Effects of Sulforaphane on SARS‑CoV‑2 infection and NF‑κB dependent expression of genes involved in the COVID‑19 ‘cytokine storm’

Gasparello,Jessica; Marzaro,Giovanni; Papi,Chiara; Gentili,Valentina; Rizzo,Roberta; Zurlo,Matteo; Scapoli,Chiara; Finotti,Alessia; Gambari,Roberto

doi:10.3892/ijmm.2023.5279

Effects of Sulforaphane on SARS‑CoV‑2 infection and NF‑κB dependent expression of genes involved in the COVID‑19 ‘cytokine storm’

Authors:
- Jessica Gasparello
- Giovanni Marzaro
- Chiara Papi
- Valentina Gentili
- Roberta Rizzo
- Matteo Zurlo
- Chiara Scapoli
- Alessia Finotti
- Roberto Gambari
View Affiliations

Affiliations: Department of Life Sciences and Biotechnology, University of Ferrara, I‑44121 Ferrara, Italy, Department of Pharmaceutical and Pharmacological Sciences, University of Padova, I‑35131 Padova, Italy, Department of Chemical and Pharmaceutical Sciences, University of Ferrara, I‑44121 Ferrara, Italy
Published online on: July 14, 2023 https://doi.org/10.3892/ijmm.2023.5279
Article Number: 76
Copyright: © Gasparello et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Since its spread at the beginning of 2020, the coronavirus disease 2019 (COVID‑19) pandemic represents one of the major health problems. Despite the approval, testing, and worldwide distribution of anti‑severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) vaccines, the development of specific antiviral agents targeting the SARS‑CoV‑2 life cycle with high efficiency, and/or interfering with the associated ‘cytokine storm’, is highly required. A recent study, conducted by the authors' group indicated that sulforaphane (SFN) inhibits the expression of IL‑6 and IL‑8 genes induced by the treatment of IB3‑1 bronchial cells with a recombinant spike protein of SARS‑CoV‑2. In the present study, the ability of SFN to inhibit SARS‑CoV‑2 replication and the expression of pro‑inflammatory genes encoding proteins of the COVID‑19 ‘cytokine storm’ was evaluated. SARS‑CoV‑2 replication was assessed in bronchial epithelial Calu‑3 cells. Moreover, SARS‑CoV‑2 replication and expression of pro‑inflammatory genes was evaluated by reverse transcription quantitative droplet digital PCR. The effects on the expression levels of NF‑κB were assessed by western blotting. Molecular dynamics simulations of NF‑kB/SFN interactions were conducted with Gromacs 2021.1 software under the Martini 2 CG force field. Computational studies indicated that i) SFN was stably bound with the NF‑κB monomer; ii) a ternary NF‑kB/SFN/DNA complex was formed; iii) the SFN interacted with both the protein and the nucleic acid molecules modifying the binding mode of the latter, and impairing the full interaction between the NF‑κB protein and the DNA molecule. This finally stabilized the inactive complex. Molecular studies demonstrated that SFN i) inhibits the SARS‑CoV‑2 replication in infected Calu‑3 cells, decreasing the production of the N‑protein coding RNA sequences, ii) decreased NF‑κB content in SARS‑CoV‑2 infected cells and inhibited the expression of NF‑kB‑dependent IL‑1β and IL‑8 gene expression. The data obtained in the present study demonstrated inhibitory effects of SFN on the SARS‑CoV‑2 life cycle and on the expression levels of the pro‑inflammatory genes, sustaining the possible use of SFN in the management of patients with COVID‑19.

