Citric acid effects on brain and liver oxidative stress in lipopolysaccharide-treated mice

Abstract

Citric acid is a weak organic acid found in the greatest amounts in citrus fruits. This study examined the effect of citric acid on endotoxin-induced oxidative stress of the brain and liver. Mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 200 μg/kg). Citric acid was given orally at 1, 2, or 4 g/kg at time of endotoxin injection and mice were euthanized 4 h later. LPS induced oxidative stress in the brain and liver tissue, resulting in marked increase in lipid peroxidation (malondialdehyde [MDA]) and nitrite, while significantly decreasing reduced glutathione, glutathione peroxidase (GPx), and paraoxonase 1 (PON1) activity. Tumor necrosis factor-alpha (TNF-α) showed a pronounced increase in brain tissue after endotoxin injection. The administration of citric acid (1-2 g/kg) attenuated LPS-induced elevations in brain MDA, nitrite, TNF-α, GPx, and PON1 activity. In the liver, nitrite was decreased by 1 g/kg citric acid. GPx activity was increased, while PON1 activity was decreased by citric acid. The LPS-induced liver injury, DNA fragmentation, serum transaminase elevations, caspase-3, and inducible nitric oxide synthase expression were attenuated by 1-2 g/kg citric acid. DNA fragmentation, however, increased after 4 g/kg citric acid. Thus in this model of systemic inflammation, citric acid (1-2 g/kg) decreased brain lipid peroxidation and inflammation, liver damage, and DNA fragmentation.

Keywords: antioxidant activity; citric acid; cytokines; dietary supplementation; peripheral infection; systemic inflammation.

FIG. 1.

(A) Brain and (B) liver tissue concentrations of malondialdehyde (MDA: nmol/g tissue) in mice given lipopolysaccharide (LPS) or LPS+citric acid (1–4 g/kg, p.o.). *P<.05 versus saline control. ⁺P<.05 versus LPS control group. ^#P<.05 versus LPS+2 g/kg of citric acid. p.o., per os.

FIG. 2.

(A) Brain and (B) liver tissue concentrations of reduced glutathione (GSH: μmol/g tissue) in mice given LPS or LPS+citric acid (1–4 g/kg, p.o.). *P<.05 versus saline control.

FIG. 3.

(A) Brain and (B) liver tissue concentrations of nitrite (μmol/g tissue) in mice after treatment with LPS or LPS+citric acid (1–4 g/kg, p.o.). *P<.05 versus saline control. ⁺P<.05 versus LPS control group. ^#P<.05 versus LPS+2 g/kg of citric acid.

FIG. 4.

Glutathione peroxidase (GPx) activity in (A) brain and (B) liver of mice after LPS or LPS+citric acid (1–4 g/kg, p.o.). *P<.05 versus saline control. ⁺P<.05 versus LPS control group. ^#P<.05 versus LPS+1 g/kg of citric acid.

FIG. 5.

Paraoxonase 1 (PON1) activity in mice (A) brain and (B) liver after treatment with LPS or LPS+citric acid (1–4 g/kg, p.o.). *P<.05 versus saline control. ⁺P<.05 versus LPS control group. ^#P<.05 versus LPS+1 g/kg of citric acid.

FIG. 6.

Brain tissue tumor necrosis factor-alpha (TNF-α; pg/g tissue) in mice given LPS or LPS+citric acid (1–4 g/kg, p.o.). *P<.05 versus the saline control. ⁺P<.05 versus LPS control group. ^#P<.05 versus the LPS+1 g/kg of citric acid.

FIG. 7.

DNA fragmentation (%) in mice liver after LPS or LPS+citric acid (1–4 g/kg, p.o.). *P<.05 versus saline control. ⁺P<.05 versus LPS control group. ^#P<.05 versus LPS+1 g/kg of citric acid.

FIG. 8.

(A) Alanine aminotransferase (ALT) and (B) aspartate aminotransferase (AST) activities in mice liver after LPS or LPS+citric acid (1–4 g/kg, p.o.). *P<.05 versus saline control. ⁺P<.05 versus LPS control group. ^#P<.05 versus LPS+1 g/kg of citric acid.

FIG. 9.

Hematoxylin and eosin (H&E)–stained liver sections from mice treated with (A) saline (control). (B) LPS: inflammatory leukocytic cell infiltration around portal area (long arrow), necrosis (arrow head), dilated and congested blood sinusoids, and marinated nuclear chromatin (star). (C) LPS: focal necrotic area (arrow head), activated Kupffer cells, dilated and congested blood sinusoids, and pyknotic nuclei (star). (D) LPS+citric acid 1 g/kg: congestion of central vein (long arrow), dilated blood sinusoids, and few necrotic cells (arrowhead). (E) LPS+citric acid 2 g/kg: normal central vein (long arrow), minimally dilated blood sinusoids, and few Kupffer cells. (F) LPS+4 g/kg of citric acid: congestion of central vein (long arrow), signs of degeneration of hepatocytes, dilated congested blood sinusoids, and few Kupffer cells (H&E×400). Color images available online at www.liebertpub.com/jmf

FIG. 10.

The effect of LPS and citric acid treatment on hepatic caspase-3 (i) and inducible nitric oxide synthase (iNOS) (ii) immunostaining: (A) control liver; (B) LPS; (C) LPS+citric acid 1 g/kg; (D) LPS+citric acid 2 g/kg; (E) citric acid 4 g/kg (caspase-3 and iNOS immunohistochemistry, hematoxylin counterstain ×400). Color images available online at www.liebertpub.com/jmf