Reshaped commensal wound microbiome via topical application of Calvatia gigantea extract contributes to faster diabetic wound healing

Abstract

Background: Calvatia gigantea (CG) is widely used as a traditional Chinese medicine for wound treatment. In this study, we aimed to determine the effects of CG extract (CGE) on diabetic wound healing and the commensal wound microbiome. Method: A wound model was established using leptin receptor-deficient db/db mice, with untreated mice as the control group and CGE-treated mice as the treatment group. The wound healing rate, inflammation and histology were analyzed. Additionally, wound microbiome was evaluated via 16S ribosomal RNA (rRNA) gene sequencing. Results: CGE significantly accelerated the healing of diabetic ulcer wounds, facilitated re-epithelialization, and downregulated the transcription levels of the inflammatory cytokines, interleukin-1β and tumor necrosis factor-α. Furthermore, CGE treatment positively affected the wound microbiome, promoting diversity of the microbial community and enrichment of Escherichia-Shigella bacteria in the CGE-treated group. Conclusions: Overall, CGE enhanced diabetic wound healing by modulating the wound microbiome and facilitating macrophage polarization during inflammation. These findings suggest modulation of the commensal wound microbiome using medicinal plants as a potential therapeutic strategy for diabetic wounds.

Keywords

Calvatia gigantea extract / Diabetic wound healing / Commensal wound microbiome / Macrophage polarization / Inflammation

References

[1] Armstrong DG, Tan TW, Boulton AJM, Bus SA. Diabetic foot ulcers: a review. JAMA 2023; 330: 62-75. https://doi.org/10.1001/jama.2023.10578.

[2] Armstrong DG, Boulton AJM, Bus SA. Diabetic foot ulcers and their recurrence. N Engl J Med 2017; 376: 2367-75. https://doi.org/10.1056/NEJMra1615439.

[3] McDermott K, Fang M, Boulton AJM, Selvin E, Hicks CW. Etiology, epidemiology, and disparities in the burden of diabetic foot ulcers. Diabetes Care 2023; 46: 209-21. https://doi.org/10.2337/dci22-0043.

[4] Zarei F, Negahdari B, Eatemadi A. Diabetic ulcer regeneration: stem cells, biomaterials, growth factors, Artif cells Nanomed. Biotechnol 2018; 46: 26-32.

[5] Zhou Z, Deng T, Tao M, Lin L, Sun L, Song X, et al. Snail-inspired AFG/GelMA hydrogel accelerates diabetic wound healing via inflammatory cytokines suppression and macrophage polarization. Biomaterials 2023; 299: 122141.

[6] Byrd AL, Belkaid Y, Segre JA. The human skin microbiome. Nat Rev Microbiol 2018; 16: 143-55. https://doi.org/10.1038/nrmicro.2017.157.

[7] Chen YE, Fischbach MA, Belkaid Y. Skin microbiota-host interactions. Nature 2018; 553: 427-36. https://doi.org/10.1038/nature25177.

[8] Scharschmidt TC, Fischbach MA. What lives on our skin: ecology, genomics and therapeutic opportunities of the skin microbiome. Drug Discov Today Dis Mech 2013; 10: e83-9. https://doi.org/10.1016/j.ddmec.2012.12.003.

[9] Wang G, Sweren E, Liu H, Wier E, Alphonse MP, Chen R, et al. Bacteria induce skin regeneration via IL-1beta signaling. Cell Host Microbe 2021; 29: 777-791.e6. https://doi.org/10.1016/j.chom.2021.03.003.

[10] Kalan LR, Meisel JS, Loesche MA, Horwinski J, Soaita I, Chen X, et al. Strain-and species-level variation in the microbiome of diabetic wounds is associated with clinical outcomes and therapeutic efficacy. Cell Host Microbe 2019; 25: 641-655.e5. https://doi.org/10.1016/j.chom.2019.03.006.

[11] Di Domizio J, Belkhodja C, Chenuet P, Fries A, Murray T, Mondejar PM, et al. The commensal skin microbiota triggers type I IFN-dependent innate repair responses in injured skin. Nat Immunol 2020; 21: 1034-45. https://doi.org/10.1038/s41590-020-0721-6.

[12] Tang Q, Xue N, Ding X, Tsai KH, Hew JJ, Jiang R, et al. Role of wound microbiome, strategies of microbiota delivery system and clinical management. Adv Drug Deliv Rev 2023; 192: 114671. https://doi.org/10.1016/j.addr.2022.114671.

[13] Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med 2014; 6: 265sr266.

[14] Sawaya AP, Stone RC, Brooks SR, Pastar I, Jozic I, Hasneen K, et al. Deregulated immune cell recruitment orchestrated by FOXM1 impairs human diabetic wound healing. Nat Commun 2020; 11: 4678. https://doi.org/10.1038/s41467-020-18276-0.

[15] Dasari N, Jiang A, Skochdopole A, Chung J, Reece EM, Vorstenbosch J, et al. Updates in diabetic wound healing, inflammation, and scarring. Semin Plast Surg 2021; 35: 153-8. https://doi.org/10.1055/s-0041-1731460.

