Pharmacological Activation of RXR-α Promotes Hematoma Absorption in a PPAR-γ-Dependent Pathway After Intracerebral Hemorrhage

doi:10.21203/rs.3.rs-67854/v1

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Pharmacological Activation of RXR-α Promotes Hematoma Absorption in a PPAR-γ-Dependent Pathway After Intracerebral Hemorrhage

https://doi.org/10.21203/rs.3.rs-67854/v1

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Background: Endogenously eliminating the hematoma is a favorable strategy in addressing intracerebral hemorrhage (ICH). This study sought to determine the role of retinoid X receptor-α (RXR-α) in the context of hematoma absorption after ICH.

Methods: ICH mouse models were established by the stereotactic injection of autologous arterial blood into the right basal ganglia. The selective RXR-α agonist bexarotene and peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist GW9662 were administered intraperitoneally at 1 h after ICH. Post-ICH assessments were in the form of magnetic resonance imaging scans, neurological tests, Western blotting, enzyme-linked immunosorbent assays, and immunofluorescence.

Results: Pharmacologically activating RXR-α with bexarotene significantly accelerated hematoma clearance and alleviated neurological dysfunction after ICH. RXR-α was expressed in the microglia/macrophages, neurons, and astrocytes. Mechanically, bexarotene promoted the nuclear translocation of RXR-α and PPAR-γ, alongside reducing neuroinflammation by modulating microglia/macrophages reprograming into the M2 phenotype from M1 phenotype. However, all the beneficial effects of RXR-α in ICH were reversed by the PPAR-γ inhibitor GW9662.

Conclusion: The pharmacological activation of RXR-α conferred robust neuroprotection against ICH by accelerating hematoma clearance and repolarizing microglia/macrophages towards the M2 phenotype through PPAR-γ-related mechanisms. Our data support the notion that RXR-α might be a promising therapeutic target for ICH.

Neurobiology of Disease

Intracerebral hemorrhage

RXR-α

PPAR-γ

polarization

phagocytosis

neuroinflammation

Intracerebral hemorrhage (ICH) is one of the most fatal cerebrovascular diseases and is linked to high morbidity and mortality rates [1, 2]. Primary brain injury caused by ICH usually occurs within the first few hours of onset and hematoma mechanically damages the adjacent tissues. Subsequently, hematoma-induced secondary brain injuries, including inflammatory responses, microglia activation, oxidative stress, and neuronal apoptosis and necrosis occurring after the primary injury can result in severe neurological deficits and death [1, 3]. A growing body of studies indicates that hematoma clearance may represent a unique target in ICH treatment [4]. Although the early removal of hematoma using surgical procedures is underway, yielding promising results, this shows unsatisfactory effects in neurological recovery and fail to address the secondary brain damage of ICH [5–7]. Therefore, new therapeutic strategies focusing on endogenous hematoma clearance for ICH are urgently needed.

In the central nervous system (CNS), microglia/macrophages serve as the main phagocytes involved in the defense against brain damage, including ICH, through the phagocytosis of red blood cells and the removal of tissue debris [4, 8]. Microglia/macrophages can be activated by hematoma components, such as thrombin and neurotoxins after tissue damage occurs [9]. It has been found that microglia/macrophages of the brain may dynamically be activated into two polarized states, termed the classical M1-like phenotype and the alternative M2-like phenotype [10]. Alternatively activated M2-like microglia/macrophages of the brain are considered to be anti-inflammatory and are generally involved in phagocytosis, and tissue repair [3]. The evidence above suggests that promoting the resolution of inflammation and increasing phagocytic activity by inhibiting the M1 phenotype and promoting the M2 phenotype may be an effective and promising therapeutic strategy for ICH as well.

Retinoid X receptors (RXRs) are members of the nuclear receptors superfamily [11, 12]. Currently, there are three different RXR isoforms (α, β, and γ). RXR-α is highly expressed in all human and rodent macrophage-type cells(brain microglia, liver Kupffer cells, and bone osteoclasts). Once activated, RXR-α translocates into the nucleus and regulate the transcription of target genes, and is involved with multiple cellular processes, such as monocyte/macrophage differentiation, phagocytosis, and metabolism [12–15]. Current studies reported that activation of RXR-α, which forms a heterodimer with peroxisome proliferator-activated receptor-γ (PPAR-γ), was implicated as a promising treatment for Alzheimer’s disease [15, 16]. Notably, a recent study has demonstrated that the activation of RXR-α may promote the nuclear accumulation and transcriptional activity of PPAR-γ, therefore modulating microglia polarization in traumatic brain injury [17]. However, the role of RXR-α in the pathological process following ICH has not yet been fully understood.

