H. sinensis mycelium inhibits epithelial--mesenchymal transition by inactivating the midkine pathway in pulmonary fibrosis

Abstract

The medical fungus Hirsutella sinensis has been used as a Chinese folk health supplement because of its immunomodulatory properties. Our previous studies established the antifibrotic action of Hirsutella sinensis mycelium (HSM) in the lung. The epithelial–mesenchymal transition (EMT) is involved in the pathogenesis of idiopathic pulmonary fibrosis. The present study investigates the role of HSM in mediating EMT during the development of pulmonary fibrosis. HSM significantly inhibits bleomycin (BLM)-induced pulmonary fibrosis by blocking the EMT. In addition, the expression levels of midkine are increased in the lungs of the BLM-induced group. Further analysis of the results indicates that the mRNA level of midkine correlated positively with EMT. HSM markedly abrogates the transforming growth factor β-induced EMT-like phenotype and behavior in vitro. The activation of midkine related signaling pathway is ameliorated following HSM treatment, whereas this extract also caused an effective attenuation of the induction of EMT (caused by midkine overexpression) in vitro. Results further confirm that oral medication of HSM disrupted the midkine pathway in vivo. Overall, findings suggest that the midkine pathway and the regulation of the EMT may be considered novel candidate therapeutic targets for the antifibrotic effects caused by HSM.

Keywords

epithelial–mesenchymal transition / H. sinensis mycelium / midkine / pulmonary fibrosis

References

[1] Richeldi L, Collard HR, Jones MG. Idiopathic pulmonary fibrosis. Lancet 2017; 389(10082): 1941–1952

[2] Barratt SL, Creamer A, Hayton C, Chaudhuri N. Idiopathic pulmonary fibrosis (IPF): an overview. J Clin Med 2018; 7(8): E201

[3] Funke M, Geiser T. Idiopathic pulmonary fibrosis: the turning point is now! Swiss Med Wkly 2015; 145: w14139

[4] Kim ES, Keating GM. Pirfenidone: a review of its use in idiopathic pulmonary fibrosis. Drugs 2015; 75(2): 219–230

[5] Richeldi L, du Bois RM, Raghu G, Azuma A, Brown KK, Costabel U, Cottin V, Flaherty KR, Hansell DM, Inoue Y, Kim DS, Kolb M, Nicholson AG, Noble PW, Selman M, Taniguchi H, Brun M, Le Maulf F, Girard M, Stowasser S, Schlenker-Herceg R, Disse B, Collard HR; INPULSIS Trial Investigators. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N Engl J Med 2014; 370(22): 2071–2082

[6] Meyer KC, Decker CA. Role of pirfenidone in the management of pulmonary fibrosis. Ther Clin Risk Manag 2017; 13: 427–437

[7] Richeldi L, Cottin V, Flaherty KR, Kolb M, Inoue Y, Raghu G, Taniguchi H, Hansell DM, Nicholson AG, Le Maulf F, Stowasser S, Collard HR. Design of the INPULSIS™ trials: two phase 3 trials of nintedanib in patients with idiopathic pulmonary fibrosis. Respir Med 2014; 108(7): 1023–1030

[8] Hajari Case A, Johnson P. Clinical use of nintedanib in patients with idiopathic pulmonary fibrosis. BMJ Open Respir Res 2017; 4(1): e000192

[9] Zhu JS, Halpern GM, Jones K. The scientific rediscovery of an ancient Chinese herbal medicine: Cordyceps sinensis: part I. J Altern Complement Med 1998; 4(3): 289–303

[10] Zhu JS, Halpern GM, Jones K. The scientific rediscovery of a precious ancient Chinese herbal regimen: Cordyceps sinensis: part II. J Altern Complement Med 1998; 4(4): 429–457

[11] Huang TT, Chong KY, Ojcius DM, Wu YH, Ko YF, Wu CY, Martel J, Lu CC, Lai HC, Young JD. Hirsutella sinensis mycelium suppresses interleukin-1b and interleukin-18 secretion by inhibiting both canonical and non-canonical inflammasomes. Sci Rep 2013; 3(1): 1374

[12] Huang TT, Lai HC, Ko YF, Ojcius DM, Lan YW, Martel J, Young JD, Chong KY. Hirsutella sinensis mycelium attenuates bleomycin-induced pulmonary inflammation and fibrosis in vivo. Sci Rep 2015; 5(1): 15282

[13] Koh JH, Yu KW, Suh HJ, Choi YM, Ahn TS. Activation of macrophages and the intestinal immune system by an orally administered decoction from cultured mycelia of Cordyceps sinensis. Biosci Biotechnol Biochem 2002; 66(2): 407–411

