofluorescent staining of Iba-1 and Sirt3 in the peri-hematoma area, scale bar = 20 μm. D The Iba-1+Sirt3+ cell number was calculated, data were presented as mean ± SD, and didn’t pass Levene's test with F = 6.13, P = 0.03. Welch's t test was performed, t(df) = 10, *P = 0.001 compared with the ICH + Veh group

Macrophage/Microglia Sirt3 Participates in the Anti-inflammatory Effects of Resveratrol

Sirt3 floxed mice were bred with CX3CR1Cre mice to produce mice down-expressing Sirt3 signaling in the M/Ms and the knockout efficiency was confirmed with southern blot (Supplementary Fig. 1). The Sirt3 M/Ms-specific knockout mice (Sirt3 cKO) were used to confirm whether Sirt3 in M/Ms took part in the regulation of RSV on neuroinflammation. After RSV treatment, Sirt3 cKO mice had more Iba-1 positive M/Ms in the peri-hematoma area compared with Sirt3f/f counterparts (Fig. 4A and B, 288 ± 54 vs 422 ± 91). Besides, the concentration of TNF was higher in Sirt3 cKO mice even treated with RSV than in Sirtf/f mice (Fig. 4C, 9.1 ± 2.8 vs 16.6 ± 4.4 pg/ml). Correspondently, the IF staining indicated that when M/Ms’ Sirt3 was knockout, the number of Iba-1+CD16+ cells was significantly less than that in Sirtf/f mice (Fig. 4D and E, 58 ± 32 vs 192 ± 23). These results demonstrated that the M/Ms-specific knockout of Sirt3 abolished the effects of RSV against neuroinflammation.

Fig. 4.

Fig. 4

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Pro-inflammatory macrophage/microglia activation and inflammatory response after RSV treatment in Sirt3f/f or Sirt3 cKO mice. A The representative images of IHC staining for detecting Iba-1 positive M/Ms in the peri-hematoma area on day 7 after ICH with RSV treatment, scale bar = 20 μm. B The number of Iba-1 positive cells was calculated, data were presented as mean ± SD, and passed Levene's test with F = 0.31, P = 0.59. Student t-test was performed, t(df) = 10, *P = 0.01 compared with the Sirt3f/f group. C TNF concentration in the brain tissue around the hematoma was measured with ELISA. Data were presented as mean ± SD, and passed Levene's test with F = 1.52, P = 0.25. Student t-test was performed, t(df) = 10, *P = 0.01 compared with the Sirt3f/f group. D The representative images of IF staining of Iba-1 and pro-inflammation marker CD16 in the peri-hematoma area, scale bar = 20 μm. E The Iba-1+CD16+ cell number was calculated, data were presented as mean ± SD, and passed Levene's test with F = 0.06, P = 0.81. Student t-test was performed, t(df) = 10, *P < 0.001 compared with the Sirt3f/f group

Macrophage/Microglia-Specific Sirt3 Knockout Abolishes the Neuroprotection Effects of Resveratrol After Experimental Intracerebral Hemorrhage

The effects of M/Ms’ Sirt3 on ICH-induced brain damage and neurological deficits were further confirmed. The results of H&E staining indicated that compared with Sirt3f/f mice, resveratrol couldn’t effectively reduce hematoma volume in Sirt3 cKO mice on day 7 after ICH (Fig. 5A and B, 3.3 ± 0.6 vs 4.9 ± 1.2 mm3). TUNEL staining showed more apoptotic cells in the brain in Sirt3 cKO mice compared with Sirtf/f mice after resveratrol treatment (Fig. 5C and D, 9.8% ± 3.4% vs 15.1% ± 3.3%). Accordantly, the results of behavioral tests also showed more severe behavioral deficits in Sirt3 cKO mice compared with that of Sirt3f/f mice after resveratrol treatment on day 7 (Fig. 5E and F, Table S2). Together, the above results indicated that the neuroprotective effects of resveratrol were limited when M/Ms’ Sirt3 was knockout.

Fig. 5.