Based on the evidence above, we aimed to determine the role of RXR-α in modulating microglia/macrophages polarization and promoting hematoma clearance and neurological function via the PPAR-γ pathway.

Animals and ethical statement

All procedures were approved by the ethics committee of Zhejiang University and followed the National Institutes of Health guidelines for the Care and Use of Laboratory Animals. Adult male C57BL/6J mice (age 8–10 weeks, weight 22–25 g,n = 341) were obtained from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The mice were kept in a humidity-controlled room (25 ± 1 °C, 12-h light/dark cycle) and were raised with free access to food and water.

ICH model

The autologous blood injection model of ICH was performed as previously described [18]. Briefly, mice were anesthetized and maintained under 1% pentobarbital sodium, after which a volume of 25 µL of autologous blood was injected 2.5 mm to the right and 3 mm below the bregma at a 5° angle toward the midline. Mice in the sham group underwent the same procedure (anesthesia and needle insertion), except for injection.

Experimental design

All mice were randomly assigned to the following experiments (Fig. 1). A total of 341 mice including the dead ones, were used in this study.

Experiment I

Bexarotene is a highly selective RXR-α agonist approved by The Food and Drug Administration (FDA or USFDA) as an antineoplastic agent for the treatment of cutaneous T-cell lymphoma, with high blood-brain barrier permeability and a good safety profile [19]. To assess the effects of RXR-α activation on hematoma clearance and neurological function after ICH, the RXR-α agonist bexarotene was used. Neurological function (as assessed by the cylinder test, corner turn test, and forelimb placement test) was tested after ICH. Mice were assigned into three groups: sham group, ICH + vehicle group (10% Dimethyl sulfoxide (DMSO), in saline), and ICH + bexarotene group (5 mg/kg) (n = 9). The sham group received the same volume of vehicle intraperitoneally at the same time points after ICH induction. A T2* weighted magnetic resonance imaging (MRI) scan was used to measure the hematoma volume at 1, 3, 7, 14, and 28 days after ICH. Mice were randomly divided into two groups: ICH + vehicle (10% DMSO, in saline), and ICH + bexarotene (5 mg/kg) (n = 6).

Experiment II

To assess the expression pattern of RXR-α and PPAR-γ after ICH, mice were assigned to five groups: a sham, ICH 1 d, ICH 3 d, ICH 7 d, and ICH 14 d group (n = 6). Whole-cell lysates Western blots(n = 6) and cytoplasmic and nuclear protein Western blots (n = 6) were performed at different time-points. Next, the cellular location of RXR-α was assessed by double immunofluorescence staining in ICH (3 days) group (n = 6). To assess the nuclear translocation of RXR-α, mice were divided into three groups: a sham group, ICH + vehicle group (10% DMSO, in saline), and ICH + bexarotene group (n = 6). Whole-cell lysate Western blots (n = 6) and cytoplasmic and nuclear protein Western blots (n = 6) in these groups were performed at 3 days after ICH. To determine whether PPAR-γ plays a role in RXR-α activation after ICH, the RXR-α agonist bexarotene and PPAR-γ antagonist GW9662 were used. To assess the nuclear translocation of PPAR-γ, mice were divided into four groups: a sham group, ICH + vehicle group (10% DMSO, in saline), ICH + bexarotene + vehicle group, and an ICH + bexarotene + GW9662(4 mg/kg) (n = 6). Whole-cell lysate Western blots (n = 6) and cytoplasmic and nuclear protein Western blots (n = 6) in these groups were performed at 3 days after ICH.

Experiment III

To assess the mechanism underlying the role of RXR-α activation in ICH, mice were randomly assigned to four groups: a sham group, ICH + vehicle group (10% DMSO, in saline), ICH + bexarotene group + vehicle group (10% DMSO, in saline), and an ICH + bexarotene (5 mg/kg) + GW9662 (4 mg/kg) group. Immunofluorescence staining (n = 4), Western blots (n = 6), and enzyme-linked immunosorbent assays (ELISAs) (n = 6) were used to assess microglia/macrophages polarization.