[14] Nordin SL, Jovic S, Kurut A, Andersson C, Gela A, Bjartell A, M�rgelin M, Olin AI, Lund M, Egesten A. High expression of midkine in the airways of patients with cystic fibrosis. Am J Respir Cell Mol Biol 2013; 49(6): 935–942

[15] Shou QY, Fu HY, Zhang LZ, Cai YQ, Chen FM, Chen ML. Study on treatment effect and mechanism of Hirsutella sinensis mycelium in idiopathic pulmonary fibrosis in rats. China J Chin Mater Med (Zhongguo Zhong Yao Za Zhi) 2012; 37(23): 3618–3623 (in Chinese)

[16] Yue H, Zhao Y, Wang H, Ma F, Liu F, Shen S, Hou Y, Dou H. Anti-fibrosis effect for Hirsutella sinensis mycelium based on inhibition of mTOR p70S6K phosphorylation. Innate Immun 2017; 23(7): 615–624

[17] Chen B, Cai HR, Xue S, You WJ, Liu B, Jiang HD. Bile acids induce activation of alveolar epithelial cells and lung fibroblasts through farnesoid X receptor-dependent and independent pathways. Respirology 2016; 21(6): 1075–1080

[18] O’Beirne SL, Walsh SM, Fabre A, Reviriego C, Worrell JC, Counihan IP, Lumsden RV, Cramton-Barnes J, Belperio JA, Donnelly SC, Boylan D, Marchal-Sommé J, Kane R, Keane MP. CXCL9 regulates TGF-b1-induced epithelial to mesenchymal transition in human alveolar epithelial cells. J Immunol 2015; 195(6): 2788–2796

[19] Wang YC, Liu JS, Tang HK, Nie J, Zhu JX, Wen LL, Guo QL. miR221 targets HMGA2 to inhibit bleomycininduced pulmonary fibrosis by regulating TGFb1/Smad3-induced EMT. Int J Mol Med 2016; 38(4): 1208–1216

[20] Willis BC, Borok Z. TGF-b-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol 2007; 293(3): L525–L534 doi:10.1152/ajplung.00163.2007

[21] Kage H, Borok Z. EMT and interstitial lung disease: a mysterious relationship. Curr Opin Pulm Med 2012; 18(5): 517–523 doi:10.1097/MCP.0b013e3283566721

[22] Xiao X, Huang C, Zhao C, Gou X, Senavirathna LK, Hinsdale M, Lloyd P, Liu L. Regulation of myofibroblast differentiation by miR-424 during epithelial-to-mesenchymal transition. Arch Biochem Biophys 2015; 566: 49–57

[23] Zhang H, Okamoto M, Panzhinskiy E, Zawada WM, Das M. PKCd/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells. Am J Physiol Cell Physiol 2014; 306(7): C648–C658

[24] Weckbach LT, Preissner KT, Deindl E. The role of midkine in arteriogenesis, involving mechanosensing, endothelial cell proliferation, and vasodilation. Int J Mol Sci 2018; 19(9): E2559

[25] Masuda T, Maeda K, Sato W, Kosugi T, Sato Y, Kojima H, Kato N, Ishimoto T, Tsuboi N, Uchimura K, Yuzawa Y, Maruyama S, Kadomatsu K. Growth factor midkine promotes T-cell activation through nuclear factor of activated T cells signaling and Th1 cell differentiation in lupus nephritis. Am J Pathol 2017; 187(4): 740–751

[26] Mitsiadis TA, Muramatsu T, Muramatsu H, Thesleff I. Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis. J Cell Biol 1995; 129(1): 267–281

[27] Erdogan S, Turkekul K, Dibirdik I, Doganlar O, Doganlar ZB, Bilir A, Oktem G. Midkine downregulation increases the efficacy of quercetin on prostate cancer stem cell survival and migration through PI3K/AKT and MAPK/ERK pathway. Biomed Pharmacother 2018; 107: 793–805

[28] Vieceli FM, Bronner ME. Leukocyte receptor tyrosine kinase interacts with secreted midkine to promote survival of migrating neural crest cells. Development 2018; 145(20): dev164046

[29] Konishi N, Nakamura M, Nakaoka S, Hiasa Y, Cho M, Uemura H, Hirao Y, Muramatsu T, Kadomatsu K. Immunohistochemical analysis of midkine expression in human prostate carcinoma. Oncology 1999; 57(3): 253–257

