The CCL11‐CCR3 Axis Regulates the Aggravation of Neuroinflammation in Astrocytic Necroptosis After Cerebral Ischemia

ABSTRACT

In ischemic stroke (IS), the programmed death of neurons is a key factor contributing to poor prognosis and neurological dysfunction. Recently, it has been discovered that necroptosis, as a form of programmed cell death, promotes neuroinflammatory damage after IS; however, its mechanism is still unclear. Recent studies have shown that CCL11, as an eosinophil chemokine, may induce neuronal cytotoxicity by triggering the production of reactive oxygen species (ROS) in microglia. Meanwhile, CCR3, serving as the major functional membrane receptor target of CCL11, plays a crucial role in transducing CCL11 signals. This study establishes a mouse model of IS using permanent middle cerebral artery ligation (pMCAL) and utilizes methods such as Western blot (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence (IF) to detect changes in necroptosis markers and chemokines. The aim was to analyze the dynamic progression of necroptosis after stroke and its potential regulatory mechanisms. In in vivo experiments, the recovery levels of necroptosis and neuroinflammation were observed by administering a C‐C motif chemokine receptor 3 (CCR3) inhibitor via cerebral stereotaxic injection. In vitro experiments utilized an oxygen–glucose deprivation (OGD) model to simulate the in vivo ischemic state of astrocytes. Exogenous CCL11 was administered, and the expression of CCR3 on astrocytes was inhibited to observe changes in necroptosis and apoptosis markers, including receptor‐interacting protein kinase 1 (RIPK1), receptor‐interacting protein kinase 1 (RIPK3), and mixed lineage kinase domain‐like protein (MLKL), as well as inflammatory factors interleukin‐1 beta (IL‐1β), NOD‐like receptor thermal protein domain‐associated protein 3 (NLRP3), and apoptosis‐associated speck‐like protein containing a CARD(ASC). The results indicated that CCL11 stimulation significantly activated necroptosis and apoptosis in astrocytes, while blocking the CCR3 receptor on astrocytes resulted in a significant inhibition of this process. By collecting supernatants from various groups of astrocytes and stimulating neurons, it was further observed that necroptosis, induced by CCL11‐CCR3 stimulation, significantly disrupts synaptic connections in neurons. We conclude that the CCL11‐CCR3 axis plays a key role in the necroptosis of astrocytes after IS.

Schematic diagram of the triggering mechanism of necroptosis after cerebral ischemia. First, CCL11 targetedly binds to CCR3 on the surface of astrocytes and upregulates the expression of CCR3. After CCR3 activation, it triggers the transcriptional activation of necroptosis. Phosphorylated RIPK1 then induces the phosphorylation of RIPK3, which in turn promotes the formation of phosphorylated MLKL oligomers. This process subsequently causes cell membrane disruption and the release of inflammatory factors (IL‐1β, NLRP3, and ASC), thereby exacerbating injury after cerebral ischemia.

1. Introduction

Ischemic stroke (IS) is a cerebrovascular disease caused by the obstruction of cerebral blood vessels, leading to localized ischemia and hypoxia that result in neurological dysfunction (1). Its mortality and disability rates have remained consistently high over the years. Currently, the treatment options for stroke are scarce and significantly limited in usage, highlighting an urgent need for new therapeutic approaches to change the current situation of stroke [1, 2]. In the pathological features of stroke, the irreversible death of nerve cells triggers a secondary immune response, which is manifested as the activation of glial cells, the recruitment of peripheral immune cells, and the release of cytokines. This secondary immune response is a key factor leading to poor prognosis and neurological dysfunction after stroke [3, 4, 5, 6]. Astrocytes are the most abundant glial cells in the central nervous system. They maintain the normal functioning of the nervous system through various mechanisms, including maintaining neuronal environments and regulating neurodevelopment [7, 8]. The latest research has found that, in addition to the common forms of cell death such as apoptosis, autophagy, and ferroptosis [3, 9, 10, 11], ATP depletion induced by ischemia and hypoxia can trigger necroptosis and apoptosis in astrocytes through the classical receptor‐interacting protein kinase‐mixed lineage kinase domain‐like protein (RIPK‐MLKL) pathway. Inhibition of MLKL can alleviate neuroinflammation in astrocytes and promote the formation of glial scars [12]. Necroptosis refers to a programmed form of cell death that occurs when the apoptotic process is suppressed. Specifically, when Caspase‐8 activity is inhibited, pattern recognition receptors (such as TLR3/4) phosphorylate RIPK1 and RIPK3. Then, p‐RIPK1 further phosphorylates and activates MLKL, along with p‐RIPK3. p‐MLKL oligomerizes and inserts into the cell membrane to form pores, leading to membrane rupture and the release of damage‐associated molecular patterns (DAMPs) [13, 14, 15, 16]. Necroptosis shares many similarities with apoptosis, but compared to the apoptotic process, necroptosis is often a more highly pro‐inflammatory mode of cell death. It exacerbates neuroinflammation by mediating the release of numerous inflammatory factors [17]. Therefore, targeting and regulating the process of necroptosis in astrocytes may provide new insights into alleviating ischemia‐induced inflammatory infiltration and promoting functional recovery. However, as a novel form of programmed cell death, research on the regulatory mechanisms that activate necroptosis is still quite limited.

Chemokines are a class of structurally similar small secreted proteins or peptides with a molecular weight of approximately 8–10 kDa. Their core function is to guide the directional migration of immune cells through concentration gradients [18, 19]. After IS, chemokines respond to ischemia and hypoxia stimuli, recruiting peripheral immune cells that infiltrate and create a pro‐inflammatory environment, exacerbating postischemic brain injury [20]. C‐C motif chemokine ligand 11 (CCL11) is a member of the eosinophil activation chemokine family, and its primary function is to recruit eosinophils to sites of inflammation or infection specifically. It plays a key role in allergic diseases (such as asthma), parasitic infections, and tissue fibrosis [21, 22]. Recent studies have found that the specific role of CCL11 in cardiovascular and cerebrovascular diseases is quite complex. On the one hand, in peripheral vascular diseases, the Gasdermin D (GSDMD)‐mediated pyroptosis process may promote the release of CCL11, thereby improving endothelial function and angiogenesis [23]. On the other hand, CCL11 can exacerbate ischemic brain injury by recruiting B and T lymphocytes and enhancing microglial polarization. CCL11, as a chemokine involved in the injury and repair processes of various diseases, plays a crucial role. Therefore, clarifying the specific mechanisms of action of CCL11 is essential. However, little is currently known about the specific mechanism by which CCL11 exerts its effects. Previous studies have found that the CCL11 receptor C‐C motif chemokine receptor 3 (CCR3) is widely expressed in the central nervous system (such as in neurons, astrocytes, and microglia). The targeted inhibition of CCL11‐CCR3 to prepare target cyclic peptides significantly improves the apoptosis of retinal neurons induced by long‐term intravitreal administration [24]. In the immune activation and inflammation induced by spontaneous abortion, CCL28 induces apoptosis of decidual stromal cells (DSCs) through the activation of CCR3 and CCR10. However, in astrocytes, there have been no studies reporting whether CCL11‐CCR3 mediates the process of necrotic apoptosis.

