Korean Circ J.  2018 Oct;48(10):933-943. 10.4070/kcj.2017.0373.

Treatment with 3-Bromo-4,5-Dihydroxybenzaldehyde Improves Cardiac Function by Inhibiting Macrophage Infiltration in Mice

• Ji N
• Lou H
• Gong X
• Fu T
• Ni S

• KMID: 2420646
• DOI: http://doi.org/10.4070/kcj.2017.0373

Abstract

BACKGROUND AND OBJECTIVES
Appropriate inflammatory response is necessary for cardiac repairing after acute myocardial infarction (MI). Three-Bromo-4,5-dihydroxybenzaldehyde (BDB) is a potent antioxidant and natural bromophenol compound derived from red algae. Although BDB has been shown to have an anti-inflammatory effect, it remains unclear whether BDB affects cardiac remolding after MI. The aim of this study was to investigate the potential role of BDB on cardiac function recovery after MI in mice.
METHODS
Mice were intraperitoneally injected with BDB (100 mg/kg) or vehicle control respectively 1 hour before MI and then treated every other day. Cardiac function was monitored by transthoracic echocardiography at day 7 after MI. The survival of mice was observed for 2 weeks and hematoxylin and eosin (H&E) staining was used to determine the infarct size. Macrophages infiltration was examined by immunofluorescence staining. Enzyme-linked immunosorbent assay (ELISA) was used to test the production of cytokines associated with macrophages. The phosphorylation status of nuclear factor (NF)-κB was determined by western blot.
RESULTS
BDB administration dramatically improved cardiac function recovery, and decreased mortality and infarcted size after MI. Treatment with BDB reduced CD68+ macrophages, M1 and M2 macrophages infiltration post-MI, and suppressed the secretion of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1, and IL-6 in the injured hearts. Furthermore, BDB inhibited the phosphorylation of NF-κB in the infarcted hearts.
CONCLUSIONS
These data demonstrate, for the first time, that BDB treatment facilitated cardiac healing by suppressing pro-inflammatory cytokine secretion, and indicate that BDB may serve as a therapeutic agent for acute MI.

Keyword

Myocardial infarction; 3-Bromo-4,5-Dihydroxybenzaldehyde; Macrophages

MeSH Terms

Animals
Blotting, Western
Cytokines
Echocardiography
Enzyme-Linked Immunosorbent Assay
Eosine Yellowish-(YS)
Fluorescent Antibody Technique
Heart
Hematoxylin
Interleukin-6
Interleukins
Macrophages*
Mice*
Monocytes
Mortality
Myocardial Infarction
Phosphorylation
Recovery of Function
Rhodophyta
Tumor Necrosis Factor-alpha
Cytokines
Eosine Yellowish-(YS)
Hematoxylin
Interleukin-6
Interleukins
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 BDB treatment improved cardiac recovery after MI in mice. (A) Schematic of protocol for adoptive transfer and administration of BDB to mice. Acute MI was induced by surgical ligation of LAD in mice. (B, C) Cardiac function of vehicle and BDB treatment mice on day 14 after MI. (D) Ratio of HW/BW of mice was measured 14 days after MI. (E) Survival rates after MI in BDB- or vehicle-treated mice. Survival curves up to 2 weeks after MI were created by Kaplan-Meier method and compared by a log-rank test. (F) Representative H&E staining of heart sections from vehicle or BDB treated-mice at 2 weeks after MI (scale bar=1,000 μm). (G) Quantitative analysis of infarct size in vehicle and BDB treated-mice at 2 weeks after MI. Data are expressed as mean±SEM (n=6–14). BDB = 3-Bromo-4,5-dihydroxybenzaldehyde; H&E = hematoxylin and eosin; HW/BW = heart weight to body weight; LAD = left anterior descending artery; LV = left ventricle; LVEF = left ventricular ejection fraction; LVFS = left ventricular fractional shortening; MI = myocardial infarction; SEM = standard error of measurement. *The p<0.05 compared with vehicle group.

