Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

ABSTRACT

Neuroinflammation plays a critical role in cerebral ischemic injury, making it an important therapeutic target for stroke treatment. Ganoderic acids (GAs), the primary bioactive compounds isolated from Ganoderma lucidum, exhibit well‐demonstrated anti‐inflammatory properties. This study aimed to investigate the neuroprotective potential of GAs in the context of ischemic stroke. Mice subjected to transient middle cerebral artery occlusion (tMCAO) served as an in vivo model of focal cerebral ischemia, while LPS‐treated microglial cells were utilized as an in vitro model to evaluate microglial activation. GAs treatment significantly alleviated cerebral ischemic injury, inhibited microglial overactivation, and decreased inflammatory cytokine expression in both in vitro and in vivo models. Mechanistically, eight principal monomers in GAs, particularly GA‐K, were found to target myeloid differentiation protein 2 (MD2), thereby preventing its interaction with Toll‐like receptor 4 (TLR4), and subsequently inhibiting MAPK and NF‐κB pathways. MD2 was found to be overexpressed under ischemic conditions. In MD2‐deficient mice, microglial activation was inhibited, and neuroprotection against ischemic injury was observed, unaffected by GAs. These findings suggest that GAs, particularly GA‐K, provide neuroprotection in ischemic stroke by modulating microglia‐mediated neuroinflammation through MD2, which may serve as a promising therapeutic target for stroke patients.

Ganoderic acids confer neuroprotection in ischemic stroke by directly targeting MD2, thereby blocking MD2/TLR4 complex formation and downstream MAPK and NF‐κB activation. This suppresses microglia‐driven neuroinflammation and mitigates cerebral ischemic injury.

Abbreviations

AP‐1

activator protein‐1

COX‐2

cyclooxygenase 2

ERK

extracellular signal‐regulated kinase

iNOS

inducible nitric oxide synthase

JNK

c‐Jun N‐terminal kinase

LPS

lipopolysaccharide

MAPK

mitogen‐activated protein kinase

MD2

myeloid differentiation factor 2

MyD88

myeloid differentiation primary response protein 88

NF‐κB

nuclear factor‐κB

p38

p38 stress‐activated protein kinase

TLR4

Toll‐like receptor 4

TNF‐α

tumor necrosis factor‐α

TTC

2,3,5‐triphenyltetrazolium chloride

1. Introduction

Ischemic stroke remains a leading cause of mortality and long‐term disability worldwide [1]. Recanalization through thrombolysis or mechanical thrombectomy is considered the standard treatment for acute ischemic stroke [2, 3]. However, the limited therapeutic window for reperfusion and the potential complications arising from ischemia/reperfusion injury prevent many patients from benefiting from these interventions [4]. Microglia‐mediated neuroinflammation plays a pivotal role in acute ischemic brain damage [5, 6]. Following ischemic stroke, microglia are rapidly activated, releasing pro‐inflammatory cytokines in both the injured area and the peri‐infarct zone. This inflammatory response not only exacerbates local injury but also compromises the integrity of the blood–brain barrier (BBB), leading to the infiltration of peripheral immune cells and further secondary damage [7, 8]. Therefore, targeting the microglia‐mediated inflammatory pathway presents a promising therapeutic strategy for ischemic stroke.

Natural products have attracted increasing attention in recent years as potential sources for drug discovery [9, 10]. Numerous studies have highlighted the beneficial effects of specific natural compounds in modulating microglia‐mediated neuroinflammation [11, 12, 13]. Despite their therapeutic potential, these compounds often suffer from limitations such as complex extraction processes, insufficient efficacy, and considerable side effects. Consequently, there is a pressing need to explore new natural substances that could overcome these barriers and provide safer and more effective therapeutic alternatives.

Ganoderic acids (GAs) are bioactive lanostane‐type triterpenoids derived from Ganoderma lucidum, have garnered significant attention due to their diverse pharmacological properties, including immunomodulatory and anti‐inflammatory effects [14, 15]. As secondary metabolites of G. lucidum, GAs have been traditionally used in East Asia for their purported health benefits, such as promoting longevity and improving immune function [16, 17, 18]. Recent studies have shown that GAs can modulate key signaling pathways, such as the TLR4‐mediated MAPK and NF‐κB pathways, which are known to play critical roles in the pathogenesis of ischemic stroke [19, 20, 21]. Furthermore, several individual GA monomers have been reported to exhibit anti‐inflammatory properties in microglial cells, suggesting their potential therapeutic applications in neuroinflammatory diseases [22, 23, 24]. However, whether GAs can offer protection against cerebral ischemic injury remains to be fully elucidated.

In this study, we aimed to evaluate the neuroprotective effects and determine the optimal dosage of GAs and their major bioactive components in a murine model of transient focal cerebral ischemia. The impact of GAs on microglial activation was assessed using both in vivo and in vitro experimental models. Advanced techniques, including surface plasmon resonance (SPR), molecular docking, and protein chip assays, were employed to identify the direct molecular targets responsible for the anti‐inflammatory actions of GAs. Furthermore, the underlying mechanisms were explored through Western blotting, immunoprecipitation, and ELISA. Our results demonstrate that GAs alleviate cerebral ischemia‐reperfusion injury by targeting myeloid differentiation factor 2 (MD2), indicating that GAs hold promise as a potential therapeutic candidate for ischemic stroke‐related cerebral damage.

2. Materials and Methods

2.1. Reagents and Chemicals

The GAs used in this study were kindly provided by Fujian Agriculture and Forestry University. These compounds were extracted from G. lucidum using a method involving hot water extraction followed by alcohol precipitation, as previously described [25]. The isolated GAs included ganoderic acid A (GA‐A), ganoderic acid B (GA‐B), ganoderic acid C2 (GA‐C2), ganoderic acid C6 (GA‐C6), ganoderic acid G (GA‐G), ganoderic acid H (GA‐H), ganoderic acid K (GA‐K), and ganoderenic acid B (GNA‐B), each with a purity exceeding 98%. For in vivo experiments, GAs were initially dissolved in Tween‐80 and subsequently diluted to a 5% concentration with saline. For in vitro assays, GAs and their individual components were prepared by dissolving in dimethyl sulfoxide (DMSO) and subsequently stored at −20°C until future use. These stock solutions were diluted in culture medium before use. Additional reagents, such as 2,3,5‐triphenyltetrazolium chloride (TTC), lipopolysaccharides (LPS), Tween‐80, and DMSO were procured from Sigma Chemical Co. (St. Louis, MO, USA).

2.2. Cell Culture

The BV‐2 murine microglial cell line was acquired from Shanghai FuHeng Biotechnology Co., Ltd. (Shanghai, China). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% (v/v) fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, USA), along with antibiotic–antimycotic solution containing 100 U/mL penicillin and 100 µg mL⁻¹ streptomycin (Gibco, Thermo Fisher Scientific, USA). Cultures were incubated at 37°C in a humidified atmosphere with 5% CO₂.

Primary microglial cells were obtained from the cerebral cortex of neonatal mice (1‐day‐old) using a previously established protocol [26]. Initially, the cortical tissue was carefully dissociated using mechanical disruption with sterile scalpel blades to ensure even fragmentation of the tissue. This was followed by a 15‐min incubation with 0.125% trypsin at 37°C to facilitate the enzymatic digestion of the tissue and release the cells into suspension. Following enzymatic digestion, the cellular suspension was centrifuged at 1000 rpm for 5 min to isolate the dissociated cell aggregates. The cell pellet was then resuspended in a complete culture medium, which included DMEM/F12 supplemented with 10% FBS, penicillin 100 (U/mL), and streptomycin (100 µg mL⁻¹), to ensure optimal cell growth conditions. The resuspended cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Following a 14‐day culture period, the flasks were transferred to a rotary shaker operating at 180 rpm for 6 h to facilitate the separation of non‐adherent cells. After this gentle agitation, the supernatant containing the detached cells was carefully aspirated and transferred to fresh culture flasks for continued culture and further analysis.

