Introduction

Intracerebral hemorrhage (ICH) is a stroke subtype, with high mortality and morbidity due to its large fatality rate and poor functional outcome (Panos et al. 2020; Ponamgi et al. 2020; Yang et al. 2008; Zhang et al. 2017). The released blood cells and blood components such as thrombin, as well as erythrocyte rupture, result in mechanical damage and secondary damage [4–6]. Therefore, the therapeutic strategy targeting inflammation as well as the blood–brain barrier (BBB) and edema is a promising approach for ICH (Chen et al. 2015; Deng et al. 2020; Hou et al. 2020; Zhu et al. 2018).

Inflammasomes are multiprotein complexes that are involved in caspase-1 activation and interleukin-1β (IL-1β) maturation as well as pyroptosis (Zhang et al. 2018). Though numerous inflammasomes have been reported, the NLRP3 inflammasome is the most extensively studied. NLRP3 inflammasome is stimulated by a variety of factors, including infection, tissue damage, and metabolic dysregulation, and then activated through an integrated cellular signal (Wynosky-Dolfi et al. 2014). Recent evidence demonstrate that NLRP3 inflammasome plays a vital role in inflammation after intracerebral hemorrhage (Feng et al. 2015).

VSIG4, also known as CRIg or Z39Ig, is a newly identified member of the B7 super family (Tanaka et al. 2012). The expression of VSIG4 is restricted to tissue macrophages, including peritoneal macrophages and liver residential Kupffer cells (Shin et al. 2018). It negatively modulates the inflammation process by causing T-cell anergy and activates macrophage immunity when binding to the C3b/iC3b (Zhang et al. 2021). VSIG4 mediates transcriptional inhibition of NLRP3 and Il-1β in macrophages (Huang et al. 2019). Nevertheless, whether VSIG4 is capable of manipulating NLRP3 after ICH remains elusive.

In this experiment, C57BL/6 mice were subjected to the ICH model, and the regulation role of VSIG4 on NLRP3 and specific mechanism were analyzed.

Methods

Animals

All experimental protocols for this study were approved by the Animal Ethics Committee of Chongqing Medical University. The study complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. C57BL/6 mice (male, weight about 25 g) were purchased from and bred at the Animal Center of Chongqing Medical University. All mice were housed in a light- and temperature-controlled room with free access to food and water.

ICH mouse model induction

Mice were anesthetized with 10% chloral hydrate (350 mg/kg) and were placed in a stereotaxic frame (Alcott Biotech, Shanghai, China). Through a hole drilled in the skull, a 32-gauge needle was implanted into the striatum, 2.0 mm lateral to the midline, 1.0 mm anterior to the coronal suture, and at a depth of 4.0 mm from the surface of the brain. Each mouse was microinjected with 25 μl of autologous whole blood (right striatum) taken from the tail vein over 10 min using a microinfusion pump (ALC-IP600, Alcott Biotech). Then, the needle was pulled out without blood reflux after 5 min of dwelling, and the wound was sutured. Only the mouse observed to have a neurological deficit was regarded as a successful model. The mice in the sham operation group had the same operation, but no blood was injected.

Intracerebroventricular injection

The in vivo transfection was performed according to the method described as follows: the stereotaxic coordinates were 0.5 mm posterior and 1.0 mm lateral to bregma and 2.5–3.0 mm ventral to the surface of the skull. The Ad-VSIG4, Ad-NLRP3 siRNA, Ad-A20 RNAi or control (1 × 109 plaque-forming units (Pfu), Sangon Biotech, Shanghai, China) were added to 2 μl of a 10-mg/ml solution prepared in 0.9% NaCl. In the inhibitor treatment experiments, mice were received intracerebroventricular injection of 0.9% NaCl (2 μl) in the presence of the STAT3 inhibitor S3I-201 (50 mM, Sigma-Aldrich), or the JAK2 inhibitor TG101348 (50 μM, Sigma-Aldrich), respectively. The solution was mixed gently, left for 15 min and then injected intracerebroventricularly (i.c.v.) using a micro syringe (Hamilton, NV, USA) under the guidance of the stereotaxic instrument (RWD Life Science).