We hypothesize that CCL11 exacerbates neuroinflammation by stimulating the activation of necroptosis in astrocytes through the CCR3 receptor. To validate the above hypothesis, we examined the changes in chemokine and necroptosis marker expression after cerebral ischemic injury in a mouse model. The study further investigated the effects of inhibiting chemokine receptor expression on the activation of necroptosis and neuroinflammation following ischemia. In the in vitro experiment, oxygen–glucose deprivation (OGD) was used to simulate ischemic conditions and verify the mechanism by which chemokines bind to astrocyte chemokine receptors, activating necroptosis. Overall, the above results demonstrate the specific mechanism by which chemokines exacerbate neuroinflammation through the activation of necroptosis. A comprehensive understanding of the forms of cell death in IS and their activation mechanisms will provide a theoretical foundation for targeted necroptosis therapy, which has significant clinical application value.

2. Methods

2.1. Animals

A total of 115 male C57BL/6 mice (8 weeks old, body weight 20–22 g) were provided by Liaoning Changsheng Biotechnology Co. Ltd. The animals were housed in an SPF‐grade animal room at 22°C–24°C with 50%–60% relative humidity, under a 12‐h light–dark cycle, and provided free access to food and water. The Ethics Committee of Harbin Medical University approved all experimental procedures.

2.2. Model Establishment and Interventions

C57BL/6 male mice (20–22 g) were used to establish a pMCAL model via electrocoagulation [25]. The left middle cerebral artery was occluded. Mice were anesthetized via intraperitoneal injection of 2.5% Avertin. After reaching deep anesthesia, the left cranial hair was shaved. Mice were fixed under a microscope, and the surgical area was disinfected with 75% ethanol. A vertical skin incision was made at the midpoint between the outer canthus and the external auditory canal. The temporalis muscle was dissected, and a cranial window was drilled to expose the MCA. The MCA, identified as a thick longitudinal vessel near the eye, was coagulated using an electrocoagulation pen. The muscle was repositioned, and the wound was sutured. Mice were placed on a heating pad until recovery. Sham‐operated controls underwent craniotomy without MCA coagulation. In the drug treatment group, on the first 2 days before model construction, 5 μL of the CCR3 inhibitor SB328437 (20 μmol) was stereotactically injected into the ventricular area. All in vivo experiments were repeated at least three times (n = 3). Mice with a Longa score of approximately 3–4 after pMCAL surgery were included in the study, while those showing signs of subarachnoid hemorrhage or basilar artery rupture were excluded. Five eligible samples were selected for statistical analysis.

2.3. TTC (Triphenyl Tetrazolium Chloride) Staining

Mice were euthanized via rapid cervical dislocation and decapitation. Brains were extracted, placed in a precooled brain slice mold, and sectioned coronally into 2 mm slices. Sections were incubated in 2% TTC solution (HY‐D0714, MCE, USA) at 37°C for 15 min. Viable tissue stained red, while infarcted areas remained white. Images were captured, and infarct volume was quantified using ImageJ software.

2.4. Primary Astrocyte (AS) Culture

Cortices from postnatal day 3 (P3) mice were isolated by dissecting and discarding the midbrain and cerebellum. The remaining cerebral cortex was transferred to a centrifuge tube, mechanically dissociated via repeated pipetting, and allowed to settle for 2 min. The suspension was filtered through a 40 μm cell strainer to remove residual tissue fragments. After centrifugation at 1600 rpm, 4°C, 5 min, cells were resuspended in culture medium and seeded at a density equivalent to 1.5 T25 cell culture flasks per mouse. Cells were cultured for 7 days prior to drug interventions and OGD experiments. The Nec‐1 treatment group received 10 ng/mL Nec‐1 pretreatment for 12 h before OGD. The CCL11‐treated group was pretreated with 1 ng/mL CCL11 for 12 h before OGD exposure. Similarly, the CCR3 inhibitor group received 5 ng/mL SB328437 for 12 h prior to OGD. All in vitro experiments must be repeated at least three times (n = 5), with the average of three repetitions used for statistical analysis.

2.5. Primary Neuron Culture and Intervention

Prepare reagents such as Neurobasal medium (21 103 049, Gibco, USA), B27 supplement (A3320201, Gibco, USA) in advance. Supplies are pretreated with poly‐L‐lysine (P6407, Sigma, USA), and animal ethics are adhered to. Collect cortices from embryos of 16‐ to 18‐day pregnant rats, mince them, and incubate with 37°C trypsin for 10–15 min. Add serum to terminate digestion, then use DNase I to disperse cells, followed by filtration and centrifugation to obtain cells. Adjust the concentration to (1–2) × 10⁵ cells/cm², seed onto PDL‐pretreated culture plates, and place in a 37°C, 5% CO₂ incubator. Replace with fresh medium 24 h later, and perform half‐medium changes every 3–4 days thereafter. Neurons will mature in 7–10 days. Subsequently, we collected the supernatant from primary astrocytes stimulated with CCL11, SB328437, and OGD. After filtering the supernatant through a 0.45 μm filter, we added it to neurons for a 4‐h co‐culture, and then performed the IF assay.

2.6. IF Staining

In this experiment, immunofluorescence analysis was conducted separately in in vivo and in vitro studies. For the in vivo arm, brain tissues flash‐frozen in liquid nitrogen were cryosectioned at 10 μm thickness, followed by fixation with 4% paraformaldehyde (PFA) for 15 min. In the in vitro arm, astrocytes or neurons cultured on coverslips were rinsed with phosphate‐buffered saline (PBS) and subsequently fixed with 4% PFA for 5 min. Subsequent procedures were identical for both experimental conditions. Permeabilization was performed using 0.3% Triton X‐100 for 15 min, blocked with 5% BSA for 1 h at room temperature (RT), and incubated overnight at 4°C with primary antibodies: rabbit anti‐p‐RIPK1 (1:200, 28 252–‐1‐AP, Proteintech, China), rabbit anti‐p‐MLKL (1:200, 82 090–‐2‐RR, Cell Signaling Technology, China), rabbit anti‐CCR3 (1:200, CY5559, Abways, China), goat anti‐GFAP (1:400, ab302644, Abcam, England). Subsequently, chicken anti‐MAP2 (1:500, ab5392, Abcam, England), rabbit anti‐Homer1 (1:500, ab316287, Abcam, England), and mouse anti‐Basson (1:500, ab82958, Abcam, England). Sections were then incubated at RT for 1 h with secondary antibodies: TRITC goat anti‐rabbit (1:400, 711–‐025‐152 Jackson, USA), FITC donkey anti‐goat (1:400, 705–‐095‐003, Jackson, USA), 647 donkey anti‐chicken (1:500,703–‐605‐155, Jackson, USA), and then with DAPI (1:1000, C1006, Beyotime, China) for 3 min at RT. Images were captured using a confocal microscope (Zeiss, Germany).

