Metformin inhibits HMGB1 release in LPS‐treated RAW 264.7 cells and increases survival rate of endotoxaemic mice

Abstract

BACKGROUND AND PURPOSE Recently, metformin, a well‐known anti‐diabetic drug, has been shown to possess anti‐inflammatory activities. This study investigated the effect of metformin on the expression of pro‐inflammatory cytokines including high mobility group box 1 (HMGB1) in lipopolysaccharide (LPS)‐treated animals and cells. EXPERIMENTAL APPROACH We investigated whether metformin inhibits the release of HMGB1 in LPS‐treated RAW 264.7 cells and increases survival rate in endotoxaemic mice (lethal endotoxaemia was induced by an i.p. injection of LPS). This was achieved by a range of techniques including Western blotting, enzyme‐linked immunosorbent assay, specific pharmacological inhibitors, knock out of α1‐subunit of AMP‐activated protein kinase (AMPK) and recombinant HMGB1. KEY RESULTS Both pre‐ and post‐treatment with metformin significantly improved survival of animals during lethal endotoxaemia (survival rate was monitored up to 2 weeks), decreased serum levels of tumour necrosis factor‐alpha (TNF‐α), interleukin‐1β, HMGB1 expression and myeloperoxidase activity in lungs. However, metformin failed to improve survival in endotoxaemic animals that had additionally been treated with recombinant HMGB1. In an in vitro study, metformin dose‐dependently inhibited production of pro‐inflammatory cytokines and HMGB1 release. Metformin activated AMPK by its phosphorylation. Compound C (pharmacological inhibitor of AMPK) and siAMPKα1 reversed the anti‐inflammatory effect of metformin in LPS‐treated cells. CONCLUSIONS AND IMPLICATIONS Our data indicate that metformin significantly attenuates the pro‐inflammatory response induced by LPS both in vivo and in vitro. Metformin improved survival in a mouse model of lethal endotoxaemia by inhibiting HMGB1 release. AMPK activation was implicated as one of the mechanisms contributing to this inhibition of HMGB1 secretion.

Store-operated Ca2+ Entry (SOCE) Induced by Protease-activated Receptor-1 Mediates STIM1 Protein Phosphorylation to Inhibit SOCE in Endothelial Cells through AMP-activated Protein Kinase and p38β Mitogen-activated Protein Kinase

“…Zhao et al, (35) have shown that AICAR-mediated AMPK activation reduced endotoxininduced acute lung injury in mice. Another study showed that administration of AMPK activator metformin (anti-diabetic drug) increased the survival rate of endotoxemic mice (36). Creighton et al (27) have demonstrated that AMPK␣1 signaling promotes the endothelial barrier repair process.…”

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

“…For example, release of HMGB1, histones and mitochondrial proteins are known to increase the sever- (68)(69)(70)(71). Of note, AMPK activators, including metformin, were shown to diminish acute inflammatory injury of lung or liver as well as decrease the release of DAMPs and improve mortality in experimental models of LPS-induced sepsis (5,29,36). Although antibiotics are sufficient to kill bacteria, the combination of metformin and antibiotics may have additional benefit in sepsis, particularly as metforminstimulated neutrophils have increased chemotaxis and bacterial uptake.…”

“…Recent studies have shown that besides their ability to regulate cellular metabolism (27), metformin and other AMPK activators can decrease the severity of organ injury in acute inflammatory states (28), including LPS-induced liver injury or acute lung injury (29,30). Metformin, berberine or aminoimidazole carboxamide ribonucleotide (AICAR) were all shown to diminish activation of the tolllike receptor 4 (TLR4)/NF-κB signaling cascade, as well as to release of proinflammatory mediators of neutrophils and macrophages, and also to enhance endothelial integrity in vitro and in models for sepsis (31)(32)(33)(34)(35)(36). In addition, metformin enhances host defense mechanisms by facilitating the chemotaxis and maturation of T cells (37,38).…”

Type 2 diabetes mellitus, drug addiction, bipolar disorder and epilepsy display overlapping aetiopathogenic mechanisms: Implication for prevention and pharmacotherapy

“…C-reactive protein levels, which are elevated in pilocarpine-induced status epilepticus (Holtman et al, 2013),and may be a biomarker of de-novo depression risk in bipolar disorder (Walker et al, 2014), is lowered by metformin (Oriaifo et al, 2014) which also decreases COX 2 levels (Luchetti et al, 2008) that may be upregulated by NF-kappa B (Crofford et al, 1997) and important for epileptogenesis (Loscher et al, 2010). Metformin and other AMPK activators such as valproate may acutely and chronically suppress TLR 4 signaling (Soraya et al, 2014) and high-mobility group box-I (HMGBI) (Tsoyi et al, 2011) important for genesis of proinflammatory cascades, insulin resistance, bipolar disorder and epileptogenesis showing a convergence of mechanisms amongst these disorders.…”