BBB permeability

To evaluate BBB permeability, Evans blue (Aladdin, China) was injected intraperitoneally (100 μl of 4% solution in saline). After 3 h of circulation, mice were transcardially perfused with cold phosphate-buffered saline (0.1 M, PBS, pH7.4) under deep anesthesia. Afterwards, the brain was removed and divided into left and right hemispheres and stored at − 80 °C immediately. The right part of the brain was homogenized in 1100 μl PBS, sonicated, and centrifuged (12,000 g, 4 °C, 30 min). The supernatant was collected and added an equal amount of trichloroacetic acid (TCA) to incubate overnight by 4 °C. After centrifugation (12,000 g, 4 °C, 30 min), Evans blue stain was measured by spectrophotometer (Thermo Fisher Scientific, USA) at 610 nm.

Immunofluorescence staining

The mice were deeply anesthetized and were transcardially perfused with 20 ml ice-cold PBS followed by 20 ml of 4% paraformaldehyde at 24 h post-ICH. The whole brain was collected and then fixed in 4% paraformaldehyde for another 24 h. Afterwards, the brain was fixed in 20% sucrose solution until the tissue sinks to the bottom followed by fixing in 30% sucrose solution for another 24 h. After being frozen at − 25 °C, the brain was cut into 10 μm-thick coronal sections using a cryostat (CM1860; Leica Microsystems, Germany). To conduct double immunohistochemistry staining, the brain sections were incubated with primary antibody of CD11b, VSIG4, or NLRP3 (1:100, Santa Cruz) overnight at 4 °C. After being incubated with the appropriate secondary antibody (1:200, Bioss) at 37 °C for 1 h, the sections were visualized and photographed with a fluorescence microscope (UHGLGPS, OLYMPUS, Japan). Microphotographs were analyzed with cellSens Standard software. The numbers of positive cells were identified and counted in three different fields in the perihematoma area from five random coronal sections per brain, and data were expressed as cells/field.

Histochemical evaluation of macrophage accumulation

The mice were deeply anesthetized and were transcardially perfused with 20 ml ice-cold PBS followed by 20 ml of 4% paraformaldehyde at 24 h post-ICH. After the mice were perfused and fixed, the perihematomal region of cerebral tissues were collected, fixed in 4% paraformaldehyde for 24 h, dehydrated in 30% sucrose solution for 48 h, embedded, frozen, and cut into 25 μm sections using a Leica CM1900 cryostat. The perihematomal region was treated with 3% H2O2 in 0.01 M phosphate-buffered saline (PBS) and preincubated in 5% normal goat serum. The samples were then incubated in a primary antibody solution containing rat anti-CD11b antibody (Serotec, Fullerton, CA, USA, 1:200) overnight at 4 °C. After washing, the samples were incubated in a secondary IgG antibody (1:200) for 1 h at room temperature (RT). Finally, the sections were incubated in horseradish peroxidase (HRP)-Streptavidin (1:200) for 1 h at RT, and the color reaction was conventionally developed with diaminobenzidine (DAB) and H2O2. For each animal, six representative sections of each brain were selected. IPP6.0 image processing software (Media Cybernetics, MD, USA) was utilized to count the number of positive cells.

Western blotting

Briefly, the mice were perfused with 0.01 mol/l phosphate-buffered saline after ICH, and the cerebral tissues from the perihematomal region were isolated (n = 5). The perihematomal tissues were lysed in 1 ml of radioimmunoprecipitation assay lysis buffer, and then the protein was extracted, electrophoresed, and transferred onto polyvinylidene fluoride membranes (Amersham Pharmacia). The polyvinylidene fluoride membranes were incubated with primary antibodies overnight, followed by incubation with peroxidase-conjugated secondary antibodies for 6 h. The same membranes were probed with an antibody for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Protein signals were detected with an enhanced chemiluminescence system. The signals were quantified by scanning densitometry and computer-assisted image analysis. Protein levels were expressed as the ratio of the values of the detected protein band to the GAPDH band.