Fig. 5

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Brain damage and neurological performance of mice after RSV treatment. A The representative images of H&E staining showed hematoma in mice brains at day 7 after ICH with RSV treatment (circled with yellow lines), scale bar = 500 μm. B Hematoma volume was calculated, data were presented as mean ± SD, and passed Levene's test with F = 0.92, P = 0.36. Student t-test was performed, t(df) = 10, *P = 0.02 compared with the Sirt3f/f group. C TUNEL staining was used to show the cell death in the peri-hematoma area, scale bar = 20 μm. D The percentage of TUNEL-positive cells was calculated, data were presented as mean ± SD, and passed Levene's test with F = 0.01, P = 0.94. Student t-test was performed, t(df) = 10, *P = 0.02 compared with the Sirt3f/f group. E Corner turn and F Forelimb use asymmetry tests were performed to measure the behavioral performance before ICH and at 1, 3, and 7 days after ICH. Data were presented as the mean ± SD. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s test. F(df1,df2) = 1,10, *P = 0.01 (E) and *P = 0.04 (F) compared with the Sirt3f/f group

Discussion

In the present study, we reported the role of M/Ms Sirt3 in the protective effects of resveratrol against ICH-induced neuroinflammation and brain damage (Fig. 6). We have demonstrated that resveratrol treatment alleviates neurological deficits, reduces cell death, and increases hematoma clearance. Resveratrol also effectively suppressed M/Ms activation and TNF release. The potential mechanism of resveratrol was partially due to Sirt3 signaling activation.

Fig. 6.

Fig. 6

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Resveratrol suppressed microglia-induced neuroinflammation after ICH by enhancing Sirt3 expression

Non-traumatic and spontaneous ICH is a devastating form of stroke with high fatal or permanently disabling risk and is usually due to a cerebrovascular event (Schrag and Kirshner 2020). The brain damages following ICH include both the primary physical insults of enlarged hematoma and the secondary brain injuries (SBI) such as blood–brain barrier disruption, brain edema, and inflammation (Su et al. 2022). At present, surgical hematoma evacuation has been the primary and preferred treatment for ICH (Kuramatsu et al. 2019). However, most clinical trials failed to show the benefits of the surgical treatment (Su et al. 2022). Furthermore, potential neuroprotective drugs did not significantly improve clinical outcomes either (Liu et al. 2022). Thus, it is urgent to develop effective treatment methods for ICH.

The pathogenesis of secondary brain injury after ICH is considered a complex and multifactorial process. During that, the inflammatory response is a significant factor inducing poor prognosis. Local microglia are the first responder cells to ICH insult within an hour, and their pro-inflammatory activation leads to the release of inflammatory cytokines, including TNF, interleukin (IL) -1, and IL-6 (Kuramoto et al. 2022; Zille et al. 2022). Activated microglia also recruit peripheral macrophages and neutrophilic infiltrates which could aggravate inflammation and cytotoxicity to exacerbate ICH. Previously published articles and our work have indicated that minocycline has shown beneficial effects against M/Ms derived inflammation in rodent and mammal ICH models, but the early-stage clinical trials failed to support its promotion in clinical practice (Bai et al. 2020). Thus, alternative approaches to effectively regulate inflammatory responses are needed to be explored.

RSV is the most studied polyphenolic stilbenoid and has possess multiple biological functions including anti-aging, anti-oxidative, anti-cancer, antiplatelet, and anti-inflammatory (Zheng et al. 2021). In both animal and human studies, RSV has beneficial effects on ischemic heart failure by decreasing the levels of inflammatory cytokines, IL-1, IL-6, and TNF (Gal et al. 2020). Recently, a study indicated RSV exerted a neuroprotective effect in a mouse ICH model and inhibited ferroptosis in HT22 cells injured by erastin (Mo et al. 2021). Correspondingly, our findings revealed that RSV suppressed M/Ms activation on day 7 after ICH in a mice model. In addition, RSV effectively decreased TNF production and ameliorated neurological deficits. Intriguingly, we didn’t observe significant effects of RSV on days 1 and 3, it may be because of its limited permeability across the blood–brain barrier. To improve the defects, several studies tried to explore a series of drug deliveries for RSV, such as polymer nanoparticles, albumin nanoparticles, and zein nanoparticles (Mo et al. 2021). Some of these carrier systems have shown improved oral bioavailability and increased efficiency in crossing biological barriers of RSV.

