Male C57BL/6 mice (8–10 weeks old, 22–25 g) were sourced from Shanghai Laboratory Animal Center and acclimatized for 7 days at Huzhou Central Hospital’s Animal Experiment Center before surgical procedures. The animal room is a barrier environment for specific pathogens (SPF) with a temperature controlled at 20–26 °C, relative humidity of 40%–70%, and a 12-hour light/dark cycle. The mice were housed in polycarbonate cages with 3–5 mice per cage, with a heat-sterilized bedding material at the bottom, which was replaced twice a week. The laboratory animals had free access to standard autoclaved laboratory mouse feed and sterile drinking water. All animal experiments complied with institutional ethical guidelines approved by the hospital’s Animal Care Committee (Approved number: 202411005).

RAW264.7 and HT22 cells were obtained from Central Laboratory of Huzhou Hospital (Huzhou, China). Cells were cultured in DMEM (Gibco, 11965, Waltham, MA, USA) supplemented with 10% heat-inactivated FBS (Gibco, A3161002) and 1% penicillin-streptomycin (Gibco, 15140122) to ensure optimal growth. The cells were incubated at 37 °C in a 5% CO₂ atmosphere. All cell lines underwent short tandem repeat profiling for authentication and tested negative for Mycoplasma.

The mice were anesthetized using a 2% isoflurane/air mixture in a general-purpose small animal anesthesia machine (RWD R500, Shenzhen, China) before being placed on a stereotaxic apparatus. After drilling a hole in the skull, a 32-gauge needle was inserted into the right striatum using coordinates of 2.0 mm from the median line, 1.0 mm from the bregma, and a depth of insertion of 4.0 mm below the brain’s surface. Autologous whole blood (obtained from the caudal vein) was injected into each mouse over 10 min using a microsyringe. After injection, the needle was left in place for 5 minutes to prevent blood reflux before being gently withdrawn. The wound was then sutured. The overall generation rate of mice within 72 h post-ICH was approximately 85%, and mice with a modified Neurological Severity Score (mNSS) greater than 2 were considered successfully modeled. The mice in the sham-operated group underwent the same procedure, except that no blood was injected.

Cell viability was assessed via the Cell Counting Kit-8‌‌ (CCK8) assay (Sigma-Aldrich, 96992, St. Louis, MO, USA) in accordance with the supplier’s standardized protocol.

We used Beyotime assay kits to measure various biochemical activities: a Superoxide Dismutase Assay Kit (Beyotime, S0101S, Shanghai, China) for SOD activity, Lipid Peroxidation MDA Assay Kit (Beyotime, S0131S, Shanghai, China) for MDA levels, and Micro Reduced Glutathione Assay Kit (Beyotime, S0053, Shanghai, China) for GSH measurement. All experimental steps were executed as specified in the manufacturer’s guidelines.

After treatment, tissues and cells were lysed using a radioimmunoprecipitation assay-based solution containing protease inhibitors. Extracted proteins were resolved by SDS-polyacrylamide gel electrophoresis, transmitted to a polyvinylidene difluoride membrane, blocked with FBS, and cleared with PBS. Subsequently, the membrane was incubated overnight at 4 °C with primary antibodies, namely rabbit anti-mouse NRF2 (1:2000; CST, 12721, Danvers, MA, USA), heme oxygenase-1 (HO-1) (1:2000; CST, 70081, USA), B-cell lymphoma 2 (Bcl-2) (1:2000; CST, 3498, USA), Bax (1:2000; CST, 2772, USA), VSIG4 (1:2000; Abcam, ab252933, Cambridge, UK), and GAPDH (1:2000; CST, 2118, USA). After three 5-minute wash cycles with PBS, the substrate was coupled to a goat anti-rabbit IgG horseradish peroxidase secondary antibody (1:3000; Affinity, S0001, USA) for 1 hour at room temperature. Protein signals were visualized using an enhanced chemiluminescence system and quantified via scanning densitometry and computer-aided image analysis. Protein levels were reported as the ratio of each detected band to that of GAPDH.

