Ethyl Pyruvate Downregulates Tumor Necrosis Factor Alpha and Interleukin (IL)-6 and Upregulates IL-10 in Lipopolysaccharide-Stimulated Canine Peripheral Blood Mononuclear Cells

Abstract

ABSTRACT. We investigated the anti-inflammatory effects of ethyl pyruvate (EP) on LPS-stimulated canine PBMCs in vitro. We found that EP treatment inhibited the mRNA expressions of proinflammatory cytokines (TNF- and IL-6), but induced mRNA expression of anti-inflammatory cytokines (IL-10). ELISA measurements revealed that EP also effectively downregulates the LPS-induced increase in proinflammatory cytokine release, while upregulating anti-inflammatory cytokine release. These data indicate that EP could be an effective anti-inflammatory agent in dogs.KEY WORDS: canine peripheral blood mononuclear cells, cytokine, ethyl pyruvate, mRNA.J. Vet. Med. Sci. 72(10): 1379-1381, 2010 Ethyl pyruvate (EP) is a simple aliphatic ester derived from the endogenous metabolite pyruvic acid and has been shown to exhibit antioxidant [5] and anti-inflammatory [1] properties. While EP is known to inhibit activation of p28 mitogen-activated protein kinase and NF-B in murine macrophages [10], its anti-inflammatory mechanism remains unclear.We hypothesized that lipopolysaccharide (LPS) treatment of canine PBMCs would induce proinflammatory cytokine expression and that EP would attenuate this increase at either the mRNA or protein level. To address this hypothesis, we selected TNF- and IL-6 as representative proinflammatory cytokines that are upregulated after LPS administration. In addition, we selected IL-10 as an anti-inflammatory cytokine.Blood was collected from six healthy beagles and peripheral blood mononuclear cells (PBMCs) were separated using Histopaque ) and 100 U/ml penicillin G and 10 g/ml streptomycin (Sigma) at 37C in 5% CO 2 and incubated overnight. The next day, the mononuclear cells were exposed to 100 ng/ml of LPS (Escherichia coli serotype O111: B4, Sigma) and simultaneously treated with various concentrations of EP (Sigma) (5 d 10 mM each) based on previous in vitro studies [9]. The cell pellets were used for RNA isolation, and the cell-free supernatants were stored at -80C until they were analyzed. Samples were harvested after 3, 6, 12 and 24 hr of cultivation. Unstimulated cells and cells in 10 mM ethyl pyruvate were used as controls. All procedures used in this study were approved by local animal welfare authorities (CBU2008-021).For the quantification of cytokine gene expression, SYBR ® green real-time PCR was used. Total RNA was extracted using an RNeasy The primers utilized in this study are summarized in Table 1. The optimal annealing temperature (T a ) for all assays ranged from 60C for GAPDH and IL-6 to 62C for TNF- and IL-10. Primer efficiency calculations [E (%) = (10 (1/-S) -1)  100, S=slope] for all of the standard lines were between 95.7 and 102.4%. Relative quantification was analyzed using the -2 CT method as previously described [4]. Tests were performed in duplicate.Our quantification of cytokine mRNA abundance demonstrated that EP inhibits TNF- and IL-6 mRNA synthesis and promotes IL-10 mRNA expression on LPS stimulation,

Pathophysiologic and Immunologic Changes in a Canine Endotoxemia over a Period of 24 Hours

“…This cytokine activation profile obtained with CRI-LL is in accordance with our previous canine peripheral blood mononuclear cell (PBMC) over a period of 24 hr in vitro study [35]. The simultaneous increase in IL-10 with TNF-α and IL-6 increment can be explained a compensatory antiinflammatory response.…”

HMGB1 in renal ischemic injury

“…EP is a pharmacological inhibitor of HMGB1 secretion (13,14,18,31,41,53,59,62,73,74,82); however, the precise mechanism(s) that mediate its inhibitory effects have not been identified as of yet. Treatment with EP has been shown to improve survival and alleviate organ dysfunction in a wide variety of preclinical models of critical illness (19,20) including the amelioration of IRI in organs (13,34,77,84) including the kidney (13).…”

“…For animal experiments using EP, animals received a one-time intraperitoneal bolus injection of 40 mg/kg body wt of EP (Sigma, St. Louis, MO) (26,31,41,73,82) 15 min before IRI surgery. Due to the acidic nature of EP, 15 mM HEPES was added and the resulting solution was set to a pH of 7.2.…”

Valproic acid attenuates the expression of pro-inflammatory cytokines lipopolysaccharide-treated canine peripheral blood mononuclear cells (in vitro) and in a canine endotoxemia model (in vivo)

“…All procedures were similar to our previous report (Yu et al, 2010b). PBMCs were isolated by centrifugation with a Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA) at 450 × g for 45 min.…”