2.3. Cell Viability Assay

Cell survival was assessed using the Cell Counting Kit‐8 (CCK‐8) assay (Dojindo, Kumamoto, Kyushu, Japan). BV‐2 microglial cells and primary microglia were seeded in 96‐well plates at a density of 1 × 10⁴ cells per well. Following cell attachment, the cells were separately treated with a range of compounds, including GA, GA‐A, GA‐B, GA‐C2, GA‐C6, GA‐G, GA‐H, GA‐K, or GNA‐B. After 24 h of incubation, the CCK‐8 reagent was added to each well according to the manufacturer's protocol. The plates were subsequently incubated at 37°C for an additional hour to facilitate the colorimetric reaction. Absorbance readings were taken at 450 nm using a microplate reader (BioTek, MQX200, Winooski, VT, USA), providing an accurate assessment of cell metabolic activity, which correlates with cell viability. Control groups, treated with the vehicle alone, were considered to have 100% viability and were used for normalization of the data.

2.4. Western Blot Analysis

Total cellular protein was extracted using RIPA lysis buffer (Applygen, Beijing, China), which was supplemented with a cocktail of protease inhibitor (Roche, Basel, Switzerland) and phosphatase inhibitors (Applygen, Beijing, China) to inhibit enzymatic degradation. Nuclear proteins were isolated through the use of a Minute Cytoplasmic and Nuclear Extraction Kit (NT‐032 and SC‐003, Inventbiotech, MN, USA) following the manufacturer's instructions. Protein concentrations were quantified using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA) to ensure precise normalization of protein loading. Proteins were separated by SDS‐PAGE (Amersham Biosciences, Boston, MA, USA) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non‐fat dry milk in TBST for 2 h at room temperature and incubated overnight at 4°C with primary antibodies against iNOS (1:1000, Cell Signaling Technology), COX‐2 (1:1000, Abclonal), TNF‐α (1:1000, Cell Signaling Technology), ZO‐1 (1:1000, Proteintech), Occludin (1:1000, Proteintech), MD2 (1:1000, Abcam), TLR4 (1:1000, Cell Signaling Technology), p‐ERK1/2 (1:1000, Cell Signaling Technology), ERK1/2 (1:1000, Cell Signaling Technology), p‐JNK (1:1000, Cell Signaling Technology), JNK (1:1000, ABclonal), p‐p38 (1:1000, Cell Signaling Technology), p38 (1:1000 Cell Signaling Technology), NF‐κB (1:1000, Cell Signaling Technology), AP‐1 (1:1000, Abclonal), Lamin B (1:1000, Cell Signaling Technology), GAPDH (1:5000, Proteintech) and β‐actin (1:10,000, Abclonal). After primary antibody incubation, membranes were washed three times with TBST to remove excess antibody. Secondary antibodies included goat anti‐rabbit IgG (1:10,000, EASYBIO) or goat anti‐mouse IgG (1:10,000, EASYBIO), and incubation was performed at room temperature for 1 h. Protein bands were visualized by enhanced chemiluminescence (ECL) using the GeneGnome XRQ System (Syngene, Cambridge, UK) and analyzed quantitatively using densitometric analysis to determine relative protein expression levels. Data were normalized to housekeeping proteins such as β‐actin or Lamin B to ensure consistency across samples.

2.5. Mouse Focal Cerebral Ischemia Model and Drug Treatment

Eight‐week‐old male C57BL/6J mice, weighing 23 ± 1 g, were procured from the Animal Center of Peking University Health Science Center in Beijing, China. In addition, MD2^−/− mice with a C57BL/6 genetic background, kindly provided by Dr. Guang Liang from Wenzhou Medical University in Zhejiang Province, were also included in the study. All mice were acclimatized in the housing facility for a minimum of three days before the commencement of experimental procedures. The mice were maintained in a controlled environment with a 12‐hour light/dark cycle, and food and water were available ad libitum.

To induce focal cerebral ischemia, a transient middle cerebral artery occlusion (tMCAO) model was utilized, as previously described [27, 28]. Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dosage of 80 mg kg⁻¹, and positioned in a supine orientation on a heating pad set to maintain body temperature at 37°C throughout the procedure. A midline incision was made on the neck to expose and dissect the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). Ischemia was induced by introducing a 60‐silicon rubber‐coated nylon monofilament (9 ± 1 mm in length) into the CCA through the ECA, advancing it until it obstructed the origin of the middle cerebral artery (MCA). The right MCA was occluded for 60 min, after which the monofilament was carefully withdrawn to initiate reperfusion. For the sham group, an identical surgical procedure was performed, but without the insertion of the monofilament. GA or GA‐K were administered immediately following the reperfusion phase. A saline‐based solution containing 5% (v/v) Tween‐80 was administered as the vehicle control in parallel experimental groups.

Mice were assigned to nine experimental groups as follows: (1) sham‐operated controls receiving vehicle treatment (n = 40), (2) sham‐operated animals administered GAs at 20 mg kg⁻¹ (n = 10), (3) tMCAO‐operated controls treated with vehicle (n = 40), (4) tMCAO‐operated mice receiving GAs at 1.25 mg kg⁻¹ (n = 15), (5) tMCAO‐operated mice receiving GAs at 5 mg kg⁻¹ (n = 15), (6) tMCAO‐operated mice treated with GAs at 20 mg kg⁻¹ (n = 25), (7) tMCAO‐operated mice receiving GA‐K at 20 mg kg⁻¹ (n = 6), (8) tMCAO‐operated MD2^−/− controls treated with vehicle (n = 40), and (9) tMCAO‐operated MD2^−/− mice administered GAs at 20 mg kg⁻¹ (n = 15).

All procedures involving animals were conducted in strict adherence to institutional ethical guidelines for animal welfare.  The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University Health Science Center (approval number: BCJB0045).

2.6. Neurological Functional Test

To evaluate neurological function following reperfusion, a neurological deficit score system was utilized as previously described [29]. At 24 h post‐reperfusion, all mice underwent a series of neurological tests performed by a single, blinded investigator to ensure unbiased scoring. The scoring criteria were as follows: a score of 0 indicating no observable deficits, 1 for the inability to extend the left forelimb, 2 for consistent circling toward the left, 3 for circling toward the left even at rest, and 4 representing a complete lack of spontaneous movement.

2.7. Assessment of Infarct Volume and Ipsilateral Edema

Following the neurological function assessment, the mice were humanely euthanized with an overdose of anesthetic. The brains were rapidly removed and frozen at −20°C for 10 min to facilitate tissue preservation. Subsequently, five coronal brain slices, each 2 mm thick, were obtained and subjected to 2% TTC staining at 37°C for 10 min. After staining, the sections were fixed overnight in 4% paraformaldehyde. Infarct volumes were then quantified using previously established methodologies [30]. The degree of cerebral edema in the affected hemisphere was quantitatively assessed by calculating the percentage volume difference between the ischemic and non‐ischemic hemispheres, employing the computational formula, ($\frac{\mathrm{ipsilateral}\mathrm{volume}-\mathrm{contralateral}\mathrm{volume}}{\mathrm{contralateral}\mathrm{volume}}$) ×100%.

2.8. Hematoxylin and Eosin (HE) Staining

Mice were euthanized 24 h after reperfusion by administering an overdose of anesthetic. Following confirmation of anesthesia, animals were placed on a surgical platform, and a midline thoracotomy was performed to expose the heart. A 20‐gauge cannula was inserted into the left ventricle, and the right atrium was punctured for drainage. Perfusion was conducted with ice‐cold 0.9% saline (4°C) at a rate of 10 mL/min, using a peristaltic pump (BT100‐3J, Longer Pump, China), followed by 4% paraformaldehyde to fix the tissues. Perfusate was drained by gravity until the outflow was free of blood.