Evaluation of neurological scores

The neurological scores were assessed by Neurological Severity Scores, according to the motor, sensory, reflex, and balance tests. Neurological function was assessed on a scale of 1–18; a score of 1 point is regarded as the inability to perform the test or for the lack of a tested reflex. The higher the score, the more severe the injury (normal score: 2–3; maximal deficit score: 18).

Measurement of brain edema

Brain hemisphere was quickly separated and weighted to assess the wet weight (wW) using an electronic analytic balance. After drying the brain hemisphere in an oven at 100 °C for 24 h, dry tissue weight (dW) was assessed. The percentage of water was calculated according to the following formula: Brain water content (%) = (wW-dW)/wW × 100%.

Statistics analysis

The data are expressed as the mean ± SD of at least 3 separate experiments performed in triplicate. The differences between groups were determined with the Student’s t tests and a one-way analysis of variance (ANOVA) using SPSS 13.0 software. Differences were declared significant at *P < 0.05.

Results

VSIG4 and NLRP3 expressions in the perihematomal brain tissues after ICH

The expression of VSIG4 and NLRP3 proteins in perihematomal brain tissues after ICH was quantified using western blotting at the indicated time points (12, 24, and 72 h after ICH). The expression of VSIG4 started decreasing at 12 h and peaked at 24 h after ICH (P < 0.05; Fig. 1A). The expression of NLRP3 started increasing at 12 h and peaked at 24 h after ICH (P < 0.05; Fig. 1B). The representative images of double immunofluorescence staining of VSIG4 and NLRP3 expressions in CD11b+ macrophage of the perihematomal brain tissues at 24 h after ICH are shown in Fig. 1C.

Fig. 1
figure 1

VSIG4 and NLRP3 expressions in the perihematomal brain tissues after ICH. After 24 h after ICH, mice (n = 5 per group) were deeply anesthetized in a transcardial manner. A, B The brains were removed and post fixed. The perihematomal region of cerebral tissue was collected, VSIG4 and NLRP3 expressions were analyzed with western blot analysis. C Representative images of double immunofluorescence staining of VSIG4 or NLRP3 colocalized with macrophage (CD11b) at 24 h after ICH. Scale bar = 50 μm. Experiments performed in triplicate showed consistent results. Data are presented as the mean ± standard error of mean (SEM) of three independent experiments. *P < 0.05

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Upregulation of VSIG4 decreased the NLRP3 expression at 24 h after ICH

Mice were received intracerebroventricular injection of Ad-VSIG4 at 24 h before ICH. The expression of VSIG4 and NLRP3 proteins in perihematomal brain tissues after ICH was quantified using western blotting at 24 h after ICH. The expression of VSIG4 significantly increased after Ad-VSIG4 injection compared with that of the control groups. However, NLRP3 significantly decreased after Ad-VSIG4 injection compared with that of the control groups (P < 0.05; Fig. 2A, B). The representative images of double immunofluorescence staining of VSIG4 or NLRP3 expression in the macrophage of the perihematomal brain tissues at 24 h after ICH are shown in Fig. 2C. CD11b+ macrophage numbers were not decreased after Ad-VSIG4 treatment, but NLRP3 + CD11b+ macrophage numbers decreased after Ad-VSIG4 treatment compared with that of the control groups (P < 0.05; Fig. 2D).

