Marinobufagenin Inhibits Neutrophil Migration and Proinflammatory Cytokines

Abstract

Cardiotonic steroids, such as ouabain and digoxin, are known to bind to Na+/K+-ATPase and to promote several biological activities, including anti-inflammatory activity. However, there are still no reports in the literature about inflammation and marinobufagenin, a cardiotonic steroid from the bufadienolide family endogenously found in mammals. Therefore, the aim of this work was to analyze, in vivo and in vitro, the role of marinobufagenin in acute inflammation. Swiss mice were treated with 0.56 mg/kg of marinobufagenin intraperitoneally (i.p.) and zymosan (2 mg/mL, i.p.) was used to induce peritoneal inflammation. Peritoneal fluid was collected and used for counting cells by optical microscopy and proinflammatory cytokine quantification (IL-1β, IL-6, and TNF-α) by immunoenzymatic assay (ELISA). Zymosan stimulation, as expected, induced increased cell migration and proinflammatory cytokine levels in the peritoneum. Marinobufagenin treatment reduced polymorphonuclear cell migration and IL-1β and IL-6 levels in the peritoneal cavity, without interfering in TNF-α levels. In addition, the effect of marinobufagenin was evaluated using peritoneal macrophages stimulated by zymosan (0.2 mg/mL) in vitro. Marinobufagenin treatment at different concentrations (10, 100, 1000, and 10000 nM) showed no cytotoxic effect on peritoneal macrophages. Interestingly, the lowest concentration, which did not inhibit Na+/K+-ATPase activity, attenuated proinflammatory cytokines IL-1β, IL-6, and TNF-α levels. To investigate the putative mechanism of action of marinobufagenin, the expression of surface molecules (TLR2 and CD69) and P-p38 MAPK were also evaluated, but no significant effect was observed. Thus, our results suggest that marinobufagenin has an anti-inflammatory role in vivo and in vitro and reveals a novel possible endogenous function of this steroid in mammals.

Neuroinflammation and Neutrophils: Modulation by Ouabain

“…In vitro studies also presented the inhibitory effect of ouabain on rabbit (Ward and Becker, 1970), human (Ray and Samanta, 1997), and mice neutrophil chemotaxis (Cavalcante-Silva et al, 2021a). The cardiotonic steroid marinobufagenin also reduces neutrophil migration during inflammation, corroborating the ouabain effect (Carvalho et al, 2019) (Figure 1B).…”

Effect of ouabain on calcium signaling in rodent brain: A systematic review of in vitro studies

“…An interesting feature is that Na + /K + -ATPase isoforms are expressed in a cell-/tissue-specific fashion ( Blanco, 2005 ), ( Cereijido et al, 2012 ), ( Geering, 2008 ): the α1 isoform is ubiquitously expressed in mammalian tissues and, in rodents, it is 100–1000-fold less sensitive to cardiotonic steroids. In contrast, it has been shown not to be the case for the bufadienolide marinobufagenin ( Fedorova and Bagrov, 1997 ), ( Fedorova et al, 2001 ), although our group reported that it behaves similar to any known cardiotonic steroid ( Godinho et al, 2017 ), ( Carvalho et al, 2019 ); the α 2 isoform is found in striated and smooth muscle, as well as in both astrocytes and neurons in the central nervous system, and adipose tissue; the α 3 isoform is basically found in neurons but not in astrocytes and should be considered a neuronal marker ( Dobretsov and Stimers, 2005 ); the α 4 isoform is specifically found in the midpiece of sperm ( Blanco, 2005 ). The β subunit is a type II glycoprotein of around 300 amino acids, that is, fundamental for a normal pumping activity.…”

Mechanisms providing effects of cardiotonic steroids in mammalian cells with special attention to blood cells

“…It was demonstrated [91] that administration of ouabain in a dose of 0.56 mg/kg to mice infected with Leishmania (L.) amazonensis resulted in a decrease of peritoneal migration of macrophages and in the production of two proinflammatory cytokines interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) and had no cytotoxic effects on macrophages. Intraperitoneal injection of marinobufagenin in a dose of 0.56 mg/kg to mice showed a decrease in zymosan-activated production of proinflammatory cytokines IL-1β and IL-6, without affecting the level of TNF-α [92]. The treatment of peritoneal macrophages with different concentration (10, 100, 1000, and 10000 nM) of marinobufagenin in vitro showed no cytotoxic effect.…”