RNA isolation from tissue and cell samples was carried out with TRIzol reagent per the manufacturer’s protocol, followed by reverse transcription of RNA into cDNA using commercial kits. RT-qPCR was performed using a specific PCR system according to the manufacturer’s instructions. The PCR cycling conditions consisted of the following steps: The initial step was a pre-denaturation phase, which was performed at 95 °C for 10 min. After that, 40 cycles were carried out. Each cycle involved denaturation at 95 °C for 15 s and then an annealing and extension phase at 60 °C for 35 s. PCR was performed in triplicate for each sample. Each experiment was repeated at least three times. Data were analyzed using the comparative CT ($\mathrm{\Delta }$$\mathrm{\Delta }$CT) method to calculate relative gene expression levels. The following primer sequences were used: VSIG4 (F-CCTGGGCCACCTAATAGTGC, R-TGTAGCCTCTCAGGGGATCAT), GAPDH (F-TTGTCATGGGAGTGAACGAGA, R-CAGGCAGTTGGTGGTACAGG).

The interleukin-1$\beta$ (IL-1$\beta$) (Epizyme, HJ177, Shanghai, China) and tumor necrosis factor-alpha (TNF-$\alpha$) (Epizyme, HJ207, Shanghai, China) ELISA kits were obtained from Epizyme. The concentrations of these molecules were determined using ELISA kits, following the manufacturer’s guidelines.

Three days post-ICH, the mice were evaluated using the mNSS as described [18, 19]. The scoring scale ranged from 0 (no visible neurological impairment) to 18 (the most severe neurological impairment). The mNSS assesses motor function, sensory responses, balance, and reflexes. Motor deficits, sensory impairments, balance dysfunction, and reflex abnormalities were scored on a scale of 0–6, 0–2, 0–6, and 0–4, respectively. All evaluations were conducted by trained investigators who were blinded to the experimental groups.

72 h after ICH, the mice were sacrificed by cervical dislocation. Immediately thereafter, the brain specimens were divided into four parts: the ipsilateral cortex (Ipsi-CX), basal ganglia on the ipsilateral side (Ipsi-BG), contralateral cortex (Cont-CX), and basal ganglia on the contralateral side (Cont-BG). First, the specimens were weighed to determine their wet weight. They were then placed in an oven at 105 °C and dried for 72 h. After drying, the specimens were weighed again to determine their dry weight. As described [20, 21], the percentage of brain water content was derived by applying the following formula: (wet weight–dry weight)/wet weight $\times$ 100%.

On day 3 after ICH, samples were obtained, and paraffin-embedded slices were prepared according to the previously described method [17]. Brain slices were incubated with 5% BSA to block unspecific binding. After overnight incubation at 4 °C with primary antibodies—anti-Iba-1 (1:500, CST 17198, USA), anti-NeuN (1:500, Abcam, ab104224, UK) and anti-GFAP (1:500, CST, 3670)—procedures proceeded to subsequent steps. The samples were then incubated with fluorescence-conjugated secondary antibodies (Abcam, ab150115 and ab150077, UK) at room temperature for 2 h. After incubation with secondary antibodies, the cells were washed three times with PBS. Next, the nuclei were stained with DAPI (Abcam, ab228549, UK). Finally, the ImageJ software (1.54f, National Institutes of Health, Bethesda, MD, USA) was used to calculate and quantify the average number of positively stained cells.

Tissue sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and rinsed with water. Antigen retrieval was performed by microwave heating in sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 minutes. After washing with TBST, sections were blocked with 5% BSA for 30 minutes at room temperature. The sections were incubated with primary antibodies against VSIG4 (1:500, Abcam, ab252933, UK), TMEM119 (1:500, CST 90840, USA), and IBA1 (1:500, CST 17198, USA) for 1 hour at room temperature, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody (absin, abs50012, Shanghai, China) for 10 minutes. Fluorescence signal amplification was achieved using tyramide conjugates (absin, abs50012, Shanghai, China) for 10 minutes. Nuclei were counterstained with DAPI (Abcam, ab228549, UK), and sections were mounted with anti-fade medium. Images were acquired using a fluorescence microscope (Olympus DP74, Olympus Corporation, Tokyo, Japan).