Experiment IV

Next, we assessed whether PPAR-γ plays a role in the protective effects linked to RXR-α activation. To this end, the cylinder test, corner turn test, and forelimb placement test were used after ICH. Mice were randomly divided into three groups: an ICH + vehicle group (10% DMSO, in saline), ICH + bexarotene + vehicle group (5 mg/kg), ICH + bexarotene (5 mg/kg) + GW9662 (4 mg/kg) (n = 6). The sham group received the same volume of vehicle intraperitoneally at the same time points after ICH induction. An MRI scan was used to measure the hematoma volume at 1, 3, 7, and 14 days after ICH. Mice were randomly divided into three groups: an ICH + vehicle group (10% DMSO, in saline), ICH + bexarotene + vehicle group (5 mg/kg), and ICH + bexarotene (5 mg/kg) + GW9662 (4 mg/kg) group (n = 6).

Drug administration

Bexarotene (MedChem Express, New Jersey, USA) was dissolved in 10% dimethyl sulfoxide (DMSO) as previously described[20]. The selective PPAR-γ antagonist GW9662 (MedChem Express, New Jersey, USA) was diluted in 10% DMSO. bexarotene solution (5 mg/kg) or an equal volume of vehicle or bexarotene solution (5 mg/kg) + GW9662 solution (4 mg/kg) was administered intraperitoneally 1 h after ICH for the first time, followed by daily injections until sacrifice. The dosage and time points of bexarotene and GW9662 were based on a previous study[21, 22].

Calculation of hematoma volume

Mice were anesthetized with 1% pentobarbital sodium for the MRI examination. An MRI was performed at days 1,3,7,14 and 28 after ICH in a 3.0-T MRI scanner. The MRI included a T2* sequence. The scanning parameters for T2* weighted imaging were: TR/TE = 2200/103.8 ms, Number of averages = 10, acquisition matrix = 208 × 208, voxel size = 0.12 × 0.12 × 1 mm, flip angle = 130°, slices = 5. MRI image datasets were obtained in the Digital Imaging and Communications in Medicine (DICOM) format. The data were transformed into the NIfTI (.nii) format and then assessed with 3D Slicer. The 3D-Slicer method is one of the software methods serving to measure the volume of a hematoma(http://www.slicer.org/). Hematomas were manually identified pixel by pixel in each slice. Next, a 3D model was established and the hematoma volume was calculated by adding up the volume of the pixels.

Behavioral tests

Neurobehavioral functions were evaluated by a forelimb placing test, forelimb use asymmetry (cylinder) test, and a corner turn test, as previously reported [19]. Baseline data were recorded for the reduction of variability and identification of the preferential side. The neurological scores were evaluated by a blinded observer.

Forelimb placement can be assessed by stimulating the mouse's vibrissae to trigger a response. To test the function of the forelimbs, the researchers held the animal's torso and hung the forelimbs freely, while brushing its vibrissae on the corner edge of a table. Non-brain-damaged animals usually respond to placing the forelimb on the table on the same side as the affected side, while ICH mice will be impaired in the placement of their paws. This test can be scored by counting the percentage of placements.

For the forelimb use asymmetry test (cylinder test), the mouse is placed in a transparent cylinder and observed the independent wall contacts. The behavior score was recorded as the number of times the ipsilateral (unimpaired) forelimb (I), contralateral (impaired) forelimb (C), and both forelimbs (B). A single overall limb use asymmetry score was calculated as follows: Limb use asymmetry score=[I/(I + C + B)]-[C/(I + C + B)].

For the corner test, a mouse was then placed between the boards facing the 30º angle corner. As the mouse approaches the corner, both sides of the vibrissae were simultaneously stimulated causing the animal to rear and turn 180º, the number of the right turns was then calculated.

