Protective Effects of Thymoquinone, an Active Compound of Nigella sativa, on Rats with Benzo(a)pyrene-Induced Lung Injury through Regulation of Oxidative Stress and Inflammation

Abstract

Benzopyrene [B(a)P] is a well-recognized environmental carcinogen, which promotes oxidative stress, inflammation, and other metabolic complications. In the current study, the therapeutic effects of thymoquinone (TQ) against B(a)P-induced lung injury in experimental rats were examined. B(a)P used at 50 mg/kg b.w. induced lung injury that was investigated via the evaluation of lipid profile, inflammatory markers, nitric oxide (NO), and malondialdehyde (MDA) levels. B(a)P also led to a decrease in superoxide dismutase (SOD) (34.3 vs. 58.5 U/mg protein), glutathione peroxidase (GPx) (42.4 vs. 72.8 U/mg protein), catalase (CAT) (21.2 vs. 30.5 U/mg protein), and total antioxidant capacity compared to normal animals. Treatment with TQ, used at 50 mg/kg b.w., led to a significant reduction in triglycerides (TG) (196.2 vs. 233.7 mg/dL), total cholesterol (TC) (107.2 vs. 129.3 mg/dL), and inflammatory markers and increased the antioxidant enzyme level in comparison with the group that was administered B(a)P only (p < 0.05). B(a)P administration led to the thickening of lung epithelium, increased inflammatory cell infiltration, damaged lung tissue architecture, and led to accumulation of collagen fibres as studied through haematoxylin and eosin (H&E), Sirius red, and Masson's trichrome staining. Moreover, the recognition of apoptotic nuclei and expression pattern of NF-κB were evaluated through the TUNEL assay and immunohistochemistry, respectively. The histopathological changes were found to be considerably low in the TQ-treated animal group. The TUNEL-positive cells increased significantly in the B(a)P-induced group, whereas the TQ-treated group showed a decreased apoptosis rate. Significantly high cytoplasmic expression of NF-κB in the B(a)P-induced group was seen, and this expression was prominently reduced in the TQ-treated group. Our results suggest that TQ can be used in the protection against benzopyrene-caused lung injury.

Keywords: TUNEL assay; antioxidant status; benzopyrene; immunohistochemistry; inflammatory markers; thymoquinone.

Figure 1

The lipid profile as cholesterol and triglyceride levels in different animal groups. The values indicate the mean ± SEM, with 8 animals/group. The animals administered with B(a)P showed these parameters at significantly higher levels than the control group. The TQ (50 mg/kg b.w.) promoted a significant reduction in lipid profile. The statistical differences are denoted with an asterisk (*), indicating significance at p < 0.05 in comparison with the control group, and a hashtag (#), signifying p < 0.05 in comparison with the disease control.

Figure 2

The protein profile (total protein and serum albumin) in different animal groups. The values indicate the mean ± SEM, with 8 animals/group. The statistical differences are denoted with an asterisk (*), indicating significance at p < 0.05 in comparison with the control group, and a hashtag (#), signifying p < 0.05 in comparison with the disease control group.

Figure 3

(a) The level of MDA was considerably increased in the animal group intoxicated with only B(a)P in comparison with the control group. TQ treatment significantly decreased the MDA level compared to the group treated with B(a)P only. (b) The NO concentration was considerably amplified in the group treated with B(a)P only compared to the control animals. TQ treatment decreased the NO level significantly in comparison with the B(a)P-treated group. The values denote the mean ± SEM, with 8 rats/group. The statistical differences are denoted with an asterisk (*), indicating significance at p < 0.05 in comparison with the control group, and a hashtag (#), signifying p < 0.05 in comparison with the disease control.

Figure 4

Catalase, SOD, GPx, and overall antioxidant capacity were considerably reduced in the group treated with only B(a)P in comparison with the control group. The activities of these enzymes significantly recovered after treatment with TQ. The numbers signify the mean ± SEM, with 8 rats/group. The statistical differences are denoted with an asterisk (*), indicating significance at p < 0.05 in comparison with the control group, and a hashtag (#), signifying p < 0.05 in comparison with the disease control.

Figure 5

Inflammatory marker levels were significantly increased in animals intoxicated with B(a)P compared to the control group. B(a)P plus TQ treatment significantly decreased these inflammatory marker levels. The graphs represent the mean ± SEM, with 8 rats/group. The numbers signify the mean ± SEM, with 8 rats/group. The statistical differences are denoted with as asterisk (*), indicating significance at p < 0.05 in comparison with the control group, and a hashtag (#), signifying p < 0.05 in comparison with the disease control.

Figure 6

Histopathological changes of lung tissues in different animal groups. (a) Typical lung architecture in control rats includes normal airway bronchi and bronchioles and blood vessels and alveolar sacs. (b,c) B(a)P mediated the lung epithelium thickening, damage of the alveolar architecture, and infiltration of inflammatory cells within lung tissues, incidence of congestion and haemorrhage. The arrows indicate infiltration of inflammatory cells and congestion. (d) Histopathological changes were found to be considerably lower with TQ plus B(a)P co-administration, as this attenuated the damage to lung epithelium and alveolar architecture and decreased the incidence of edema. The arrow indicates less edema. (e) Thymoquinone-only treated group shows normal lung architecture (Scale bar = 100 μm).

Figure 7

Effect of TQ on lung fibrosis. (a) Normal fibre architecture of lung in control animals; (b) B(a)P administration induces severe collagen deposition; the arrow indicates high deposition of collagen fibers (c) TQ shows suppression of B(a)P-induced fibrosis; the arrow indicates less deposition of collagen fibers. (d) normal collagen in the group treated with TQ only. (Scale bar = 100 μm).

Figure 8

Effect of TQ on lung fibrosis. (a) Normal fibre architecture of lung in control animals; (b) B(a)P administration induces severe fiber deposition; the arrow indicates deposition of fibers (c) TQ shows suppression of B(a)P-induced fibrosis, the arrow indicates less deposition of fibers and (d) normal fibre architecture observed in the group treated with TQ only. (Scale bar = 100 μm).

Figure 9

NF-κB protein expressional analysis. (a) The control rats did not express this protein; (b) the group treated with B(a)P only displayed high expression of NF-κB, the arrow indicates cytoplasmic positivity of NF-κB (c) the expression of this marker protein was reduced in animals co-treated with both B(a)P and TQ together, the arrow indicates cytoplasmic positivity of NF-κB (d) the animals treated with only TQ did not show any expression. (Scale bar = 100 μm.)

Figure 10

Effect of TQ on terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive cells. (a) Brown-stained nuclei were not noticed in the control group; (b) large numbers of positive cells were stained brown in the B(a)P-induced lung injury group, the arrow indicates brown stained nuclei (c) the group treated with TQ plus B(a)P showed a significantly decreased apoptosis rate, the arrow indicates brown stained nuclei (d): the group treated with only TQ did not show any signs of apoptosis. (Scale bar = 100 μm.)