Icariin ameliorates viral myocarditis by inhibiting TLR4-mediated ferroptosis

Abstract

Objective: To explore the mechanism by which icariin alleviates viral myocarditis. Methods: CVB3-induced cardiomyocytes were used as an in vitro model of viral myocarditis to assess the effects of icariin treatment on cell viability, inflammation, and apoptosis. Moreover, the effects of icariin on ferroptosis and TLR4 signaling were assessed. After AC16 cells were transfected with TLR4 overexpression plasmids, the role of TLR4 in mediating the regulatory effect of icariin in viral myocarditis was investigated. Results: Icariin significantly elevated cell viability and reduced inflammatory factors TNF-α, IL-1β, IL-6, and IL-18. Flow cytometry revealed that icariin decreased apoptosis rate, and the protein expression of Bax and cleaved caspase 3 and 9 in CVB3-induced cardiomyocytes. Additionally, it suppressed ferroptosis including lipid peroxidation and ferrous ion, as well as the TLR4 signaling. However, TLR4 overexpression abrogated the modulatory effects of icariin. Conclusions: Icariin mitigates CVB3-induced myocardial injury by inhibiting TLR4-mediated ferroptosis. Further animal study is needed to verify its efficacy.

Keywords

Viral myocarditis / CVB3 / TLR4 / Ferroptosis / Icariin

Conflict of interest statement

The authors declare that there is no conflict of interest.

Funding

This study was supported by Affiliated Hospital of Youjiang Medical University for Nationalities (No. Y20212615).

Data availability statement

The data supporting the findings of this study are available from the corresponding authors upon request.

Authors’ contributions

WL and YL contributed to conceptualization, investigation, visualization, and draft. JHD, PL, YH, WXL, and CLH contributed to investigation and analysis. All authors approved the final version.

References

[1] Xu D, Wang P, Yang J, Qian Q, Li M, Wei L, et al. Gr-1+ cells other than Ly6G+ neutrophils limit virus replication and promote myocardial inflammation and fibrosis following coxsackievirus B3 infection of mice. Front Cell Infect Microbiol 2018; 8: 157.

[2] Olejniczak M, Schwartz M, Webber E, Shaffer A, Perry TE. Viral myocarditis-incidence, diagnosis and management. J Cardiothorac Vasc Anesth 2020; 34(6): 1591-1601.

[3] Pollack A, Kontorovich AR, Fuster V, Dec GW. Viral myocarditis-diagnosis, treatment options, and current controversies. Nat Rev Cardiol 2015; 12(11): 670-680.

[4] Patel T, Kelleman M, West Z, Peter A, Dove M, Butto A, et al. Comparison of multisystem inflammatory syndrome in children-related myocarditis, classic viral myocarditis, and COVID-19 vaccine-related myocarditis in children. J Am Heart Assoc 2022; 11(9). doi: 10.1161/JAHA.121.024393.

[5] Rout A, Suri S, Vorla M, Kalra DK. Myocarditis associated with COVID-19 and its vaccines - a systematic review. Prog Cardiovasc Dis 2022; 74: 111-121.

[6] Tschöpe C, Ammirati E, Bozkurt B, Caforio ALP, Cooper LT, Felix SB, et al. Myocarditis and inflammatory cardiomyopathy: Current evidence and future directions. Nat Rev Cardiol 2021; 18(3): 169-193.

[7] Luo W, Deng J, Xie J, Liang D, Lu J, Lu Y. The protective effect of icariin on myocardial injury in viral myocarditis based on the cyclic adenosine monophosphate-protein kinase a signaling pathway. Nanosci Nanotechnol Lett 2020; 12: 1378-1385.

[8] He C, Wang Z, Shi J. Pharmacological effects of icariin. Adv Pharmacol 2020; 87: 179-203.

[9] Martiniakova M, Babikova M, Omelka R. Pharmacological agents and natural compounds: Available treatments for osteoporosis. J Physiol Pharmacol 2020; 71(3). doi: 10.26402/jpp.2020.3.01.

[10] Koenig A, Buskiewicz I, Huber SA. Age-associated changes in estrogen receptor ratios correlate with increased female susceptibility to coxsackievirus B3-induced myocarditis. Front Immunol 2017; 8: 1585.

[11] Jin J, Wang H, Hua X, Chen D, Huang C, Chen Z. An outline for the pharmacological effect of icariin in the nervous system. Eur J Pharmacol 2019; 842: 20-32.

