Microglial Annexin A3 Downregulation Alleviates Ischemic Injury by Inhibiting NF-κB/NLRP3-mediated Inflammation

Abstract

Microglial inflammation is a hallmark of ischemic stroke. Annexin A3 (ANXA3) is expressed in microglia and plays a detrimental role in stroke. However, the role of ANXA3 in microglial inflammation after ischemic stroke is unclear. In this study, an ischemic stroke model was established in mice via middle cerebral artery occlusion (MCAO). The adeno-associated virus shANXA3 (AAV-shANXA3) was injected into ipsilateral cortex ischemic lesion, and the infarction volume, neurological score, and neuronal injury were examined. Moreover, primary microglia were transfected with a lentivirus (LV-shANXA3) and subjected to oxygen–glucose deprivation (OGD). Neuron viability and lactose dehydrogenase (LDH) levels of neurons cocultured with microglia were analyzed. Additionally, microglial activation and ANXA3, p-NF-κB, NLRP3 and downstream proteins of NLRP3 inflammasome (cleaved caspase-1, N-GSDMD and IL-1β) expression levels were measured. We found that microglial ANXA3 expression was increased after ischemic injury and that ANXA3 knockdown reduced the infarction volume, mitigated neurological deficits, and alleviated neuronal injuries. Additionally, ANXA3 knockdown ameliorated microglial activation and reduced the levels of p-NF-κB and inhibited NLRP3 inflammasome signaling. Furthermore, ANXA3 upregulation resulted in decreased IκBα levels, whereas ANXA3 downregulation resulted in increased IκBα levels. Notably, IκBα knockdown blocked the neuroprotective effects of AAV-shANXA3 against ischemic injury. In conclusion, microglial ANXA3 downregulation alleviates ischemic stroke by inhibiting NF-κB/NLRP3-mediated microglial inflammation, which indicates that ANXA3 may be a potential therapeutic target for ischemic stroke.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02287-4.

Introduction

Stroke ranks among the top three prevalent medical conditions worldwide, with ischemic stroke constituting approximately 85% of stroke cases [1, 2]. Ischemic stroke significantly contributes to the disease burden, resulting in high rates of illness and premature death [3]. While there have been improvements in the management of ischemic stroke, conventional methods such as antithrombotic treatment, neuroprotective agents, and surgical options frequently fall short in their effectiveness [4]. As a result, the identification of novel treatment strategies and targets for ischemic stroke remains a primary research focus.

An increasing number of studies have indicated that the inflammatory response linked to microglial activation plays significant roles in neurological impairments following ischemic stroke [5, 6]. Research has shown that microglial activation aggravates ischemic injury, whereas inhibiting microglia-mediated neuroinflammation has neuroprotective effects [7, 8]. NF-κB, a crucial factor in neuroinflammation, promotes the transcription of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) after its activation [9]. Furthermore, NF-κB regulates the secretion of proinflammatory cytokines, including TNF-α and IL-1β, which ultimately mediate the activation of microglia [10, 11]. Recent studies have shown that NLRP3 is highly upregulated in the ischemic penumbra and that an NLRP3 inhibitor exerts neuroprotective effects after stroke [12, 13]. Moreover, suppressing the NF-κB/NLRP3 signaling cascade in microglia enhances recovery outcomes in a middle cerebral artery occlusion (MCAO) mouse model [14, 15]. Thus, the development of effective approaches that target NF-κB/NLRP3-mediated microglial activation may offer promising directions for ischemic stroke therapy.

Annexin A3 (ANXA3) has been identified as an upregulated protein in activated microglia [16, 17], and ANXA3 levels have been reported to be increased in the serum of patients who have experienced ischemic stroke and in septic mice [18–20]. The absence of ANXA3 leads to a reduction in inflammation and cell death associated with sepsis-induced acute lung injury (ALI) [21], suggesting that ANXA3 is associated with inflammatory responses in the central and peripheral immune systems. Recent studies have shown that ANXA3 is strongly upregulated and that ANXA3 inhibition effectively suppresses pyroptosis after ischemic stroke [16, 17]. Moreover, miR-18b provides a protective effect against ischemic damage through the downregulation of ANXA3 expression [22], further supporting the potential role of ANXA3 in exacerbating neuronal damage following stroke. Nevertheless, the precise molecular mechanisms remain ambiguous. Notably, previous research has indicated that ANXA3 knockdown inhibits NF-κB signaling in breast and esophageal cancer cell lines [23, 24], suggesting that ANXA3 may regulate inflammatory pathways. Therefore, we explored the involvement of microglial ANXA3 in the context of ischemic stroke and investigated the potential participation of the NF-κB/NLRP3 signaling pathways.

