Activating Ferroptosis of M1 Macrophages: A Novel Mechanism of Asiaticoside Encapsuled in GelMA for Anti-Inflammation in Diabetic Wounds

Abstract

Diabetic wounds are characterized by chronic inflammation, partly due to the persistent accumulation of pro-inflammatory M1 macrophages. Asiaticoside (AS), a triterpenoid extracted from Centella asiatica, has known anti-inflammatory effects in several diseases, but the underlying mechanisms in diabetic wounds are still unclear. This study reveals that AS alleviates inflammation in diabetic wounds by activating ferroptosis of M1 macrophages. In vitro, AS reduces the number of M1 macrophages in a high glucose microenvironment and their secretion of proinflammatory cytokines with concurrent induction of ferroptosis. Further investigation shows that AS-activated ferroptosis is attributed to the downregulation of ferroportin 1 (FPN1) and ferritinophagy-induced degradation of ferritin heavy chain 1 (FTH1), which together increase the amount of intracellular free ferrous ions (Fe²⁺). In vivo, AS-encapsulated gelatin-methacryloyl hydrogels accelerates diabetic wound healing and shortens the inflammatory period by activating ferroptosis of M1 macrophages with the reduced expression of FPN1 and FTH1. These results suggest a promising AS-based strategy for treating inflammatory diseases associated with excessive activation of M1 macrophages.

Keywords

asiaticoside / anti-inflammation / diabetic wounds / ferroptosis / macrophages

References

[1] A. Zacharias, T. A. Schwann, C. J. Riordan, S. J. Durham, A. S. Shah, and R. H. Habib, “Obesity and Risk of New-Onset Atrial Fibrillation After Cardiac Surgery,” Circulation112 (2005): 3247-3255, https://doi.org/10.1161/CIRCULATIONAHA.105.553743.

[2] X. Liu, Q. Wei, Z. Q. Sun, et al., “Small Extracellular Vesicles: Yields, Functionalization and Applications in Diabetic Wound Management,” Interdisciplinary Medicine1, no. 4 (2023): 0230019, https://doi.org/10.1002/INMD.20230019.

[3] T. F. Ma, X. Y. Zhai, M. D. Jin, et al., “Multifunctional Wound Dressing for Highly Efficient Treatment of Chronic Diabetic Wounds,” View3, no. 6 (2022): 20220045, https://doi.org/10.1002/VIW.20220045.

[4] Q. Li, W. Hu, Q. Huang, et al., “MiR146a-Loaded Engineered Exosomes Released From Silk Fibroin Patch Promote Diabetic Wound Healing by Targeting IRAK1,” Signal Transduction and Targeted Therapy8 (2023): 62, https://doi.org/10.1038/s41392-022-01263-w.

[5] Z. Chu, Q. Huang, K. Ma, et al., “Novel Neutrophil Extracellular Trap-Related Mechanisms in Diabetic Wounds Inspire a Promising Treatment Strategy With Hypoxia-Challenged Small Extracellular Vesicles,” Bioactive Materials27 (2023): 257-270.

[6] Y. Wang, Z. Cao, Q. Wei, et al., “VH298-Loaded Extracellular Vesicles Released From Gelatin Methacryloyl Hydrogel Facilitate Diabetic Wound Healing by HIF-1α-Mediated Enhancement of Angiogenesis,” Acta Biomaterialia147 (2022): 342-355, https://doi.org/10.1016/j.actbio.2022.05.018.

[7] X. L. Qi, E. Cai, Y. J. Xiang, et al., “An Immunomodulatory Hydrogel by Hyperthermia-Assisted Self-Cascade Glucose Depletion and ROS Scavenging for Diabetic Foot Ulcer Wound Therapeutics,” Advanced Materials35, no. 48 (2023): 2306632.

[8] S. J. Lee, C. K. Lee, S. Kang, et al., “Angiopoietin-2 Exacerbates Cardiac Hypoxia and Inflammation After Myocardial Infarction,” Journal of Clinical Investigation128 (2018): 5018-5033, https://doi.org/10.1172/JCI99659.

[9] L. C. Chan, M. Rossetti, L. S. Miller, et al., “Protective immunity in recurrent Staphylococcus aureus infection reflects localized immune signatures and macrophage-conferred memory,” PNAS115 (2018): E11111-E11119.