[12] Bi Z, Zhang W, Yan X. Anti-inflammatory and immunoregulatory effects of icariin and icaritin. Biomed Pharmacother 2022; 151. doi: 10.1016/j.biopha.2022.113180.

[13] Nickel J, Gohlke BO, Erehman J, Banerjee P, Rong WW, Goede A, et al. SuperPred: Update on drug classification and target prediction. Nucleic Acids Res 2014; 42(Web Server issue): W26-W31.

[14] Di Florio DN, Sin J, Coronado MJ, Atwal PS, Fairweather D. Sex differences in inflammation, redox biology, mitochondria and autoimmunity. Redox Biol 2020; 31. doi: 10.1016/j.redox.2020.101482.

[15] Yi L, Hu Y, Wu Z, Li Y, Kong M, Kang Z, et al. TFRC upregulation promotes ferroptosis in CVB3 infection via nucleus recruitment of Sp1. Cell Death Dis 2022; 13(7): 592.

[16] Liu XJ, Lv YF, Cui WZ, Li Y, Liu Y, Xue YT, et al. Icariin inhibits hypoxia/reoxygenation-induced ferroptosis of cardiomyocytes via regulation of the Nrf2/HO-1 signaling pathway. FEBS Open Bio 2021; 11(11): 2966-2976.

[17] Kim SH, Shin HH, Kim JH, Park JH, Jeon ES, Lim BK. Protein kinase B2 (PKB2/AKT2) is essential for host protection in CVB3-induced acute viral myocarditis. Int J Mol Sci 2022; 23(3). doi: 10.3390/ijms23031489.

[18] Fuse K, Chan G, Liu Y, Gudgeon P, Husain M, Chen M, et al. Myeloid differentiation factor-88 plays a crucial role in the pathogenesis of Coxsackievirus B3-induced myocarditis and influences type I interferon production. Circulation 2005; 112(15): 2276-2285.

[19] Garmaroudi FS, Marchant D, Hendry R, Luo H, Yang D, Ye X, et al. Coxsackievirus B3 replication and pathogenesis. Future Microbiol 2015; 10(4): 629-653.

[20] Schultheiss HP, Piper C, Sowade O, Waagstein F, Kapp JF, Wegscheider K, et al. Betaferon in chronic viral cardiomyopathy (BICC) trial: Effects of interferon-β treatment in patients with chronic viral cardiomyopathy. Clin Res Cardiol 2016; 105(9): 763-773.

[21] Amdani SM, Kim HS, Orvedahl A, John AO, Said A, Simpson K. Successful treatment of fulminant neonatal enteroviral myocarditis in monochorionic diamniotic twins with cardiopulmonary support, intravenous immunoglobulin and pocapavir. BMJ Case Rep 2018; 2018. doi: 10.1136/bcr-2017-224133.

[22] Abzug MJ, Michaels MG, Wald E, Jacobs RF, Romero JR, Sánchez PJ, et al. A randomized, double-blind, placebo-controlled trial of pleconaril for the treatment of neonates with enterovirus sepsis. J Pediatric Infect Dis Soc 2016; 5(1): 53-62.

[23] Putschoegl A, Auerbach S. Diagnosis, evaluation, and treatment of myocarditis in children. Pediatr Clin North Am 2020; 67(5): 855-874.

[24] Tatsumi K, Schmedes CM, Houston ER, Butler E, Mackman N, Antoniak S. Protease-activated receptor 4 protects mice from Coxsackievirus B3 and H1N1 influenza A virus infection. Cell Immunol 2019; 344. doi: 10.1016/j.cellimm.2019.103949.

[25] Becher PM, Hinrichs S, Fluschnik N, Hennigs JK, Klingel K, Blankenberg S, et al. Role of Toll-like receptors and interferon regulatory factors in different experimental heart failure models of diverse etiology: IRF7 as novel cardiovascular stress-inducible factor. PLoS One 2018; 13(3). doi: 10.1371/journal.pone.0193844.

[26] Jacobs W, Lammens M, Kerckhofs A, Voets E, Van San E, Van Coillie S, et al. Fatal lymphocytic cardiac damage in coronavirus disease 2019 (COVID-19): Autopsy reveals a ferroptosis signature. ESC Heart Fail 2020; 7(6): 3772-3781.

[27] Kung YA, Chiang HJ, Li ML, Gong YN, Chiu HP, Hung CT, et al. Acyl-coenzyme A synthetase long-chain family member 4 is involved in viral replication organelle formation and facilitates virus replication via ferroptosis. mBio 2022; 13(1). doi: 10.1128/mbio.02717-21.