Materials and Methods

Animals

C57BL/6 J male mice, aged 8 to −10 weeks, were obtained from Beijing Vital River Laboratory Animal Technology Co. [Institutional License: SCXK (Jing) −2019–0009]. The experimental protocols involving animals were reviewed and sanctioned by the Animal Ethics Committee at Tianjin Medical University Cancer Institute and Hospital and followed the guidelines set forth by the National Institutes of Health of China Guide for the Care and Use of Laboratory Animals. All the mice were housed in cages equipped with ventilation and maintained at 22 °C ± 1 °C under a 12-h light/dark cycle and had unrestricted access to food and water.

MCAO

Left middle cerebral artery (MCA) ischemia was induced using the MCAO model as detailed in earlier studies [25]. The mice were sedated with a solution of 1% pentobarbital sodium at a dose of 100 mg/kg via intraperitoneal injection, and the body temperature was maintained at a stable level of 37.0 °C. A 6.0-mm nylon monofilament (Doccol Corp., Redlands, CA, USA) was inserted via the left common carotid artery (CCA) to occlude blood flow to the MCA and was removed after 1 h. The animals that underwent a sham operation experienced the same surgical technique but did not have the artery occluded. The assessment of regional cerebral blood flow was conducted via laser Doppler flowmetry (Periflux system 5000), and a decrease in rCBF ≥ 70% was considered successful occlusion. The physiological parameters are provided in Additional file 1.

Neurobehavioral Evaluation and Infarct Volume Measurement

Three days following reperfusion, neurological assessments were performed via a double-blinded method using the following grading scale: 0, no neurological deficit; 1, failure to extend the left forepaw fully; 2, circling to the left; 3, inability to bear weight on the left; and 4, no spontaneous walking with a depressed level of consciousness. Following neurological assessment, the brains of the mice were subjected to 2,3,5-triphenyltetrazolium chloride (TTC) staining. In brief, the brain samples were sectioned into 1-mm-thick coronal slices and placed in a 2% solution of TTC, followed by immersion overnight in 4% paraformaldehyde. The brain slices were then photographed and analyzed via ImageJ software by a blinded observer. The percentage (%) infarct volume = [(VC-VL)/VC] × 100 (VC: the volume of the control hemisphere; VL: the volume of the noninfarcted tissue in the lesioned hemisphere).

Stereotaxic Microinjection

To reduce the expression of the ANXA3 protein within ipsilateral cortex ischemic lesions in mice, an adeno-associated viral vector targeting ANXA3 (AAV-shANXA3, serotype 9, 5’-CGGCCATCCAATCAGATACTT-3’) and a negative sequence (AAV-NC, 5’-CGCTGAGTACTTCGAAATGTC-3’) manufactured by Shanghai Genechem (China) were administered 14 days prior to the induction of MCAO. In brief, the mice were placed under anesthesia in a stereotactic apparatus (RWD Life Science). A volume of 500 nl of either AAV-shANXA3 or AAV-NC was then administered to the ipsilateral cortex ischemic lesions at a rate of 50 nl/min via a microsyringe (Gaoge, Shanghai, China) with a microelectrode via a microsyringe pump (Kd Scientific, Holliston, MA, USA). Ischemic lesions in the left cortical region were targeted 0.3 mm anterior to the bregma, −3 mm lateral, 2 mm deep, and 1.9 mm posterior to the bregma, and −3 mm lateral, 2 mm deep, as previously reported [26]. For IκBα and ANXA3 knockdown experiments, a mixture of 500 nl AAV-NC/AAV-shIκBα and 500 nl AAV-shANXA3 was injected into the above regions 14 days before MCAO. The target sequence of the adeno-associated virus for IκBα shRNA (AAV-shIκBα) was 5’-CTCCACTTCTCGAGAAGTGGA-3’. The effectiveness of AAV-shANXA3 and AAV-shIκBα was evaluated via Western blot analysis and Immunofluorescence staining assay (refer to Fig. S2 in the Additional file 1).

Primary Microglia Culture and Neuron‒Microglia Cocultures

Primary microglia from neonatal C57BL/6 mice aged 1–2 days were isolated following established protocols [25]. Briefly, the meninges and blood vessels were carefully excised. Then, enzymatic digestion and mechanical separation of the cortical hemispheres were performed. The cells were then harvested by filtering. Following centrifugation, the cells were then suspended in Dulbecco’s modified Eagle’s medium (DMEM, HyClone) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin. Two weeks later, after a period of 1 h on an orbital shaker at 220 rpm, the separated microglia were collected and transferred back into the cell cultivation containers. The presence of microglia was validated through staining with the Iba1 microglial marker (refer to Fig. S1 in the Additional file 1).