The brains were promptly removed and immersed in 4% paraformaldehyde solution for 12–16 h at 4°C for complete fixation. Following fixation, the tissues were equilibrated in a phosphate‐buffered solution containing 30% sucrose. Once saturated, the brains were sectioned coronally into 8 µm slices using a vibrating microtome (Leica, Wetzlar, Germany). Sections were stained with hematoxylin for 10 min, followed by differentiation in 1% hydrochloric acid ethanol for 5–10 s. After differentiation, sections were immersed in 1% ammonia water for 1–2 min, then rinsed under running water for 3 min. Finally, sections were stained with 1% eosin for 1 min. Histological examination of the stained sections was performed to evaluate morphological changes and tissue integrity.

2.9. Evaluation of BBB Disruption

BBB integrity was assessed by measuring the extravasation of Evans blue (EB) dye [31]. Twenty‐four hours post‐reperfusion, mice were injected with 4 mL/kg of a 5% EB solution via the tail vein and allowed to circulate for 1 h. Following circulation, animals were euthanized with an overdose of anesthesia, and intracardiac perfusion with saline was performed to remove any intravascular dye and prevent pooling within the blood vessels. The brains were coronally sectioned using a precision tissue slicer to obtain five sequential sections, each with a uniform thickness of 2 mm, followed by digital image acquisition. The extent of EB leakage in each section was quantified using an image analysis system, calculating the leakage area (mm²) and multiplying by the section thickness (2 mm) to determine the total area of leakage. The cumulative volume of EB leakage across all brain slices was calculated as the total leakage volume (mm³). To quantify Evans blue extravasation, the ipsilateral cerebral hemisphere was carefully weighed and subjected to tissue processing. The weighed tissue samples were homogenized using a 50% (w/v) trichloroacetic acid solution (IKA, Staufen, Germany), followed by centrifugation at 3000 × g for 15 min at 4°C. The resulting supernatant was collected for spectrophotometric analysis, with absorbance measurements performed at 620 nm wavelength. The concentration of Evans blue dye was calculated based on a standard calibration curve and normalized to tissue weight, expressed as micrograms of dye per gram of wet tissue mass (µg/g tissue).

2.10. Immunofluorescence

Brain tissue sections with a thickness of 8 µm, preserved at low temperatures, were initially treated for 60 min at ambient temperature with a solution comprising phosphate‐buffered saline (PBS), 0.3% Triton X‐100, and 5% normal goat serum to inhibit non‐specific antibody interactions. Subsequently, the sections were exposed to primary antibodies, specifically rabbit‐derived anti‐Iba‐1 (dilution 1:200; Wako, Tokyo, Japan), rabbit‐derived anti‐MD2 (dilution 1:200; Abcam, Cambridge, UK), and maintained at 4°C for approximately 16 h. Following multiple rinses with PBS, the sections were treated with Alexa Fluor 488‐conjugated donkey anti‐rabbit IgG secondary antibody (dilution 1:400; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 561‐conjugated goat anti‐mouse IgG secondary antibody (dilution 1:400; Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 647‐conjugated goat anti‐rat IgG secondary antibody (dilution 1:400; Invitrogen, Carlsbad, CA, USA) to facilitate fluorescence labeling. The density of positive cells was assessed using a high‐resolution confocal microscope (Carl Zeiss, Lsm880 equipped with Airyscan, Zena, Germany), and quantitative analysis was performed utilizing Image‐Pro Plus 6.0 software to determine cellular distribution and intensity.

2.11. Identification and Analysis of Principal Compounds in GAs

GAs were isolated from the extract‐like residue obtained at the bottom of the concentration tank during the water extraction process of G. lucidum. The isolated GAs were dissolved in ethanol, subjected to ultrasonic treatment for 5 min, and then analyzed using ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) [32]. For quantification, an Agilent UPLC system (1290 Infinity II) was coupled with an Agilent 6495 Triple Quadrupole mass spectrometer.

The chromatographic separation of GAs was performed using an Agilent ZORBAX Eclipse Plus C18 column (dimensions: 2.1 × 150 mm; particle size: 1.8 µm), maintained at a constant temperature of 35°C. A gradient elution was performed using a binary mobile phase, consisting of water (mobile phase A) with 0.01% acetic acid and acetonitrile (ACN, mobile phase B). The gradient program was as follows: ≈0–18 min, ≈26%–27% B; ≈18–28 min, ≈27%–35% B; ≈28–31 min, ≈35%–60% B; ≈31–36 min, ≈60%–90% B, ≈36–40 min, ≈90%–100% B. The mass spectrometric analysis was conducted in negative electrospray ionization (ESI) mode, utilizing dynamic multiple‐reaction monitoring for targeted quantification. Instrument parameters were carefully optimized to ensure optimal sensitivity and reproducibility: the drying gas temperature was maintained at 200°C with a flow rate of 16 L/min, while the sheath gas temperature was set to 320°C with a flow rate of 12 L/min. The capillary voltage was adjusted to 3500 V, complemented by a capillary outlet voltage of 380 V and a nozzle voltage of 2000 V. High‐purity nitrogen served as the curtain gas, nebulizer gas, heater gas, and collision gas throughout the analytical process, ensuring stable ionization and efficient fragmentation.

2.12. Elisa Assay

BV‐2 microglial cells were plated in 12‐well culture plates at a density of 5 × 10⁵ cells per well and allowed to adhere for 24 h in standard growth medium. Following this, cells were treated with the following compounds for 1 h: GA at a concentration of 12.5 µg mL⁻¹, and GA‐A, GA‐B, GA‐C2, GA‐C6, GA‐G, GA‐H, GA‐K, and GNA‐B at 25 µM. DMSO was used as the vehicle control. After pretreatment, the cells were exposed to 10 ng mL⁻¹ LPS for 12 h. The concentrations of cytokines TNF‐α and IL‐6 in the conditioned media were quantified using a commercially available ELISA kit (Abclonal, Wuhan, China) in strict accordance with the manufacturer's standardized instructions. The total protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL, USA). Results were normalized to the total protein amount in the cell lysates and expressed as a percentage relative to the LPS‐vehicle control group in the cell‐based experiments.

2.13. Surface Plasmon Resonance Analysis for GA Monomers and MD2 Protein Interaction

To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed. SPR experiments were conducted on a Biacore T200 instrument (GE Healthcare Inc., Piscataway, NJ, USA) utilizing a CM5 sensor chip (GE, #10248879). Prior to loading, the sensor surface was functionalized by activating it with a mixture of N1‐((ethylimino)methylene)‐N3,N3‐dimethylpropane‐1,3‐diamine and N‐hydroxysuccinimide to immobilize rhMD2. GA monomers were diluted to concentrations between 0 and 200 µM in a running buffer containing PBS, 0.05% (v/v) polysorbate 20 (P20), and 5% (v/v) DMSO. The analyte solutions were injected over both reference and target sensor surfaces at a constant flow rate of 30 µL min⁻¹ for 60 s to allow binding, followed by a 60‐s dissociation phase at 25°C. Data were processed by subtracting the baseline signal from both the reference and blank controls, providing sensorgrams for further analysis. Kinetic parameters, including the dissociation constant (KD), were calculated using Biacore T200 evaluation software (Version 3.x) by globally fitting the sensorgram data to a 1:1 Langmuir binding model, which assumes a single binding site interaction between GA monomers and rhMD2 within the tested concentration range.