Fig. 2
figure 2

Upregulation of VSIG4 decreased the NLRP3 expression at 24 h after ICH. Mice were received intracerebroventricular injection of Ad-VSIG4 at 24 h before ICH. After 24 h after ICH, mice (n = 5 per group) were deeply anesthetized in a transcardial manner. A, B The brains were removed and post fixed. The perihematomal region of cerebral tissue was collected, VSIG4 and NLRP3 expressions were analyzed with western blot analysis. C Representative images of double immunofluorescence staining of VSIG4 or NLRP3 colocalized macrophage (CD11b) at 24 h after ICH. Scale bar = 50 μm. D CD11b+ or NLRP3 + CD11b+ macrophage numbers after Ad-VSIG4 or Ad-LacZ treatment. Experiments performed in triplicate showed consistent results. Data are presented as the mean ± standard error of mean (SEM) of three independent experiments. *P < 0.05

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Upregulation of VSIG4 improved neurological deficits and reduced brain edema and BBB permeability after ICH

To further evaluate the role of VSIG4 on the ICH-induced neurological impairment, we injected Ad-VSIG4 at 24 h before ICH. The neurological deficits and brain edema were worse at 24 h post-ICH compared with the sham group. However, administration of Ad-VSIG4 significantly improved the neurological deficits and reduced brain edema (P < 0.05; Fig. 3A, B). BBB permeability was assessed by EB extravasation assay. EB extravasation in the ICH group was significantly increased at 24 h after ICH; however, Ad-VSIG4 significantly decreased EB dye leakage compared with that of the control group (Fig. 3C).

Fig. 3
figure 3

Upregulation of VSIG4 improved neurological deficits and reduced brain edema and BBB permeability after ICH. Mice were received intracerebroventricular injection of Ad-VSIG4 at 24 h before ICH. After 24 h after ICH, mice (n = 5 per group) were deeply anesthetized in a transcardial manner. A The brains were removed and post fixed. The perihematomal region of cerebral tissue was collected, VSIG4 expression was analyzed with western blot analysis. B The BBB disruption of mice was analyzed. C The cerebral water content of mice was also analyzed. D The neurological deficit tests were performed by behavioral measurement, including composite of motor, sensory, reflex, and balance tests. Experiments performed in triplicate showed consistent results. Data are presented as the mean ± standard error of mean (SEM) of three independent experiments. *P < 0.05

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Knockdown of NLRP3 aggravated neurological deficits, brain edema, and BBB disruption after ICH

To further investigate the protective role of VSIG4, NLRP3 siRNA was administered by i.c.v injection to knock down the expression of endogenous NLRP3. Western blot showed that NLRP3 expression was inhibited by NLRP3 siRNA at 24 h after injection (Fig. 4A). The knockdown of NLRP3 abolished the protective effect of VSIG4 on neurological functions, brain edema, and BBB integrity at 24 h after ICH (Fig. 4B–D).

Fig. 4
figure 4

Knockdown of NLRP3 aggravated neurological deficits, brain edema, and BBB disruption after ICH. Mice were received intracerebroventricular injection of Ad-NLRP3 siRNA at 24 h before ICH. After 24 h after ICH, mice (n = 5 per group) were deeply anesthetized in a transcardial manner. A The brains were removed and post fixed. The perihematomal region of cerebral tissue was collected, NLRP3 expression was analyzed with western blot analysis. B The BBB disruption of mice was analyzed. C The cerebral water content of mice was also analyzed. D The neurological deficit tests were performed by behavioral measurement, including composite of motor, sensory, reflex, and balance tests. Experiments performed in triplicate showed consistent results. Data are presented as the mean ± standard error of mean (SEM) of three independent experiments. *P < 0.05

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Upregulation of VSIG4 inhibited macrophage accumulation and downstream molecule expression in ICH