Primary neuronal cells were extracted from the cerebral cortices of mouse embryos at embryonic days 16 to 17. The harvested dissociated cells were subsequently placed into culture vessels that had been treated with poly-D-lysine. The neurobasal medium consisted of 2% B27 supplement, along with 1% glutamine and a combination of penicillin and streptomycin.

For indirect neuron‒microglia cocultures, neurons were seeded in 24-well plates and incubated for two days. Then, primary microglia (microglia:neurons = 1:2) were added to 0.4-μm pore-sized Transwell inserts. The cells were cocultured for two days before they were subjected to oxygen‒glucose deprivation/reperfusion (OGD/R) treatment.

OGD/R

The cells were subjected to two wash cycles with glucose-free DMEM before being maintained in glucose-free DMEM. The samples were subsequently placed in a modular chamber for incubation, where a gas mixture consisting of 95% N₂ and 5% CO₂ was circulated at a rate of 3 L/min for 15 min, after which they were placed in a 37 °C incubator for 4 h (microglia) or 1 h (neurons) to induce OGD injury. After OGD exposure, the cells were allowed to recover in regular media under standard conditions for 12 h to mimic reperfusion injury.

Lentiviral Transfection

To reduce the expression of the ANXA3 protein in microglia, an shRNA lentivirus targeting the mouse ANXA3 sequence (5’-CGGCCATCCAATCAGATACTT-3’) and a negative control lentivirus (LV-NC; 5’-TTCTCCGAACGTGTCACGT-3’) produced by Suzhou Genepharma (China) were used [27, 28]. In brief, microglia were cultured in Transwell inserts with 0.4-μm pores for coculture experiments or in 6-well plates for subsequent Western blot analysis. The lentivirus was diluted in DMEM and used to infect microglia for 24 h. Afterward, the microglia were cocultured with neurons and exposed to OGD/R. Western blot analysis was conducted to evaluate the efficiency of LV-shANXA3.

CCK-8 Cell Viability Assay

To evaluate the viability of the neuronal cells, a cytotoxicity assay utilizing a Cell Counting Kit (CCK)−8 (Seven Sea Biotech, Shanghai, China) was performed. In brief, following the removal of the cell culture medium from neuronal cells in a 24-well plate, 50 µl of CCK-8 reagent was mixed with 500 µl of new medium in each well and incubated at 37 °C for 4 h. The absorbance was subsequently measured at 450 nm via a microplate reader (Infinite M200, TECAN).

LDH Release Assay

To evaluate the degree of injury to neuronal cells, an LDH-Cytotoxicity Colorimetric Assay Kit II (#K313-500, BioVision, USA) was utilized. In this procedure, a 10 µl aliquot from the culture medium of neuronal cells grown in a 24-well plate was transferred to a transparent 96-well plate. Next, 100 µl of LDH reaction mixture was added to each well and allowed to incubate for 30 min at 37 °C. The absorbance at 450 nm was subsequently measured via a microplate reader (Infinite M200).

Enzyme-Linked Immunosorbent Assay (ELISA)

The protein expression levels of TNF-α and IL-1β in the microglial supernatant were assessed via ELISA (R&D Systems, Minneapolis, MN, USA) following the instructions provided by the manufacturer.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Staining

An In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany) was utilized for TUNEL staining according to the manufacturer.

Immunofluorescence Staining Assay

The immunofluorescence assay was conducted as previously described [29, 30]. The brain sections were incubated overnight with several primary antibodies, including a rabbit anti-ANXA3 antibody (1:200, Sigma, USA), a goat anti-Iba1 antibody (1:200, Abcam, England), and a goat anti-NeuN antibody (1:200, Invitrogen, USA). The samples were subsequently treated with either an Alexa 594-conjugated antibody (1:200, Bioss, Germany) or an Alexa 488-conjugated antibody (1:200, Beyotime, China). Photographs of the sections were captured with an Olympus BX51.

Western Blot Analysis

ANXA3, p-NF-κB, NLRP3, cleaved caspase-1, N-GSDMD p31 and IL-1β p17, IκBα and Iba1 protein expression was assessed in both penumbra tissues and primary microglia [31]. Following extraction, the proteins were subjected to electrophoresis and subsequently transferred to PVDF membranes. These membranes were then incubated with specific primary antibodies, including a rabbit anti-ANXA3 antibody (1:200, Sigma, USA), a mouse anti-NLPR3 antibody (1:1,000, Cell Signaling Technology, USA), a rabbit anti-cleaved caspase-1 antibody (1:1,000, Cell Signaling Technology, USA), anti-cleaved N-terminal GSDMD antibody (1:1,000, Abcam, UK), a goat anti-IL-1β p17 antibody (1:1,000, R&D Systems, USA), a rabbit anti-Iba1 antibody (1:500, Wako, Japan), a rabbit anti-IκBα antibody (1:1,000, Beyotime, China), a mouse anti-p-NF-κB antibody (1:1,000, Cell Signaling Technology, USA), and a rabbit anti-β-tubulin antibody (1:1,000, Sangon Biotech, China). The membranes were subsequently incubated with secondary anti-rabbit or anti-mouse antibodies (1:5,000, Absin, China). The density values of each band were subsequently analyzed and adjusted relative to the optical density of β-tubulin.

