Nitro-oleic acid protects against endotoxin-induced endotoxemia and multiorgan injury in mice

Abstract

Nitroalkene derivatives of nitro-oleic acid (OA-NO2) are endogenous lipid products with potent anti-inflammatory properties in vitro. The present study was undertaken to evaluate the in vivo anti-inflammatory effect of OA-NO2 in mice given LPS. Two days before LPS administration, C57BL/6J mice were chronically infused with vehicle (LPS vehicle) or OA-NO2 (LPS OA-NO2) at 200 microg x kg(-1) x day(-1) via osmotic minipumps; LPS was administered via a single intraperitoneal (ip) injection (10 mg/kg in saline). A third group received an ip injection of saline without LPS or OA-NO2 and served as controls. At 18 h of LPS administration, LPS vehicle mice displayed multiorgan dysfunction as evidenced by elevated plasma urea and creatinine (kidney), aspartate aminotransferase (AST) and alanine aminotransferase (ALT; liver), and lactate dehydrogenase (LDH) and reduced ejection fraction (heart). In contrast, the severity of multiorgan dysfunction was less in LPS OA-NO2 animals. The levels of circulating TNF-alpha and renal TNF-alpha mRNA expression, together with renal mRNA expression of monocyte chemoattractant protein-1, ICAM-1, and VCAM-1, and with renal mRNA and protein expression of inducible nitric oxide synthase and cyclooxygenase 2, and renal cGMP and PGE2 contents, were greater in LPS vehicle vs. control mice, but were attenuated in LPS OA-NO2 animals. Similar patterns of changes in the expression of inflammatory mediators were observed in the liver. Together, pretreatment with OA-NO2 ameliorated the inflammatory response and multiorgan injury in endotoxin-induced endotoxemia in mice.

Fig. 1.

Effect of nitro-oleic acid (OA-NO₂) on body temperature and hematocrit in LPS-induced endotoxemia in mice. Male C57/BL6 mice were pretreated with vehicle (LPS vehicle) or OA-NO₂ (LPS OA-NO₂) at 200 μg·kg⁻¹·day⁻¹ via osmotic minipumps, followed by LPS administration. LPS dissolved in saline was administered at 10 mg/kg via a single intraperitoneal (ip) injection. Body temperature and hematocrit was analyzed 18 h post-LPS. Control mice (Cont) received an ip injection of saline. Control: n = 4; LPS: n = 8; LPS+OA-NO₂: n = 9. Values are means ± SE.

Fig. 2.

Plasma blood urea nitrogen (BUN; A) and creatinine (B) levels in control, LPS vehicle, and LPS OA-NO₂ mice. Control: n = 4; LPS: n = 8; LPS+OA-NO₂: n = 9. Values are means ± SE.

Fig. 3.

Renal TNF-α mRNA expression and circulating TNF-α levels in control, LPS vehicle, and LPS OA-NO₂ mice. Renal TNF-α mRNA expression was determined by qRT-PCR and normalized by β-actin. Plasma TNF-α was measured by ELISA. Control: n = 4; LPS: n = 8; LPS+OA-NO₂: n = 9. Values are means ± SE.

Fig. 4.

qRT-PCR analysis of renal mRNA expression of monocyte chemoattractant protein-1 (MCP-1; A), ICAM-1 (B), and VCAM-1 (C) in control, LPS vehicle, and LPS OA-NO₂ mice. Control: n = 4; LPS: n = 8; LPS+OA-NO₂: n = 9. Values are means ± SE.

Fig. 5.

Renal inducible nitric oxide synthase (iNOS) expression in control, LPS vehicle, and LPS OA-NO₂ mice. A: quantitative (q)RT-PCR. B: immunoblot analysis (top, immunoblots; bottom, densitometry). C: renal concentration of cGMP. Control: n = 4; LPS: n = 8; LPS+OA-NO₂: n = 9. Values are means ± SE.

Fig. 6.

Renal cyclooxygenase 2 (COX-2) expression in control, LPS vehicle, and LPS OA-NO₂ mice. A: qRT-PCR analysis of COX-2 mRNA, B: immunoblot analysis of COX-2 protein (top, immunoblots; bottom, densitometry). C: ELISA analysis of kidney PGE₂ content. (top, immunoblots; bottom, densitometry). Control: n = 4; LPS: n = 8; LPS+OA-NO2: n = 9. Values are means ± SE.

Fig. 7.

The activities of plasma aspartate aminotransferase (AST; A), alanine aminotransferase (ALT; B), and liver TNF-α (C) in control, LPS vehicle, and LPS OA-NO₂ mice. Tissue TNF-α content was determined by ELISA. Control: n = 4; LPS: n = 8; LPS+OA-NO₂: n = 9. Values are means ± SE.

Fig. 8.

qRT-PCR analysis of hepatic mRNA expression of TNF-α (A), IL-1 (B), ICAM-1 (C), MCP-1 (D), iNOS (E), and COX-2 (F) in control, LPS vehicle, and LPS OA-NO₂ mice. Control: n = 4; LPS: n = 8; LPS+OA-NO₂: n = 9. Values are means ± SE.

Fig. 9.

Evaluation of endotoxin-induced cardiac injury in control, LPS vehicle, and LPS OA-NO₂ mice. A: echocardiography analysis of ejection fraction (EF). B: plasma LDH. C: qRT-PCR analysis of heart TNF-α. Control: n = 4–5; LPS: n = 8–11; LPS+OA-NO₂: n = 9–12. Values are means ± SE.

Fig. 10.

Radiotelemetric determination of mean arterial pressure (MAP; A) and heart rate (B) in LPS vehicle and LPS OA-NO₂ mice. The arrow indicates the time of LPS injection. The immediate rise of MAP and heart rate following LPS injection reflects the influence from stress. LPS: n = 8; LPS+OA-NO₂: n = 10. Values are means ± SE. *P < 0.05 vs. basal values in the same group. #P < 0.05 vs. LPS alone at the corresponding period.

Fig. 11.

Comparison of the effects of OA-NO₂ vs. oleic acid (OA) on body temperature (A), hematocrit (B), plasma BUN (C), and creatinine (D) in endotoxemic C57/BL6 mice; n = 5/group. Values are means ± SE.