2.7. WB

Proteins from mouse cortices or cells were lysed in buffer containing PMSF (ST505, Beyotime, China) and phosphatase inhibitors (P1045, Beyotime, China). Lysates were sonicated, vortexed for 30 min, and centrifuged (12,000 g, 4°C, 15 min). Protein concentrations were normalized using the BCA assay (KTD3001, Abbkine, China). Proteins were separated on 10% SDS‐PAGE gels and transferred to PVDF membranes (PR05509, Milipore, America). Membranes were blocked with 5% skim milk (1 h, RT) and incubated overnight at 4°C with primary antibodies: rabbit anti‐NLRP3 (1:500, 15101, Proteintech, China), rabbit anti‐ASC (1:200, DF6304, Affinity, America), mouse anti‐β‐actin (1:1000, TA‐09, ZSBIO, China), rabbit anti‐MLKL (1:200, 82090‐2‐RR, Cell Signaling Technology, China), rabbit anti‐RIPK1 (1:200, 28252‐1‐AP, Proteintech, China), rabbit anti‐RIPK3 (1:1000, Immunoway, China), rabbit anti‐p‐MLKL (1:500, Cell Signaling Technology), rabbit anti‐p‐RIPK1 (1:1000, Proteintech), rabbit anti‐p‐RIPK3 (1:1000, Immunoway), rabbit anti‐CCL11 (1:500, Abcam, UK), rabbit anti‐CCR3 (1:1000, Affinity, USA), and mouse anti‐IL‐1β (1:500, Cell Signaling Technology). HRP‐conjugated goat anti‐mouse (1:1000, ZSGB‐BIO) or goat anti‐rabbit (1:1000, ZSGB‐BIO) secondary antibodies were applied. Bands were visualised using an ECL system and analysed using ImageJ, with normalisation to β‐actin.

2.8. qPCR

Total RNA was extracted from ischemic brain tissues using Trizol (108–95‐2, Takara, Japan). cDNA was synthesized using reverse transcriptase M‐MLV (2641A, Takara, Japan). qPCR was performed with Hieff SYBR Green Master Mix (11141E510, Yeasen, China) and gene‐specific primers (Table S1). Relative mRNA levels were calculated using the 2 − ΔΔCt method, with 18 s as the internal control.

2.9. CCK8

Add 100 μL of cell suspension containing 5 × 10³ cells to each well of the 96‐well plate. Place the plate in a 37°C, 5% CO₂ incubator and incubate for 24 h. Set up blank control groups, negative control groups, and drug treatment groups. For the drug treatment group, add 10 μL of drug solution to each duplicate well, with specific concentrations set at 0, 0.01, 0.1, 0.5, 1, 5, 10, 50, 500, and 100 ng/mL. After treating cells with OGD‐2 h, measure the absorbance of each well using a microplate reader at 450 nm wavelength.

2.10. Proteomics

The proteomics study employed a method established in conjunction with Shanghai Biotree Biomedical Technology Co. Ltd. (Shanghai, China) on the Thermo Scientific Orbitrap Fusion Lumos platform, combined with EASY‐nLC 1200. This approach utilized label‐free quantitative proteomics technology to assess the proteome of astrocytes. Collect astrocytes in a centrifuge tube, add 50 μL RIPA lysis buffer, and use a cell sonicator (Beijing Dequan Keji Company, Beijing, China) to extract proteins on ice for 30 s ultrasonically. At 4°C, after centrifugation at 12000 g for 20 min, collect the supernatant and determine the protein concentration using the BCA method. The protein is precipitated with acetone, then dissolved, reduced, alkylated, enzymatically hydrolyzed, and removed from SDC and peptide residues for subsequent detection.

The separation and analysis of peptides were conducted using the EASY‐nLC1200 UPLC system (Thermo Fisher Scientific, San Jose, CA, USA) in combination with a Q Exactive HFX Orbitrap instrument (Thermo Fisher Scientific, San Jose, CA, USA), equipped with a nano‐electrospray ion source. The peptides were separated on a reversed‐phase column (100 μm ID × 15 cm, resil‐pur120C18AQ, 1.9 μm, Dr. Maisch) at a flow rate of 300 nL/min. The mobile phase A is H2O‐0.1% formic acid‐2% acetonitrile, and the mobile phase B is 80% acetonitrile‐0.1% formic acid. The solvent gradient parameters are set as follows: mobile phase B 2%–5% (2 min), 5%–22% (88 min), 22%–45% B (26 min), 45%–95% (2 min), and at 95% (2 min). The scanning sequence utilizes MS1 spectra (Orbitrap analysis: resolution 120 000 (@200 m/z); mass range 350–1600 m/z; automatic gain control (AGC) target of 3E6, maximum IT of 50 ms) and MS2 spectra (dynamic first mass; resolution 15 000; AGC target of 1E5, maximum IT of 110 milliseconds) in both negative and positive modes. The top 20 ions were fragmented by HCD (collision energy 27%; isolation window, 1.2 m/z). The previous target ion exclusion of 35S, single‐charge peaks, and peaks with charges exceeding six are excluded from the data‐dependent acquisition (DDA) process.

The mass spectrometry data were retrieved using Proteome Discoverer (PD) software (version 2.4.0.305) with the built‐in SequestHT search engine, which searched the raw data files of MS/MS spectra against the UniProt FASTA database (uniprot‐Mus_musculus‐10 090‐2020‐10). The translation is: “fasta”. The main search parameters are as follows: select carbamoyl (C) for fixed modifications; LFQ project type; oxidation (M), acetylation (protein n terms); protease used is trypsin; peptide tolerance set to 10 ppm; MS/MS tolerance set to 0.02 Da; false discovery rate (FDR) at both the PSM and peptide levels is 0.01. Translate to English: Quantify proteins using Unique peptide and Razor peptide, and normalize with the total amount of peptides.

2.11. Statistical Analysis

Data were analyzed using GraphPad Prism 10.0 and expressed as mean ± SD. All experiments were performed at least three times. Normality was assessed via the Shapiro–Wilk test. For normally distributed data, one‐way ANOVA with Tukey's post hoc test was used. Nonparametric data were analyzed using the Kruskal–Wallis test, followed by Dunn's multiple comparison post hoc test. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001.

3. Result

3.1. Cerebral Ischemic Injury Promotes the Occurrence of Necroptosis in the Brain

Cerebral ischemia leads to significant neuronal cell death in the affected brain tissue. As a novel mode of programmed cell death, necroptosis has been confirmed to play an important pathological role in ischemic brain injury. To observe the changes in necroptosis and apoptosis of neural cells in the ischemic hemisphere during cerebral ischemia, we used WB experiments to detect the levels and phosphorylation status of proteins related to necroptosis pathways—MLKL, RIPK1, and RIPK3—in brain tissue at different time points after cerebral ischemia. The results shown in Figure 1A indicate that, compared to the sham group, the expression of pMLKL, pRIPK1, and pRIPK3 initially increased and then decreased as the duration of cerebral ischemia was prolonged. Statistical data analysis revealed that pMLKL, pRIPK1, and pRIPK3 significantly increased on days 1, 2, and 3 after brain ischemia. Among these proteins, the expression levels of pMLKL and pRIPK1 peaked at day three postbrain ischemia, while the expression level of pRIPK3 reached its peak on day 2 (Figure 1B). At the same time, we used qPCR experiments to detect changes in the gene levels of MLKL, RIPK1, and RIPK3. The results shown in Figure S1 indicate that, compared to the sham group, the mRNA levels of MLKL, RIPK1, and RIPK3 were significantly increased after cerebral ischemia and peaked at 1 day postischemia. Previous reports have suggested that the activation of necroptosis signaling pathways is closely linked to inflammatory responses. To investigate the impact of necroptosis on the inflammatory response in ischemic brain tissue, we employed WB and quantitative PCR (qPCR) to assess the expression levels of ASC, NLRP3, and IL‐1β. The results, as shown in Figure 1C, indicate that compared to the sham group, the protein expression levels of ASC, NLRP3, and IL‐1β initially increased and then decreased with prolonged brain ischemia duration. Statistical data analysis revealed that the protein expression levels of ASC, NLRP3, and IL‐1β increased significantly after cerebral ischemia. Among these, the protein expression levels of ASC and IL‐1β peaked at 5 days postcerebral ischemia, whereas the peak for NLRP3 was observed at 1 day postcerebral ischemia. At the same time, we used qPCR experiments to detect changes in the gene levels of ASC, NLRP3, and IL‐1β. The statistical analysis results shown in Figure 1E indicate that compared to the sham group, mRNA levels of ASC, NLRP3, and IL‐1β significantly increased at 1d, 2d, and 3d after cerebral ischemia; moreover, NLRP3 and IL‐1β reached their peaks at 1 day postcerebral ischemia. Next, we used TTC staining to detect changes in the volume of ischemic necrotic brain tissue after 1, 2, and 3 days of cerebral ischemia. The results are shown in Figure 1E, indicating that the volume of necrotic brain tissue in the ischemic hemisphere increases with prolonged ischemia time. Meanwhile, as shown in Figure 1G, statistical analysis of neurological behavior scores demonstrates that compared to the sham group, there is a significant increase in neurological dysfunction at days 1, 2, and 3 postischemia. The above results suggest that during cerebral ischemia, the occurrence of necroptosis in nerve cells of the ischemic hemisphere may exacerbate brain injury and inflammatory responses within the brain.