Immunofluorescence double labeling

Coronal sections were blocked with 5% Bovine serum albumin (BSA) and 0.3% Triton X-100 and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were mouse anti–NeuN (1:500, ab-104224, Abcam, MA, USA), goat anti–Iba-1 (1:500, ab-5076, Abcam, MA, USA), mouse anti–GFAP protein (1:500, ab10062, Abcam, MA, USA), rabbit anti-RXR-alpha antibody (1:250, ab125001, Abcam, MA, USA), rabbit anti-Arg1 (1:500, 16001-1-AP, Proteintech, Hubei, China), rabbit anti-Nitric oxide synthase (iNOS) Antibody (18985-1-AP,1:500, Proteintech, Hubei, China), and rabbit anti-PPAR-γ antibody (1:250, ab178860, Abcam, MA, USA). The sections were incubated with secondary antibodies at room temperature for 2 h. Finally, the sections were observed and analyzed using a fluorescence microscope (Olympus, Tokyo, Japan). Photomicrographs were saved and merged using the Image-Pro Plus software.

Enzyme-linked immunosorbent assay

Brain samples were homogenized in chilled lysis buffer containing protease and phosphatase inhibitor cocktails (P1005, Beyotime, Shanghai, China). ELISA kits for mouse TNF-α (Boster, EK0527, Wuhan, China) were used to assess the levels of TNF-α in the brain. The total protein content of each sample was determined by a bicinchoninic acid (BCA) assay (ThermoFisher, Waltham, MA USA). An equal amount of protein brain homogenates were diluted at a 1:10 ratio with the sample diluent provided with the kits, and all procedures were performed per the manufacturer’s instructions.

Western blot analysis

A Western blot analysis was performed as previously described [23]. Briefly, the basal ganglia were homogenized and centrifuged for 15 min (13,000 g, 4 °C). For the whole cell lysates, tissue proteins from basal ganglia were lysed using RIPA lysis buffer. For the extraction of cytoplasmic and nuclear protein, the cytoplasmic and nuclear proteins were extracted using Cytoplasmic and Nuclear Protein Extraction Kit ( P0027, Beyotime, Shanghai, China). Proteins were assessed using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA USA). An equal amount of protein (40 µg) was suspended in a loading buffer (denatured at 95 °C for 5 min), loaded on an SDS-PAGE, and transferred to nitrocellulose membranes. Next, the membranes were blocked with a nonfat dry milk buffer for 1 h and incubated overnight with the primary antibody. The membranes were then incubated with the secondary antibody for 1 h at room temperature. Bands were visualized using the ECL Plus chemiluminescence reagent kit (Amersham Bioscience, Arlington Heights, IL). The band densities were quantified using Image J software. The primary antibodies used in this study were rabbit anti-PPAR-γ (1:1000, Abcam, Cambridge, UK, ab45036), rabbit anti-RXR-α antibody (1:1000, ab125001, Abcam, MA, USA), rabbit anti-Arg1 (1:1000, 16001-1-AP, Proteintech, Hubei, China), mouse anti-GAPDH (1:5000, ab8245, Abcam, Cambridge, UK), mouse anti-β actin (1:5000, ab8227, Abcam, Cambridge, UK), and rabbit anti-histone H3 (1:2000, #9715, Cell Signaling Technology, MA, USA).

Statistical analysis

All data in this study are expressed as the mean ± standard error of the mean. For data that meet a normal distribution and homogeneity of variance, differences among the groups were analyzed using a one-way analysis of variance (ANOVA) followed by a Tukey’s multiple comparison test. A Kruskal-Wallis test with a Bonferroni correction was used for the non-normally distributed data. All statistical analyses were performed in SPSS (version 22.0). A p-value < 0.05 was considered statistically significant.

Animal mortality rate

A total of 341 mice were used, of which 74 underwent a sham procedure and 267 mice underwent an ICH induction. None of the sham mice died, and the mortality rate in the ICH group was 8.99% (24/267). There was no significant difference in mortality rate across the modeling groups.

Pharmacological activation of RXR-α significantly alleviated neurological dysfunction and promoted hematoma clearance after ICH

Bexarotene, an FDA-approved antineoplastic agent, which functions by selectively activating RXR-α, was introduced in the current study to determine the effects of RXR-α under ICH conditions. Three different behavioral tests, including cylinder test, corner turn test, and forelimb placement test, were used to assess the effects of RXR-α on behavioral recovery at days 1, 3, 7, 14, and 28 after ICH. There was no significant difference in the behavioral tests of mice in the four groups at baseline (pre-ICH). Animals in the modeling groups suffered from a serious neurological deficit at 24 h after ICH when compared with sham-operated mice (p < 0.05, Fig. 2A-C). The activation of RXR-α significantly improved the neurological function in all the tests carried out on days 3 and 7 after ICH, as compared to the vehicle group. A T2*-weighted MRI scan (coronal sections) was used to measure intracranial hematoma volume in parenchyma and ventricles at 1, 3, 7, 14, and 28 days after ICH. No significant differences in hematoma volume were noted at 24 h post-modeling, suggesting that the models yield consistent hematoma volumes. Compared with the vehicle group, the activation of RXR-α with bexarotene significantly decreased the hematoma volume at day 3 to day 7 after ICH (p < 0.05, Fig. 2D).

