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Objective <p>This study aimed to investigate the molecular mechanism and regulatory effect by which lentinan (LNT) alleviates autoimmune myocarditis via suppressing CD40 expression and GSDMD-mediated cardiomyocyte pyroptosis.</p> Methods <p>Fifty 8-week-old male BALB/c mice were randomly divided into 5 groups (n = 10): Control, experimental autoimmune myocarditis (EAM) model, EAM + low-dose LNT (5 mg/kg), EAM + high-dose LNT (20 mg/kg), and EAM + dexamethasone (DEX, 2.5 mg/kg). EAM was established by immunization with cardiac myosin on D0 and D7 using ‌α-MyHC. LNT and DEX were administered intraperitoneally from D8. Additionally, 40 CD40fl/flLysMCre+/− (CD40mye−KO) mice and 16 CD40WT littermates were used. CD40mye−KO mice were divided into 4 groups (n = 10): CD40mye−KO, CD40mye−KO EAM model, CD40mye−KO EAM model + High-dose LNT (20 mg/kg), CD40mye−KO EAM model + DEX (2.5 mg/kg). CD40WT mice divided into 2 groups (n = 10): CD40WT, CD40WT EAM model. The HW/BW ratio was measured, and LVEF, LVFS, LVESD, and LVEDD were assessed by echocardiography. Cardiac histopathological changes were evaluated using inflammatory scoring after H&amp;E staining. Bone marrow-derived macrophages were pre-treated with 0.1 µg/mL LPS to induce cell damage, followed by treatment with 5 µg/mL LNT, 20 µg/mL LNT, 100 nM DEX, or 100 ng/mL CD40 activator (anti-mouse CD40 antibody fgk4.5/fgk45) for 24 h. Primary mouse cardiomyocytes were isolated from hearts, and supernatants from pyroptotic macrophages were used to treat cardiomyocytes for 24 h. Cell viability was measured using CCK-8 assay. LDH content in culture supernatant was detected to evaluate cell membrane damage. Serum and cell culture supernatants were analyzed for cTnT, CK-MB, IL-1β and IL-18 using ELISA. Protein expression of CD40, TRAF6, NF-κB p65, p-NF-κB p65, cleaved caspase-1, NLRP3, and GSDMD-N was assessed by WB. The spatial distribution of CD40 and GSDMD-N fluorescent signals and the expression of GSDMD-N in cardiomyocytes were analyzed by IF.</p> Results <p>LNT significantly improved LVEF and LVFS while reducing LVESD and LVEDD in EAM mice. Both LNT doses reduced serum levels of cTnT, CK-MB, IL-1β and IL-18, with low-dose LNT exerting beneficial effects less pronounced than high-dose LNT and DEX. H&amp;E staining demonstrated increased cardiac inflammation and immune cell infiltration in EAM mice, which was significantly alleviated by LNT and DEX treatment. WB analysis showed that LNT significantly downregulated protein expression of CD40, TRAF6, p-NF-κB p65/NF-κB p65, cleaved caspase-1, NLRP3, and GSDMD-N in myocardial tissues of EAM mice. IF staining showed that the spatial overlap and distribution of CD40 and GSDMD-N fluorescent signals was significantly reduced after LNT treatment. Reversal experiments demonstrated that LNT counteracted the effects of CD40 activator on cardiomyocyte pyroptosis. Experiments in CD40mye−KO mice confirmed that CD40 is a critical target for LNT-mediated cardiac protection. In vitro studies demonstrated that LNT regulates CD40 expression and downstream GSDMD-related pyroptosis signaling in macrophages, while a CD40 activator could partially counteract its protective effect against cardiomyocyte pyroptosis, further highlighting the central role of CD40 in LNT-regulated pyroptosis.</p> Conclusion <p>Lentinan alleviates autoimmune myocarditis, and its protective effect is closely related to the inhibition of CD40 expression and the subsequent suppression of GSDMD-mediated cardiomyocyte pyroptosis.</p> Clinical trial number <p>Not applicable.</p>

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Lentinan alleviates autoimmune myocarditis via inhibiting CD40 expression and GSDMD-mediated cardiomyocyte pyroptosis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月29日

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  • Xiao Huang,
  • Yongfang Huang,
  • Qi Lin,
  • Yiheng Yang,
  • Peng Yang,
  • Qingshan Tian

摘要

Objective

This study aimed to investigate the molecular mechanism and regulatory effect by which lentinan (LNT) alleviates autoimmune myocarditis via suppressing CD40 expression and GSDMD-mediated cardiomyocyte pyroptosis.

