Galangin Activates Nrf2 Signaling and Attenuates Oxidative Damage, Inflammation, and Apoptosis in a Rat Model of Cyclophosphamide-Induced Hepatotoxicity

Abstract

Cyclophosphamide (CP) is a widely used chemotherapeutic agent; however, its clinical application is limited because of its multi-organ toxicity. Galangin (Gal) is a bioactive flavonoid with promising biological activities. This study investigated the hepatoprotective effect of Gal in CP-induced rats. Rats received Gal (15, 30 and 60 mg/kg/day) for 15 days followed by a single dose of CP at day 16. Cyclophosphamide triggered liver injury characterized by elevated serum transaminases, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH), and histopathological manifestations. Increased hepatic reactive oxygen species, malondialdehyde, nitric oxide, and oxidative DNA damage along with declined glutathione and antioxidant enzymes were demonstrated in CP-administered rats. CP provoked hepatic nuclear factor-kappaB (NF-κB) phosphorylation and increased mRNA abundance of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) both expression and serum levels. Gal prevented CP-induced liver injury, boosted antioxidants and suppressed oxidative stress, DNA damage, NF-κB phosphorylation and pro-inflammatory mediators. Gal diminished Bax and caspase-3, and increased B-cell lymphoma-2 (Bcl-2) in liver of CP-administered rats. In addition, Gal increased peroxisome proliferator-activated receptor gamma (PPARγ) expression and activated hepatic nuclear factor erythroid 2-related factor 2 (Nrf2) signaling showed by the increase in Nrf2, NAD(P)H: quinone acceptor oxidoreductase-1 (NQO-1) and heme oxygenase 1 (HO-1) in CP-administered rats. These findings suggest that Gal prevents CP hepatotoxicity through activation of Nrf2/HO-1 signaling and attenuation of oxidative damage, inflammation and cell death. Therefore, Gal might represent a promising adjuvant therapy to prevent hepatotoxicity in patients on CP treatment.

Keywords: cyclophosphamide; galangin; hepatotoxicity; inflammation; nuclear factor erythroid 2-related factor 2; reactive oxygen species.

Figure 1

Galangin (Gal) prevents liver injury in cyclophosphamide (CP)-intoxicated rats. Gal ameliorated (A) alanine aminotransferase (ALT), (B) aspartate aminotransferase (AST), (C) alkaline phosphatase (ALP), and (D) lactate dehydrogenase (LDH) in serum of rats received CP. Data are mean ± SEM, (N = 6). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 2

Photomicrographs of liver sections of (A) control and (B) Gal-treated rats showing normal liver architecture, (C–E) CP-intoxicated rats showing degenerative changes and cytoplasmic vacuolations (black arrow), leukocyte infiltration (arrowhead), hemorrhage (green arrow), and congestions (blue arrow), and CP-induced rats pre-treated with (F) 15 mg/kg, (G) 30 mg/kg, and (H) 60 mg/kg Gal showing mild congestion (blue arrow), hemorrhage (green arrow), and cytoplasmic vacuolations (black arrow). (Hematoxylin and eosin (H&E); X200) (Scale bar = 100 µm).

Figure 3

Galangin (Gal) prevents CP-induced alterations in the expression of cytochromes (CYPs). (A) Gal down-regulated CYPs 2B1, 2B2, 2E1, and 3A2 mRNA abundance in CP-induced rats. Data are mean ± SEM, (N = 6). *** P < 0.001 versus Control, and ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 versus CP. (B) Heat map showing the effect of Gal on the expression of CYPs in CP-induced rats.

Figure 4

Galangin (Gal) suppresses oxidative stress in CP-intoxicated rats. Gal suppressed hepatic (A) reactive oxygen species (ROS), (B) malondialdehyde (MDA), (C) nitric oxide (NO), and (D) 8-Oxo-dG. Data are mean ± SEM, (N = 6). *** P < 0.001.

Figure 5

Galangin (Gal) enhances cellular antioxidants in CP-intoxicated rats. Gal increased (A) Reduced glutathione (GSH), (C) GSH/glutathione disulfide (GSSG) ratio, and activity of (D) superoxide dismutase (SOD), (E) catalase (CAT), and (F) glutathione peroxidase (GPx), and decreased GSSG (B) in the liver of CP-intoxicated rats. Data are mean ± SEM, (N = 6). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 6

Galangin (Gal) suppresses inflammation in CP-intoxicated rats. Gal reduced hepatic (A) NF-κB p65, mRNA abundance of (B) iNOS, (C) COX-2, (D) TNF-α, and (E) IL-1β, and serum levels of (F) TNF-α and (G) IL-1β. Data are mean ± SEM, (N = 6). * P < 0.05 and *** P < 0.001.

Figure 7

Galangin (Gal) attenuates CP-induced hepatic apoptosis in CP-intoxicated rats. Gal increased BCL-2 (B) and decreased (A) BAX, (C) BAX/BCL-2 ratio, (D) caspase-3 mRNA, and (E) caspase-3 activity. Data are mean ± SEM, (N = 6). * P < 0.05 and *** P < 0.001.

Figure 8

Galangin (Gal) activates Nrf2/HO-1 signaling in CP-intoxicated rats. Gal increased (A) Nrf2 mRNA, (B) Nrf2 protein, (C) NQO-1 mRNA, and (D) HO-1 mRNA in liver of CP-induced rats. Data are mean ± SEM, (N = 6). * P < 0.05 and *** P < 0.001.

Figure 9

Galangin (Gal) increases peroxisome proliferator activated receptor gamma (PPARγ) expression in liver of CP-intoxicated rats. Data are mean ± SEM, (N = 6). *** P < 0.001.

Figure 10

A schematic diagram illustrating the protective effect of galangin against cyclophosphamide hepatotoxicity. Galangin up-regulated Nrf2/HO-1 signaling and PPARγ, enhanced antioxidants, and suppressed ROS production, inflammation, and apoptosis in CP-induced rats. RXR, retinoid X receptor; CYP450, cytochrome P450; sMAF, small MAF proteins.