Brain tissues from each group of mice were collected and fixed in 4% paraformaldehyde at 4 °C for one week. Following paraffin embedding and sectioning, HE staining was performed according to the manufacturer’s instructions (Solarbio, G1120, Beijing, China). Tissue sections were dehydrated using graded ethanol, and staining results were observed under an inverted fluorescence microscope (Olympus DP74, Olympus Corporation, Japan).

Quantitative data are presented as mean $\pm$ SD. Intergroup differences were analyzed using Student’s t-test (two groups) or one-way ANOVA with Tukey’s post hoc analysis (multiple groups). Non-parametric data were evaluated via the Kruskal-Wallis test. Statistical significance was set at p 0.05.

Microglia and macrophages have similar functions and play essential roles in neuroinflammation after ICH [22]. It has been reported that VSIG4 can be expressed in peripheral macrophages [23]. To investigate VSIG4 expression in microglia/macrophages in brain tissue after ICH, we constructed an ICH mouse model and performed immunostaining (Fig. 1A). Immunostaining revealed VSIG4 expression exclusively in IBA1⁺ TMEM119^– cells (peripheral macrophages) post-ICH, with no detection in IBA1⁺ TMEM119⁺ microglia (Fig. 1B). In addition, compared to 1 day after ICH, the fluorescence intensity of VSIG4 in IBA1⁺ cells decreased 3 days after ICH (p 0.01), indicating that VSIG4 expression in macrophages after ICH was reduced, which is consistent with existing research (Fig. 1C) [24].

Fig. 1.

Illustrates the expression characteristics of VSIG4 following ICH and the expression of VSIG4 in macrophages following LPS stimulation. (A) A schematic diagram outlining the experimental protocol carried out in mice. (B) Representative immunofluorescence images of VSIG4-positive cells, IBA1-positive cell and TMEM119-positive cells in the tissue surrounding the hematoma on days 1 and 3 after ICH, n = 5, scale 50 µm. The red arrow shows VSIG4⁺IBA⁺TMEM119^– cells. (C) The relative fluorescence intensity of VSIG4-positive and IBA1-positive cells in the tissue surrounding the hematoma on day 1 and day 3 after ICH, with n = 5. (D) The RT-qPCR results of VSIG4 mRNA in RAW264.7 cells after LPS stimulation. (E,F) Western blotting analysis of VSIG4 in RAW264.7 cells after LPS stimulation. VSIG4, V-set and immunoglobulin domain-containing 4; ICH, intracranial hemorrhage; LPS, lipopolysaccharide.

Inflammatory stimulation is an important factor that causes changes in macrophages. To examine VSIG4 expression in macrophages under inflammatory stimulation, RAW264.7 cells were treated with LPS (1 µg/mL) in vitro. RT-qPCR findings revealed that, compared to the control group, LPS treatment resulted in a time-dependent decrease in VSIG4 mRNA levels (p 0.01) (Fig. 1D). Moreover, we measured VSIG4 protein levels, and found that the changes in VSIG4 protein levels following LPS stimulation were consistent with the changes in mRNA levels (p 0.01) (Fig. 1E,F). These findings imply that upon exposure to an inflammatory stimulus, the VSIG4 expression level in macrophages progressively decreases.