FIGURE 1.

Ischemic brain injury promotes the occurrence of necroptosis and apoptosis in the brain. (A‐B) Western blot analysis of RIPK3, pRIPK3, RIPK1, pRIPK1, MLKL, and pMLKL protein expression at different time points after ischemia (A) and statistical analysis (B); (C‐D) Western blot analysis of IL‐1β, NLRP3, and ASC protein expression at different time points after ischemia (C) and statistical analysis (D); (E) QPCR analysis of mRNA expression levels for ASC, IL‐1β, and NLRP3 at different times postischemia; (F) TTC staining showing changes in infarct volume over various time points following ischemia; (G) Neurological function score analyses at varying times postischemia; N = 5; *p < 0.05, **p < 0.01, ***p < 0.001.

3.2. Cerebral Ischemic Injury Induces Necroptosis of Astrocytes

Research has shown that the inflammatory response triggered by neurological diseases is linked to necroptosis events in various brain cells. To identify the type of cell that undergoes necroptosis after cerebral ischemic injury, we cultured primary astrocytes, the BV2 microglial cell line, N2A neurons, and bEnd.3 brain microvascular endothelial cell lines, and the RAW264.7 monocyte/macrophage cell line under OGD conditions. We assessed the expression levels of key necroptosis proteins MLKL and pMLKL through WB experiments.

The results shown in Figure 2A indicate that, compared to several other types of brain cells, the phosphorylated protein pMLKL is expressed at the highest level in astrocytes. At the same time, we employed IF experiments to detect changes in pMLKL expression in astrocytes following OGD. The results shown in Figure 2B indicate that OGD 2 h can upregulate the expression level of pMLKL in the nuclei of astrocytes. Although MLKL is considered a key protein in regulating the occurrence of necroptosis, it also plays a crucial role in apoptosis and inflammatory signaling pathways. Therefore, to further verify the impact of OGD 2 h conditions on the necroptosis‐apoptosis signaling pathway of astrocytes, we conducted a proteomic analysis to examine the changes in apoptotic and inflammatory‐related signaling pathways in astrocytes under OGD 2 h stimulation. The results shown in Figure 2C indicate that compared to the Control group, the OGD 2 h group had an upregulation of 106 proteins and a downregulation of 37 proteins in astrocytes. Next, we will conduct KEGG clustering analysis on the upregulated proteins. The results are shown in Figure 2D, indicating a significant upregulation of the apoptosis, TNF signaling pathway, and necroptosis signaling pathway. At the same time, an analysis of the genes in the differential pathways was conducted. The results shown in Figure 2E indicate that, compared to the Control group, MLKL, Cflar, Xiap, and Rbck1 significantly increased in the necroptosis signaling pathway of the OGD 2 h group. The results above indicate that OGD 2 h conditions can upregulate the necroptosis signaling pathway in astrocytes. To investigate the dynamic changes in astrocytes following cerebral ischemia in vitro, we simulated in vivo ischemic conditions at various time points using the OGD model. We randomly divided the astrocytes into the Control group, the OGD 2 h group, the OGD 4 h group, and the OGD 6 h group. We detected changes in necroptosis‐related proteins RIPK1, RIPK3, and MLKL, as well as their phosphorylated forms, using WB experiments for each group. The results shown in Figure 2F indicate that, compared to the Control group, the protein levels of pMLKL, pRIPK1, and pRIPK3 significantly increased under OGD conditions and peaked at 2 h of OGD. The expression levels of NLRP3, ASC, and IL‐1β in each group were detected using WB and qPCR experiments. The results shown in Figure 2G,H indicate that, compared to the Control group, the gene levels of NLRP3, ASC, and IL‐1β significantly increased under OGD conditions. The above results indicate that OGD conditions can activate necroptosis events in astrocytes through the RIPK1/RIPK3/MLKL signaling pathway, promoting their production of pro‐inflammatory cytokines.

FIGURE 2.

Ischemic brain injury induces necroptosis in astrocytes. (A) Western blot analysis of the necroptosis markers MLKL and pMLKL protein expression in primary astrocytes, endothelial cells (bEND.3), microglia (BV2), neurons (N2A), and macrophages (RAW264) with statistical analysis. (B) Immunofluorescence analysis showing colocalization of pMLKL with astrocytes (GFAP) between control and OGD groups, along with statistical analysis. GFAP (green), p‐MLKL (red), and DAPI (blue). 400×, scale bar = 50 μm. (C) Volcano plot displaying differential protein expression in astrocytes between control and OGD groups. (D) KEGG cluster analysis of 106 upregulated differential genes; (E) Heatmap showing significantly changed genes in the necroptosis pathway; (F) Western blot analysis of RIPK3, pRIPK3, RIPK1, pRIPK1, MLKL, and pMLKL protein expression at different time points in astrocytes following OGD along with statistical analysis; (G) Western blot analysis of IL‐1β, NLRP3, and ASC protein expression at different time points in astrocytes following OGD along with statistical analysis; (H) QPCR analysis of mRNA expressions for ASC, IL‐1β, and NLRP3 in astrocytes at different time points after OGD. N = 5; *p < 0.05, **p < 0.01, ***p < 0.001.