• 

  Figure 2 BDB treatment did not affect cardiomyocytes apoptosis after MI. (A) Representative TUNEL staining of heart sections from vehicle- and BDB-treated mice at day 3 post-MI (scale bar, 20 μm). (B) Quantification of TUNEL+ cardiomyocytes from (A) (n=6–8). BDB = 3-Bromo-4,5-dihydroxybenzaldehyde; DAPI = 4′,6-diamidino-2-phenylindole; MI = myocardial infarction; ns = not significant; TUNEL = terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling.

• 

  Figure 3 BDB treatment reduced macrophages infiltration after MI in mice. The representative CD68 (red) staining images of the infarcted hearts from BDB- or vehicle-treated mice at day 3 (A) and day 7 (C) post-MI (scale bar, 50 μm). (B) Quantitation of CD68+ cells in injured hearts as shown in (A). (D) Quantitation of CD68+ cells of infarcted hearts as shown in (C). Data are expressed as mean±SEM (n=8–10). BDB = 3-Bromo-4,5-dihydroxybenzaldehyde; BZ = ischemic border zone; DAPI = 4′,6-diamidino-2-phenylindole; DZ = non-ischemic distant zone; MI = myocardial infarction; SEM = standard error of measurement. *The p<0.05 compared with vehicle group.

• 

  Figure 4 Treatment with BDB suppresses both M1 and M2 macrophage recruitment in hearts of mice after MI. (A) Gating strategy for CD11b+CD45+Ly6G−F4/80+CD206+ and CD11b+CD45+Ly6G−F4/80+CD206− macrophages in hearts from BDB-treated mice after MI. (B-D) Effect of BDB treatment on the recruitment of (B) total macrophages, (C) M1, and (D) M2 macrophages in injured hearts from vehicle or BDB-treated mice on day 3 and 7 after MI. Data are expressed as mean±SEM (n=6–8). BDB = 3-Bromo-4,5-dihydroxybenzaldehyde; MI = myocardial infarction. *The p<0.05 compared with vehicle group.

• 

  Figure 5 Administration of BDB reduced the levels of inflammatory cytokines in infarcted hearts. (A-D) Hearts from 3 days MI mice treated with vehicle and BDB (100 mg/kg) were evaluated for protein expressions by ELISA analysis; (A) TNF-α, (B) MCP-1, (C) IL-1β, and (D) IL-6. (E-H) Hearts from 7 days MI mice treated with vehicle and BDB (100 mg/kg) were evaluated for protein expressions by ELISA analysis: (E) TNF-α, (F) MCP-1, (G) IL-1β, and (H) IL-6. Data are expressed as mean±SEM (n=6–8). BDB = 3-Bromo-4,5-dihydroxybenzaldehyde; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; MCP = monocyte chemoattractant protein; MI = myocardial infarction; TNF = tumor necrosis factor; SEM = standard error of measurement. *The p<0.05 compared with vehicle group.

• 

  Figure 6 Effect of BDB on the NF-κB p65 phosphorylation in mice hearts of MI. (A) The protein level of phosphorylated NF-κB p65 in sham or infarcted hearts was analyzed by western blotting. (B) Quantification of phosphorylated NF-κB p65. Data are expressed as mean±SEM (n=4). BDB = 3-Bromo-4,5-dihydroxybenzaldehyde; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; MI = myocardial infarction; NF = nuclear factor. *The p<0.05 compared with vehicle group.

Reference

1. Aurora AB, Porrello ER, Tan W, et al. Macrophages are required for neonatal heart regeneration. J Clin Invest. 2014; 124:1382–1392.

2. Maruotti N, Annese T, Cantatore FP, Ribatti D. Macrophages and angiogenesis in rheumatic diseases. Vasc Cell. 2013; 5:11.

3. Hofmann U, Frantz S. Role of lymphocytes in myocardial injury, healing, and remodeling after myocardial infarction. Circ Res. 2015; 116:354–367.

4. Fan X, Xu NJ, Shi JG. Bromophenols from the red alga Rhodomela confervoides . J Nat Prod. 2003; 66:455–458.

5. Li K, Li XM, Ji NY, Wang BG. Bromophenols from the marine red alga Polysiphonia urceolata with DPPH radical scavenging activity. J Nat Prod. 2008; 71:28–30.