Time course and localization of RXR-α in the ipsilateral basal ganglia after ICH

Dual-label immunofluorescence staining showed that RXR-α was expressed in microglia/macrophages (Iba-1), neurons (NeuN), and astrocytes (GFAP) in the perihematomal region on the third-day post-ICH (Fig. 3A). To observe the temporal profiles of RXR-α after ICH in mice, we examined their total and subcellular expression patterns by Western blotting on the 1st, 3rd, 7th, and 14th days after ICH. The expression patterns of RXR-α in whole-cell of the ipsilateral hemispheres did not show any significant differences across these groups (p > 0.05, Fig. 3B). The cytoplasmic expression of RXR-α was significantly reduced on the 3rd and 7th day after ICH (p < 0.05, Fig. 3C), whereas the nuclear level showed an increase on the 3rd and 7th day in ICH-injured mice as compared to the sham group (p < 0.05, Fig. 3D). To further explore the effect of bexarotene on the nuclear-cytoplasmic shuttling of RXR-α, the expression of RXR-α was assessed in the ipsilateral basal ganglia at 3 days after ICH by Western blotting. The total expression of RXR-α did not show any significant changes across the three groups (p > 0.05, Fig. 3E). The RXR-α expression level of the bexarotene group was increased in the nuclei and decreased in the cytoplasm as compared with the vehicle group (p < 0.05, Fig. 3F-G). In sum, the nuclear translocation of RXR-α was induced after ICH, and the administration of bexarotene further enhanced the nuclear translocation of RXR-α.

Effect of RXR-α activation on the nuclear translocation of PPAR-γ after ICH

To determine the nuclear-cytoplasmic translocation of PPAR-γ after ICH, we tested the total and subcellular expressions of PPAR-γ by Western blotting at days 1, 3, 7, and 14 after ICH. The endogenous expression of PPAR-γ in the whole cells increased on the 3rd day and remained significantly higher on the 14th day(p < 0.05, Fig. 4A). The expression of the PPAR-γ in the cytoplasm was reduced on the 7th day, and the expression of the PPAR-γ in nuclei increased on the 3rd and 7th days after ICH (p < 0.05, Fig. 4B-C). To explore whether PPAR-γ played a role in the process of RXR-α activation, the nuclear-cytoplasmic translocation of PPAR-γ was assessed 3 days after ICH among the four experimental groups. The total expression of PPAR-γ did not show any significant changes between the vehicle group, the bexarotene group, and the bexarotene + GW9662 group (p > 0.05, Fig. 4D). The PPAR-γ expression in the bexarotene group showed a remarkable reduction in the cytoplasm and was increased in nuclei as compared with the vehicle group, while the PPAR-γ antagonism GW9662, in contrast, decreased the PPAR-γ expression in nuclei and increase the PPAR-γ expression in the cytoplasm (p < 0.05, Fig. 4E-F).

RXR-α activation regulates microglia/macrophage polarization through PPAR-γ

To determine whether the PPAR-γ played a role in microglia/macrophages polarization, we examined microglia/macrophages polarization markers iNOS (a pro-inflammatory state (M1) marker) and Arg1 (an anti-inflammatory state (M2) marker) on the day 3 after ICH by immunofluorescence. It was found that the expression of iNOS and Arg1 were all increased after ICH compared to the sham group (p < 0.05, Fig. 5A-B). Furthermore, the expression of iNOS and Arg1 was high in Iba1 + cells (microglia/macrophage) in the perihematomal area of mouse brain slices. The co-expression of iNOS and Iba1 was less pronounced in the presence of bexarotene while GW9662 reversed this effect (p < 0.05, Fig. 5A). In contrast, Bexarotene led to a significant increase in the number of M2-like polarized microglia/macrophages, which was reversed by GW9662 administration (p < 0.05, Fig. 5B). An ELISA revealed that the TNF-α levels were increased after ICH (p < 0.05, Fig. 5C). Bexarotene reduced TNF-α, and the effect was attenuated by GW9662 (p < 0.05, Fig. 5C). The expression of the M2-like marker Arg1 was measured by Western blot. Bexarotene stimulated the M2-like marker while GW9662 reversed this reduction (p < 0.05, Fig. 5D).