To explore the effects of VSIG4 on macrophages, we introduced recombinant VSIG4 (100 µg/mL) into RAW264.7 cells. Western blot analysis showed that VSIG4 treatment significantly upregulated the expression of NRF2 and HO-1 in RAW264.7 cells (p 0.05), potentially affecting cellular antioxidant and stress response pathways (Fig. 2A–C). Prior research indicates that the NRF2/HO-1 axis modulates oxidative stress responses [25]. Therefore, we examined oxidative stress levels in macrophages. Compared to the IgG group, VSIG4 administration markedly elevated GSH levels and decreased SOD activity (p 0.01) (Fig. 2D,E). Conversely, MDA levels notably decreased following VSIG4 intervention under LPS stimulation (p 0.01) (Fig. 2F). These results indicate that VSIG4 can enhance the antioxidant capacity of cells, thereby effectively counteracting LPS-induced oxidative stress damage.

Fig. 2.

Recombinant VSIG4 activates the NRF2 signaling pathway to inhibit oxidative stress and inflammatory responses in macrophages. (A–C) Western blotting analysis of NRF2 and HO-1 in RAW264.7 cells after the addition of recombinant VSIG4, n = 3. (D–F) Levels of GSH, SOD, and MDA in RAW264.7 cells after the addition of recombinant VSIG4, n = 3. (G,H) Levels of IL-1$\beta$ and TNF-$\alpha$ in RAW264.7 cells after the addition of recombinant VSIG4, n = 9. (I) Survival of HT22 cells after co-culture with RAW264.7 cells treated with different treatments. (J–L) Western blot analyses were performed to examine Bcl-2 and Bax in HT22 cells following co-culture with RAW264.7 cells exposed to different treatments, n = 3. NRF2, nuclear factor erythroid 2 related factor 2; HO-1, heme oxygenase-1; GSH, glutathione; SOD, superoxide dismutase; MDA, malondialdehyde; IL-1$\beta$, interleukin-1$\beta$; TNF-$\alpha$, tumor necrosis factor-alpha; Bcl-2, B-cell lymphoma 2. “ns” means no significant difference.

Oxidative stress has also been shown to activate inflammatory cells and release pro-inflammatory cytokines. As expected, ELISA findings demonstrated a notable increase in TNF-$\alpha$ and IL-1$\beta$ levels in macrophages treated with LPS. In contrast, treatment with VSIG4 resulted in a notable decrease in TNF-$\alpha$ and IL-1$\beta$ levels compared to the IgG group (p 0.01) (Fig. 2G,H). This suggests that VSIG4 plays a key role in inhibiting excessive inflammatory responses during inflammatory induction. Collectively, these findings indicate that VSIG4 activates the NRF2/HO-1 pathway, thereby inhibiting oxidative stress and inflammatory responses in macrophages.

Subsequently, we conducted a co-culture experiment with HT22 and RAW264.7 cells treated with various treatments to investigate the potential protective effect of VSIG4-induced macrophages in vitro. According to the CCK-8 assay results, no significant difference was observed in cell viability between the two groups without LPS exposure. However, under LPS stimulation, VSIG4-treated RAW264.7 cells displayed improved viability compared to the IgG group (p 0.05) (Fig. 2I). Bcl-2 and Bax are apoptosis-related proteins [26]. Western blot analysis showed that VSIG4-treated RAW264.7 cells partially reversed the changes in Bcl-2 and Bax expression levels under LPS stimulation (p 0.05) (Fig. 2J–L). These findings revealed that VSIG4 helps preserve the equilibrium of anti-apoptotic signals, thereby protecting cells from LPS-induced damage and apoptosis.

Next, we evaluated the potential protective effects of VSIG4 in ICH mice. To this end, VSIG4 or IgG was injected into the mouse brain ventricle after ICH induction, and neurological function scoring was performed on day 3 after ICH. Parameters including cerebral edema severity, antioxidant/apoptotic protein expression, and inflammatory marker dynamics were further analyzed. Neurological function scores in the VSIG4 group showed significant improvement relative to the IgG controls (p 0.01) (Fig. 3A). Further examination of brain water content revealed that VSIG4 markedly reduced brain tissue edema following ICH compared to the IgG group, indicating that it exerts a beneficial effect on the reduction of brain tissue damage and swelling (p 0.01) (Fig. 3B). Western blot analysis demonstrated that VSIG4 treatment markedly elevated NRF2 and HO-1 protein levels in brain tissue (p 0.01). Furthermore, VSIG4 therapy significantly upregulated Bcl-2 expression and reduced Bax expression (Bcl-2, p 0.01; Bax, p 0.05) (Fig. 3C–G).