Methods

Fifty 8-week-old male BALB/c mice were randomly divided into 5 groups (n = 10): Control, experimental autoimmune myocarditis (EAM) model, EAM + low-dose LNT (5 mg/kg), EAM + high-dose LNT (20 mg/kg), and EAM + dexamethasone (DEX, 2.5 mg/kg). EAM was established by immunization with cardiac myosin on D0 and D7 using ‌α-MyHC. LNT and DEX were administered intraperitoneally from D8. Additionally, 40 CD40fl/flLysMCre+/− (CD40mye−KO) mice and 16 CD40WT littermates were used. CD40mye−KO mice were divided into 4 groups (n = 10): CD40mye−KO, CD40mye−KO EAM model, CD40mye−KO EAM model + High-dose LNT (20 mg/kg), CD40mye−KO EAM model + DEX (2.5 mg/kg). CD40WT mice divided into 2 groups (n = 10): CD40WT, CD40WT EAM model. The HW/BW ratio was measured, and LVEF, LVFS, LVESD, and LVEDD were assessed by echocardiography. Cardiac histopathological changes were evaluated using inflammatory scoring after H&E staining. Bone marrow-derived macrophages were pre-treated with 0.1 µg/mL LPS to induce cell damage, followed by treatment with 5 µg/mL LNT, 20 µg/mL LNT, 100 nM DEX, or 100 ng/mL CD40 activator (anti-mouse CD40 antibody fgk4.5/fgk45) for 24 h. Primary mouse cardiomyocytes were isolated from hearts, and supernatants from pyroptotic macrophages were used to treat cardiomyocytes for 24 h. Cell viability was measured using CCK-8 assay. LDH content in culture supernatant was detected to evaluate cell membrane damage. Serum and cell culture supernatants were analyzed for cTnT, CK-MB, IL-1β and IL-18 using ELISA. Protein expression of CD40, TRAF6, NF-κB p65, p-NF-κB p65, cleaved caspase-1, NLRP3, and GSDMD-N was assessed by WB. The spatial distribution of CD40 and GSDMD-N fluorescent signals and the expression of GSDMD-N in cardiomyocytes were analyzed by IF.

Results

LNT significantly improved LVEF and LVFS while reducing LVESD and LVEDD in EAM mice. Both LNT doses reduced serum levels of cTnT, CK-MB, IL-1β and IL-18, with low-dose LNT exerting beneficial effects less pronounced than high-dose LNT and DEX. H&E staining demonstrated increased cardiac inflammation and immune cell infiltration in EAM mice, which was significantly alleviated by LNT and DEX treatment. WB analysis showed that LNT significantly downregulated protein expression of CD40, TRAF6, p-NF-κB p65/NF-κB p65, cleaved caspase-1, NLRP3, and GSDMD-N in myocardial tissues of EAM mice. IF staining showed that the spatial overlap and distribution of CD40 and GSDMD-N fluorescent signals was significantly reduced after LNT treatment. Reversal experiments demonstrated that LNT counteracted the effects of CD40 activator on cardiomyocyte pyroptosis. Experiments in CD40mye−KO mice confirmed that CD40 is a critical target for LNT-mediated cardiac protection. In vitro studies demonstrated that LNT regulates CD40 expression and downstream GSDMD-related pyroptosis signaling in macrophages, while a CD40 activator could partially counteract its protective effect against cardiomyocyte pyroptosis, further highlighting the central role of CD40 in LNT-regulated pyroptosis.

Conclusion

Lentinan alleviates autoimmune myocarditis, and its protective effect is closely related to the inhibition of CD40 expression and the subsequent suppression of GSDMD-mediated cardiomyocyte pyroptosis.

Clinical trial number

Not applicable.