2.14. Immunoprecipitation Assay to Detecting MD2/TLR4 Complexes

To evaluate the influence of GAs or their monomers on the formation of MD2/TLR4 complexes, co‐immunoprecipitation (Co‐IP) assays were performed. BV‐2 cells (1 × 10⁶) were pretreated with 25 µM of various GA monomers or DMSO for 1 h. Subsequently, cells were stimulated with or without LPS at 10 ng mL⁻¹ for 15 min. Brain tissues were homogenized to prepare protein lysates. The lysates from both BV‐2 cells and brain tissues were incubated overnight at 4°C with an anti‐TLR4 antibody (1 µg per 250 µg total protein; Santa Cruz Biotechnology). The antibody‐protein complexes were then captured using protein A/G plus‐agarose beads (SC‐2003, Santa Cruz Biotechnology) and subjected to incubation at 4°C for a duration of 6 h. Following incubation, the immune complexes were thoroughly rinsed three times with ice‐cold PBS. The bound proteins were then eluted by heating in sample buffer, and the resulting eluates were subjected to Western blot analysis to identify MD2 as a co‐precipitated protein.

2.15. Proteome Microarray Assay

The proteome microarray assay and subsequent data analysis were carried out following previously established protocols [33, 34]. HuProt v4.0 microarrays (Arrayit) were first incubated with a blocking buffer at room temperature for 1 h to prevent non‐specific binding. The biotinylated GA‐A ligand was initially prepared at a concentration of 10 µM by dilution in the identical blocking buffer. The diluted solution was then carefully applied to the microarray surfaces and allowed to incubate for 1 h at room temperature. During this time, the array was gently agitated to promote even interaction between the ligand and the array surface. After incubation, the arrays were rinsed with wash buffer and subsequently treated with Cy3‐labeled streptavidin (diluted 1:1000) for a further 1‐h incubation at room temperature. The microarrays were then spun dry and scanned. Data analysis was performed using GenePix Pro 6.0, with a cutoff for the signal‐to‐noise ratio set at 1.1 to identify significant signals.

2.16. Computational Analysis of GA Monomer Binding to MD2

To explore the molecular interactions between GA monomers and the MD2 protein, computational docking studies were conducted using AutoDock software (version 4.2.6). The three‐dimensional structure of human MD2 (retrieved from the Protein Data Bank, PDB ID: 2E56) served as the receptor model for the docking simulations. Before the docking process, the GA monomer ligand underwent energy minimization and was converted into the PDBQT format to ensure compatibility with the docking software. A grid box with dimensions of 60 × 60 × 60 points and a spacing of 0.375 Å was set up to map the binding site. The AutoGrid module was utilized to generate the affinity maps for the MD2 receptor. The necessary input files for the docking simulations were generated using AutoDockTools (version 1.5.6). The resulting docking outcomes were analyzed by comparing the docking scores and examining the intermolecular interactions between the ligand and the receptor. This analysis adhered to the default parameters set by the software, with special emphasis on analyzing hydrogen bonding interactions and interatomic distances in the protein‐ligand complex.

2.17. Statistical Analysis

All experimental data were processed and analyzed using GraphPad Prism software (version 8.0). For comparisons between two groups, an independent two‐tailed Student's t‐test was applied. In cases involving multiple group comparisons, one‐way analysis of variance was performed, followed by Tukey's post‐hoc test for pairwise comparisons. Data are presented as mean values ± standard error of the mean (SEM). A threshold of p < 0.05 was used to define statistical significance. To ensure reliability, each experiment was repeated in triplicate under identical conditions.

3. Results

3.1. GA Attenuated LPS‐Induced Inflammatory Activation in Microglial Cells

Both the BV‐2 mouse microglial cell line and primary mouse microglial cells were utilized to assess the impact of GAs on neuroinflammation. Initial screening revealed that GAs exhibited no toxicity to either primary mouse microglial cells or BV‐2 cells at concentrations up to 200 µg mL⁻¹ (Figure 1A,D). Subsequently, BV‐2 cells or primary mouse microglial cells were pretreated with or without GA for 1 h before being stimulated with LPS (10 ng mL⁻¹) for a duration of 12 h. It was observed that GA at concentrations of 3.125, 12.5, or 50 µg mL⁻¹ dose‐dependently inhibited LPS‐induced expression levels of iNOS, COX‐2, and TNF‐α in BV‐2 cells. Notably, treatment with 50 µg mL⁻¹ GA resulted in more than a 50% reduction in the expression of these cytokines (Figure 1B,C). Interestingly, similar effects were noted when using GA at a concentration of 50 µg mL⁻¹ on primary mouse microglial cells as well (Figure 1E,F). These findings suggest that GAs possess significant anti‐neuroinflammatory activity within a safe dosage range.

FIGURE 1.

GAs suppress LPS‐induced inflammatory mediator expression in BV‐2 and primary mouse microglial cells. (A) BV‐2 cell viability. (B) Representative Western blots showing the inflammatory mediators iNOS, COX‐2, and TNF‐α in total BV‐2 cell lysates. (C) Quantification of protein expression relative to the control group. (D) Primary microglial cell viability. (E) Representative Western blots of iNOS, COX‐2, and TNF‐α in total lysates of primary mouse microglial cells. (F) Quantification of protein expression relative to the control group. The data are presented as the mean ± SEM (n = 3–6). Statistical significance: ^##P < 0.01, ^###P < 0.001 compared to the control group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared to the LPS‐treated group, NS, no significant difference.

3.2. GA Attenuated Acute Cerebral Ischemic Injury in Mice

To assess the neuroprotective potential of GAs against cerebral ischemic injury, a mouse model of transient middle cerebral artery occlusion (tMCAO) was employed. Mice were subjected to 1 h of occlusion followed by immediate GA administration upon reperfusion. Neurological deficits, infarct volume, and cerebral edema were assessed 24 h post‐reperfusion. In the tMCAO model, mice treated with GA (5 and 20 mg kg⁻¹) demonstrated significantly improved neurological function compared to the vehicle‐treated group (Figure 2A). TTC staining showed that GA significantly reduced cerebral infarct volume in tMCAO mice (Figure 2B,C). As shown in Figure 2D, tMCAO induced severe cerebral edema, increasing the volume of the ischemic hemisphere by 22.5% relative to the sham group. GA treatment at 20 mg kg⁻¹ reduced edema by approximately 62.3%. The results suggest that GAs played a significant neuroprotective role in a dose‐dependent manner against cerebral ischemic injury.

FIGURE 2.

GAs protect against acute cerebral ischemic injury in the mouse tMCAO model. GA (0, 1.25, 5, or 20 mg kg⁻¹, i.p.) was administered immediately after reperfusion. Experiments were conducted 24 h post‐reperfusion. (A) Quantification of neurological deficit scores. (B) Representative coronal brain sections stained with TTC, with typical infarct areas shown in white. Scale bar = 5 mm. (C) Measurement of infarct volume. (D) Assessment of ipsilateral edema percentage. (E) Representative histopathological images of the cerebral cortex and hippocampus following H&E staining (upper panel, magnification ×100; lower panel, magnification ×50). Scale bars = 50 and 100 µm, respectively. (F) Representative images of brain tissues with coronal sections showing BBB leakage at 24 h post‐reperfusion. The blue area indicates Evans blue dye extravasation. Scale bar = 5 mm. (G) Quantification of Evans blue leakage. (H) Volume measurement in the ipsilateral hemisphere of mice. The data are expressed as mean ± SEM (n = 3–8). Statistical significance: ^##P < 0.01, ^###P < 0.001 compared to the sham group; ^*P < 0.05, ^**P < 0.01 compared to the tMCAO group.