Statistical Analysis

The statistical analyses were conducted via GraphPad Prism version 9.0 software. The experiments conformed to the principles of blinding. Neurological deficit scores are reported as medians and were evaluated via two-tailed Mann‒Whitney U tests. For additional data, the data are reported as the means ± SDs. Two groups were compared via unpaired Student’s t tests with two tails, and comparisons among multiple groups were carried out via one-way ANOVA with Tukey’s post hoc test. A P value of less than 0.05 was considered statistically significant.

Results

Microglial ANXA3 Expression is Increased after Ischemic Injury

Our previous study indicated that ANXA3 is specifically expressed in microglia in the central nervous system (CNS) [32]. As shown in Fig. 1a-b, in the penumbra, the immunofluorescence intensity of ANXA3 and Iba1 markedly increased 3 days following ischemia–reperfusion compared with that in the sham group. Analyses via Western blotting further revealed that ANXA3 protein levels in the penumbra gradually increased, peaking at 24 h and remaining elevated compared with those in the sham group three days after ischemia–reperfusion (Fig. 1c, d). These findings indicate that microglial activation is triggered by MCAO/R injury, leading to increased microglial ANXA3 expression in the penumbra.

Fig. 1.

Microglial ANXA3 protein expression increases after ischemic injury. (a-b) Representative immunofluorescence images showing ANXA3 expression in Iba1-labeled microglia in the penumbras of sham group mice and MCAO group mice 3 days after reperfusion. Scale bar = 50 μm. The histogram shows the immunofluorescence intensity analysis results. The data are expressed as the means ± standard deviations (SDs) and were analyzed via two-tailed unpaired Student’s t tests. **p < 0.01 compared with the Sham group. n = 6 per group. c, d Western blot analysis of ANXA3 protein expression levels in the ischemic penumbras of MCAO model mice at 6 h, 12 h, 1 day, 2 days, and 3 days after reperfusion. The panel shows ANXA3 and the corresponding tubulin bands. The histogram shows the results of the densitometric analysis. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Dunnett’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the Sham group. n = 4 per group

ANXA3 Knockdown Exerts Neuroprotective Effects against Ischemic Stroke

To assess the involvement of ANXA3 in the context of ischemic stroke, ipsilateral cortical ischemic lesions were subjected to injections of either adeno-associated virus shANXA3 (AAV-shANXA3) or a negative control vector (AAV-NC) 14 days before MCAO/R injury. As shown in Fig. 2a, b, following reperfusion, a substantial infarct volume was observed in the MCAO group of mice 3 days after reperfusion. Compared with the infarct volume observed in the mice treated with AAV-NC (MCAO + AAV-NC group), those receiving AAV-shANXA3 (MCAO + AAV-shANXA3 group) exhibited a significant reduction in infarct size, along with noticeable improvements in neurological function (Fig. 2c).

Fig. 2.

ANXA3 knockdown exerts neuroprotective effects against ischemic stroke. (a) Representative photographs of brain slices showing the infarct volume 3 days after reperfusion in mice subjected to MCAO/R injury. b Infarction volume as a percentage of the sham group volume. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 compared with the Sham group. ^##p < 0.01 compared with the MCAO + AAV-NC group. n = 12 per group. (c) Neurological deficit scores were evaluated 3 days after reperfusion. The data are expressed as medians and were analyzed via a two-tailed Mann‒Whitney U test. ***p < 0.001 compared with the Sham group. ^##p < 0.01 compared with the MCAO + AAV-NC group. n = 12 per group. AAV: adeno-associated virus; MCAO/R: middle cerebral artery occlusion/reperfusion

Following MCAO/R injury, the ischemic penumbra presented an increase in the number of TUNEL-positive neurons (apoptotic neurons) and a decrease in the number of NeuN-positive neurons (surviving neurons) 3 days after reperfusion (Fig. 3). Compared with the MCAO + AAV-NC group, the MCAO + AAV-shANXA3 group presented a marked reduction in the population of TUNEL-positive apoptotic neurons (Fig. 3a, b), and a notable increase in the number of surviving NeuN-positive neurons was observed in the MCAO + AAV-shANXA3 group (Fig. 3c, d). Together, these findings suggest that ANXA3 knockdown induced neuroprotection against ischemic stroke.

Fig. 3.