Role of PPAR-γ on RXR-α-mediated neuroprotection and hematoma absorption against ICH

In the next part of this study, the PPAR-γ antagonist GW9662 was introduced to further determine the role of PPAR-γ in RXR-α-mediated protective effects under ICH conditions. Three behavioral tests were used to examine the role of PPAR-γ on behavioral recovery on days 1, 3, 7, 14, and 28 after ICH. The activation of RXR-α significantly improved the neurological function, while the protective effects of the bexarotene treatment were reversed by PPAR-γ inhibition (p < 0.05, Fig. 6A-C). A T2*-weighted MRI scan (coronal sections) was used to measure the hematoma volume on days 1, 3, 7, and 14 after ICH. Similarly, RXR-α activation significantly promoted hematoma absorption on days 3 and 7 after ICH (p < 0.05, Fig. 6D). However, the blockade of PPAR-γ with GW9662 reversed bexarotene-mediated hematoma absorption (p < 0.05, Fig. 6D).

In this present study, we investigated the role of RXR-α in the pathological process following ICH and assessed the relevant underlying mechanisms. Our foundings are as follows: (1) The activation of RXR-α alleviated ICH-induced neurological deficits and promote hematoma absorption. (2) RXR-α nuclear translocation was observed after ICH, and this effect was further enhanced by the pharmacological activation of RXR-α. (3) A similar nuclear translocation of PPAR-γ following injury was also observed. Moreover, RXR-α activation could enhance PPAR-γ translocation, whereas the inhibition of PPAR-γ reversed this effect. (4) RXR-α activation could tend to make microglia/macrophages polarize into M2-like rather than M1-like cells through PPAR-γ signaling. (5) The PPAR-γ antagonist GW9662 abolished the protective effect of RXR-α activation after ICH. Based on the evidence above, the pharmacological activation of RXR-α significantly alleviated ICH-induced neurological deficits and promoted hematoma absorption. The protective effect of RXR-α activation was at least partly mediated by modulating M1/M2 macrophage polarization through PPAR-γ signaling.

ICH is a cerebrovascular disease linked to high mortality and morbidity rates [24]. A hematoma within brain parenchyma leads to secondary injuries and severe neurological deficits [25]. To date, the efficient removal of the hematoma is a clinically promising intervention for intracerebral hemorrhage [26, 27]. Successful elimination of deposited blood and apoptotic cells by phagocytosis is essential for the resolution of inflammation [4]. RXR is a member of the superfamily of nuclear hormone receptors and consists of three distinct functional domains: an amino-terminal domain, involved in its ligand-independent basal transcriptional activity, a DNA-binding domain (DBD), and a ligand-binding domain (LBD). The LBD contains the ligand-binding regions that coordinate the formation of dimers and the recruiting of transcriptional coregulators [28]. RXR mediates their ligand-dependent transcriptional activities via LBD and DBD. It regulates metabolic and immune responses, including the regulation of macrophage immune phenotypes and the clearance of apoptotic cells [12, 29]. Additionally, several studies have highlighted that RXR indeed plays an important role in optimizing the phagocytic function in the CNS [30, 31]. Despite the role of RXR-α have been studied for years, little is known about the role of RXR-α in the pathological processes following ICH.

Therefore, in the first part of this study, we assessed the effects of RXR-α in the pathological process of ICH. RXR-α agonist bexarotene can significantly attenuate neurological function, which is negatively related to the volume of the existent hematoma after ICH in humans and rodents [32, 33]. Additionally, bexarotene significantly promotes hematoma resolution in the experimental mice model, which was consistent with the results of Chang et al. [34]. All of the above data suggest that the activation of RXR-α promoted hematoma clearance while conferring a neuroprotective effect against ICH. However, the mechanisms underlying the protective effects of RXR-α remain unclear.