[10] Y. Guo, C. Lin, P. Xu, et al., “AGEs Induced Autophagy Impairs Cutaneous Wound Healing via Stimulating Macrophage Polarization to M1 in Diabetes,” Scientific Reports6 (2016): 36416, https://doi.org/10.1038/srep36416.

[11] S. Devaraj, S. K. Venugopal, U. Singh, and I. Jialal, “Hyperglycemia Induces Monocytic Release of Interleukin-6 via Induction of Protein Kinase C-α and -β,” Diabetes54 (2005): 85-91, https://doi.org/10.2337/diabetes.54.1.85.

[12] S. Pavlou, J. Lindsay, R. Ingram, H. Xu, and M. Chen, “Sustained High Glucose Exposure Sensitizes Macrophage Responses to Cytokine Stimuli but Reduces Their Phagocytic Activity,” BMC Immunology19 (2018): 24, https://doi.org/10.1186/s12865-018-0261-0.

[13] E. Cai, X. L. Qi, Y. Z. Shi, et al., “Immunomodulatory Melanin@Pt Nanoparticle-Reinforced Adhesive Hydrogels for Healing Diabetic Oral Ulcers,” Chemical Engineering Journal488 (2024): 150372, https://doi.org/10.1016/j.cej.2024.150372.

[14] X. Qi, E. Cai, Y. Xiang, et al., “An Immunomodulatory Hydrogel by Hyperthermia-Assisted Self-Cascade Glucose Depletion and ROS Scavenging for Diabetic Foot Ulcer Wound Therapeutics,” Advanced Materials35, no. 48 (2023): e2306632, https://doi.org/10.1002/adma.202306632.

[15] Y. J. Xiang, X. L. Qi, E. Cai, et al., “Highly Efficient Bacteria-Infected Diabetic Wound Healing Employing a Melanin-Reinforced Biopolymer Hydrogel,” Chemical Engineering Journal460 (2023): 141852-141852, https://doi.org/10.1016/j.cej.2023.141852.

[16] J. He, W. Zhang, Y. Cui, L. Cheng, X. L. Chen, and X. Wang, “Multifunctional Cu 2 Se/F127 Hydrogel With SOD-Like Enzyme Activity for Efficient Wound Healing,” Advanced Healthcare Materials13, no. 16 (2024): e2303599, https://doi.org/10.1002/adhm.202303599.

[17] Y. Cui, W. Zhang, J. Shan, et al., “Copper Nanodots-Based Hybrid Hydrogels With Multiple Enzyme Activities for Acute and Infected Wound Repair,” Advanced Healthcare Materials13, no. 8 (2024): e2302566, https://doi.org/10.1002/adhm.202302566.

[18] H. Tian, J. Yan, W. Zhang, et al., “Cu-GA-Coordination Polymer Nanozymes With Triple Enzymatic Activity for Wound Disinfection and Accelerated Wound Healing,” Acta Biomaterialia167 (2023): 449-462, https://doi.org/10.1016/j.actbio.2023.05.048.

[19] J. Han, X. Jin, C. Zhang, et al., “Metal Natural Product Complex Ru-procyanidins With Quadruple Enzymatic Activity Combat Infections From Drug-Resistant Bacteria,” Acta Pharmaceutica Sinica B14, no. 5 (2024): 2298-2316, https://doi.org/10.1016/j.apsb.2023.12.017.

[20] X. Jin, W. Zhang, J. Shan, et al., “Thermosensitive Hydrogel Loaded With Nickel–Copper Bimetallic Hollow Nanospheres With SOD and CAT Enzymatic-Like Activity Promotes Acute Wound Healing,” ACS Applied Materials and Interfaces14, no. 45 (2022): 50677-50691, https://doi.org/10.1021/acsami.2c17242.

[21] A. P. Veith, K. Henderson, A. Spencer, A. D. Sligar, and A. B. Baker, “Therapeutic Strategies for Enhancing Angiogenesis in Wound Healing,” Advanced Drug Delivery Reviews146 (2019): 97-125, https://doi.org/10.1016/j.addr.2018.09.010.

[22] J. Jin, Z. Zhang, J. Chen, Y. Liu, Q. Chen, and Q. Wang, “Jixuepaidu Tang-1 Inhibits Epithelial-Mesenchymal Transition and Alleviates Renal Damage in DN Mice Through Suppressing Long Non-Coding RNA LOC498759,” Cell Cycle18 (2019): 3125-3136, https://doi.org/10.1080/15384101.2019.1669986.