ANXA3 knockdown alleviates neuronal injury in the ischemic penumbra, (a) Representative photomicrographs showing TUNEL staining of the ischemic penumbra 3 days after reperfusion in mice subjected to MCAO/R injury. Scale bars = 20 μm. b The percentage of TUNEL-positive cells (apoptotic neurons) in the ischemic penumbra. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 compared with the Sham group. ^###p < 0.001 compared with the MCAO + AAV-NC group. n = 12 per group. c Representative photomicrographs showing NeuN staining (surviving neurons) in the ischemic penumbra 3 days after reperfusion in mice subjected to MCAO/R injury. Scale bars = 20 μm. d The percentage of surviving neurons in the ischemic penumbra. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 compared with the Sham group. ^###p < 0.001 compared with the MCAO + AAV-NC group. n = 12 per group. TUNEL: terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nick-end labeling

ANXA3 Knockdown Ameliorates Microglial Activation and Inhibits the NF-κB/NLRP3 Pathway after MCAO/R Injury

Compared with those in the MCAO + AAV-NC group, ANXA3 protein levels in the penumbra were notably lower in the MCAO + AAV-shANXA3 group 3 days after ischemia–reperfusion (Fig. 4a, b). In addition, compared with those in the sham group, the protein expression levels of Iba1 and the proinflammatory cytokines TNF-α and IL-1β in the penumbra of the MCAO group were significantly increased 3 days after ischemia–reperfusion (Fig. 4). Compared with those in the MCAO + AAV-NC group, the protein levels of Iba1, TNF-α and IL-1β in the MCAO + AAV-shANXA3 group were significantly lower in the penumbra (Fig. 4), suggesting that ANXA3 knockdown ameliorated microglial activation. Furthermore, MCAO/R injury increased the protein levels of ANXA3, p-NF-κB and NLRP3 in the penumbra 3 days after ischemia–reperfusion (Fig. 4a, c). In addition to a reduction in ANXA3 protein levels in the penumbra, the protein levels of p-NF-κB and NLRP3 in the penumbra were notably lower in the MCAO + AAV-shANXA3 group than in the MCAO + AAV-NC group 3 days after ischemia–reperfusion (Fig. 4a, c), indicating that ANXA3 knockdown resulted in suppression of the NF-κB/NLRP3 signaling pathway.

Fig. 4.

ANXA3 knockdown ameliorates microglial activation and inhibits the NF-κB/NLRP3 pathway. a-c Western blot analysis of ANXA3, Iba1, p-NF-κB and NLRP3 protein expression in the ischemic penumbra 3 days after reperfusion. The left panel shows ANXA3, Iba1, p-NF-κB, NLRP3 and the corresponding tubulin bands. The middle and right panels show the results of the densitometric analysis. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 compared with the Sham group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the MCAO + AAV-NC group. n = 5 per group. (d-e) The levels of the proinflammatory cytokines TNF-α and IL-1β in the ischemic penumbra 3 days after reperfusion in MCAO/R injury model mice were detected via ELISA. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 compared with the Sham group. ^#p < 0.05 compared with the MCAO + AAV-NC group. n = 5 per group. f-i Western blot analysis of GSDMD-N, cleaved caspase-1 and IL-1β p17 protein expression in the ischemic penumbra 3 days after reperfusion. The panel shows GSDMD-N, cleaved caspase-1, IL-1β p17 and the corresponding tubulin bands. The histogram shows the results of the densitometric analysis. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. *p < 0.05, ***p < 0.001 compared with the Sham group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the MCAO + AAV-NC group. n = 5 per group

Considering that canonical NLRP3 inflammasome activation results in cleaved caspase-1 mediated release of N-GSDMD p31 and IL-1β p17, the protein expression levels of cleaved caspase-1, N-GSDMD p31 and IL-1β p17 were assessed. As shown, compared with those in the sham group, the protein expression levels of cleaved caspase-1, N-GSDMD p31 and IL-1β p17 in the penumbra of the MCAO group were significantly increased 3 days after ischemia–reperfusion (Fig. 4f-i). Compared with those in the MCAO + AAV-NC group, the protein levels of cleaved caspase-1, N-GSDMD p31 and IL-1β p17 in the MCAO + AAV-shANXA3 group were significantly lower in the penumbra (Fig. 4f-i), which further confirmed that ANXA3 knockdown inhibited NLRP3 inflammasome signaling.

