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.

Keywords: Toll‐like receptor 4; ganoderic acid; ischemic stroke; microglia; myeloid differentiation protein 2; neuroinflammation.

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.

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.

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.

FIGURE 4

Identification of composition of GA by HPLC.

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.

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.

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.

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.

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.

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.

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.