[30] Netsu S, Shishido T, Kitahara T, Honda Y, Funayama A, Narumi T, Kadowaki S, Takahashi H, Miyamoto T, Arimoto T, Nishiyama S, Watanabe T, Woo CH, Takeishi Y, Kubota I. Midkine exacerbates pressure overload-induced cardiac remodeling. Biochem Biophys Res Commun 2014; 443(1): 205–210

[31] Nordin SL, Jovic S, Kurut A, Andersson C, Gela A, Bjartell A, M�rgelin M, Olin AI, Lund M, Egesten A. High expression of midkine in the airways of patients with cystic fibrosis. Am J Respir Cell Mol Biol 2013; 49(6): 935–942

[32] Siempos II, Choi AM. Midkine: in the middle of the pathogenesis of acute respiratory distress syndrome-associated lung fibrosis? Am J Respir Crit Care Med 2015; 192(3): 271–272

[33] Zhang R, Pan Y, Fanelli V, Wu S, Luo AA, Islam D, Han B, Mao P, Ghazarian M, Zeng W, Spieth PM, Wang D, Khang J, Mo H, Liu X, Uhlig S, Liu M, Laffey J, Slutsky AS, Li Y, Zhang H. Mechanical stress and the induction of lung fibrosis via the midkine signaling pathway. Am J Respir Crit Care Med 2015; 192(3): 315–323

[34] G�ng�r C, Zander H, Effenberger KE, Vashist YK, Kalinina T, Izbicki JR, Yekebas E, Bockhorn M. Notch signaling activated by replication stress-induced expression of midkine drives epithelial-mesenchymal transition and chemoresistance in pancreatic cancer. Cancer Res 2011; 71(14): 5009–5019

[35] Zhao G, Nie Y, Lv M, He L, Wang T, Hou Y. ERb-mediated estradiol enhances epithelial mesenchymal transition of lung adenocarcinoma through increasing transcription of midkine. Mol Endocrinol 2012; 26(8): 1304–1315

[36] Huang Y, Hoque MO, Wu F, Trink B, Sidransky D, Ratovitski EA. Midkine induces epithelial-mesenchymal transition through Notch2/Jak2-Stat3 signaling in human keratinocytes. Cell Cycle 2008; 7(11): 1613–1622

[37] Misa K, Tanino Y, Wang X, Nikaido T, Kikuchi M, Sato Y, Togawa R, Tanino M, Tanaka S, Kadomatsu K, Munakata M. Involvement of midkine in the development of pulmonary fibrosis. Physiol Rep 2017; 5(16): e13383

[38] Wang QL, Wang H, Zhao SL, Huang YH, Hou YY. Over-expressed and truncated midkines promote proliferation of BGC823 cells in vitro and tumor growth in vivo. World J Gastroenterol 2008; 14(12): 1858–1865

[39] Rajasekaran K, Xiong V, Fong L, Gorski J, Malarkannan S. Functional dichotomy between NKG2D and CD28-mediated co-stimulation in human CD8⁺ T cells. PLoS One 2010; 5(9): e12635

[40] Gardner A, Fisher AJ, Richter C, Johnson GE, Moisey EJ, Brodlie M, Ward C, Krippner-Heidenreich A, Mann DA, Borthwick LA. The critical role of TAK1 in accentuated epithelial to mesenchymal transition in obliterative bronchiolitis after lung transplantation. Am J Pathol 2012; 180(6): 2293–2308

[41] Tian R, Zhu Y, Yao J, Meng X, Wang J, Xie H, Wang R. NLRP3 participates in the regulation of EMT in bleomycin-induced pulmonary fibrosis. Exp Cell Res 2017; 357(2): 328–334

[42] Vyas-Read S, Wang W, Kato S, Colvocoresses-Dodds J, Fifadara NH, Gauthier TW, Helms MN, Carlton DP, Brown LA. Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition. Am J Physiol Lung Cell Mol Physiol 2014; 306(4): L326–L340

[43] Suzuki N, Shibata Y, Urano T, Murohara T, Muramatsu T, Kadomatsu K. Proteasomal degradation of the nuclear targeting growth factor midkine. J Biol Chem 2004; 279(17): 17785–17791

[44] Phan SH. The myofibroblast in pulmonary fibrosis. Chest 2002; 122(6 Suppl): 286S–289S

[45] Zhao H, Wu QQ, Cao LF, Qing HY, Zhang C, Chen YH, Wang H, Liu RY, Xu DX. Melatonin inhibits endoplasmic reticulum stress and epithelial-mesenchymal transition during bleomycin-induced pulmonary fibrosis in mice. PLoS One 2014; 9(5): e97266