ANXA3 Downregulation Ameliorates Microglial Activation and Inhibits the NF-κB/NLRP3 Pathway after OGD/R Injury

Western blot analysis validated the effectiveness of LV-shANXA3 (Fig. 5). Compared with those in the Con group, the levels of the Iba1 protein in microglia, as well as the levels of the proinflammatory cytokines TNF-α and IL-1β in the microglial supernatant, were notably increased 12 h after OGD/R injury (Fig. 5a, b, d, e). Additionally, in contrast with those in the OGD + LV-NC group, the protein levels of Iba1, TNF-α and IL-1β in the OGD + LV-shANXA3 group were notably lower 12 h after OGD/R injury (Fig. 5a, b, d, e), indicating that ANXA3 downregulation ameliorated microglial activation after OGD/R injury. Furthermore, OGD/R injury resulted in increased protein expression levels of ANXA3, p-NF-κB and NLRP3 in microglia (Fig. 5a, c). In addition to the decreased protein expression of ANXA3, the protein expression levels of both p-NF-κB and NLRP3 in microglia were significantly lower in the OGD + LV-shANXA3 group than in the OGD + LV-NC group 12 h after OGD/R injury (Fig. 5a, c), indicating that ANXA3 downregulation in microglia inhibited the NF-κB/NLRP3 pathway.

Fig. 5.

ANXA3 downregulation inhibits microglial activation and the NF-κB/NLRP3 pathway and alleviates neuronal injury. a-c Western blot analysis of ANXA3, Iba1, p-NF-κB and NLRP3 protein expression in primary microglia 12 h after the reintroduction of oxygen and glucose. The left panel shows ANXA3, Iba1, p-NF-κB, NLRP3 and the corresponding tubulin bands. The middle and right panels show the results of the densitometric analysis. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. **p < 0.01, ***p < 0.001 compared with the Con group. ^#p < 0.05, ^###p < 0.001 compared with the OGD + LV-NC group. The data were pooled from five independent experiments. d, e The levels of the proinflammatory cytokines TNF-α and IL-1β in the supernatants of primary microglia 12 h after OGD/R injury were measured via ELISA. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 compared with the Con group. ^#p < 0.05, ^##p < 0.01 compared with the OGD + LV-NC group. The data were pooled from five independent experiments. (f-g) CCK-8 cell viability and LDH release assays were performed to assess neuronal injury 12 h after the reintroduction of oxygen and glucose. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 compared with the Con group. ^#p < 0.05, ^##p < 0.01 compared with the OGD + LV-NC group. The data were pooled from five independent experiments. OGD/R: oxygen‒glucose deprivation/reintroduction; LDH: lactate dehydrogenase

ANXA3 Downregulation in Microglia Exerts Neuroprotective Effects against OGD/R Injury

A neuron-microglia coculture system was prepared to explore the neuroprotective effects of ANXA3 downregulation in microglia. Briefly, microglia and neurons were cocultured for 2 days before the microglia were subjected to OGD for 4 h, whereas the neurons were subjected to OGD for 1 h in designated separate chambers. As shown, ANXA3 downregulation in microglia substantially increased the viability of neurons (Fig. 5f). Moreover, the release of LDH from neurons cocultured with microglia from the OGD + LV-shANXA3 group was considerably lower than that from neurons cocultured with microglia from the OGD + LV-NC group (Fig. 5g). Collectively, these findings indicate that ANXA3 downregulation in microglia has neuroprotective effects on cocultured neurons.

ANXA3 Knockdown Inhibits the NF-κB Signaling Pathway by Increasing IκBα Levels

Considering that ANXA3 is a cytoplasmic protein while NF-κB migrates into the nucleus after phosphorylation, the possibility of a direct interaction between ANXA3 and p-NF-κB may be unlikely. Therefore, to explore the molecular mechanisms underlying the suppression of the NF-κB signaling pathway caused by ANXA3 knockdown, the protein expression levels of IκBα were assessed, as IκBα is a cellular protein that serves to suppress the transcriptional activity of NF-κB. The downregulation and overexpression of microglial ANXA3 were achieved through the application of lentiviral vectors containing ANXA3-specific shRNA (LV-shANXA3) and LV-ANXA3, respectively. Compared with those in the LV-NC group, the protein levels of ANXA3 were notably lower in the LV-shANXA3 group and significantly greater in the LV-ANXA3 group (Fig. 6a, b). Furthermore, the reduced expression of ANXA3 in microglia treated with LV-shANXA3 led to a marked increase in IκBα protein levels; in contrast, the increased expression of ANXA3 in microglia treated with LV-ANXA3 led to a notable reduction in IκBα protein expression compared with that in microglia treated with LV-NC (Fig. 6a, b).

Fig. 6.