View Figures

View References

1	Walker PGT, Whittaker C, Watson OJ, Baguelin M, Winskill P, Hamlet A, Djafaara BA, Cucunubá Z, Olivera Mesa D, Green W, et al: The impact of COVID-19 and strategies for mitigation and suppression in low- and middle-income countries. Science. 369:413–422. 2020. View Article : Google Scholar : PubMed/NCBI
2	Khan S, Siddique R, Ali A, Bai Q, Li Z, Li H, Shereen MA, Xue M and Nabi G: The spread of novel coronavirus has created an alarming situation worldwide. J Infect Public Health. 13:469–471. 2020. View Article : Google Scholar : PubMed/NCBI
3	Killerby ME, Biggs HM, Midgley CM, Gerber SI and Watson JT: Middle east respiratory syndrome coronavirus transmission. Emerg Infect Dis. 26:191–198. 2020. View Article : Google Scholar : PubMed/NCBI
4	Watson OJ, Barnsley G, Toor J, Hogan AB, Winskill P and Ghani AC: Global impact of the first year of COVID-19 vaccination: A mathematical modelling study. Lancet Infect Dis. 22:1293–1302. 2022. View Article : Google Scholar : PubMed/NCBI
5	Tulimilli SV, Dallavalasa S, Basavaraju CG, Kumar Rao V, Chikkahonnaiah P, Madhunapantula SV and Veeranna RP: Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and vaccine effectiveness. Vaccines (Basel). 10:17512022. View Article : Google Scholar : PubMed/NCBI
6	Villamagna AH, Gore SJ, Lewis JS and Doggett JS: The need for antiviral drugs for pandemic coronaviruses from a global health perspective. Front Med (Lausanne). 7:5965872020. View Article : Google Scholar
7	Takashita E, Kinoshita N, Yamayoshi S, Sakai-Tagawa Y, Fujisaki S, Ito M, Iwatsuki-Horimoto K, Halfmann P, Watanabe S, Maeda K, et al: Efficacy of antiviral agents against the SARS-CoV-2 omicron subvariant BA.2. N Engl J Med. 386:1475–1477. 2022. View Article : Google Scholar : PubMed/NCBI
8	Kumari M, Lu RM, Li MC, Huang JL, Hsu FF, Ko SH, Ke FY, Su SC, Liang KH, Yuan JP, et al: A critical overview of current progress for COVID-19: Development of vaccines, antiviral drugs, and therapeutic antibodies. J Biomed Sci. 29:682022. View Article : Google Scholar
9	Zhu C, Lee JY, Woo JZ, Xu L, Nguyenla X, Yamashiro LH, Ji F, Biering SB, Van Dis E, Gonzalez F, et al: An intranasal ASO therapeutic targeting SARS-CoV-2. Nat Commun. 13:45032022. View Article : Google Scholar : PubMed/NCBI
10	Mondal S, Quintili AL, Karamchandani K and Bose S: Thromboembolic disease in COVID-19 patients: A brief narrative review. J Intensive Care. 8:702020. View Article : Google Scholar : PubMed/NCBI
11	Zong X, Gu Y, Yu H, Li Z and Wang Y: Thrombocytopenia is associated with COVID-19 severity and outcome: An updated meta-analysis of 5637 patients with multiple outcomes. Lab Med. 52:10–15. 2021. View Article : Google Scholar
12	Smilowitz NR, Kunichoff D, Garshick M, Shah B, Pillinger M, Hochman JS and Berger JS: C-reactive protein and clinical outcomes in patients with COVID-19. Eur Heart J. 42:2270–2279. 2021. View Article : Google Scholar
13	Santa Cruz A, Mendes-Frias A, Oliveira AI, Dias L, Matos AR, Carvalho A, Capela C, Pedrosa J, Castro AG and Silvestre R: Interleukin-6 is a biomarker for the development of fatal severe acute respiratory syndrome coronavirus 2 pneumonia. Front Immunol. 12:6134222021. View Article : Google Scholar : PubMed/NCBI
14	Hu B, Huang S and Yin L: The cytokine storm and COVID-19. J Med Virol. 93:250–256. 2021. View Article : Google Scholar
15	Iwasaki M, Saito J, Zhao H, Sakamoto A, Hirota K and Ma D: Inflammation triggered by SARS-CoV-2 and ACE2 augment drives multiple organ failure of severe COVID-19: Molecular mechanisms and implications. Inflammation. 44:13–34. 2021. View Article : Google Scholar
16	Yang J, Petitjean SJL, Koehler M, Zhang Q, Dumitru AC, Chen W, Derclaye S, Vincent SP, Soumillion P and Alsteens D: Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor. Nat Commun. 11:45412020. View Article : Google Scholar :