To visually observe the effect of GAs on the tissue structure of the cortex and hippocampus in mice, the brain slices were stained with HE (Figure 2E). In the sham group, the neurons were well arranged in a normal pattern in the brain cortex and the dentate gyrus (DG) region of the hippocampus. In contrast, in the tMCAO group, neurons were disarranged, most nuclei became pyknotic, and were surrounded by swollen cells. However, GAs alleviated the disordered arrangement of neurons and kept the number of intact neurons compared to the tMCAO group.

To investigate the potential protective effects of GAs on BBB function, EB extravasation was measured 24 h post‐reperfusion as an indicator of BBB integrity. The results showed significant EB extravasation in tMCAO mice compared to the sham group, indicating severe disruption of the BBB after ischemic stroke (Figure 2F). GA treatment dose‐dependently reduced both EB leakage content (Figure 2G) and volume (Figure 2H) in brain tissue after ischemic stroke. We further assessed the effects of GAs on the expression of tight junction proteins ZO‐1 and Occludin in ischemic brain microvascular tissue following ischemic stroke, as these proteins are pivotal in maintaining the integrity of the BBB after such events. As demonstrated in Figure S2, Supporting Information, the expression levels of ZO‐1 and Occludin were significantly reduced in the tMCAO group compared to the sham‐operated controls, indicating compromised BBB function. Remarkably, GA treatment led to a significant upregulation of both ZO‐1 and Occludin expression in the tMCAO group. These findings suggest that GA exerts a protective effect on BBB integrity, potentially mitigating early‐stage ischemic stroke‐induced disruption of the BBB.

3.3. GA Suppressed Excessive Activation of Microglia and Reduced Inflammation in the Brain of Ischemic Mice

To determine whether GAs also possess anti‐neuroinflammation effect in vivo, microglia specific marker Iba‐1 was examined in the peri‐infarct area of cortex (Figure 3A) and the DG area of the hippocampus (Figure 3B) at 24 h after reperfusion. Immunohistochemistry staining clearly showed that only a few Iba‐1 positive cells in the cerebral cortex and hippocampus and microglia cells were at a resting state in the sham group. In contrast, in the tMCAO group, the number of Iba‐1 positive cells strikingly increased in the ipsilateral peri‐infarct cerebral cortex and hippocampus and microglia cells were activated, which was characterized by an increased arborization. Administration of GAs at a dosage of 20 mg kg⁻¹ markedly decreased the number of Iba‐1‐positive cells in both the cortical ischemic penumbra and hippocampal regions of tMCAO mice.

FIGURE 3.

GAs inhibit microglial activation and inflammatory mediator expression in mice post‐tMCAO. GAs (0 or 20 mg kg⁻¹, i.p.) were administered at the same time after reperfusion. (A) Representative micrographs (upper panels, magnification of ×100, scale bars: 100 µm; lower panels, magnified views of the rectangular regions in the upper panels, scale bar: 50 µm) showing Iba‐1 (green) immunofluorescence and quantification (right) of Iba‐1 positive cells in the peri‐infarct region of the cortex at 24 h post‐reperfusion. (B) Representative micrographs (upper panels, magnification ×100, scale bar: 100 µm; lower panels, magnified views of the rectangular regions in the upper panels, scale bar: 50 µm) showing Iba‐1 (green) immunofluorescence and quantification (right) of Iba‐1 positive cells in the DG region of the hippocampus at 24 h post‐reperfusion. (C) Representative Western blot images of iNOS, COX‐2, and TNF‐α in the cortical penumbra at 24 h after reperfusion. (D) Quantification of iNOS, COX‐2, and TNF‐α protein expression. The data are presented as the mean ± SEM (n = 3–5). Statistical significance: ^###P < 0.001 compared to the sham group; ^**P < 0.01 compared to the tMCAO group.

Microglia activation can continually release various inflammatory mediators such as iNOS, COX‐2 and TNF‐α. Western blotting analysis showed that the protein expressions of iNOS, COX‐2, and TNF‐α, were significantly increased in the injured cortical tissue of tMCAO mice, which were remarkably attenuated by GA treatment (Figure 3C,D). These results suggest that GAs can suppress microglia‐mediated neuroinflammation in the brain after ischemic stroke.

3.4. GA Monomers Suppress Pro‐Inflammatory Cytokine Release in LPS‐Induced Microglial Cells

To determine which components in GA play main anti‐neuroinflammatory and neuroprotective roles, we first analyzed the composition of GAs and then isolated principal compounds from GAs to examine their anti‐inflammatory activities in LPS‐induced microglial cells by ELISA. According to HPLC and UPLC‐MS/MS analysis, 19 compounds were identified from GAs (Figure 4 and Table 1).

FIGURE 4.

Identification of composition of GA by HPLC.

TABLE 1.

GA monomers identified by UPLC‐MS/MS.

No.	Name	Concentration (ng mL−1)	Mass content (mg g−1)	Proportion (%)
1	Ganoderic acid G	1573.92	108.55	10.85
2	Ganoderic acid H	1014.27	69.95	6.99
3	Ganoderic acid B	973.09	67.11	6.71
4	Ganoderic acid C2	786.84	54.26	5.43
5	Ganoderic acid K	600.57	41.42	4.14
6	Ganoderic acid A	556.23	38.36	3.84
7	Ganoderic acid C6	505.27	34.85	3.48
8	Ganoderenic acid B	491.28	33.88	3.39
9	Ganoderenic acid A	197.9	13.65	1.36
10	Ganoderenic acid C	79.18	5.46	0.55
11	Ganoderic acid N	30.42	2.1	0.21
12	Ganoderic acid D	30.29	2.09	0.21
13	Ganoderic acid I	22.03	1.52	0.15
14	Ganoderenic acid H	18.7	1.29	0.13
15	Ganoderenic acid D	16.43	1.13	0.11
16	Ganoderic acid F	16.15	1.11	0.11
17	3‐o‐Acetyl‐16a‐hydroxytrametenolic acid	12.06	0.83	0.08
18	Lucidenic acid A	11.79	0.81	0.08
19	Ganoderic acid DM	5.51	0.38	0.04

The compounds with a ratio higher than 2% in GAs included ganoderic acid A (GA‐A), ganoderic acid B (GA‐B), ganoderic acid C2 (GA‐C2), ganoderic acid C6 (GA‐C6), ganoderic acid G (GA‐G), ganoderic acid H (GA‐H), ganoderic acid K (GA‐K), and ganoderenic acid B (GNA‐B) (Figure 5A). Subsequently, we assessed their cytotoxicity in BV‐2 cells utilizing the CCK8 method. As illustrated in Figure S1, Supporting Information, all GA monomers at concentrations ranging from 12.5 to 100 µM exhibited no cytotoxic effects. BV‐2 cells were pretreated with either 25 µM of various GA monomers or 12.5 µg mL⁻¹ of GA (approximately equivalent to 25 µM) for 1 h before being exposed to LPS at a concentration of 10 ng mL⁻¹ for 12 h. Previously identified anti‐inflammatory GA mixtures served as positive controls. The results indicated that LPS significantly enhanced the production of both TNF‐α (Figure 5B) and IL‐6 (Figure 5C) and all GA monomers pretreatment significantly decreased LPS‐induced cytokine production. Among these compounds, GA‐A inhibited both TNF‐α and IL‐6 by nearly 50%, while GA‐G and GA‐K demonstrated over a 50% reduction in TNF‐α release. Additionally, both GA‐C2 and GA‐K exhibited high inhibitory rates against IL‐6 release. Notably, GA‐K achieved more than a 50% inhibition of both TNF‐α and IL‐6 release, showcasing the most pronounced inhibitory effect among the eight examined GA monomers.

FIGURE 5.