6. Kim SY, Kim SR, Oh MJ, Jung SJ, Kang SY. In vitro antiviral activity of red alga, Polysiphonia morrowii extract and its bromophenols against fish pathogenic infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus. J Microbiol. 2011; 49:102–106.

7. Piao MJ, Kang KA, Ryu YS, et al. The red algae compound 3-bromo-4,5-dihydroxybenzaldehyde protects human keratinocytes on oxidative stress-related molecules and pathways activated by UVB irradiation. Mar Drugs. 2017; 15:E268.

8. Hyun YJ, Piao MJ, Zhang R, Choi YH, Chae S, Hyun JW. Photo-protection by 3-bromo-4, 5-dihydroxybenzaldehyde against ultraviolet B-induced oxidative stress in human keratinocytes. Ecotoxicol Environ Saf. 2012; 83:71–78.

9. Kim KC, Hyun YJ, Hewage SR, et al. 3-Bromo-4,5-dihydroxybenzaldehyde enhances the level of reduced glutathione via the Nrf2-mediated pathway in human keratinocytes. Mar Drugs. 2017; 15:E291.

10. Kang NJ, Han SC, Kang HJ, et al. Anti-inflammatory effect of 3-bromo-4,5-dihydroxybenzaldehyde, a component of polysiphonia morrowii, in vivo and in vitro. Toxicol Res. 2017; 33:325–332.

11. Kong D, Shen Y, Liu G, et al. PKA regulatory IIα subunit is essential for PGD2-mediated resolution of inflammation. J Exp Med. 2016; 213:2209–2226.

12. Vlasov IA, Volkov AM. Dependence of heart weight on body weight in patients with cardiovascular diseases. Fiziol Cheloveka. 2004; 30:62–68.

13. Gaffney L, Warren P, Wrona EA, Fisher MB, Freytes DO. Macrophages' role in tissue disease and regeneration. Results Probl Cell Differ. 2017; 62:245–271.

14. Lavine KJ, Epelman S, Uchida K, et al. Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart. Proc Natl Acad Sci USA. 2014; 111:16029–16034.

15. Zhang JY, Yang Z, Fang K, Shi ZL, Ren DH, Sun J. Oleuropein prevents the development of experimental autoimmune myocarditis in rats. Int Immunopharmacol. 2017; 48:187–195.

16. Speranskii AI, Kostyuk SV, Kalashnikova EA, Veiko NN. Enrichment of extracellular DNA from the cultivation medium of human peripheral blood mononuclears with genomic CpG rich fragments results in increased cell production of IL-6 and TNF-a via activation of the NF-kB signaling pathway. Biomed Khim. 2016; 62:331–340.

17. Lee JC, Hou MF, Huang HW, et al. Marine algal natural products with anti-oxidative, anti-inflammatory, and anti-cancer properties. Cancer Cell Int. 2013; 13:55.

18. Nahrendorf M, Swirski FK, Aikawa E, et al. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med. 2007; 204:3037–3047.

19. Nahrendorf M, Swirski FK. Monocyte and macrophage heterogeneity in the heart. Circ Res. 2013; 112:1624–1633.

20. Gilmore TD. Introduction to NF-kappaB: players, pathways, perspectives. Oncogene. 2006; 25:6680–6684.

21. Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol. 2007; 8:49–62.

22. Capece D, Verzella D, Tessitore A, Alesse E, Capalbo C, Zazzeroni F. Cancer secretome and inflammation: the bright and the dark sides of NF-κB. [Epub ahead of print]. Semin Cell Dev Biol. 2017.

23. Catrysse L, van Loo G. Inflammation and the metabolic syndrome: the tissue-specific functions of NF-κB. Trends Cell Biol. 2017; 27:417–429.

24. Han Y, Englert JA, Yang R, Delude RL, Fink MP. Ethyl pyruvate inhibits nuclear factor-kappaB-dependent signaling by directly targeting p65. J Pharmacol Exp Ther. 2005; 312:1097–1105.