17	Song P, Li W, Xie J, Hou Y and You C: Cytokine storm induced by SARS-CoV-2. Clin Chim Acta. 509:280–287. 2020. View Article : Google Scholar :
18	Hazeldine J and Lord JM: Neutrophils and COVID-19: Active participants and rational therapeutic targets. Front Immunol. 12:6801342021. View Article : Google Scholar : PubMed/NCBI
19	Veenith T, Martin H, Le Breuilly M, Whitehouse T, Gao-Smith F, Duggal N, Lord JM, Mian R, Sarphie D and Moss P: High generation of reactive oxygen species from neutrophils in patients with severe COVID-19. Sci Rep. 12:104842022. View Article : Google Scholar : PubMed/NCBI
20	Sefik E, Qu R, Junqueira C, Kaffe E, Mirza H, Zhao J, Brewer JR, Han A, Steach HR, Israelow B, et al: Inflammasome activation in infected macrophages drives COVID-19 pathology. Nature. 606:585–593. 2022. View Article : Google Scholar : PubMed/NCBI
21	Moss P: The T cell immune response against SARS-CoV-2. Nat Immunol. 23:186–193. 2022. View Article : Google Scholar
22	Mohammed RN, Tamjidifar R, Rahman HS, Adili A, Ghoreishizadeh S, Saeedi H, Thangavelu L, Shomali N, Aslaminabad R, Marofi F, et al: A comprehensive review about immune responses and exhaustion during coronavirus disease (COVID-19). Cell Commun Signal. 20:792022. View Article : Google Scholar : PubMed/NCBI
23	Majidpoor J and Mortezaee K: Interleukin-6 in SARS-CoV-2 induced disease: Interactions and therapeutic applications. Biomed Pharmacother. 145:1124192022. View Article : Google Scholar
24	Zizzo G, Tamburello A, Castelnovo L, Laria A, Mumoli N, Faggioli PM, Stefani I and Mazzone A: Immunotherapy of COVID-19: Inside and beyond IL-6 signalling. Front Immunol. 13:7953152022. View Article : Google Scholar : PubMed/NCBI
25	Khan S, Shafiei MS, Longoria C, Schoggins JW, Savani RC and Zaki H: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway. Elife. 10:e685632021. View Article : Google Scholar
26	Matsuyama T, Kubli SP, Yoshinaga SK, Pfeffer K and Mak TW: An aberrant STAT pathway is central to COVID-19. Cell Death Differ. 27:3209–3225. 2020. View Article : Google Scholar : PubMed/NCBI
27	Kaiser AE, Baniasadi M, Giansiracusa D, Giansiracusa M, Garcia M, Fryda Z, Wong TL and Bishayee A: Sulforaphane: A broccoli bioactive phytocompound with cancer preventive potential. Cancers (Basel). 13:47962021. View Article : Google Scholar
28	Barba FJ, Nikmaram N, Roohinejad S, Khelfa A, Zhu Z and Koubaa M: Bioavailability of glucosinolates and their breakdown products: Impact of processing. Front Nutr. 3:242016. View Article : Google Scholar : PubMed/NCBI
29	Narbad A and Rossiter JT: Gut glucosinolate metabolism and isothiocyanate production. Mol Nutr Food Res. 62:e17009912018. View Article : Google Scholar : PubMed/NCBI
30	Lenzi M, Fimognari C and Hrelia P: Sulforaphane as a promising molecule for fighting cancer. Cancer Treat Res. 159:207–223. 2014. View Article : Google Scholar
31	Kubo E, Chhunchha B, Singh P, Sasaki H and Singh DP: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress. Sci Rep. 7:141302017. View Article : Google Scholar : PubMed/NCBI
32	Haristoy X, Angioi-Duprez K, Duprez A and Lozniewski A: Efficacy of sulforaphane in eradicating Helicobacter pylori in human gastric xenografts implanted in nude mice. Antimicrob Agents Chemother. 47:3982–3984. 2003. View Article : Google Scholar
33	Schepici G, Bramanti P and Mazzon E: Efficacy of sulforaphane in neurodegenerative diseases. Int J Mol Sci. 21:86372020. View Article : Google Scholar :
34	Li YP, Wang SL, Liu B, Tang L, Kuang RR, Wang XB, Zhao C, Song XD, Cao XM, Wu X, et al: Sulforaphane prevents rat cardiomyocytes from hypoxia/reoxygenation injury in vitro via activating SIRT1 and subsequently inhibiting ER stress. Acta Pharmacol Sin. 37:344–353. 2016. View Article : Google Scholar : PubMed/NCBI