Main compounds in GA and their anti‐inflammatory effects in LPS‐ stimulated BV‐2 cells. (A) Chemical structures of Ganoderic acid A, B, C2, C6, G, H, K, and Ganoderenic acid B. (B) TNF‐α levels. (C) IL‐6 levels. The data are expressed as the mean ± SEM (n = 6). Statistical significance: ^###P < 0.001 compared to the control group; ^**P < 0.01, ^***P < 0.001 compared to the LPS‐induced group.

3.5. GA Monomers Directly Bound to MD2 and Inhibited MD2/TLR4 Complex Formation and Signaling Cascade in LPS‐Induced Microglia Cells

To identify the molecular mechanism by which GAs suppress inflammatory response in microglial cells, we screened for potential GA monomers binding proteins. GAs show structural similarities to zhankuic acid A isolated from Taiwanofungus camphoratus, which is a specific inhibitor of MD2/TLR4, inducing anti‐inflammatory properties [35]. We therefore hypothesized that the anti‐inflammatory ability of GA was exerted by selectively targeting MD2 and inhibiting the TLR4 signaling pathway. To test this, we first examined the direct interaction of GA monomers with recombinant MD2 proteins by SPR analysis. The results showed that eight purified GA monomers bound to recombinant MD2 proteins (Figure 6A–H), especially GA‐K, GA‐G, and GA‐A with K_d values of 0.47, 1.20, and 4.74 µM, respectively.

FIGURE 6.

Interaction of GA monomers with the MD2/TLR4 complex. (A–H) Surface plasmon resonance (SPR) analysis showing direct binding of Ganoderic acid A, B, C2, C6, G, H, K, and Ganoderenic acid B to MD2. (I) Binding of GA‐A to MD2 as assessed by protein microarray analysis. (J) Identification of MD2/TLR4 complexes via immunoprecipitation.

GA‐A, as one of the main compounds in GA, has demonstrated potential biological effects in combating inflammation and infectious diseases. [36, 37]. To further confirm MD2 as the target of GA monomers, we used biotin‐labeled GA‐A (Bio‐GA‐A, Figure 6I) and assayed for the binding of GA‐A to recombinant proteins fabricated on HuProtTM human protein using Cy3‐streptavidin (Cy3‐SA). The experimental results confirmed that GA‐A could directly interact with MD2.

Next, we wanted to verify the interaction between GA monomers and MD2 through functional assays. BV‐2 cells were exposed to LPS, which caused the formation of MD2‐TLR4‐LPS complex (Figure 6J). The addition of eight different GA monomers could suppress this complex formation. Interestingly, the results are consistent with SPR analysis and anti‐inflammatory effect assays, GA‐K, GA‐G, and GA‐A showed excellent inhibitory effect among eight GA monomers (Figure 6J).

Since LPS activates TLR4 to trigger the release of pro‐inflammatory cytokines via MAPK/AP‐1 and NF‐κB pathway activation, we evaluated the impact of GA on these signaling mediators. The results indicated that LPS stimulation significantly elevated the phosphorylation of ERK1/2, JNK, and P38 in whole‐cell lysates (Figure 7A,B), as well as the activation of NF‐κB and AP‐1 in the nuclear fraction (Figure 7C,D). However, LPS‐induced MAPKs/AP‐1 and NF‐κB signaling activation were significantly blocked by GAs. These results indicate that GA monomers play anti‐neuroinflammatory effects associated with directly binding to MD2 and inhibiting MD2/TLR4 complex formation.

FIGURE 7.

GAs inhibit MAPK and NF‐κB signaling activation in LPS‐induced BV‐2 microglial cells. BV‐2 cells were pretreated with or without GA for 1 h, followed by stimulation with LPS (10 ng mL⁻¹) for 12 h. (A) Representative Western blot images showing proteins involved in the MAPK signaling pathway from total cell lysates of BV‐2 cells. (B) Quantification of phosphorylation levels. (C) Representative Western blots of NF‐κB and AP‐1 expression in the nuclear fraction. (D) Quantification of protein expression levels. The data are expressed as mean ± SEM (n = 4). Statistical significance: ^##p < 0.01, ^###p < 0.001 compared to the control group; ^**p < 0.01 compared to the LPS‐treated group.

3.6. GA‐K With Strong Affinity to MD2 Attenuated Cerebral Ischemic Injury in Mice

To investigate the underlying structural mechanism of GA monomer‐MD2 interaction, we used a molecular docking assay. As shown in Figure 8A and Figure S3, Supporting Information, GA monomer molecule was buried inside the lipid binding pocket of MD2 and interacted with the inside hydrophobic residues ARG‐90. Consistent with SPR analysis and co‐immunoprecipitation assays, molecular docking analysis also showed that GA‐K had the highest docking score among eight GA monomers (Table 2).

FIGURE 8.

Molecular docking of GA‐K with MD2 and its effect on cerebral ischemic injury in the mouse tMCAO model. (A) Molecular docking of GA‐K (yellow) with the MD2 protein (green), analyzed using the Trips molecular modeling software. (B) Representative coronal brain sections stained with TTC, showing typical infarct areas in white. Scale bar = 5 mm. (C) Quantification of infarct volume. (D) Neurological deficit scores quantification. The data are presented as the mean ± SEM (n = 8). Statistical significance: ^**p < 0.01 compared to the tMCAO group.

TABLE 2.

Docking score of GA monomers in the active site pocket of MD2.

No.	Name	Docking score
1	Ganoderic acid K	−8.206
2	Ganoderic acid G	−7.897
3	Ganoderic acid B	−7.418
4	Ganoderic acid A	−7.393
5	Ganoderic acid C6	−7.364
6	Ganoderenic acid B	−7.076
7	Ganoderic acid C2	−7.065
8	Ganoderic acid H	−5.624

To further evaluate whether GA‐K was able to attenuate cerebral ischemic injury, mice were subjected to tMCAO and then administered with GA‐K (20 mg kg⁻¹). TTC staining showed that GA‐K significantly reduced the infarct volume compared with the vehicle‐treated tMCAO group (Figure 8B,C) and the neurological deficit score also showed that GA‐K treated mice exhibited significantly less neurological dysfunction than those treated with vehicle control (Figure 8D). These results suggest that, as the monomer with the strongest affinity to MD2 in GA, GA‐K is an effective component of GA.

3.7. GA Prevented MD2/TLR4 Complex Formation and Downregulated the MAPK and NF‐κB Signaling Pathways in the Brain of Ischemic Mice

Since GAs inhibited the formation of the MD2/TLR4 complex in cultured microglia cells, the same action was expected in lysates prepared from brain tissues. Co‐immunoprecipitation revealed that the amounts of MD2/TLR4 complex were significantly increased in the cortical penumbra after ischemia attack (Figure 9A,B). While GA treatment blocked the interaction between TLR4 and MD2 in the brain after ischemia onset.

FIGURE 9.

GAs suppress MD2/TLR4 complex formation and inhibit MAPK and NF‐κB signaling pathways in a mouse model of tMCAO. GA (administered at doses of 0 or 20 mg kg⁻¹, i.p.) was given immediately after reperfusion. At 24 h post‐reperfusion, total and nuclear proteins were isolated from the cortical penumbra for analysis by Western blotting. (A) Immunoprecipitation analysis of the MD2/TLR4 complex in the ischemic hemisphere. (B) Quantification of MD2 expression levels. (C) Representative Western blot images showing proteins involved in the MAPK signaling pathway. (D) Quantitative analysis of phosphorylation levels. (E) Representative Western blot images of nuclear NF‐κB and AP‐1. (F) Quantification of protein expression. The data are presented as the mean ± SEM (n = 4). Statistical significance is indicated as follows: ^###p < 0.001 compared to the sham group, ^*p < 0.05, ^**p < 0.01 compared to the vehicle‐treated tMCAO group.