3.3. During Cerebral Ischemia, CCL11 Promotes Necroptosis of Astrocytes

Research has shown that necroptosis and the initiation of inflammatory responses are regulated by various cytokines, particularly those belonging to the chemokine family. To investigate whether chemokines can influence the occurrence of necroptosis in ischemic brain tissue during cerebral ischemia, we conducted qPCR experiments to detect changes in the expression of chemokine genes in ischemic brain tissue at various time points. The statistical results shown in Figure 3A indicate that, compared to the sham group, the mRNA expression levels of CCL2, CCL11, and CXCL9 are significantly increased after cerebral ischemia. However, only the mRNA expression level of CCL11 peaked at 1 day after cerebral ischemia, which is consistent with the peak time of necroptosis‐related protein expression in ischemic brain tissue. At the same time, we also examined the mRNA expression changes of CCL1, CCL6, CCL7, CCL8, CCL19, and CCL24. The statistical results shown in Figure S2 indicate that, compared to the sham group, there is no significant change in mRNA expression levels of these chemokines following cerebral ischemia. Next, we conducted WB experiments to detect the protein expression level of CCL11 in ischemic brain tissue during cerebral ischemia. The results shown in Figure 3B indicate that compared to the sham group, the protein expression level of CCL11 initially increased and then decreased as the duration of cerebral ischemic injury extended. At the same time, qPCR experiments were conducted to detect changes in CCL11 mRNA expression in ischemic brain tissue at various time points after cerebral ischemia. The statistical results shown in Figure 3C indicate that, compared to the sham group, CCL11 mRNA levels increased significantly in the early stages after brain ischemic injury. To observe the spatial relationship between astrocytes and CCL11 in ischemic brain tissue, we conducted IF experiments to detect the expression of CCL11 around astrocytes. The results shown in Figure 3D indicate that, compared to the sham group, the expression level of CCL11 around activated astrocytes in ischemic brain tissue is significantly increased. To further verify whether CCL11 affects the occurrence of necroptosis in astrocytes, first, the present study aimed to screen the optimal concentration of CCL11 for subsequent experiments by evaluating the viability of astrocytes under different CCL11 concentrations using the Cell Counting Kit‐8 (CCK8) assay. The results shown in Figure S3 indicate that compared to the zero ng/mL CCL11 group, CCL11 at concentrations of 50 ng/mL or higher significantly inhibits the activity of astrocytes. In contrast, CCL11 at concentrations of 10 ng/mL or lower does not have an inhibitory effect on the activity of astrocytes under normal and OGD 2 h conditions. Based on the principle of minimum drug dosage, we selected 0.1 ng/mL CCL11 as the optimal concentration to stimulate astrocytes in subsequent in vitro experiments. Next, astrocytes from different groups were treated with 0.1 ng/mL CCL11, and WB experiments were performed to detect the changes in necroptosis‐related proteins under normal and OGD 2 h conditions. The results are shown in Figure 3E. Compared to the None group, 0.1 ng/mL CCL11 significantly upregulated the expression levels of pMLKL, pRIPK1, and pRIPK3 proteins under both normal and OGD 2 h conditions; furthermore, the expression levels of these proteins in astrocytes from the OGD 2 h + CCL11 group were significantly higher than those in the Control+CCL11 group. At the same time, we used WB to detect changes in the expression of ASC, NLRP3, and IL‐1β proteins in astrocytes from each group. The results are shown in Figure 3F. Compared to the None group, 0.1 ng/mL CCL11 significantly upregulated the expression levels of ASC and NLRP3 proteins under both normal and OGD 2 h conditions; furthermore, the expression levels of ASC, NLRP3, and IL‐1β proteins in astrocytes from the OGD 2 h + CCL11 group were markedly higher than those in the Control + CCL11group.

FIGURE 3.

CCL11 promotes necroptosis of astrocytes during cerebral ischemia; (A) QPCR analysis of chemokines CCL2, CCL11, and CXCL9 mRNA expression at different time points after ischemia; (B‐C) Western blot analysis for protein (B) and mRNA (C) expression levels of CCL11 at different time points postischemia along with statistical analyses; (D) Immunofluorescence analysis showing colocalization of CCL11 with astrocytes (GFAP) at various time points following ischemia and corresponding statistical evaluation. GFAP (red), CCL11 (green), and DAPI (blue). 400×, scale bar = 50 μm; (E) Western blot assessment analyzing RIPK3, pRIPK3, RIPK1, pRIPK1, MLKL, and pMLKL protein expressions in astrocytes under treatment with both CCL11 and OGD 2 h stimulus accompanied by quantitative outcomes; (F) Western blot examination evaluating ASC, IL‐1β, NLRP3 protein expressions in response to stimulation from both CHL11and OGD 2 h alongside analytic statistics; N = 5; *p < 0.05, **p < 0.01, ***p < 0.001.

3.4. Cerebral Ischemic Injury Induces Excessive Expression of CCL11 Receptor CCR3 in Astrocytes

Typically, chemokines regulate cell functions by binding to chemokine receptors on the cell surface. Existing reports indicate that CCL11 primarily targets CCR3 to regulate neuronal function in central nervous system diseases [26]. To investigate whether cerebral ischemic injury affects the expression level of the CCL11 receptor CCR3 in the ischemic hemisphere, we conducted WB experiments to detect changes in CCR3 protein expression levels in brain tissue at various time points following cerebral ischemia. The results shown in Figure 4A indicate that the expression level of CCR3 protein increased and then decreased as the duration of cerebral ischemia was prolonged. Statistical analysis revealed that, compared to the sham group, there was a significant increase in CCR3 protein levels in brain tissue subjected to ischemia for 1, 2, and 3 days, with the peak level reached on day 1 postischemia. Concurrently, qPCR experiments were conducted to determine the mRNA expression level of CCR3 in ischemic brain tissue at various time points following cerebral ischemia. The results shown in Figure 4B indicate that compared with the sham group, there was a significant increase in CCR3 mRNA expression levels in brain tissues subjected to ischemia for 12 h, 1 day, and 2 days. Next, in order to observe which type of cells highly express CCR3 protein in ischemic brain tissue induced by cerebral ischemia injury, we cultured different cell types capable of expressing CCR3 under OGD 2 h conditions in vitro [27, 28]. We measured the expression levels of CCR3 protein using WB experiments. The results shown in Figure 4C indicate that cerebral ischemic injury can significantly upregulate the expression level of CCR3 protein in astrocytes and BV2 cells, whereas the expression level of CCR3 in N2A and bEnd.3, and RAW264.7 cells has significantly lower levels than those in astrocytes. Next, to verify whether cerebral ischemic injury can affect the expression changes of CCR3 on astrocytes, we conducted IF experiments to detect the expression levels of CCR3 on astrocytes in ischemic brain tissue. The results shown in Figure 4D indicate that compared to the sham group, the expression level of CCR3 on astrocytes significantly increased in ischemic brain tissue at days 1, 2, and 3 following cerebral ischemia, peaking at day 1. At the same time, we used WB experiments to detect the expression levels of CCR3 protein in astrocytes at different times after OGD 2 h. The results shown in Figure 4E indicate that, compared to the Control group, the expression level of CCR3 protein in astrocytes initially increases and then decreases with the extension of OGD for 2 h. Furthermore, at OGD 2 h, the expression level of CCR3 protein in astrocytes reaches its peak. At the same time, we used qPCR experiments to detect changes in CCR3 mRNA expression in astrocytes at different time points after OGD 2 h. The results, as shown in Figure 4F, indicate that compared to the Control group, the mRNA expression level of CCR3 in astrocytes was significantly increased at 2 and 4 h after OGD 2 h. Next, to observe the effect of different CCL11 concentrations on CCR3 protein expression levels in astrocytes under OGD 2 h conditions, we adjusted the administration concentration of CCL11 based on previous results, ranging from 10 mg/mL to 0.01 mg/mL. We used WB experiments to detect changes in CCR3 protein levels among the groups. The results are shown in Figure 4G. Compared to the OGD 2 h + 0 mg/mL group, CCL11 at concentrations of 10 mg/mL and 0.01 mg/mL significantly increased the expression levels of CCR3 protein in astrocytes under OGD 2 h conditions, with a peak observed at 0.1 mg/mL. At the same time, compared to the N‐OGD 2 h + 0.1 mg/mL group, there was a significant increase in CCR3 expression in astrocytes of the OGD 2 h + 0.1 mg/mL group. The above results indicate that CCL11 can significantly upregulate the expression level of CCR3 in astrocytes following cerebral ischemia.

FIGURE 4.