ANXA3 knockdown inhibits the NF-κB signaling pathway by upregulating IκBα. a, b Western blot analysis of ANXA3 and IκBα protein expression in primary microglia. The left panel shows ANXA3, IκBα and the corresponding tubulin bands. The right panel shows the results of the densitometric analysis. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Dunnett’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the LV-NC group. The data were pooled from five independent experiments. c Representative photographs of brain slices showing the infarct volume 3 days after reperfusion in MCAO/R injury mice. d Infarction volume as a percentage of the sham value. The data are expressed as the means ± SDs and were analyzed via one-way ANOVA with Tukey’s post hoc test. **p < 0.01 compared with the AAV-shANXA3 + NC group. n = 12 per group. e Neurological deficit scores were evaluated 3 days after reperfusion in mice subjected to MCAO/R injury. The data are expressed as medians and were analyzed via the Mann‒Whitney U test. *p < 0.05 compared with the AAV-shANXA3 + NC group. n = 12 per group. AAV: adeno-associated virus; MCAO/R: middle cerebral artery occlusion/reperfusion

To explore whether ANXA3 knockdown alleviates ischemic injury via NF-κB signaling pathway regulation, a mixture of AAV-shANXA3 and AAV-shIκBα was injected 14 days before MCAO to knockdown ANXA3 and IκBα protein expression. Compared with those of the mice treated with AAV-shANXA3 + NC (AAV-shANXA3 + NC group), the infarct volumes and neurological deficit scores of the mice treated with AAV-shANXA3 + shIκBα (AAV-shANXA3 + shIκBα group) were significantly greater (Fig. 6c-e), suggesting that IκBα knockdown blocked the effect of AAV-shANXA3 on ischemic stroke. Collectively, these findings indicate that ANXA3 knockdown may alleviate ischemic injury by inhibiting the NF-κB/NLRP3 signaling pathway to some degree through the upregulation of IκBα protein expression.

Discussion

Increasing evidence suggests that the inflammatory response is essential in the pathogenesis of ischemic stroke. In this study, we initially reported that microglial ANXA3 expression was increased after ischemic injury and that ANXA3 knockdown notably alleviated neurological deficits and mitigated neuronal damage. In addition, ANXA3 knockdown ameliorated microglial activation and reduced the levels of p-NF-κB and inhibited NLRP3 inflammasome signaling. The upregulation of ANXA3 resulted in decreased IκBα levels, whereas the downregulation of ANXA3 resulted in increased IκBα levels in microglia. Additionally, IκBα knockdown blocked the neuroprotective effects of AAV-shANXA3 against ischemic injury. Therefore, ANXA3 knockdown may alleviate ischemic injury by increasing IκBα expression and suppressing the NF-κB/NLRP3 signaling cascade, indicating that targeting ANXA3 may be a promising strategy for treating ischemic stroke.

ANXA3 is an immune-related protein associated with ischemic stroke, as ANXA3 levels are elevated in the serum of patients who have experienced ischemic stroke [19, 20]. Research indicates that the downregulation of ANXA3 in myocardial cells promotes the regeneration and recovery of myocardial tissues following acute myocardial infarction (AMI) [33]. In septic mice, elevated levels of ANXA3 are observed in both lung tissue and serum, while the depletion of ANXA3 significantly alleviates inflammation and reduces apoptosis in sepsis-induced ALI model mice [18, 21]. These findings suggest that ANXA3 is pivotal in regulating the peripheral immune response. Within the CNS, ANXA3 is specifically expressed in microglia [34]. Our previous research revealed that ANXA3 participates in the proliferation and migration of microglia [32]. Furthermore, we reported that ANXA3 downregulation in the spinal cord alleviates both the pain associated with bone cancer and the pain resulting from chronic constriction injury [27, 35]. Recent investigations suggest a possible link between ANXA3 and the alterations noted in the membrane dynamics of microglial ruffles following nerve injury [36]. Additionally, ANXA3 expression is strongly upregulated in the rat brain under ischemic conditions [16], and ANXA3 inhibition effectively suppresses pyroptosis after ischemic stroke [17]. Furthermore, inhibition of ANXA3 expression by miR-18b has been shown to confer protection against damage caused by ischemia [22]. Consistent with these findings, we found that ANXA3 protein levels increased progressively and peaked at 24 h in the penumbra following reperfusion. ANXA3 knockdown led to notable improvements in neurological function and a reduction in neuronal damage, as confirmed by smaller infarct sizes, lower scores on neurological deficit assessments, and a greater number of surviving neurons within the ischemic penumbra. In vitro, ANXA3 downregulation in microglia notably increased neuronal viability and decreased LDH release in the supernatant of neurons cocultured with microglia. Together, these results indicate that ANXA3 knockdown exerts neuroprotective effects against ischemic stroke.