[16] Landen NX, Li D, Stahle M. Transition from inflammation to proliferation: a critical step during wound healing. Cell Mol Life Sci 2016; 73: 3861-85. https://doi.org/10.1007/s00018-016-2268-0.

[17] Louiselle AE, Niemiec SM, Zgheib C, Liechty KW. Macrophage polarization and diabetic wound healing. Transl Res 2021; 236: 109-16. https://doi.org/10.1016/j.trsl.2021.05.006.

[18] Mirza R, Koh TJ. Dysregulation of monocyte/macrophage phenotype in wounds of diabetic mice. Cytokine 2011; 56: 256-64. https://doi.org/10.1016/j.cyto.2011.06.016.

[19] Kivrak I, Kivrak S, Harmandar M. Bioactive compounds, chemical composition, and medicinal value of the Giant puffball, Calvatia gigantea (higher basidiomycetes), from Turkey. Int J Med Mushrooms 2016; 18: 97-107. https://doi.org/10.1615/IntJMedMushrooms.v18.i2.10.

[20] Sari M, Prange A, Lelley JI, Hambitzer R. Screening of beta-glucan contents in commercially cultivated and wild growing mushrooms. Food Chem 2017; 216: 45-51. https://doi.org/10.1016/j.foodchem.2016.08.010.

[21] Guillamon E, Garcia-Lafuente A, Lozano M, D’Arrigo M, Rostagno MA, Villares A, et al. Edible mushrooms: role in the prevention of cardiovascular diseases. Fitoterapia 2010; 81: 715-23. https://doi.org/10.1016/j.fitote.2010.06.005.

[22] Rop O, Mlcek J, Jurikova T. Beta-glucans in higher fungi and their health effects. Nutr Rev 2009; 67: 624-31. https://doi.org/10.1111/j.1753-4887.2009.00230.x.

[23] Yang A. Effect of Mabo oil ointment on wound healing and angiogenesis related factors VEGF and PEDF in rabbits with chronic skin ulcer. Chin Arch Tradit Chin Med 2023; 41: 168-73.

[24] Yang J. Comparative study on Hemostasis and adsorption force of different parts of Lasiosphaera fenzlii Reich. Chin Arch Tradit Chin Med 2020; 38: 157-61.

[25] He T, Sun P, Liu B, Wan S, Fang P, Chen J, et al. Puffball spores improve wound healing in a diabetic rat model. Front Endocrinol (Lausanne) 2022; 13: 942549. https://doi.org/10.3389/fendo.2022.942549.

[26] Ogbole OO, Nkumah AO, Linus AU, Falade MO. Molecular identification, in vivo and in vitro activities of Calvatia gigantea (macro-fungus) as an antidiabetic agent. Mycology 2019; 10: 166-73. https://doi.org/10.1080/21501203.2019.1595204.

[27] Eroglu C, Secme M, Atmaca P, Kaygusuz O, Gezer K, Bagci G, et al. Extract of Calvatia gigantea inhibits proliferation of A549 human lung cancer cells. Cytotechnology 2016; 68: 2075-81. https://doi.org/10.1007/s10616-016-9947-4.

[28] Chen S, Zhou Y, Chen Y, Gu J. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018; 34: i884-90. https://doi.org/10.1093/bioinformatics/bty560.

[29] Li D, Liu CM, Luo R, Sadakane K, Lam TW. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 2015; 31: 1674-6. https://doi.org/10.1093/bioinformatics/btv033.

[30] Noguchi H, Park J, Takagi T. MetaGene: prokaryotic gene finding from environmental genome shotgun sequences. Nucleic Acids Res 2006; 34: 5623-30. https://doi.org/10.1093/nar/gkl723.

[31] Fu L, Niu B, Zhu Z, Wu S, Li W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 2012; 28: 3150-2. https://doi.org/10.1093/bioinformatics/bts565.

[32] Li R, Li Y, Kristiansen K, Wang J. SOAP: short oligonucleotide alignment program. Bioinformatics 2008; 24: 713-4. https://doi.org/10.1093/bioinformatics/btn025.

[33] Xu C, Bentinger M, Savu O, Moshfegh A, Sunkari V, Dallner G, et al. Mono-epoxy-tocotrienol-alpha enhances wound healing in diabetic mice and stimulates in vitro angiogenesis and cell migration. J Diabetes Complicat 2017; 31: 4-12. https://doi.org/10.1016/j.jdiacomp.2016.10.010.

[34] Lu Y, Li H, Wang J, Yao M, Peng Y, Liu T, et al. Engineering bacteria-activated multifunctionalized hydrogel for promoting diabetic wound healing. Adv Funct Mater 2021; 31: 2105749. https://doi.org/10.1002/adfm.202105749.

[35] Wu X, He W, Mu X, Liu Y, Deng J, Liu Y, et al. Macrophage polarization in diabetic wound healing. Burns Trauma 2022; 10: tkac051. https://doi.org/10.1093/burnst/tkac051.

