Neuroprotective effect of tangeretin against chromium-induced acute brain injury in rats: targeting Nrf2 signaling pathway, inflammatory mediators, and apoptosis

Abstract

Potassium dichromate (PD) is an environmental xenobiotic commonly recognized as teratogenic, carcinogenic, and mutagenic in animals and humans. The present study was conducted to investigate the role of tangeretin (TNG) as a neuro-protective drug against PD-induced brain injury in rats. Thirty-two male adult Wistar rats were blindly divided into four groups (8 rats/group). The first group received saline intranasally (i.n.). The second group received a single dose of PD (2 mg/kg, i.n.). The third group received TNG (50 mg/kg; orally), for 14 days followed by i.n. of PD on the last day of the experiment. The fourth group received TNG (100 mg/kg; orally) for 14 days followed by i.n. of PD on the last day of the experiment. Behavioral indices were evaluated 18 h after PD administration. Neuro-biochemical indices and histopathological studies were evaluated 24 h after PD administration. Results of the present study revealed that rats intoxicated with PD induced- oxidative stress and inflammation via an increase in malondialdehyde (MDA) and a decrease in nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and glutathione(GSH) levels with an increase in brain contents of tumor necrosis factor-alpha (TNF-α) and interleukin (IL-6). Pre-treatment with TNG (100 mg/kg; orally) ameliorated behavior, cholinergic activities, and oxidative stress and decreased the elevated levels of pro-inflammatory mediators; TNF-α and IL-6 with a decrease in brain content of chromium residues detected by Plasma-Optical Emission Spectrometer. Also, the histopathological picture of the brain was improved significantly in rats that received TNG (100 mg/kg). Additionally, TNG decreased caspase-3 expression in the brain of PD rats. In conclusion, TNG possesses a significant neuroprotective role against PD-induced acute brain injury via modulating the Nrf2 signaling pathway and quenching the release of inflammatory mediators and apoptosis in rats.

Keywords: Brain Injury; Caspase-3; Chromium; Inflammation; Nrf2; Tangeretin.

Fig. 1

SchemaLtic representation of the experimental design

Fig. 2

Effect of tangeretin on motor activity and motor coordination in rats received PD induced - acute brain injury. Pre-treatment of acute brain injury induced by i.n dose of PD with TNG orally either 50 or 100 mg/kg for 14 days. (a) Motor activity; (b) Motor coordination were measured 18h after the last dose of the drug. Results are expressed as mean ± SEM (n = 8). ^aSignificant difference from normal group P < 0.05. ^bSignificant difference from PD group P < 0.05. ^cSignificant difference from PD+TNG (50 mg/kg) group P < 0.05

Fig. 3

Effect of tangeretin on open field test (rearing & crossing) in rats received PD induced - acute brain injury. Pre-treatment of acute brain injury induced by i.n dose of PD with TNG orally either 50 or 100 mg/kg for 14 days. (a) Rearing; (b) Cossing counts were measured 18h after the last dose of the drug. Results are expressed as mean ± SEM (n = 8). ^aSignificant difference from normal group P < 0.05. ^bSignificant difference from PD group P < 0.05. ^cSignificant difference from PD+TNG (50 mg/kg) group P < 0.05

Fig. 4

Effect of tangeretin on the serum levels of S100β in rats received PD induced - acute brain injury. Pre-treatment of acute brain injury induced by i.n dose of PD with TNG orally either 50 or 100 mg/kg for 14 days. Serum levels of S100β were evaluated 24h after the last dose of the drug. Results are expressed as mean ± SEM (n = 8). ^aSignificant difference from normal group P < 0.05. ^bSignificant difference from PD group P < 0.05. ^cSignificant difference from PD+TNG (50 mg/kg) group P < 0.05

Fig. 5

Effect of tangeretin on AChE and ATP levels in rats received PD induced - acute brain injury. Pre-treatment of acute brain injury induced by i.n dose of PD with TNG orally either 50 or 100 mg/kg for 14 days. (a) AChE; (b) ATP levels were measured 24h after the last dose of the drug. Results are expressed as mean ± SEM (n = 8). ^aSignificant difference from normal group P < 0.05. ^bSignificant difference from PD group P < 0.05. ^cSignificant difference from PD+TNG (50 mg/kg) group P < 0.05