Ischemic brain injury induces excessive expression of chemokine receptor CCR3 in astrocytes; (A) Western blot analysis of CCR3 protein expression and statistical analysis at different time points after ischemia; (B) QPCR analysis of CCR3 mRNA expression at different time points postischemia; (C) Immunofluorescence analysis showing colocalization and statistical assessment between CCR3 and astrocyte marker GFAP at various times following ischemia. GFAP (green), CCR3 (red), and DAPI (blue).400×, scale bar = 50 μm; (D) Western blot analysis comparing the expression levels of CCR3 protein in primary astrocytes, endothelial cells (bEND.3), microglial cells (BV2), neurons (N2A) and macrophages (RAW264) with corresponding statistics; (E) Western blot evaluation of CCR3 protein expressions over time during OGD 2 h along with statistical analyses; (F) QPCR examination on transcript levels for CCCR mRNA from astrocytes collected under different duration conditions due to OGD 2 h; (G) Western blot analysis and statistical evaluation of CCR3 protein expression in response to 10, 5, 1, 0.1, 0.01 ng/mL of CCL11 Stimulation under Control (C) and OGD2h (O) Conditions. The 0 ng CCL11 group used the solvent DMSO as the control. N = 5, *p < 0.05, **p < 0.01, ***p < 0.001.

3.5. CCR3 Inhibitor SB328437 Reduced Astrocytic Necroptosis and Reduced Cerebral Ischemia Injury

To investigate whether the excessive expression of CCR3 induced by cerebral ischemia contributes to brain ischemic injury, we employed the CCR3 inhibitor SB328437 in vivo to reduce CCR3 protein expression levels in ischemic brain tissue. Mice in the drug treatment group were administered CCR3 inhibitor SB328437 via stereotaxic intracerebral injection at a concentration of 20 μmol and a dose of 5 μL, 24 h prior to model establishment. We then assessed changes in cerebrovascular damage among different groups using TTC staining experiments and neurological behavioral scoring methods. The results shown in Figure 5A,B indicate that, compared to the ischemia for 1 day group, the volume of necrotic brain tissue in the ischemic hemisphere was significantly reduced in the CCR3 inhibitor group. Additionally, as demonstrated by the statistical outcomes of neurological behavioral scores illustrated in Figure 5C, mice treated with CCR3 inhibitors exhibited a significant recovery of neurological function following cerebral ischemia.

FIGURE 5.

CCR3 inhibitor SB328437 reduces necroptosis and apoptosis in astrocytes and alleviates brain ischemic injury. (A) Schematic diagram of intraperitoneal injection of CCR3 inhibitor SB328437; (B) TTC staining shows changes in infarct volume after inhibiting CCR3 and statistical analysis; (C) Neurological function score analysis in mice after inhibiting CCR3; (D) Western blot analysis showing protein expression levels of RIPK3, pRIPK3, RIPK1, pRIPK1, MLKL, and pMLKL following inhibition of CCR3 along with statistical analysis; (E) Western blot analysis indicating protein expression levels for ASC, IL‐1β, NLRP3 postinhibition of CCR3 along with statistical analyses; F‐G: Immunofluorescence analyses demonstrating colocalization expressions between the necroptosis markers pMLKL (F) pRIPK3 (G) and astrocyte marker GFAP alongside their respective statistical analyses. GFAP (green), p‐MLKL (red), and DAPI (blue). 400×, scale bar = 50 μm. N = 5; *p < 0.05, **p < 0.01, ***p < 0.001.

Next, to further verify whether the reversal of cerebral ischemic injury by CCR3 inhibitors is closely related to the occurrence of necroptosis in ischemic brain tissue. We examined the expression levels of necroptosis‐related proteins in ischemic brain tissues from normal mice and CCR3 inhibitor‐treated mice via WB experiments. The results shown in Figure 5D indicate that compared to the pMCAL1d group, the protein expression levels of CCR3, pMLKL, pRIPK1, and pRIPK3 were significantly reduced in the ischemic brain tissue of the pMCAL1d + SB328437 group. At the same time, to verify the effect of CCR3 inhibitors on inflammation in ischemic brain tissue, we examined the expression changes of inflammation‐related proteins in each group of ischemic brain tissue using WB analysis. The results shown in Figure 5E indicate that compared to the pMCAL1d group, the expression levels of ASC, NLRP3, and IL‐1β proteins in the ischemic brain tissue of the pMCAL1d + SB328437 group were significantly reduced. Next, to clarify that the CCR3 inhibitor SB328437 downregulates necroptosis signaling pathways in ischemic brain tissue by inhibiting the expression level of astrocytic CCR3, we conducted IF experiments to detect the expression levels of necrosis‐related proteins in astrocytes from different groups of ischemic brain tissue. The results shown in Figure 5F,G indicate that, compared to the pMCAL1d group, the expression levels of pMLKL and pRIPK3 in astrocytes in the ischemic brain tissue of the pMCAL1d + SB328437 group were significantly reduced. The above results indicate that the CCR3 inhibitor SB328437 reduces inflammatory responses in ischemic brain tissue and alleviates cerebral ischemic injury by inhibiting the necroptosis signaling pathway of astrocytes after brain ischemia.

3.6. Cerebral Ischemic Injury Induces CCL11 Activation of CCR3/RIPK3/MLKL Signaling Pathway to Promote Astrocytic Necroptosis and Inflammatory Response

Subsequently, we further investigated whether CCL11 acts through the astrocytic CCR3 receptor to activate the necroptosis signaling pathway. To determine the optimal concentration of SB328437 for in vitro administration, we conducted CCK‐8 assays to evaluate its impact on astrocyte viability at 100, 50, 10, 5, 1, 0.1, and 0.01 ng/mL. As illustrated in Figure S4A,B, compared with the 0 ng/mL group, SB328437 administration at concentrations up to 100 ng/mL exhibited no significant effect on astrocyte viability. Accordingly, 100 ng/mL SB328437 was selected as the optimal treatment concentration for astrocytes. The concentration of CCL11 was maintained at 1 ng/mL, which had been previously established to significantly activate CCR3. To assess the effect of 1 ng/mL CCL11 and 100 ng/mL SB328437 on CCR3 expression in astrocytes following OGD 2 h, we administered 1 ng/mL CCL11 alone or in combination with 100 ng/mL SB328437 to different experimental groups. After 12 h of stimulation, the cells were subjected to OGD for 2 h. As shown in Figure S4C, compared to the OGD + CCL11 group, the expression level of CCR3 protein in astrocytes was significantly reduced in the OGD + CCL11 + SB328437 group. Additionally, relative to the Control+CCL11 group, CCR3 protein expression was markedly elevated in the OGD + CCL11 group. These results indicate that OGD conditions can promote the upregulation of CCR3 protein expression in CCL11‐stimulated astrocytes, whereas the application of SB328437 effectively reverses CCR3 expression under combined OGD and CCL11 treatment. Subsequently, we further examined the effects of CCL11 and SB328437 on the changes in necroptosis‐related proteins in astrocytes following 2 h of OGD. As shown in Figure 6A, WB results revealed that, compared to the OGD + CCL11 group, the protein expression levels of pMLKL, pRIPK1, and pRIPK3 were significantly reduced in the OGD + CCL11 + SB328437 group. IF assays were performed to observe the colocalization of pMLKL and pRIPK3 with astrocytes. The results in Figure 6B demonstrated a marked decrease in the expression levels of pMLKL and pRIPK3 colocalized with astrocytes in the OGD + CCL11 + SB328437 group compared to the OGD + CCL11 group. Furthermore, we assessed the impact of CCL11 and SB328437 on the expression levels of inflammatory proteins in astrocytes after 2 h of OGD. WB analysis in Figure 6C showed that, relative to the OGD + CCL11 group, the expression levels of NLRP3, ASC, and IL‐1β proteins were significantly downregulated in the OGD + CCL11 + SB328437 group.