[23] C. Puglia, M. R. Lauro, G. G. Tirendi, et al., “Modern Drug Delivery Strategies Applied to Natural Active Compounds,” Expert Opinion on Drug Delivery14 (2017): 755-768, https://doi.org/10.1080/17425247.2017.1234452.

[24] Y. Y. Huang, C. W. Lin, N. C. Cheng, et al., “Effect of a Novel Macrophage-Regulating Drug on Wound Healing in Patients with Diabetic Foot Ulcers,” JAMA Network Open4, no. 9 (2021): e2122607, https://doi.org/10.1001/jamanetworkopen.2021.22607.

[25] D. Paolino, D. Cosco, F. Cilurzo, et al., “Improved In Vitro and In Vivo Collagen Biosynthesis by Asiaticoside-Loaded Ultradeformable Vesicles,” Journal Control Release162 (2012): 143-151, https://doi.org/10.1016/j.jconrel.2012.05.050.

[26] H. Li, Q. Peng, Y. Guo, X. Wang, and L. Zhang, “Preparation and In Vitro and In Vivo Study of Asiaticoside-Loaded Nanoemulsions and Nanoemulsions-Based Gels for Transdermal Delivery,” The International Journal of Nanomedicine15 (2020): 3123-3136, https://doi.org/10.2147/IJN.S241923.

[27] J. Wan, X. Gong, R. Jiang, Z. Zhang, and L. Zhang, “Antipyretic and Anti-Inflammatory Effects of Asiaticoside in Lipopolysaccharide-Treated Rat Through Up-Regulation of Heme Oxygenase-1,” Phytotherapy Research27 (2013): 1136-1142, https://doi.org/10.1002/ptr.4838.

[28] J. Qiu, L. Yu, X. Zhang, et al., “Asiaticoside Attenuates Lipopolysaccharide-Induced Acute Lung Injury via Down-Regulation of NF-κB Signaling Pathway,” International Immunopharmacology26 (2015): 181-187, https://doi.org/10.1016/j.intimp.2015.03.022.

[29] D. K. Li and G. H. Wang, “Asiaticoside Reverses M2 Phenotype Macrophage Polarization-Evoked Osteosarcoma Cell Malignant Behaviour by TRAF6/NF-κB Inhibition,” Pharmaceutical Biology60 (2022): 1635-1645, https://doi.org/10.1080/13880209.2022.2109688.

[30] S. Cui, X. Liu, Y. Liu, et al., “Autophagosomes Defeat Ferroptosis by Decreasing Generation and Increasing Discharge of Free Fe²⁺ in Skin Repair Cells to Accelerate Diabetic Wound Healing,” Advanced Science10 (2023): e2300414, https://doi.org/10.1002/advs.202300414.

[31] R. Kim, A. Hashimoto, N. Markosyan, et al., “Ferroptosis of Tumour Neutrophils Causes Immune Suppression in Cancer,” Nature612 (2022): 338-346, https://doi.org/10.1038/s41586-022-05443-0.

[32] Y. Wu, H. Chen, N. Xuan, et al., “Induction of Ferroptosis-Like Cell Death of Eosinophils Exerts Synergistic Effects With Glucocorticoids in Allergic Airway Inflammation,” Thorax75 (2020): 918-927, https://doi.org/10.1136/thoraxjnl-2020-214764.

[33] Y. Cui, Z. Zhang, X. Zhou, et al., “Microglia and Macrophage Exhibit Attenuated Inflammatory Response and Ferroptosis Resistance After RSL3 Stimulation via Increasing Nrf2 Expression,” Journal Neuroinflammation18 (2021): 249, https://doi.org/10.1186/s12974-021-02231-x.

[34] Y. Yang, Y. Wang, L. Guo, W. Gao, T. L. Tang, and M. Yan, “Interaction Between Macrophages and Ferroptosis,” Cell Death & Disease13 (2022): 355, https://doi.org/10.1038/s41419-022-04775-z.

[35] A. A. Kapralov, Q. Yang, H. H. Dar, et al., “Redox Lipid Reprogramming Commands Susceptibility of Macrophages and Microglia to Ferroptotic Death,” Nature Chemical Biology16 (2020): 278-290, https://doi.org/10.1038/s41589-019-0462-8.

[36] F. Yu, Q. Zhang, H. Liu, et al., “Dynamic O-GlcNAcylation Coordinates Ferritinophagy and Mitophagy to Activate Ferroptosis,” Cell Discovery8 (2022): 40, https://doi.org/10.1038/s41421-022-00390-6.