[36] White EK, Grice EA. The wound microbiome. Cold Spring Harb Perspect Biol 2023; 15: a0412185. https://doi.org/10.1101/cshperspect.a041218.

[37] Huang L, Cai HA, Zhang MS, Liao RY, Huang X, Hu FD. Ginsenoside Rg1 promoted the wound healing in diabetic foot ulcers via miR-489-3p/Sirt1 axis. J Pharmacol Sci 2021; 147: 271-83. https://doi.org/10.1016/j.jphs.2021.07.008.

[38] Li G, Ko CN, Li D, et al. A small molecule HIF-1alpha stabilizer that accelerates diabetic wound healing. Nat Commun 2021; 12: 3363. https://doi.org/10.1038/s41467-021-23448-7.

[39] Luo MJ, Rao SS, Tan YJ, Yin H, Hu XK, Zhang Y, et al. Fasting before or after wound injury accelerates wound healing through the activation of pro-angiogenic SMOC1 and SCG2. Theranostics 2020; 10: 3779-92. https://doi.org/10.7150/thno.44115.

[40] Patel BK, Patel KH, Huang RY, Lee CN, Moochhala SM. The gut-skin microbiota Axis and its role in diabetic wound healing-a review based on current literature. Int J Mol Sci 2022; 23: 2375. https://doi.org/10.3390/ijms23042375.

[41] Ding X, Tang Q, Xu Z, Xu Y, Zhang H, Zheng D, et al. Challenges and innovations in treating chronic and acute wound infections: from basic science to clinical practice. Burns Trauma 2022; 10: tkac014.

[42] Spragge F, Bakkeren E, Jahn MT, Araujo EBN, Pearson CF, Wang X, et al. Microbiome diversity protects against pathogens by nutrient blocking. Science 2023; 382: eadj3502. https://doi.org/10.1126/science.adj3502.

[43] Gardiner M, Vicaretti M, Sparks J, Bansal S, Bush S, Liu M, et al. A longitudinal study of the diabetic skin and wound microbiome. PeerJ 2017; 5: e3543. https://doi.org/10.7717/peerj.3543.

[44] Brockway B. Introducing Escherichia coli (E. coli) part 1. 2020. Available from: http://thesecretlifeofskin.com/2020/11/10/introducing-e-coli/#:~:text=Yes%2C%20E.%20coli%20strains%20are%20isolated%20from%20skin,bacteria%20as%20well%20as%20its%20ability%20to%20adapt.

[45] Chen H, Lei P, Ji H, Yang Q, Peng B, Ma J, et al. Advances in Escherichia coli Nissle 1917 as a customizable drug delivery system for disease treatment and diagnosis strategies. Mater Today Bio 2023; 18: 100543. https://doi.org/10.1016/j.mtbio.2023.100543.

[46] Reid G, Younes JA, Van der Mei HC, Gloor GB, Knight R, Busscher HJ. Microbiota restoration: natural and supplemented recovery of human microbial communities. Nat Rev Microbiol 2011; 9: 27-38. https://doi.org/10.1038/nrmicro2473.

[47] Wang G, Sweren E, Andrews W, Li Y, Chen J, Xue Y, et al. Commensal microbiome promotes hair follicle regeneration by inducing keratinocyte HIF-1α signaling and glutamine metabolism. Sci Adv 2023; 9: eabo7555. https://doi.org/10.1126/sciadv.abo7555.

[48] Hamidzadeh K, Christensen SM, Dalby E, Chandrasekaran P, Mosser DM. Macrophages and the recovery from acute and chronic inflammation. Annu Rev Physiol 2017; 79: 567-92. https://doi.org/10.1146/annurev-physiol-022516-034348.

[49] Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol 2008; 8: 958-69. https://doi.org/10.1038/nri2448.

[50] Brancato SK, Albina JE. Wound macrophages as key regulators of repair: origin, phenotype, and function. Am J Pathol 2011; 178: 19-25. https://doi.org/10.1016/j.ajpath.2010.08.003.

[51] Basu Mallik S, Jayashree BS, Shenoy RR. Epigenetic modulation of macrophage polarization-perspectives in diabetic wounds. J Diabetes Complicat 2018; 32: 524-30. https://doi.org/10.1016/j.jdiacomp.2018.01.015.

[52] Atri C, Guerfali FZ, Laouini D. Role of human macrophage polarization in inflammation during infectious diseases. Int J Mol Sci 2018; 19: 1801. https://doi.org/10.3390/ijms19061801.

[53] Tomic-Canic M, Burgess JL, O’Neill KE, Strbo N, Pastar I. Skin microbiota and its interplay with wound healing. Am J Clin Dermatol 2020; 21: 36-43. https://doi.org/10.1007/s40257-020-00536-w.

[54] Witherel CE, Abebayehu D, Barker TH, Spiller KL. Macrophage and fibroblast interactions in biomaterial-mediated fibrosis. Adv Healthc Mater 2019; 8: e1801451. https://doi.org/10.1002/adhm.201801451.