[46] Marudamuthu AS, Bhandary YP, Shetty SK, Fu J, Sathish V, Prakash Y, Shetty S. Role of the urokinase-fibrinolytic system in epithelial-mesenchymal transition during lung injury. Am J Pathol 2015; 185(1): 55–68

[47] Kyung SY, Kim DY, Yoon JY, Son ES, Kim YJ, Park JW, Jeong SH. Sulforaphane attenuates pulmonary fibrosis by inhibiting the epithelial-mesenchymal transition. BMC Pharmacol Toxicol 2018; 19(1): 13

[48] Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN, Sheppard D, Chapman HA. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci USA 2006; 103(35): 13180–13185

[49] Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest 2003; 112(12): 1776–1784

[50] Tanjore H, Xu XC, Polosukhin VV, Degryse AL, Li B, Han W, Sherrill TP, Plieth D, Neilson EG, Blackwell TS, Lawson WE. Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis. Am J Respir Crit Care Med 2009; 180(7): 657–665

[51] Kim KK, Wei Y, Szekeres C, Kugler MC, Wolters PJ, Hill ML, Frank JA, Brumwell AN, Wheeler SE, Kreidberg JA, Chapman HA. Epithelial cell a3b1 integrin links b-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis. J Clin Invest 2009; 119(1): 213–224

[52] Huang Y, Hoque MO, Wu F, Trink B, Sidransky D, Ratovitski EA. Midkine induces epithelial-mesenchymal transition through Notch2/Jak2-Stat3 signaling in human keratinocytes. Cell Cycle 2008; 7(11): 1613–1622

[53] Misa K, Tanino Y, Wang X, Nikaido T, Kikuchi M, Sato Y, Togawa R, Tanino M, Tanaka S, Kadomatsu K, Munakata M. Involvement of midkine in the development of pulmonary fibrosis. Physiol Rep 2017; 5(16): e13383

[54] Liu F, Gomez Garcia AM, Meyskens FL Jr. NADPH oxidase 1 overexpression enhances invasion via matrix metalloproteinase-2 and epithelial-mesenchymal transition in melanoma cells. J Invest Dermatol 2012; 132(8): 2033–2041

[55] Kesanakurti D, Maddirela D, Banasavadi-Siddegowda YK, Lai TH, Qamri Z, Jacob NK, Sampath D, Mohanam S, Kaur B, Puduvalli VK. A novel interaction of PAK4 with PPARg to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma. Oncogene 2017; 36(37): 5309–5320

[56] Das SJ, Lovicu FJ, Collinson EJ. Nox4 plays a role in TGF-b-dependent lens epithelial to mesenchymal transition. Invest Ophthalmol Vis Sci 2016; 57(8): 3665–3673

[57] Rao YK, Fang SH, Tzeng YM. Evaluation of the anti-inflammatory and anti-proliferation tumoral cells activities of Antrodia camphorata, Cordyceps sinensis, and Cinnamomum osmophloeum bark extracts. J Ethnopharmacol 2007; 114(1): 78–85

[58] Ko YF, Liau JC, Lee CS, Chiu CY, Martel J, Lin CS, Tseng SF, Ojcius DM, Lu CC, Lai HC, Young JD. Isolation, culture and characterization of Hirsutella sinensis mycelium from Caterpillar fungus fruiting body. PLoS One 2017; 12(1): e0168734

[59] Datta A, Kim GA, Taylor JM, Gugino SF, Farrow KN, Schumacker PT, Berkelhamer SK. Mouse lung development and NOX1 induction during hyperoxia are developmentally regulated and mitochondrial ROS dependent. Am J Physiol Lung Cell Mol Physiol 2015; 309(4): L369–L377

[60] Zanetti F, Giacomello M, Donati Y, Carnesecchi S, Frieden M, Barazzone-Argiroffo C. Nicotine mediates oxidative stress and apoptosis through cross talk between NOX1 and Bcl-2 in lung epithelial cells. Free Radic Biol Med 2014; 76: 173–184

Acknowledgements

The present work is supported by the Six Talent Peaks Project in Jiangsu Province (No. YY-021), the Fundamental Research Funds of the Central Universities (No. 021414380342), and the Fund of State Key Laboratory of Trauma Burns and Combined Injury (No. SKLKF201602). We are also very grateful to Dr. Lizhi Xu (Medical school, Nanjing University) for her technical support.

Compliance with ethics guidelines

Li Lu, Haiyan Zhu, Hailin Wang, Huaping Liang, Yayi Hou, and Huan Dou declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11684-020-0737-1 and is accessible for authorized users.

RIGHTS & PERMISSIONS

2020 Higher Education Press