FIGURE 6.

Cerebral ischemic injury induces CCL11 to activate the CCR3/RIPK3/MLKL signaling pathway, promoting astrocyte necroptosis and inflammatory response. (A) For each cell group, cells were treated with 1 ng/mL CCL11 or 1 ng/mL CCL11 + 100 ng/mL SB328437 for 12 h, followed by OGD2h. Western blot was performed to detect the protein expression of RIPK3, phosphorylated RIPK3 (pRIPK3), RIPK1, phosphorylated RIPK1 (pRIPK1), MLKL, and phosphorylated MLKL (pMLKL) in astrocytes, with subsequent statistical analysis. (B) Immunofluorescence was used to detect the colocalization of necroptosis markers (pMLKL, pRIPK3) and the astrocyte marker (GFAP) in the groups treated with “OGD 2h + 1 ng/ml CCL11” and “OGD 2h + 1 ng/ml CCL11 + 100 ng/ml SB328437”. GFAP (green), p‐MLKL (red), and DAPI (blue). 400×, scale bar = 50 μm. (C) Western blot was conducted to detect the protein expression of neuroinflammatory markers (ASC, IL‐1β, and NLRP3) in astrocytes from the “OGD 2 h + 1 ng/mL CCL11” and “OGD 2 h + 1 ng/mL CCL11 + 100 ng/mL SB328437” groups, with statistical analysis. (D) Each cell group was treated with 1 ng/mL CCL11 or 1 ng/mL CCL11 + 500 ng/mL Nec‐1 for 12 h, followed by OGD for 2 h. Western blot was used to detect the protein expression of necroptosis markers (RIPK3, pRIPK3, RIPK1, pRIPK1, MLKL, pMLKL) in astrocytes, accompanied by statistical analysis. E: Western blot was performed to detect the protein expression of neuroinflammatory markers (ASC, IL‐1β, NLRP3) in astrocytes from the “OGD for 2 h + 1 ng/ml CCL11” and “OGD for 2 h + 1 ng/ml CCL11 + 500 ng/ml Nec‐1” groups, with statistical analysis. (F) ELISA (enzyme‐linked immunosorbent assay) was used to detect the levels of IL‐1β in the astrocyte culture supernatant after inhibition of the CCL11‐CCR3 axis. (G) Each group of astrocytes was treated with 1 ng/mL CCL11 or 1 ng/mL CCL11 + 100 ng/mL SB328437 for 12 h, followed by OGD for 2 h. The cell culture medium was collected, filtered through a 0.45 μm filter, and directly added to primary neurons for 4‐h co‐culture. Immunofluorescence was then used to analyze the colocalization of the neuronal marker (MAP2) with the postsynaptic density (Homer) and presynaptic active zone (Basson). MAP2 (white), Homer (green), and Bassoon (red). 400×, scale bar = 50 μm N = 5; *p < 0.05, **p < 0.01, ***p < 0.001.

As demonstrated above, under the OGD condition, CCL11 can promote necroptosis in astrocytes and the expression of inflammatory factors via CCR3. Subsequently, to verify that CCL11 regulates the RIPK/MLKL signaling pathway through CCR3 to mediate necroptosis in astrocytes, astrocytes were treated with the RIPK1 inhibitor Necrostatin‐1 (Nec‐1). Astrocytes were incubated with 500 ng/mL Nec‐1 for 12 h and then subjected to OGD for 2 h. The WB results are presented in Figure 6D. Compared with the OGD + CCL11 group, the protein expression levels of pMLKL, pRIPK1, and pRIPK3 in astrocytes of the OGD + CCL11 + Nec‐1 group were significantly reduced. Simultaneously, we further investigated the changes in the expression of inflammatory factors in astrocytes following the application of Nec‐1. The WB results are shown in Figure 6E. Compared with the OGD + CCL11 group, the protein expression levels of NLRP3, ASC, and IL‐1β in astrocytes of the OGD + CCL11 + Nec‐1 group were significantly diminished. Given that the reduction of CCR3 protein expression can downregulate the necroptosis events induced by CCL11, we employed WB experiments to detect the expression of CCR3 protein in astrocytes of each group under the influence of Nec‐1. The results are shown in Figure S4D. Compared with the OGD + CCL11 group, there was no significant change in the expression level of CCR3 protein in astrocytes of the OGD + CCL11 + Nec‐1 group. The above results indicate that under OGD conditions, CCL11 can activate the CCR3/RIPK/MLKL signaling pathway, promoting the occurrence of necrotic apoptosis in astrocytes and the expression of inflammatory factors. Finally, to observe whether the activation of the necroptosis signaling pathway in astrocytes by CCL11 under OGD conditions could affect the function of neurons after cerebral ischemia, we first used ELISA experiments to detect the expression level of IL‐1β in the supernatant of astrocyte culture in each group. The results are shown in Figure 6F. Compared with the OGD+ CCL11 group, the expression level of IL‐1β in the culture supernatant of the OGD + 1 ng/mL CCL11 + 100 ng/mL SB328437 group was significantly increased. Subsequently, the culture medium of astrocytes was transferred to primary neurons for co‐culture for 4 h after being stimulated with either OGD2h + 1 ng/mL CCL11 or OGD2h + 1 ng/mL CCL11 + 100 ng/mL SB328437. The changes in the expression levels of presynaptic and postsynaptic proteins in neurons were detected using IF experiments. The results are shown in Figure G. Compared with the OGD + CCL11 intervention group, the expression levels of presynaptic and postsynaptic proteins in neurons in the OGD + CCL11 + SB328437 intervention group were significantly increased. These results indicate that SB328437 can specifically reduce the expression of CCR3 on astrocytes induced by OGD, inhibit the RIPK/MLKL necrotic apoptosis signaling pathway mediated by CCL11, reduce the secretion of pro‐inflammatory cytokines by activated astrocytes, and alleviate the synaptic damage of neurons after cerebral ischemia.

4. Discussion

In stroke research, multiple studies have confirmed that the polarization of astrocytes and chemokine‐mediated infiltration of immune cells are associated with the occurrence and development of IS. However, the specific mechanisms by which chemokines affect astrocytes remain unknown. This study integrates existing research and utilizes proteomics, IF, and WB techniques to discover, for the first time, a novel pathway through which chemokines promote necroptosis of astrocytes following cerebral ischemia. The specific mechanism is that CCL11 stimulates the surface CCR3 receptors on astrocytes, which in turn activate the RIPK1/RIPK3/MLKL pathway, promoting necroptosis and the release of inflammatory factors. Moreover, the inhibition of astrocytic CCL11‐CCR3 significantly improved the extent of neuronal damage after IS (Figure 6).

