Flavocoxid, a dual inhibitor of COX-2 and 5-LOX of natural origin, attenuates the inflammatory response and protects mice from sepsis

Abstract

Introduction: Cecal ligation and puncture (CLP) is an inflammatory condition that leads to multisystemic organ failure. Flavocoxid, a dual inhibitor of cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX), has been shown in vitro to possess antiinflammatory activity in lipopolysaccharide (LPS)-stimulated rat macrophages by reducing nuclear factor (NF)-κB activity and COX-2, 5-LOX and inducible nitric oxide synthase (iNOS) expression. The aim of this study was to evaluate the effects of flavocoxid in a murine model of CLP-induced polymicrobial sepsis.

Methods: C57BL/6J mice were subjected to CLP or sham operation. In a first set of experiments, an intraperitoneal injection of flavocoxid (20 mg/kg) or vehicle was administered 1 hour after surgery and repeated every 12 hours. Survival rate was monitored every 24 hours throughout 120 hours. Furthermore, additional groups of sham and CLP mice were killed 18 hours after surgical procedures for blood-sample collection and the lung and liver were collected for biomolecular, biochemical and histopathologic studies.

Results: COX-2, 5-LOX, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-10, extracellular-regulated-kinase 1/2 (ERK), JunN-terminal kinase (JNK), NF-κB, and β-arrestin 2 protein expression were evaluated in lung and liver with Western blot analysis. In addition, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), cytokines, and lipoxin A4 serum content were measured with an enzyme-linked immunosorbent assay (ELISA). Flavocoxid administration improved survival, reduced the expression of NF-κB, COX-2, 5-LOX, TNF-α and IL-6 and increased IL-10 production. Moreover, flavocoxid inhibited the mitogen-activated protein kinases (MAPKs) pathway, preserved β-arrestin 2 expression, reduced blood LTB4, PGE2, TNF-α and IL-6, and increased IL-10 and lipoxin A4 serum levels. The treatment with flavocoxid also protected against the histologic damage induced by CLP and reduced the myeloperoxidase (MPO) activity in the lung and liver.

Conclusions: Flavocoxid protects mice from sepsis, suggesting that this dual inhibitor may represent a promising approach in such a life-threatening condition.

Figure 1

Effect of flavocoxid on survival in mice with cecal ligation and puncture (CLP). C57BL/6J mice were subjected to CLP (n = 15 per group) or sham operation (n = 10 per group). All the CLP animals were fluid resuscitated with sterile NaCl 0.9% saline solution (1 ml/mouse). Animals were treated with flavocoxid (20 mg/kg, ip) with and without gentamicin (3.2 mg/kg, sc), or baicalin (16 mg/kg, ip), or cathechin (4 mg/kg, ip), or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Mouse survival was monitored every 24 hours for a total of 120 hours, and expressed as percentage of mice survived at each time point. *P < 0.01 compared with sham + vehicle or flavocoxid; ^#P < 0.01 compared with CLP + vehicle; ^§P < 0.01 compared with CLP + vehicle.

Figure 2

Western blot analysis of TNF-α, IL-6, and IL-10 in the lung (a through c) and liver (d through f) of CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; ^#P < 0.005 compared with CLP + vehicle.

Figure 3

Western blot analysis of COX-2 and 5-LOX in the lung (a and b) and liver (c and d) of CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; ^#P < 0.005 compared with CLP + vehicle.

Figure 4

Serum TNF-α (a), IL-6 (b), and IL-10 (c) levels obtained from CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; ^#P < 0.005 compared with CLP + vehicle.

Figure 5

Serum PGE₂ (a), LTB₄ (b), and lipoxin A₄ (c) levels obtained from CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; ^#P < 0.005 compared with CLP + vehicle.

Figure 6

Western blot analysis of NF-κB and β-arrestin 2 in lung (a, b) and liver (c, d) of CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, and repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; ^#P < 0.005 compared with CLP + vehicle.

Figure 7

Western blot analysis of p-ERK 1/2 and p-JNK in lung (a, b) and liver (c, d) of CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; ^#P < 0.005 compared with CLP + vehicle.

Figure 8

Histology of treated and untreated mice. (a) Normal histology of lung obtained from sham mice. (b) Representative cecal ligation and puncture (CLP)-induced lung damage. (c) Representative lung of CLP mice treated with flavocoxid (20 mg/kg, ip, administered 1 hour after surgery, repeated every 12 hours). (d) Normal histology of liver obtained from sham mice. (e) Representative CLP-induced liver damage. (f) Representative liver of CLP mice treated with flavocoxid (20 mg/kg, ip, administered 1 hour after surgery, repeated every 12 hours). Sham and CLP mice were killed 18 hours after surgery. Histopathology was performed on lung and liver. Original magnification, ×20.

Figure 9

Myeloperoxidase (MPO) activity in the lung (a) and liver (b) obtained from CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; ^#P < 0.005 compared with CLP + vehicle.