[37] F. Wang, H. Lv, B. Zhao, et al., “Iron and Leukemia: New Insights for Future Treatments,” Journal of Experimental & Clinical Cancer Research38 (2019): 406, https://doi.org/10.1186/s13046-019-1397-3.

[38] P. Qin, J. Tang, D. Sun, et al., “Zn²⁺ Cross-Linked Alginate Carrying Hollow Silica Nanoparticles Loaded With RL-QN15 Peptides Provides Promising Treatment for Chronic Skin Wounds,” ACS Applied Materials and Interfaces14, no. 26 (2022): 29491-29505, https://doi.org/10.1021/acsami.2c03583.

[39] C. B. Ibberson, A. Stacy, D. Fleming, et al., “Co-Infecting Microorganisms Dramatically Alter Pathogen Gene Essentiality During Polymicrobial Infection,” Nature Microbiology2 (2017): 17079, https://doi.org/10.1038/nmicrobiol.2017.79.30.

[40] C. Chen, L. Chen, C. Mao, et al., “Natural Extracts for Antibacterial Applications,” Small20, no. 9 (2024): e2306553, https://doi.org/10.1002/smll.202306553.

[41] G. Courties, T. Heidt, M. Sebas, et al., “In Vivo Silencing of the Transcription Factor IRF5 Reprograms the Macrophage Phenotype and Improves Infarct Healing,” Journal of the American College of Cardiology63 (2014): 1556-1566, https://doi.org/10.1016/j.jacc.2013.11.023.

[42] D. G. Armstrong, T. W. Tan, A. J. M. Boulton, and S. A. Bus, “Diabetic Foot Ulcers,” JAMA330 (2023): 62, https://doi.org/10.1001/jama.2023.10578.

[43] J. T. Paige, M. Kremer, J. Landry, et al., “Modulation of Inflammation in Wounds of Diabetic Patients Treated With Porcine Urinary Bladder Matrix,” Regenerative Medicine14 (2019): 269-277, https://doi.org/10.2217/rme-2019-0009.

[44] K. D. Mangum and K. A. Gallagher, “Obesity Confers Macrophage Memory,” Science379 (2023): 28-29, https://doi.org/10.1126/science.adf6582.

[45] M. Morey, P. O'Gaora, A. Pandit, and C. Helary, “Hyperglycemia Acts in Synergy With Hypoxia to Maintain the Pro-Inflammatory Phenotype of Macrophages,” PLoS ONE14 (2019): e0220577, https://doi.org/10.1371/journal.pone.0220577.

[46] S. Basu Mallik, B. S. Jayashree, and R. R. Shenoy, “Epigenetic Modulation of Macrophage Polarization-Perspectives in Diabetic Wounds,” Journal of Diabetes and Its Complications32 (2018): 524-530, https://doi.org/10.1016/j.jdiacomp.2018.01.015.

[47] F. Bohm, U. A. Kohler, T. Speicher, and S. Werner, “Regulation of Liver Regeneration by Growth Factors and Cytokines,” EMBO Molecular Medicine2 (2010): 294-305, https://doi.org/10.1002/emmm.201000085.

[48] S. Lakoh, L. Yi, S. Sevalie, et al., “Incidence and Risk Factors of Surgical Site Infections and Related Antibiotic Resistance in Freetown, Sierra Leone: A Prospective Cohort Study,” Antimicrobial Resistance and Infection Control11 (2022): 39, https://doi.org/10.1186/s13756-022-01078-y.

[49] N. E. Paul, C. Skazik, M. Harwardt, et al., “Topographical Control of Human Macrophages by a Regularly Microstructured Polyvinylidene Fluoride Surface,” Biomaterials29 (2008): 4056-4064, https://doi.org/10.1016/j.biomaterials.2008.07.010.

[50] C. Li, Y. Xiong, Z. Fu, et al., “The Direct Binding of Bioactive Peptide Andersonin-W1 to TLR4 Expedites the Healing of Diabetic Skin Wounds,” Cellular & Molecular Biology Letters29 (2024): 24, https://doi.org/10.1186/s11658-024-00542-4.

[51] C. Li, Z. Fu, T. Jin, et al., “A Frog Peptide Provides New Strategies for the Intervention Against Skin Wound Healing,” Cellular & Molecular Biology Letters28 (2023): 61, https://doi.org/10.1186/s11658-023-00468-3.