Previous studies have found that CCL11 primarily plays a role in mediating allergic diseases, including asthma. In recent years, an increasing number of studies have revealed the key role of CCL11 in the central nervous system. After systemic delivery of CCL11 through the circulatory system, microglial cell polarization in the hippocampal region is enhanced, and neurogenesis is impaired [29]. CCL11 has also been found to be significantly upregulated in traumatic brain injury, playing a key role in inhibiting neurogenesis and exacerbating neuroinflammation [24, 30]. Furthermore, the increased infiltration of inflammatory factors promotes cell apoptosis. In our study, we found that CCL11 stimulation induces not only the apoptotic process. After the exogenous administration of CCL11, necroptosis in astrocytes was significantly upregulated. Necroptosis, as a new form of programmed cell death, has been confirmed to occur after stroke [31]. Research shows that necroptosis disrupts the cell membrane through a phosphorylation cascade involving RIPK1/RIPK3/MLKL, leading to the release of DAMPs and inflammatory factors. Neuroinflammation is a key pathological mechanism causing neurologic dysfunction after stroke [32, 33]. Our research findings confirm that, following CCL11 stimulation, the inflammatory factor IL‐1β is upregulated in astrocytes, along with the inflammasome components ASC and NLRP3. By inhibiting RIPK1 with Nec‐1, the necroptosis apoptosis pathway is disrupted, leading to a significant improvement in neuroinflammation. Overall, our results provide strong evidence that CCL11 activates the necroptosis process in astrocytes and exacerbates neuroinflammatory injury.

It is well known that receptor activation permeates various injury pathways in the IS. N‐methyl‐D‐aspartate (NMDA) receptors are excitatory neurotransmitter receptors in the central nervous system. Under physiological conditions, they participate in the formation of synaptic connections between neurons. However, after a stroke, ischemia and hypoxia lead to a significant accumulation of glutamate within cells, resulting in excessive activation of NMDA receptors and triggering excitotoxicity [34]. Similarly, the activation of the TLR family serves as a key driving factor mediating the occurrence and development of poststroke neuroinflammation. Chemokines, a key class of cytokines, primarily exert their effects by binding to specific receptors on the surface of target cells. Previous studies have confirmed that CCR3 is the key receptor through which CCL11 exerts its effects, and our results also corroborate this after IS. We found that CCR3 was significantly upregulated in the damaged area 1 day after cerebral ischemic injury. Interestingly, dynamic expression analysis of CCR3 and CCL11 genes and proteins after stroke revealed that the mRNA levels of both peaked 12 h postischemia. In contrast, their protein levels showed a significant peak within 1–3 days. This suggests that both are likely to play a significant role in the early stages of ischemia. Based on the above results, the CCL11‐CCR3 axis may play a role in mediating necroptosis of astrocytes. To this end, we stimulated astrocytes in vitro with CCL11 and SB328437 separately and observed changes in proteins associated with necroptosis. The results indicated that CCL11 significantly enhanced necroptosis levels after OGD 2 h, while the application of SB328437 notably inhibited necroptosis. In summary, our results demonstrate that astrocytic CCR3 is a key receptor mediating CCL11‐activated necroptosis.

In the frontline clinical treatment of various diseases, receptor blockers have been widely used. It is especially common in cardiovascular diseases and neurological disorders. For instance, beta‐1 adrenergic receptor antagonists represented by Metoprolol have been proven to be the only antiarrhythmic drugs capable of reducing cardiac sudden death [35]. In contrast, multiple receptor antagonist olanzapine has become an important option for the treatment of mental illnesses [36]. Therefore, the feasibility of developing drugs that target receptor antagonism is strong, with a high success rate. Current research has found that inhibitors targeting CCR3 have shown positive therapeutic effects in various diseases. In asthma, CCR3 inhibitors effectively impede the migration and activation of eosinophils to airway inflammatory sites by blocking the CCR3 receptors on their surface, significantly alleviating eosinophilic airway inflammation and airway hyperresponsiveness [37]. In breast cancer, the inhibition of CCR3 reduces CCL11‐mediated cell proliferation and migration, thereby decreasing the ability of breast cancer cells to migrate to the lungs [38]. Our experiments have found that inhibiting CCR3 appears to play a critical role after IS as well. The study conducted comprehensive evaluations of the effects of CCR3 inhibitors on IS injury through behavioral experiments, pathological tests, IF, and WB for molecular function validation both in vivo and in vitro. We found that ischemic mice treated with CCR3 inhibitors exhibited smaller cerebral infarct volumes and improved neurological function. Through in vitro culture of astrocytes, it was found that the application of CCR3 inhibitors blocked the expression of necroptosis proteins and inflammatory factors induced by CCL11. From this, we can conclude that CCL11‐CCR3 significantly improves ischemic brain injury in mice by mitigating the necroptosis of astrocytes and the neuroinflammation it mediates. This discovery reveals for the first time the mechanism by which chemokines regulate programmed cell death in astrocytes.

However, this study still has some limitations. First, we only considered the regulatory effect of the chemokine CCL11‐CCR3 on necroptosis in astrocytes and did not investigate other forms of programmed cell death in depth. Second, our study did not delve into the specific interaction pattern between CCL11 and CCR3. The subsequent research will delve into the macroscopic regulatory role of CCL11‐CCR3 in the programmed cell death of astrocytes. In‐depth exploration of programmed cell death after stroke and its specific mechanisms will promote clinical treatment for IS.

Author Contributions

H.Y., X.Z., M.L., Y.L., YY.Z., Y.Z.: conceptualization, resources, literature survey, writing – original draft, methodology, and visualization. H.C., K.L.: literature survey, methodology, and visualization. G.W., S.Y.: supervision and funding acquisition. G.W., T.Z.: supervision, writing – review and editing, and funding acquisition. The authors declare that they have not used AI‐generated work in this manuscript.

Funding

This work was supported by 黑龙江省科技厅 | Natural Science Foundation of Heilongjiang Province (Heilongjiang Natural Science Foundation) (ZD2022H001) and MOST | National Natural Science Foundation of China (NSFC) (82101409) (32071036).

Ethics Statement

All experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of Harbin Medical University and were carried out in accordance with the guidelines for the care and use of laboratory animals of the National Institutes of Health of the United States. All authors agreed to the final version and to submit the manuscript to this journal.

Consent

All participants obtained written informed consent for publication. Department of Neurobiology, School of Basic Medical Sciences, Harbin Medical University, Harbin (150 081), Heilongjiang Province.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: A–C qPCR analysis of RIPK1 (A) RIPK3 (B) MLKL (C) mRNA levels at different ischemia time points.

Figure S2: A‐F:qPCR analysis of the mRNA levels of CCL1 (A), CCL6 (B), CCL7 (C), CCL8 (D), CCL9 (E), and CCL24 (F) at different ischemic times.

Figure S3A:,B: Cell viability was detected using the CCK8 kit. A‐B: Changes in astrocyte viability were detected under non‐OGD conditions (n‐OGD) and OGD conditions (OGD 2 h) following stimulation with CCL11 at concentrations of 100, 50, 10, 5, 1, 0.5, 0.1, and 0.01 ng/mL for 12 h.

Figure S4A:,B: The CCK‐8 assay was used to screen the effects of SB328437 at concentrations of 100, 50, 10, 5, 1, 0.5, 0.1, and 0.01 ng/mL on the viability of astrocytes. C‐D: The Western blot assay was used to evaluate the CCR3 protein levels in different groups and conduct statistical analysis.

Data S1: Supporting Information.

Yin H., Zhang X., Li M., et al., “The CCL11‐CCR3 Axis Regulates the Aggravation of Neuroinflammation in Astrocytic Necroptosis After Cerebral Ischemia,” The FASEB Journal 40, no. 1 (2026): e71373, 10.1096/fj.202502723RR.

Data Availability Statement

Included in article.