Next, we tested the effect of GA on the MAPK/AP‐1 and NF‐κB pathways in vivo. Western blot analysis revealed that the phosphorylations of ERK1/2, JNK, and P38 were increased in the vehicle‐treated tMCAO mice, while GAs remarkably reduced ischemia‐induced phosphorylation of these MAPK signal proteins, respectively (Figure 9C,D). Moreover, we also confirmed that GAs significantly suppressed NF‐κB and AP‐1 expression in the nucleus compared with the vehicle‐treated tMCAO group (Figure 9E,F). These in vivo results are in accordance with the in vitro experimental data. The results obtained so far suggest that MD2 plays a critical role in the progression of stroke.

3.8. MD2 Knockout Ameliorated Microglia Activation and Cerebral Ischemic Injury and Neuroprotective Effects of GA Dependent on MD2

To confirm the in vivo role of MD2 as a key target protecting the brain from cerebral ischemic injury. We evaluated the protein expression of MD2 in the brains of mice subjected to ischemic stroke by immunofluorescence staining. The results showed that, compared with the sham group, the MD2 protein expression was significantly increased in the cortical ischemic penumbra and hippocampus after ischemia attack (Figure 10A).

FIGURE 10.

MD2 knockout reduces microglia activation and improves acute cerebral ischemic injury in the tMCAO mouse model. (A) Representative micrographs (magnification ×100) showing immunofluorescent staining of MD2 (red) in the peri‐infarct area of the cortex and the dentate gyrus of the hippocampus, 24 h after reperfusion. Scale bars: 50 µm. WT and MD2‐KO mice underwent 1 h of tMCAO, followed by 24 h of reperfusion. GA (0 or 20 mg kg⁻¹, i.p.) was administered immediately post‐reperfusion. (B) Representative micrographs depicting immunofluorescence for Iba‐1 (green). Primary microglial cells were isolated from WT and MD2‐KO mice, pretreated with GA (50 µg mL⁻¹) or vehicle for 1 h, then stimulated with LPS (10 ng mL⁻¹) for 12 h. (C) Representative Western blots illustrating levels of p‐JNK, p‐ERK, p‐P38, and p‐NF‐κB. (D) Representative Western blots for inflammatory mediators iNOS, COX‐2, and TNF‐α (n = 4). (E) Representative coronal brain sections stained with TTC. Infarct areas appear white. Bar = 5 mm. (F) Infarction volume assessment. (G) Neurological deficit score quantification. The data are presented as the mean ± SEM (n = 8). Statistical significance is indicated as follows: ^{* *}P < 0.01, ^{** *}P < 0.001 compared to the WT tMCAO group.

Increased levels of MD2 in the brain tissue of mice with cerebral ischemic injury prompted us to examine the effect of MD2 deficiency in this process. MD2 knockout mice were subjected to tMCAO. Immunofluorescence staining clearly showed that less Iba‐1 positive cells in the cerebral cortex and hippocampus in MD2 knockout tMCAO mice than in wild‐type tMCAO mice (Figure 10B), which indicates that MD2 knockout suppressed the activation of microglia in mice after stroke onset. To examine the contribution of microglia MD2 in neuroinflammation, we isolated primary mouse microglia cells from wild‐type and MD2 knockout mice. Similarly, Western blot analysis revealed that microglia MD2 deficiency reduced phosphorylation of ERK1/2, JNK, P38, and NF‐κB (Figure 10C) and expression of inflammatory factors iNOS, COX‐2, and TNF‐α (Figure 10D) in the LPS‐induced inflammation model. Moreover, TTC staining and neurological deficit score results also showed that a smaller infarct volume (Figure 10E,F) and less neurological dysfunction (Figure 10G) in MD2 knockout mice than in wild‐type mice. These results strongly support that MD2 is critical in acute cerebral ischemia‐induced neuroinflammation and brain injury.

To further confirm whether the neuroprotective effects of GAs were mediated by MD2, we treated MD2‐deficient mice and primary microglia with GAs. Immunofluorescence staining revealed that GAs could not further reduce Iba‐1‐positive cells in the cerebral cortex and hippocampus in MD2 knockout tMCAO mice (Figure 10B). While GA treatment was unable to further enhance the inhibitory effect of MD2 knockout on neuroinflammation and the protective effects on cerebral ischemic injury (Figure 10C–G). These results clearly demonstrate that GAs exhibited anti‐inflammatory and neuroprotective effects on ischemic stroke dependent on MD2.

4. Discussion

Ischemic stroke is a severe cerebrovascular disorder characterized by the rapid onset of symptoms, including muscle weakness, mental confusion, loss of balance or coordination, sudden fainting, paralysis, and other significant manifestations [28]. Despite considerable efforts to address ischemic stroke, only recombinant tissue plasminogen activator (r‐tPA) has received approval from the U.S. Food and Drug Administration (FDA) for its treatment [38]. In China, numerous traditional Chinese medicines, such as G. lucidum, have been extensively utilized to mitigate brain injury [39]. However, the active components and underlying mechanisms of action remain poorly understood. In this study, we present evidence for the first time that GA, identified as a class of natural antagonists of MD2, exhibits neuroprotective effects against acute cerebral ischemic injury. Our data indicate that GAs, particularly GA‐K, can specifically interact with MD2. This interaction reduces the formation of the MD2/TLR4 complex and downstream activation of MAPK and NF‐κB pathways. Consequently, this leads to a decrease in inflammatory mediator production and inhibits excessive microglial activation. Furthermore, our findings suggest that MD2 plays a critical role in the progression of ischemic stroke (Figure 11).

FIGURE 11.

Proposed mechanism of GA in alleviating cerebral ischemic injury. GA monomers interact directly with MD2, preventing the dimerization of MD2 and TLR4, as well as the subsequent activation of downstream MAPK and NF‐κB signaling pathways. This process lowers inflammatory mediator production and reduces microglial overactivation.

Neuroinflammation plays a crucial role in the progression of brain ischemic injury [40]. Microglial cells serve as the initial immune responders in the detection of ischemic stroke, and their activation leads to the release of numerous pro‐inflammatory mediators, including iNOS, COX‐2, TNF‐α, and IL‐6. These substances contribute to the disruption of the BBB [41], which facilitates the infiltration of peripheral immune cells. This, in turn, exacerbates inflammation and results in vasogenic edema, further worsening disabilities. This inflammatory cascade represents a fundamental pathological mechanism underlying cerebral ischemic injury [27, 42]. Consequently, identifying effective drugs to control these harmful inflammatory responses could offer a promising therapeutic strategy for ischemic stroke. Numerous natural products were reported to exhibit significant anti‐neuroinflammatory effects. However, many of them have toxicity at therapeutic doses with a narrow safety margin [43, 44, 45, 46]. GAs, a primary bioactive component derived from G. lucidum, have emerged as a promising candidate. Studies indicate that GA monomers, such as GA‐A and deacetyl GA‐F, can inhibit LPS‐activated microglia in vitro, suggesting that GAs may have therapeutic potential for ischemic stroke [23, 24, 47]. To date, there has been no document concerning the toxicity of GAs. In this study, we demonstrated that GAs significantly reduced the activation of microglia in mice following acute cerebral ischemic injury while simultaneously inhibiting the production of inflammatory factors both in vivo and in vitro. Given that microglial polarization into M1/M2 phenotypes critically regulates neuroinflammation and tissue repair after ischemia, we evaluated the effect of GAs on this process. We found that GAs treatment reduced the proportion of M1 microglia in both the cortical and hippocampal regions post‐ischemia, without affecting the proportion of M2 microglia (Figure S3, Supporting Information). Furthermore, GAs provided dose‐dependent protection against acute cerebral ischemic injury, with effective doses ranging from 1.25 to 20 mg kg⁻¹. These findings highlight GAs as a potent therapeutic agent for the treatment of ischemic stroke.