To determine whether VSIG4 could attenuate ICH-induced neuroinflammation in mice, macrophage accumulation and downstream molecules in the hippocampus were examined 24 h after ICH. Macrophage accumulation in the perihematomal brain tissues significantly increased compared with control mice after ICH. However, administration of Ad-VSIG4 could attenuate macrophage accumulation (Fig. 5A). ZO-1, occludin, and Lama5 in the perihematomal brain tissues significantly decreased, but TNF-α, IL-1β, and IL-6 protein levels in the perihematomal brain tissues significantly increased compared with those of the control mice 24 h after ICH. However, administration of Ad-VSIG4 could enhance ZO-1, occludin, and Lama5 and attenuate TNF-α, IL-1β, and IL-6 protein levels (Fig. 5B–H). These results suggest that upregulation of VSIG4 could inhibit ICH-induced neuroinflammation.

Fig. 5
figure 5

Upregulation of VSIG4 inhibited macrophage accumulation and downstream molecule expression in ICH. Mice were received intracerebroventricular injection of Ad-VSIG4 at 24 h before ICH. After 24 h after ICH, mice (n = 5 per group) were deeply anesthetized in a transcardial manner. The brains were removed and post fixed. A Representative images of histochemical evaluation of macrophage (CD11b) at 24 h after ICH. Scale bar = 50 μm. B–H ZO-1, occludin, Lama5, TNF-α, IL-1β, and IL-6 protein levels in the perihematomal brain tissues were analyzed with western blot analysis. Experiments performed in triplicate showed consistent results. Data are presented as the mean ± standard error of mean (SEM) of three independent experiments. *P < 0.05

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Upregulation of VSIG4 attenuated NLRP3 and ameliorated neuroinflammation by activating JAK2-STAT3-A20 pathway

We further investigated the underlying signaling pathways of VSIG4 inhibiting NLRP3. A20, a protein capable of down-modulating LPS, induced NLRP3 transcription through inhibiting NF-kβ activation. Treatment with Ad-VSIG4 increased the expression of JAK2, STAT3, and A20 in the perihematoma tissue at 24 h post-ICH compared with the control group (Fig. 6A). However, STAT3 inhibitor (S31-201), JAK2 inhibitor (TG101348), and Ad-A20 siRNA enhanced NLRP3 levels in the perihematoma tissue at 24 h post-ICH compared with the control group (Fig. 6B). In addition, STAT3 inhibitor (S31-201), JAK2 inhibitor (TG101348) inhibitor (TG10ion1348), and Ad-A20 siRNA exacerbated neurological impairments and increased brain water content at 24 h after ICH (Fig. 6C, D). Furthermore, STT3 inhibitor (S31-201), JAK2 inhibitor (TG101348) inhibitor (TG10ion1348), and Ad-A20 siRNA increased the expression of TNF-α, IL-1β, and IL-6 protein levels in the perihematomal brain tissues (Fig. 6E).

Fig. 6
figure 6

Upregulation of VSIG4 attenuated NLRP3 and ameliorated brain injury by activating JAK2-STAT3-A20 pathway. Mice were received intracerebroventricular injection of Ad-VSIG4 at 24 h before ICH. After 24 h after ICH, mice (n = 5 per group) were deeply anesthetized in a transcardial manner. The brains were removed and post fixed. A p-JAK2, p-STAT3, and A20 in the perihematoma tissue were analyzed with western blot analysis. B STAT3 inhibitor (S31-201), JAK2 inhibitor (TG101348), and Ad-A20 siRNA were pretreated at 24 h before ICH, NLRP3 in the perihematoma tissue were analyzed with western blot analysis. C The BBB disruption of mice was analyzed. D The cerebral water content of mice was also analyzed. E The neurological deficit tests were performed by behavioral measurement, including composite of motor, sensory, reflex, and balance tests. Experiments performed in triplicate showed consistent results. Data are presented as the mean ± standard error of mean (SEM) of three independent experiments. *P < 0.05

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Discussion

In this experiment, we presented the following evidences: (1) VSIG4 was significantly decreased, and NLRP3 was significantly increased in the perihematoma tissue after ICH; (2) upregulation of VSIG4 decreased the NLRP3 expression following ICH; (3) upregulation of VSIG4 improved neurological deficits and reduced brain edema and BBB permeability after ICH; (4) knockdown of NLRP3 aggravated neurological deficits, brain edema, and BBB disruption after ICH; (5) upregulation of VSIG4 inhibited macrophage accumulation and downstream molecule expression in ICH; (6) upregulation of VSIG4 attenuated NLRP3 and ameliorated neuroinflammation by activating the JAK2-STAT3-A20 pathway.