Fig. 6

Effect of tangeretin on GSH and MDA levels in rats received PD induced - acute brain injury. Pre-treatment of acute brain injury induced by i.n dose of PD with TNG orally either 50 or 100 mg/kg for 14 days. (a) GSH; (b) MDA levels were measured 24h after the last dose of the drug in brain homogenate. Results are expressed as mean ± SEM (n = 8). ^aSignificant difference from normal group P < 0.05. ^bSignificant difference from PD group P < 0.05. ^cSignificant difference from PD + TNG (50 mg/kg) group P < 0.05

Fig. 7

Effect of tangeretin on SOD and CAT levels in rats received PD induced - acute brain injury. Pre-treatment of acute brain injury induced by i.n dose of PD with TNG orally either 50 or 100 mg/kg for 14 days. (a) SOD; (b) CAT levels were measured 24h after the last dose of the drug in brain homogenate. Results are expressed as mean ± SEM (n = 8). ^aSignificant difference from normal group P < 0.05. ^bSignificant difference from PD group P < 0.05. ^cSignificant difference from PD + TNG (50 mg/kg) group P < 0.05

Fig. 8

Effect of tangeretin on protein carbonyl levels and DNA fragmentation % in rats received PD induced - acute brain injury. Pre-treatment of acute brain injury induced by i.n dose of PD with TNG orally either 50 or 100 mg/kg for 14 days. (a) Protein carbonyl levels; (b) DNA fragmentation % were evaluated 24h after the last dose of the drug in brain. Results are expressed as mean ± SEM (n = 8). ^aSignificant difference from normal group P < 0.05. ^bSignificant difference from PD group P < 0.05. ^cSignificant difference from PD + TNG (50 mg/kg) group P < 0.05

Fig. 9

Effect of tangeretin on the expression levels of TNF-α and IL-6 in rats received PD induced - acute brain injury. Pre-treatment of acute brain injury induced by i.n dose of PD with TNG orally either 50 or 100 mg/kg for 14 days. (a) TNF-α; (b) IL-6 levels were evaluated 24h after the last dose of the drug in brain. Results are expressed as mean ± SEM (n = 8). ^aSignificant difference from normal group P < 0.05. ^bSignificant difference from PD group P < 0.05. ^cSignificant difference from PD + TNG (50 mg/kg) group P < 0.05

Fig. 10

Effect of tangeretin on the histopathological examination of cerebellum of rats received PD induced—acute brain injury. Normal control group showed normal histological structure of the cerebellum with normal Purkinje cells (a). PD group revealed shrinkage in the granule layer of cerebellum and necrosis of Purkinje cells with aggregation of microglia around the degenerated neurons (b). PD + TNG (50 mg/kg) group showed less scattered degenerated neurons and less shrinkage in granular layer of the cerebellum (c). PD + TNG (100 mg/kg) group succeeded to show normal histological structure of the cerebellum (d)

Fig. 11

Effect of tangeretin on the expression levels of caspase-3 in the cerebellum of rats received PD induced—acute brain injury. Normal control group revealed no positive stained caspase-3 cells in the cerebellum (a), PD group revealed a marked increment in the % of positive stained caspase-3 cells within the granular layer and Purkinje cells of the cerebellum (b), PD + TNG (50 mg/kg) group showed a decrement in the % of positive stained caspase-3 cells (c), PD + TNG (100 mg/kg) group revealed sparse % in positively stained caspase-3 cells (d) (scale bar, 100 µm)

Fig. 12

Effect of tangeretin on the expression levels of Nrf-2 in the cerebellum of rats received PD induced—acute brain injury. Normal control group revealed cytoplasmic localization of weakly positive stained Nrf-2 cells in the cerebellum (a), PD group revealed a significant decrement in the % of positive stained Nrf-2 cells within the granular layer and Purkinje cells of the cerebellum (b), PD + TNG (50 mg/kg) group showed a decrement in the % of positive stained Nrf-2 cells (c), PD + TNG (100 mg/kg) group revealed sparse % in positively stained Nrf-2 cells with nuclear staining (d) (scale bar, 100 µm)