Fig. 3.

Effect of VSIG4 on ICH mouse outcomes. (A) Neurological scores, n = 5. (B) Brain water content, n = 5. (C–G) Western blot assays were conducted in ICH mice to examine NRF2, HO-1, Bcl-2, and Bax, n = 3. (H–L) Levels of GSH, SOD, MDA, and the pro-inflammatory factors IL-1$\beta$ and TNF-$\alpha$ measured in ICH mice, n = 5. “ns” means no significant difference.

Correspondingly, analysis of antioxidant indicators showed that VSIG4 treatment significantly increased GSH and SOD levels in brain tissue (GSH, p 0.05; SOD, p 0.01) (Fig. 3H,I), while MDA levels significantly decreased (p 0.01) (Fig. 3J). Moreover, VSIG4 significantly reduced the expression of the pro-inflammatory cytokines IL-1$\beta$ and TNF-$\alpha$in brain tissue (p 0.01) (Fig. 3K,L).

Additionally, we observed that VSIG4 therapy reduced Iba-1⁺ and GFAP⁺ cell counts while increasing the NeuN⁺ cell population in mouse brain tissue compared to the IgG group (p 0.01) (Fig. 4A–F). These results confirm that VSIG4 also has an anti-oxidative stress effect and inhibits inflammatory responses in vivo, thereby providing favorable conditions for improving neurological function and promoting tissue repair.

Fig. 4.

Effect of VSIG4 on IBA1-positive cells and GFAP-positive cells in ICH mice. (A,B) Representative immunofluorescence images and cell numbers of IBA1-positive cells in the tissue surrounding the hematoma after ICH, n = 5, scale 50 µm. (C,D) Illustrative immunofluorescence images and corresponding cell counts of GFAP-positive cells in the tissue surrounding the hematoma after ICH, n = 5, scale 50 µm. (E,F) Illustrative immunofluorescence images and corresponding cell counts of NeuN-positive cells in the tissue surrounding the hematoma after ICH, n = 5, scale 50 µm. “ns” means no significant difference.

To verify whether VSIG4 alleviates oxidative stress and inflammatory responses in macrophages in vitro through the NRF2/HO-1 pathway, we treated RAW264.7 cells with the NRF2 inhibitor brusatol [27]. Western blot results showed that brusatol reduced NRF2 and HO-1 expression and inhibited VSIG4-induced upregulation of NRF2 and HO-1 expression (p 0.05) (Fig. 5A–C). Compared to VSIG4 treatment alone, VSIG4 and brusatol co-treatment resulted in lower GSH and SOD levels and higher MDA levels in RAW264.7 cells (p 0.05) (Fig. 5D–F). ELISA results also suggested that brusatol increased IL-1$\beta$ and TNF-$\alpha$ production (p 0.01) (Fig. 5G,H). Our data indicated that brusatol treatment abrogated the inhibitory effects of VSIG4 on oxidative stress and inflammatory responses following LPS stimulation.

Fig. 5.

The NRF2 inhibitor Brusatol inhibits the protective effect of VSIG4 in vitro. (A–C) Western blotting analysis of NRF2 and HO-1 in RAW264.7 cells exposed to Brusatol, n = 3. (D–F) Levels of GSH, SOD and MDA levels in Brusatol-exposed RAW264.7 cells, n = 3. (G,H) Concentrations of IL-1$\beta$ and TNF-$\alpha$ in RAW264.7 cells treated with Brusatol, n = 9. (I) Survival of HT22 cells after co-culture with RAW264.7 cells treated with different treatments. (J–L) Western blot analysis was performed to detect Bcl-2 and Bax protein expression in HT22 cells following co-culture with RAW264.7 cells subjected to various treatments, n = 3. “ns” means no significant difference.