Numerous data indicated that inflammation and BBB disruption are important points to induce secondary brain damage following ICH (Cheng et al. 2016; Li et al. 2020; Wang et al. 2019). Following ICH, blood components rapidly enter the cerebral parenchyma, leading to an inflammatory reaction. Furthermore, intensive inflammatory cascades enhance BBB disruption, resulting in blood component infiltration into the brain, and subsequent exacerbation of brain damage after ICH (Cai et al. 2015; Wu et al. 2020; Xi et al. 2002).

The NLRP3 inflammasome amplified the inflammatory response by releasing IL-1β and promoting neutrophil infiltration following ICH (Chen et al. 2014; Gicquel et al. 2015; Ma et al. 2014). The activation of the NLRP3 inflammasome is tightly regulated at the transcriptional and posttranslational levels to inhibit excessive inflammation in the host (Karasawa et al. 2018; Surabhi et al. 2020; Zheng et al. 2020). Therefore, identification and exploring of unknown factors regulating the NLRP3 inflammasome might represent a promising strategy for ICH. VSIG4 is a member of the B7 family-related protein which is well-known for its inhibition of T-cell activation and works as a co-inhibitory ligand that inhibits T-cell activation and cytokine production through cell cycle arrest at the G0/G1 phase and induces T-cell anergy (Deng et al. 2019; Wang et al. 2015; Zhai et al. 2018). The relationship between VSIG4 and NLRP3 after ICH has not been studied. In our experiment, we firstly detected VSIG4 and NLRP3 levels in the macrophage after ICH, and found that upregulation of VSIG4 decreased the NLRP3 expression at 24 h after ICH. Secondly, we evaluated the role of VSIG4 on the ICH and found that VSIG4 improved neurological deficits and reduced brain edema and BBB permeability after ICH.

A20 is an endogenous anti-inflammatory factor that can attenuate the destructive proinflammatory factor production such as IL-1β and TNF-α by inhibiting NF-kβ activation (Yao et al. 2018). A20 also interacts with IL-10 and TGF-β and inhibits inflammation in diabetes and arthritis (Liuwantara et al. 2006; Vereecke et al. 2009). A20 has been shown to attenuate NLRP3 inflammasome activity, and macrophage A20-deficient mice show exacerbated CNS inflammation due to hyperactivation of the NLRP3 inflammasome that leads to increased IL-1β secretion and CNS inflammation (Gupta et al. 2017; Mohebiany et al. 2020). Our data demonstrated that treatment with Ad-VSIG4 increased the expression of JAK2, STAT3, and A20 in the perihematoma tissue at 24 h post-ICH compared with that of the control group. However, STAT3 inhibitor (S31-201), JAK2 inhibitor (TG101348) inhibitor (TG101348), and Ad-A20 siRNA enhanced NLRP3 levels in the perihematoma tissue, proinflammatory cytokine production and BBB disruption, exacerbated neurological impairments and increased brain water content at 24 h after ICH.

Conclusion

VSIG4 could attenuate neuroinflammation and BBB disruption and improve neurological impairments after ICH in mice. The neuroprotective role of VSIG4 activated the cascade JAK2-STAT3-A20 signaling pathway, leading to NLRP3 inhibition. Therefore, VSIG4-NLRP3 might serve as a potential therapeutic agent against neuroinflammation for ICH patients.