35	Qi T, Xu F, Yan X, Li S and Li H: Sulforaphane exerts anti-inflammatory effects against lipopolysaccharide-induced acute lung injury in mice through the Nrf2/ARE pathway. Int J Mol Med. 37:182–188. 2016. View Article : Google Scholar
36	Folkard DL, Marlow G, Mithen RF and Ferguson LR: Effect of sulforaphane on NOD2 via NF-κB: Implications for Crohn's disease. J Inflamm (Lond). 12:62015. View Article : Google Scholar
37	Negi G, Kumar A and Sharma SS: Nrf2 and NF-κB modulation by sulforaphane counteracts multiple manifestations of diabetic neuropathy in rats and high glucose-induced changes. Curr Neurovasc Res. 8:294–304. 2011. View Article : Google Scholar : PubMed/NCBI
38	Xu C, Shen G, Chen C, Gélinas C and Kong ANT: Suppression of NF-kappaB and NF-kappaB-regulated gene expression by sulforaphane and PEITC through IkappaBalpha, IKK pathway in human prostate cancer PC-3 cells. Oncogene. 24:4486–4495. 2005. View Article : Google Scholar : PubMed/NCBI
39	Liu T, Zhang L, Joo D and Sun SC: NF-κB signaling in inflammation. Signal Transduct Target Ther. 2:170232017. View Article : Google Scholar
40	Libermann TA and Baltimore D: Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol Cell Biol. 10:2327–2334. 1990.PubMed/NCBI
41	McFarland BC, Hong SW, Rajbhandari R, Twitty GB Jr, Gray GK, Yu H, Benveniste EN and Nozell SE: NF-κB-induced IL-6 ensures STAT3 activation and tumor aggressiveness in glioblastoma. PLoS One. 8:e787282013. View Article : Google Scholar
42	Elliott CL, Allport VC, Loudon JA, Wu GD and Bennett PR: Nuclear factor-kappa B is essential for up-regulation of interleukin-8 expression in human amnion and cervical epithelial cells. Mol Hum Reprod. 7:787–790. 2001. View Article : Google Scholar
43	Fahey JW and Kensler TW: The challenges of designing and implementing clinical trials with broccoli sprouts… and turning evidence into public health action. Front Nutr. 8:6487882021. View Article : Google Scholar
44	Yagishita Y, Fahey JW, Dinkova-Kostova AT and Kensler TW: Broccoli or sulforaphane: Is it the source or dose that matters? Molecules. 24:35932019. View Article : Google Scholar : PubMed/NCBI
45	Yagishita Y, Gatbonton-Schwager TN, McCallum ML and Kensler TW: Current landscape of NRF2 biomarkers in clinical trials. Antioxidants (Basel). 9:7162020. View Article : Google Scholar : PubMed/NCBI
46	Zimmerman AW, Singh K, Connors SL, Liu H, Panjwani AA, Lee LC, Diggins E, Foley A, Melnyk S, Singh IN, et al: Randomized controlled trial of sulforaphane and metabolite discovery in children with autism spectrum disorder. Mol Autism. 12:382021. View Article : Google Scholar : PubMed/NCBI
47	Gasparello J, D'Aversa E, Papi C, Gambari L, Grigolo B, Borgatti M, Finotti A and Gambari R: Sulforaphane inhibits the expression of interleukin-6 and interleukin-8 induced in bronchial epithelial IB3-1 cells by exposure to the SARS-CoV-2 spike protein. Phytomedicine. 87:1535832021. View Article : Google Scholar : PubMed/NCBI
48	Ordonez AA, Bullen CK, Villabona-Rueda AF, Thompson EA, Turner ML, Merino VF, Yan Y, Kim J, Davis SL, Komm O, et al: Sulforaphane exhibits antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses in vitro and in mice. Commun Biol. 5:2422022. View Article : Google Scholar : PubMed/NCBI
49	Bortolotti D, Gentili V, Rizzo S, Schiuma G, Beltrami S, Strazzabosco G, Fernandez M, Caccuri F, Caruso A and Rizzo R: TLR3 and TLR7 RNA sensor activation during SARS-CoV-2 infection. Microorganisms. 9:18202021. View Article : Google Scholar : PubMed/NCBI
50	Papi C, Gasparello J, Zurlo M, Manicardi A, Corradini R, Cabrini G, Gambari R and Finotti A: Combined treatment of bronchial epithelial Calu-3 cells with peptide nucleic acids targeting miR-145-5p and miR-101-3p: Synergistic enhancement of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Int J Mol Sci. 23:93482022. View Article : Google Scholar