Subsequently, we co-cultured brusatol-treated RAW264.7 cells with HT22 cells to determine whether VSIG4 confers a protective effect on neurons through the NRF2/HO-1 signaling pathway. As shown in Fig. 5I, brusatol treatment reduced the survival rate of LPS-treated HT22 cells (p 0.01). However, the combination of brusatol and VSIG4 downregulated Bcl-2 levels but had no significant effect on Bax levels (p 0.05) (Fig. 5J–L). These results indicate that brusatol application impaired the neuroprotective ability of VSIG4-treated macrophages under LPS stimulation.

To further elucidate the protective effect of VSIG4 in the ICH model and its relationship with the NRF2/HO-1 pathway, we conducted a comparative analysis of neurological function, histopathological changes, and molecular-level changes between the VSIG4-only treatment group and the brusatol intervention group following ICH.

The results demonstrated that brusatol administration, which inhibits NRF2 signaling, worsened the neurological deficits in ICH mice, increased brain tissue water content (p 0.05), and significantly weakened the beneficial effects of VSIG4 on neurological function recovery and cerebral edema reduction (p 0.01) (Fig. 6A,B). These results indicate that NRF2 plays a pivotal role in regulating the protective effects of VSIG4.

Fig. 6.

The NRF2 inhibitor Brusatol inhibits the protective effect of VSIG4 in vivo. (A) Neurological scores, n = 5. (B) Brain water content, n = 5. (C–G) Western blot analysis was conducted to measure the protein levels of NRF2, HO-1, Bcl-2, and Bax in brain tissues from ICH mice treated with Brusatol, n = 3. (H–J) Levels of GSH, SOD and MDA levels in ICH mice exposed to Brusatol, n = 5. (K–M) Representative immunofluorescence images and quantification of cells positive for IBA1, GFAP, and NeuN in the perihematomal region following Brusatol treatment, n = 5, scale 50 µm. “ns” means no significant difference.

Consistent with the in vitro findings, brusatol administration resulted in a significant reduction in the increased expression of NRF2 (p 0.05) and HO-1 (p 0.05) in brain tissue after VSIG4 treatment (Fig. 6C–E). Detection of apoptosis-related proteins revealed that adding brusatol alone significantly reduced Bcl-2 expression levels (p 0.05) and significantly increased Bax expression levels (p 0.05). The VSIG4 group demonstrated the capacity to upregulate Bcl-2 and downregulate Bax, thereby reducing apoptosis. However, the addition of brusatol significantly weakened this protective effect (p 0.05) (Fig. 6F,G). Among the antioxidant markers, VSIG4 treatment resulted in a significant increase in GSH and SOD levels and a reduction in MDA content. However, the improvement in these antioxidant markers was impaired after inhibition of the NRF2 pathway (p 0.01) (Fig. 6H–J).

Tissue immunofluorescence staining and cell counting demonstrated that VSIG4 markedly diminished the aberrant proliferation of IBA1⁺ and GFAP⁺ cells, thereby attenuating the pro-inflammatory response and increasing NeuN⁺ cells survival. Conversely, brusatol increased the number of inflammatory cells (p 0.05) and reduced NeuN⁺ cells survival (p 0.01) (Fig. 6K–M, Supplementary Fig. 1A–C). The results of the inflammatory factor detection were also consistent with this conclusion. VSIG4 inhibited the overexpression of IL-1$\beta$ and TNF-$\alpha$ following ICH. However, this inhibitory effect was significantly weakened following brusatol treatment (p 0.01) (Supplementary Fig. 1D,E).

In conclusion, VSIG4 exerts notable neuroprotective effects in vivo by upregulating the NRF2/HO-1 pathway. These effects include improving neurological function, reducing cerebral edema, inhibiting apoptosis and oxidative stress, and suppressing excessive inflammatory responses. However, adding the NRF2 inhibitor brusatol significantly diminished the protective effects of VSIG4.