[52] F. X. Zhu, G. G. Nie, and C. S. Liu, “Engineered Biomaterials in Stem Cell-Based Regenerative Medicine,” Life Medicine2, no. 4 (2023): 1-22, https://doi.org/10.1093/lifemedi/lnad027.20.

[53] C. Li, Z. Fu, T. Jin, et al., “A Frog Peptide Provides New Strategies for the Intervention Against Skin Wound Healing,” Cellular & Molecular Biology Letters28, no. 1 (2023): 61, https://doi.org/10.1186/s11658-023-00468-3.

[54] C. Li, Y. Xiong, Z. Fu, et al., “The Direct Binding of Bioactive Peptide Andersonin-W1 to TLR4 Expedites the Healing of Diabetic Skin Wounds,” Cellular & Molecular Biology Letters29, no. 1 (2024): 24, https://doi.org/10.1186/s11658-024-00542-4.5.

[55] Y. Wang, B. Tang, J. Zhu, et al., “Emerging Mechanisms and Targeted Therapy of Ferroptosis in Neurological Diseases and Neuro-Oncology,” International Journal of Biological Sciences18 (2022): 4260-4274, https://doi.org/10.7150/ijbs.72251.

[56] S. Xu, J. Min, and F. Wang, “Ferroptosis: An Emerging Player in Immune Cells,” Science Bulletin66 (2021): 2257-2260, https://doi.org/10.1016/j.scib.2021.02.026.

[57] Y. S. Sohn, A. M. Mitterstiller, W. Breuer, G. Weiss, and Z. I. Cabantchik, “Rescuing Iron-Overloaded Macrophages by Conservative Relocation of the Accumulated Metal,” British Journal of Pharmacology164 (2011): 406-418, https://doi.org/10.1111/j.1476-5381.2010.01120.x.

[58] Y. Yuan, Y. Chen, T. Peng, et al., “Mitochondrial ROS-induced Lysosomal Dysfunction Impairs Autophagic Flux and Contributes to M1 Macrophage Polarization in a Diabetic Condition,” Clinical Science133 (2019): 1759-1777, https://doi.org/10.1042/CS20190672.

[59] B. Levine and G. Kroemer, “Biological Functions of Autophagy Genes: A Disease Perspective,” Cell176 (2019): 11-42, https://doi.org/10.1016/j.cell.2018.09.048.

[60] J. Füllgrabe, D. J. Klionsky, and B. Joseph, “The Return of the Nucleus: Transcriptional and Epigenetic Control of Autophagy,” Nature Reviews Molecular Cell Biology15 (2014): 65-74, https://doi.org/10.1038/nrm3716.

[61] F. Zheng, R. Li, Q. He, et al., “The Electrostimulation and Scar Inhibition Effect of Chitosan/Oxidized Hydroxyethyl Cellulose/Reduced Graphene Oxide/Asiaticoside Liposome Based Hydrogel on Peripheral Nerve Regeneration In Vitro,” Materials Science & Engineering C Materials for Biological Applications109 (2020): 110560, https://doi.org/10.1016/j.msec.2019.110560.

[62] H. Bunzen and D. Jirák, “Recent Advances in Metal–Organic Frameworks for Applications in Magnetic Resonance Imaging,” ACS Applied Materials & Interfaces14, no. 45 (2022): 50445-50462, https://doi.org/10.1021/acsami.2c10272.

[63] T. Wu, C. Cui, C. Fan, Z. Xu, Y. Liu, and W. Liu, “Tea Eggs-Inspired High-Strength Natural Polymer Hydrogels,” Bioactive Materials6 (2021): 2820.

[64] M. Xie, Y. Shi, C. Zhang, et al., “In Situ 3D Bioprinting With Bioconcrete Bioink,” Nature Communications13 (2022): 3597, https://doi.org/10.1038/s41467-022-30997-y.

[65] Z. Zhuang, Y. Zhang, S. Sun, et al., “Control of Matrix Stiffness Using Methacrylate–Gelatin Hydrogels for a Macrophage-Mediated Inflammatory Response,” ACS Biomaterials Science and Engineering6, no. 5 (2020): 3091-3102, https://doi.org/10.1021/acsbiomaterials.0c00295.

[66] S. Butenko, R. R. Nagalla, C. F. Guerrero-Juarez, et al., “Hydrogel Crosslinking Modulates Macrophages, Fibroblasts, and Their Communication, During Wound Healing,” Nature Communications15, no. 1 (2024): 6820, https://doi.org/10.1038/s41467-024-50072-y.

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