Both in clinical studies and rodent stroke model, abundant microglia are immediately activated by detecting the release of different damage‐associated molecular patterns, via their receptors Toll‐like receptor 4 (TLR4) [48]. This ligand‐receptor link leads to the activation of MAPK and NF‐κB signaling pathways, promoting production of inflammatory cytokines [49]. Several previous studies showed that GAs exhibit multiple pharmacological effects in various disease models, though altering TLR4 mediated MAPK/NF‐κB pathways. For example, GAs retarded the 5‐FU‐induced central fatigue‐like behavior, accompanied by down‐regulating inflammation cytokines’ expression in the hippocampus through inhibiting TLR4/Myd88/NF‐κB pathway [19]. In a mouse model of autosomal dominant polycystic kidney disease, GA treatment was found to significantly attenuate renal cystogenesis through dual molecular mechanisms: suppression of Ras/MAPK signaling pathway activity and enhancement of cellular differentiation processes [14]. Similar results were obtained when GAs exerted an anti‐atherosclerosis effect [20]. Our findings suggest that GAs exert regulatory effects on both MAPK and NF‐κB signaling cascades in the cortical regions surrounding ischemic lesions in mouse models of cerebral ischemia, as well as in LPS‐stimulated BV‐2 microglial cells. These molecular mechanisms provide substantial evidence supporting GA's therapeutic potential in mitigating neuroinflammatory responses following ischemic brain injury. However, there is limited information regarding the key target protein through which GAs exerts its effects. In this study, we employed SPR, protein microarray analysis, and computational docking to identify that GA monomers can inhibit TLR4‐specific accessory protein MD2. This inhibition serves as a mechanism for blocking the MAPK and NF‐κB pathways. These findings may also partially elucidate the therapeutic mechanisms by which GA operates across various disease models.

Furthermore, we investigated the potential mechanism by which GA monomers bind to MD2 through molecular docking studies. The results indicated that GA monomers are deeply embedded within the hydrophobic pocket of MD2. Notably, Arg‐90 appears to be a critical residue involved in the interaction between GA monomers and MD2. The natural compound shikonin has been shown to modulate inflammatory responses through specific molecular interactions with the Arg‐90 residue of MD2 [50]. Additionally, structural analyses of chalcone derivatives (L6H21, L6H20, and L2H21) have revealed a conserved binding pattern involving both Arg‐90 and Tyr‐102 residues within the MD2 binding pocket [51, 52, 53]. The consistent involvement of Arg‐90 in these molecular recognition events highlights its critical role in MD2‐ligand interactions, suggesting that this residue represents a key structural determinant for rational drug design of MD2‐specific inhibitors.

MD2, which interacts with the extracellular domain of TLR4, was initially recognized as essential for LPS to activate the TLR4 signaling pathway [54]. MD2 plays a pivotal role not only in LPS‐mediated inflammatory responses but also in the recognition of non‐microbial ligands, including saturated fatty acids, oxidized LDL, high‐mobility group protein 1 (HMGB1), and heat shock proteins, among others [55, 56, 57, 58]. In recent years, numerous studies have explored the therapeutic potential of MD2 antagonists across various autoimmune and inflammatory diseases. However, there is a paucity of research evaluating the impact of MD2 antagonists on neuroinflammation‐related disorders [59]. Our study revealed that MD2 was significantly overexpressed in the brain following cerebral ischemic injury. The knockout of MD2 inhibited microglia‐induced neuroinflammation and mitigated ischemia‐induced brain damage. Furthermore, GAs demonstrated potential for providing neuroprotection post‐ischemia by reducing the formation of the MD2‐TLR4 complex in the brain after focal cerebral ischemia. These findings suggest that MD2 may serve as a promising drug target for the treatment of ischemic stroke.

To clarify the structural requirement for MD2 inhibition, eight GA monomers were isolated and purified from GAs. They showed different affinities to MD2 and different anti‐inflammatory activities. The analysis of structure and activity revealed that the carboxyl group in their side chain is essential, as it has the capacity to form hydrogen bonds and salt bridges with Arg‐90 of MD2. GNA‐B showed the weakest anti‐inflammatory activity among eight monomers, which hints that the chirality at C6 of the side chain might be necessary. The conjugative effect due to the unsaturated ketone fragment weakened the flexibility of the side chain of GNA‐B, consequently increasing the thermodynamic instability of binding to MD2. It was found that monomers which R2 position replaced with carbonyl group (GA‐C6 and GA‐H) showed lower affinity to MD2 and lower anti‐inflammatory activity than those which R2 position was β‐OH (GA‐G, GA‐K, GA‐B). Furthermore, among GA‐B (R1, R2 = β‐OH, R3 = H), GA‐C2 (R1, R3 = β‐OH, R2 = H), and GA‐G (R1, R2, R3 = β‐OH), GA‐G was the most efficient to inhibit inflammatory responses, which implied introducing hydrogen bond acceptors (OH) might enhance GA monomers’ affinity to MD2 and anti‐inflammatory effect. Most notably, GA‐K (R1, R2 = β‐OH, R3 = β‐OAc) showed the most stable binding state to MD2 (docking score = −8.206, ranked first), the strongest affinity, and the highest anti‐inflammatory activity among eight monomers. The reason might be improved lipophilic property by inducing Ac moiety into R3 position (PSA: 158.1 vs. 152.03; TPAS: 798.76 vs. 747.27, predicted by Schrodinger 2018 qikprop).

Our data indicate that compounds in GAs exhibiting high affinity for MD2 and low docking scores demonstrate significant anti‐inflammatory activity, such as GA‐K, GA‐G, and GA‐B. Conversely, compounds with low affinity for MD2 and high docking scores exhibit minimal anti‐inflammatory effects, including GNA‐B, GA‐H, and GA‐C6. Notably, among all the tested compounds, GA‐K possesses the highest docking score along with the strongest affinity for MD2 and exhibits superior anti‐inflammatory activity. These findings further reinforce the notion that the anti‐inflammatory effect of GA‐K is linked to its interaction with MD2. Furthermore, they suggest that GA‐K serves as a potent inhibitor of MD2 and holds promise for development as a therapeutic agent aimed at protecting against cerebral ischemic injury.

There are several limitations in the present study. As a potential drug target for stroke treatment, while MD2, in conjunction with TLR4, is primarily expressed in microglia, this complex formation also occurs in astrocytes, oligodendrocytes, neurons, and endothelial cells within the central nervous system [60]. In this study, we have not explored the role of MD2 in cell types other than microglia following cerebral ischemic injury. Further investigations are necessary to elucidate the role of MD2 in ischemic stroke. Another limitation of our research is that MD2 may not be the sole target of GAs for treating acute ischemic stroke, GAs could potentially interact with multiple target proteins. Consequently, it remains to be determined whether GAs participate in other signaling pathways to exert their neuroprotective effects following a stroke. Finally, to validate our preclinical findings, further investigation into the anti‐inflammatory effects of GA should be conducted using immune cells from primary stroke patients.

5. Conclusion

In summary, our findings indicate that the natural product GA provides protection against cerebral ischemic injury in the mouse tMCAO model. Molecular mechanism studies demonstrated that GA monomers directly interact with the hydrophobic pocket of MD2, thereby inhibiting the formation and activation of the MD2/TLR4 complex and subsequently blocking downstream MAPK and NF‐κB pathways. This action effectively suppresses neuroinflammation. Our study suggests that GA or its monomer, GA‐K, may be developed as potential therapeutic agents for ischemic stroke by targeting MD2.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: exp270131‐sup‐0001‐SuppMat.pdf.

Supporting File 2: exp270131‐sup‐0002‐FiguresS1‐S3.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.