However, it should be taken into consideration that the activation of microglia is a dynamic progress and is highly plastic according to different microenvironments (Sarkar 2022; Yang et al. 2022). M1 M/Ms are always considered a pro-inflammatory subtype activated at a very early stage of ICH; at a late stage of ICH, M2 M/Ms subtypes might exert protective effects by promoting hematoma clearance or white matter integrity (Fu et al. 2021). But the overactivation of M2 M/Ms also induces chronic inflammation (Li et al. 2022b). To further analyze the phenotype of M/Ms, pro-inflammation marker CD16 was double stained with Iba-1. We found that RSV reduced the number of CD16+Iba-1+ cells around the hematoma area, indicating the anti-inflammatory effects of RSV at the early stage of ICH.

Mechanically, the RSV administration increased the expression of Sirt3 in M/Ms and the specific knockout of Sirt3 in M/Ms abolished the protective effects of RSV. At present, most of the work about the role of Sirtuins in RSV focuses on Sirt1, a well-known epigenetic regulator of various gene transcription (Yuan et al. 2022). In a rat model of glucocorticoid-induced osteonecrosis of the femoral head, RSV has been proved to enhance Sirt3 expression and reduced oxidative stress (Chen et al. 2021). Accumulating studies show that the mitochondrial NAD-dependent deacetylase Sirt3 can deacetylate and modify manganese superoxide dismutase (MnSOD), superoxide dismutase 2 (SOD2), glutamate dehydrogenase 1 (GLUD1), and other histones and enzymes regulating cellular oxidative or inflammatory response (Ilari et al. 2020; Hao et al. 2022; Zhou et al. 2022). To date, little is known about the effects of Sirt3 on neuroinflammation following ICH. In the current study, we used the transgenic mice to verify the specific role of Sirt3 in M/Ms, but we didn’t explore the downstream signaling of Sirt3 which was also part of the limitation of our work. Our previous studies indicated that after ICH, M/Ms-specific Sirt3 knockout reversed the protective effects of intermittent fasting against neuroinflammation through activating Nrf2/HO-1 signaling pathway (Dai et al. 2022). Recent studies have demonstrated that RSV can regulate the activation of Nrf2 in various cell types and disease models (Herrera-Arozamena et al. 2022; Wang et al. 2022). Thus, it is reasonable to hypothesize that RSV might regulate Sirt3/Nrf2 signaling pathway after ICH, which is also our future work.

In conclusion, RSV administration after ICH markedly alleviated neuroinflammation and pro-inflammatory effects of M/Ms by enhancing M/Ms Sirt3 expression. Through the above effects, RSV mitigated neurological deficits induced by ICH on mice. Based on its anti-inflammatory property, RSV possesses a potential promising treating strategy for ICH.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (TIF 754 kb) (755KB, tif)
Supplementary file2 (DOCX 32 kb) (32.9KB, docx)

Author Contributions

SHD and XFJ were the conceivers and designers of this study. They directed and oversaw the development and implementation of studies in this work. PL and XL directed and advised the writing and editing of the manuscript. JDS, PC and EWY designed and conducted all the experiments and wrote the manuscript. YF, HCZ and YFY collected all the samples and performed the behavioral tests. LZ assisted in analyzing the data.

Funding

This study was supported by the National Natural Science Foundation of China (No.81901186, No.82171458, No.82101374, and No.81974188).

Data Availability

The datasets used and/or analyzed during the current study are available in the Additional file 1: Data Supplement except for these listed in the article. Only the representative images from H&E and IHC/IF stains are available in the article.

Declarations

Conflict of interest

The authors have declared that they have no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jidong Sun, Chen Pu and ErWan Yang have contributed equally to this work.

Xiaofan Jiang and Shuhui Dai shared the last authorship.

Contributor Information

Xiaofan Jiang, Email: jiangxf@fmmu.edu.cn.

Shuhui Dai, Email: daishneuo@fmmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (TIF 754 kb) (755KB, tif)
Supplementary file2 (DOCX 32 kb) (32.9KB, docx)

Data Availability Statement

The datasets used and/or analyzed during the current study are available in the Additional file 1: Data Supplement except for these listed in the article. Only the representative images from H&E and IHC/IF stains are available in the article.

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