51	Gasparello J, Papi C, Zurlo M, Gambari L, Rozzi A, Manicardi A, Corradini R, Gambari R and Finotti A: Treatment of human glioblastoma U251 cells with sulforaphane and a peptide nucleic acid (PNA) targeting miR-15b-5p: Synergistic effects on induction of apoptosis. Molecules. 27:12992022. View Article : Google Scholar : PubMed/NCBI
52	Heiss E, Herhaus C, Klimo K, Bartsch H and Gerhäuser C: Nuclear factor kappa B is a molecular target for sulforaphane-mediated anti-inflammatory mechanisms. J Biol Chem. 276:32008–32015. 2001. View Article : Google Scholar
53	Caruso A, Caccuri F, Bugatti A, Zani A, Vanoni M, Bonfanti P, Cazzaniga ME, Perno CF, Messa C and Alberghina L: Methotrexate inhibits SARS-CoV-2 virus replication 'in vitro'. J Med Virol. 93:1780–1785. 2021. View Article : Google Scholar
54	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar
55	Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE and Berendsen HJC: GROMACS: Fast, flexible, and free. Comput Chem. 26:1701–1718. 2005. View Article : Google Scholar
56	Pronk S, Páll S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, Shirts MR, Smith JC, Kasson PM, van der Spoel D, et al: GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics. 29:845–854. 2013. View Article : Google Scholar : PubMed/NCBI
57	Marrink SJ, Risselada HJ, Yefimov S, Tieleman DP and de Vries AH: The MARTINI force field: Coarse grained model for biomolecular simulations. J Phys Chem B. 111:7812–7824. 2007. View Article : Google Scholar : PubMed/NCBI
58	Bereau T and Kremer K: Automated parametrization of the coarse-grained Martini force field for small organic molecules. J Chem Theory Comput. 11:2783–2791. 2015. View Article : Google Scholar
59	Lamprakis C, Andreadelis I, Manchester J, Velez-Vega C, Duca JS and Cournia Z: Evaluating the efficiency of the Martini force field to study protein dimerization in aqueous and membrane environments. J Chem Theory Comput. 17:3088–3102. 2021. View Article : Google Scholar : PubMed/NCBI
60	Souza PCT, Thallmair S, Conflitti P, Ramírez-Palacios C, Alessandri R, Raniolo S, Limongelli V and Marrink SJ: Protein-ligand binding with the coarse-grained Martini model. Nat Commun. 11:37142020. View Article : Google Scholar : PubMed/NCBI
61	Honorato RV, Roel-Touris J and Bonvin AMJJ: MARTINI-based protein-DNA coarse-grained HADDOCKing. Front Mol Biosci. 6:1022019. View Article : Google Scholar : PubMed/NCBI
62	Periole X, Cavalli M, Marrink SJ and Ceruso MA: Combining an elastic network with a coarse-grained molecular force field: Structure, dynamics, and intermolecular recognition. J Chem Theory Comput. 5:2531–2543. 2009. View Article : Google Scholar : PubMed/NCBI
63	Zhang X, Wu K, Wang D, Yue X, Song D, Zhu Y and Wu J: Nucleocapsid protein of SARS-CoV activates interleukin-6 expression through cellular transcription factor NF-kappaB. Virology. 365:324–335. 2007. View Article : Google Scholar : PubMed/NCBI
64	du Preez HN, Aldous C, Kruger HG and Johnson L: N-Acetylcysteine and other sulfur-donors as a preventative and adjunct therapy for COVID-19. Adv Pharmacol Pharm Sci. 2022:45554902022.PubMed/NCBI
65	Zinovkin RA and Grebenchikov OA: Transcription factor Nrf2 as a potential therapeutic target for prevention of cytokine storm in COVID-19 patients. Biochemistry (Mosc). 85:833–837. 2020. View Article : Google Scholar : PubMed/NCBI
66	Cuadrado A, Pajares M, Benito C, Jiménez-Villegas J, Escoll M, Fernández-Ginés R, Garcia Yagüe AJ, Lastra D, Manda G, Rojo AI and Dinkova-Kostova AT: Can activation of NRF2 Be a strategy against COVID-19? Trends Pharmacol Sci. 41:598–610. 2020. View Article : Google Scholar :
67	Kow CS, Ramachandram DS and Hasan SS: Use of sulforaphane in COVID-19: Clinical trials are needed. Mol Immunol. 145:78–79. 2022. View Article : Google Scholar :