Microglia are important immune cells that initiate the inflammatory response [37], and microglial activation is closely associated with poor outcomes in ischemic stroke patients [38]. In support of these observations, we found that MCAO/R injury induced microglial activation, as manifested by elevated levels of Iba1 and the proinflammatory cytokines TNF-α and IL-1β in the ischemic penumbra 3 days after reperfusion. Moreover, we found that ANXA3 downregulation in microglia abrogated these changes in both MCAO model mice and control cells in the in vitro OGD/R experiments, indicating that ANXA3 downregulation decreased microglial activation. NF-κB and its downstream target, NLRP3, have been shown to mediate the activation of microglia. Recent studies have shown that approaches targeting NF-κB/NLRP3-mediated microglial activation have neuroprotective effects against stroke. For example, argon alleviates ischemic injury by suppressing microglial polarization and the NF-κB/NLRP3 signaling cascade [14]. Both curcumin and meisoindigo improve the functional outcomes of stroke model animals and attenuate microglial pyroptosis via the inhibition of the NF-κB/NLRP3 pathway [12, 13]. Moreover, as an inhibitor of NLRP3, MCC950 has been shown to exert neuroprotective effects during stroke episodes [39]. Our research revealed that following stroke there was a notable increase in the protein levels of p-NF-κB, NLRP3 and downstream proteins of NLRP3 inflammasome signaling (such as cleaved caspase-1, N-GSDMD p31 and IL-1β p17), and ANXA3 downregulation in microglia significantly reduced these protein expression levels, suggesting that ANXA3 knockdown inhibited the NF-κB/NLRP3 signaling pathway. Collectively, our findings suggest that ANXA3 downregulation enhances functional recovery and attenuates microglial activation, at least partially, by inhibiting the NF-κB/NLRP3 signaling pathway, which further indicates that ANXA3 could serve as a viable target for stroke treatment.

The roles of ANXA3 are associated with the regulation of downstream factors. A decrease in ANXA3 levels facilitates the restoration and recovery of heart tissues during AMI by stimulating the PI3K/Akt pathway [33]. ANXA3 silencing blocks ERK/ELK1 signaling in septic mice [21]. Moreover, studies have demonstrated that ANXA3 interacts with F-actin [36]. Our previous study revealed that ANXA3 effectively ameliorates bone cancer-induced pain by regulating Hif-1α transactivation and activity in the spinal cord [27]. ANXA3 knockdown can also inhibit pyroptosis through the NLRC4/AIM2 pathway [17]. To elucidate the possible molecular pathways through which ANXA3 exerts neuroprotective effects, we focused on NF-κB, which mediates the activation of microglia and can bind the ANXA1 protein, another member of the annexin family [40]. Research has demonstrated that ANXA3 knockdown inhibits NF-κB activation in breast and esophageal cancer cell lines [23, 24]. Our results revealed a positive correlation between ANXA3 expression and activation of the NF-κB/NLRP3 signaling pathway, which was demonstrated by lower levels of ANXA3 along with decreases in p-NF-κB, NLRP3, cleaved caspase-1, N-GSDMD p31 and IL-1β p17 levels. Furthermore, we found that the upregulation of ANXA3 resulted in decreased IκBα levels, whereas the downregulation of ANXA3 resulted in increased IκBα levels in microglia. Considering that IκBα inhibits NF-κB transcription activity [41], these results suggest that ANXA3 knockdown may inhibit the NF-κB/NLRP3 signaling pathway by increasing IκBα protein expression, which was further supported by the findings that IκBα knockdown blocked the neuroprotective effects of AAV-shANXA3 against ischemic injury.

However, our research has certain limitations. First, an ANXA3 knockout model in mice should be established in future studies to provide a clearer understanding of the function of microglial ANXA3 in the context of stroke. Additionally, the long-term effects of ANXA3 downregulation on ischemic stroke require more in-depth examination. Furthermore, the pathways responsible for the ANXA3-induced increase in IκBα protein levels require additional study.

In conclusion, this research highlights the pivotal function of microglial ANXA3 in ischemic stroke, and ANXA3 downregulation may alleviate ischemic stroke damage by inhibiting NF-κB/NLRP3-mediated microglial inflammation, which broadens the understanding of the role of ANXA3 within the CNS. Notably, these findings suggest that ANXA3 could serve as a promising target for ischemic stroke therapies.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Zengli Zhang and Mengxue Zhang wrote the main manuscript text. Zengli Zhang, Mengxue Zhang, Dan Li, Ruichen Shu, and Qian Pan performed the material preparation, data collection and analysis. Wangyuan Zou, Kaiyuan Wang and Yiqing Yin reviewed and edited the manuscript. All authors reviewed the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China [NO. 82101351, 82401435]; the Tianjin Municipal Science and Technology Plan Project [NO. 22JCQNJC01050]; the Science & Technology Development Fund of Tianjin Education Commission for Higher Education [NO. 2022KJ226]; and the Pioneer Incubator Fund (NO. GWRX-2023–6).

Data Availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval

All animal experiments were performed according to institutional guidelines and approved by the Animal Care and Use Committee of Tianjin Medical University Cancer Institute and Hospital (reference number: YYJC-AE-2022023).

Competing Interests

The authors declare no competing interests.