Microglia and monocytes/macrophages are vital to maintaining homeostasis, acting as phagocytes to scavenge for debris in the CNS after ICH. Targeting these cells may be an ideal therapeutic strategy for stroke [9]. Besides, immunofluorescence staining showed that RXR-α was expressed in microglia/macrophages, astrocytes, and neurons. Notably, recent studies have demonstrated that PPAR-γ in microglia/macrophages can increase phagocytosis and modulate inflammation by downregulating the expression of pro-inflammatory mediators’ expression [27, 35]. Furthermore, to activate transcription, PPAR-γ needs to heterodimerize with RXR-α, which is an important signaling hub in nuclear receptor controlled transcription [36, 37]. More importantly, recent studies have confirmed that the PPAR-γ signaling pathway is upregulated after exposure to bexarotene, suggesting an eminent role for PPAR-γ in RXR-α transcriptional regulation [20, 31]. Mechanism underlying RXR transcriptional activities are related to its nucleoplasmic sublocalization to the nuclear splicing factor compartment [38]. Yasmin et al. reported that enhanced RXR nuclear translocation was enabled by importin-β after RXR activation [39]. Given the essential role of RXR-α in regulating PPAR-γ transcription and the expression of RXR-α observed in microglia/macrophages after ICH, we hypothesize that RXR-α acts as an important regulator mediating the nuclear translocation of PPAR-γ.

Therefore, we then investigated the nuclear translocation of RXR-α and PPAR-γ after ICH. We noted the enhanced translocation to the nucleus of RXR-α and PPAR-γ after injury. Our findings were consistent with a previous study that reported enhanced translocation of RXR-α and PPAR-γ after traumatic brain injury [17]. Several studies on intracellular RXR shuttling were performed, showing that nuclear translocation of RXR was triggered by inflammatory cytokines such as IL-1β, IL-6, and TNF-α [40, 41]. The local inflammatory response following injury may, therefore, contribute to the activation and subcellular translocation of the retinoid X receptor, which had been shown to have anti-inflammatory properties [41]. Even though the translocation of RXR-α and PPAR-γ into the nucleus was increased after ICH, this may not be sufficient to reduce the neuroinflammation after ICH. The pharmacological activation of RXR-α may further potentiate protective pathways against neuroinflammation after ICH. We found that bexarotene significantly increased the nuclear translocation of RXR-α and PPAR-γ, while the PPAR-γ inhibitor GW9662 reversed this effect of PPAR-γ. Notably, RXR-α and PPAR-γ showed a similar trend towards nuclear translocation, and pharmacologically activating RXR-α could further promote their nuclear translocation, suggesting that RXR-α could synergistically with PPAR-γ through its interaction with it.

Various mechanisms are involved in the regulation of tissue healing, hematoma removal, and inflammation resolution. One of the essential mechanisms, especially concerning the neuroinflammation that is incurred by post-hemorrhagic secondary brain injuries, is related to the conversion of the classical activation phenotype (M1) to the alternative activation phenotype (M2) [9]. In the CNS, M2-like microglia/macrophages are efficient phagocytes [42]. Nuclear receptors play a key role in balancing M1/M2 polarization and controlling phagocytosis by regulating the transcription of functional genes [36]. It was demonstrated that PPAR-γ is an important transcriptional factor that plays a crucial role in switching on M1-like or M2-like marker genes, and thereby mediating the priming of microglia/macrophages towards M2 polarization [43]. Additionally, PPAR-γ activation mediates the transcription of downstream genes, such as ABCA1 and Cd36, which are also considered as biomarkers of scavenger receptors that contribute to the phagocytic ability of microglia/macrophages [3, 31]. A previous study has shown that the activation of the RXR/PPAR heterodimer exerts neuroprotective effects by modulating microglia polarization [17]. Given the involvement of RXR-α and PPAR-γ in microglia/macrophages-type cell polarization and differentiation, we assessed the expression of M1/M2 markers in microglia/macrophages. M1 marker proteins (iNOS, TNF-α) and M2 marker protein (ARG1) were used, revealing that microglia/macrophages were significantly polarized into the classical activation phenotype (M1) after ICH. This finding was consistent with a previous study [17], as our data indicated that the activation of RXR-α could inhibit M1 activation and driving microglia/macrophages towards the M2 phenotype while PPAR-γ inhibitor GW9662 reversed these effects. In the rest of this study, we further explored the role of PPAR-γ in neurological function and hematoma volume in response to RXR-α activation after ICH. Consistent with our previous result, RXR-α agonists significantly alleviated neurological dysfunction and promoted hematoma resolution. However, all these beneficial effects were abolished by PPAR-γ inhibitor GW9662. Taken together, we deduced that the activation of RXR-α promoted microglia/macrophages polarization towards the M2 phenotype through PPAR-γ after ICH, therefore facilitating hematoma clearance and improving neurological function.

However, our research has some undeniable limitations. Firstly, as aforementioned, RXR-α expression was also observed in neurons and astrocytes, suggesting it may play a potential role in other cells. Secondly, in addition to forming heterodimers with PPAR-γ, recent findings indicated that a separate RXR-α homodimer could also affect the immune system’s function and participate in the regulation of innate immunity [44]. We solely focused on the RXR-α/PPAR-γ signaling pathway in the current study. Lastly, resident microglia and peripheral microglia may both contribute to neuroinflammation; however, which of these mediate the beneficial effects has not yet been assessed [45]. Therefore, further efforts are required to obtain more details with regard to other possible mechanisms mediated by RXR-α in ICH.

In summary, our data indicated that pharmacological activation of RXR-α promoted hematoma clearance, improved neurological function, and polarized microglia/macrophages towards the M2 phenotype via regulation of the nuclear translocation of PPAR-γ after ICH. Altogether, the current study supports the notion that targeting RXR-α might be a novel and promising therapeutic strategy for ICH.

BCA	Bicinchoninic acid
CNS	Central nervous system
DBD	DNA-binding domain
DMSO	Dimethyl sulfoxide
ELISA	Enzyme-linked immunosorbent assay
ICH	Intracerebral hemorrhage
LBD	Ligand-binding domain
MRI	Magnetic resonance imaging
PPAR-γ	Peroxisome proliferator-activated receptor-γ
RXR-α	Retinoid X receptor-α

Acknowledgments:

Part of the figures were created using BioRender (www.BioRender.com), to which we are grateful.

Authors’ contributions:

CRX, HJC, SJZ, CJS, and XLX performed the ICH model and Western blots. YCP, JFZ, and HHZ prepared the figures. JRL, QY, and YC performed the immunostaining. YL, GYZ, XJF, and PYH performed the MRI scanning, HZ, and HJC performed data analysis. PYH, FY, and GC designed experiments. CRX, HJC, and SJZ contributed to the writing and editing of the manuscript. All authors read and approved the manuscript.

Funding:

This work was supported by the National Key R&D Program of China (2018YFC1312600, 2018YFC1312603), the Key Research and Development Project of Zhejiang Province (No.2018C03011), National Natural Science Foundation of China (No.81771246，81971099，81870908), Scientific Research Fund of Zhejiang Provincial Education Department (No.Y201941838).

Availability of Data and Materials:

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate:

All procedures involved animals were conformed to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Zhejiang University.

Consent for publication:

Not applicable.

Competing Interests:

The authors declare that they have no competing interests.

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Pharmacological Activation of RXR-α Promotes Hematoma Absorption in a PPAR-γ-Dependent Pathway After Intracerebral Hemorrhage

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Abstract

Figures

Background

Materials And Methods

Animals and ethical statement

ICH model

Experimental design

Experiment I

Experiment II

Experiment III

Experiment IV

Drug administration

Calculation of hematoma volume

Behavioral tests

Immunofluorescence double labeling

Enzyme-linked immunosorbent assay

Western blot analysis

Statistical analysis

Results

Animal mortality rate

Pharmacological activation of RXR-α significantly alleviated neurological dysfunction and promoted hematoma clearance after ICH

Time course and localization of RXR-α in the ipsilateral basal ganglia after ICH

Effect of RXR-α activation on the nuclear translocation of PPAR-γ after ICH

RXR-α activation regulates microglia/macrophage polarization through PPAR-γ

Role of PPAR-γ on RXR-α-mediated neuroprotection and hematoma absorption against ICH

Discussion

Conclusion

List Of Abbreviations

Declarations

Acknowledgments:

Authors’ contributions:

Funding:

Availability of Data and Materials:

Ethics approval and consent to participate:

Consent for publication:

Competing Interests:

References

Status:

Version 1