Curcumin, an Active Constituent of Turmeric Spice: Implication in the Prevention of Lung Injury Induced by Benzo(a) Pyrene (BaP) in Rats

Abstract

Benzo(a)pyrene (BaP) is a well-known carcinogen and enhances oxidative stress and apoptosis and also alters several molecular pathways. Curcumin is an active ingredient of Curcuma longa, and it has potent anti-inflammatory, antioxidant activity that defends cells from oxidative stress and cell death. The objectives of the present study were to explore the protective effects of curcumin against long-term administration of BaP induced disturbances in lungs of rats. Male rats were randomly divided into four groups: saline control, BaP only, BaP + curcumin, and curcumin only. Lung histopathology, electron microscopy, inflammatory cytokine release, antioxidant levels, apoptosis, and cell cycle were examined. Instillation of BaP significantly increased infiltration of inflammatory cells in alveolar space and inflammatory cytokine in blood. BaP induced lung tissue alterations including mild bronchitis, scant chronic inflammatory cell infiltrate in the wall of the respiratory bronchiole, and mild intra-alveolar haemorrhage. However, these alterations were found to be significantly less as mild inflammatory cell infiltrate in curcumin plus BaP treated group. Furthermore, electron microscopy results also showed necrotic changes and broken cell membrane of Type-II epithelial cell of alveoli in BaP group, which was reduced after adding curcumin treatment. In addition, we found BaP plus curcumin treatment effectively reduced inflammatory cytokines Tumour Necrosis Factor alpha (TNF-α), Interleukin 6 (IL-6), and C-reactive protein (CRP) levels in blood serum. Moreover, the levels of tunnel staining and p53 expression were significantly increased by BaP, whereas these changes were noticeably modulated after curcumin treatment. BaP also interferes in normal cell cycle, which was significantly improved with curcumin treatment. Overall, our findings suggest that curcumin attenuates BaP -induced lung injury, probably through inhibiting inflammation, oxidative stress and apoptosis in lung epithelial cells, and improving cell proliferation and antioxidants level. Thus, curcumin may be an alternative therapy for improving the outcomes of Benzo(a)pyrene-induced lung injury.

Keywords: antioxidant; apoptosis; benzo(a)pyrene; inflammation; lung injury.

Figure 1

Effects of curcumin treatment on histopathological changes in BaP -induced lung tissue: Control group (a); BaP -induced group (b–f); (b) mild bronchitis, scant chronic inflammatory cell infiltrate in the wall of the respiratory bronchiole. Mild intra-alveolar haemorrhage as well, (c) moderate interstitial inflammatory cell infiltrate. Acute lung injury as intra-alveolar haemorrhage is also noted and acute on chronic changes; (d) moderate to marked mixed interstitial inflammatory cell infiltrate, suggestive of interstitial pneumonitis. Interstitial fibrosis is also appreciated; (e) interstitial chronic inflammatory cell infiltrate, suggestive of interstitial pneumonitis. Type 2 pneumocytes are also prominent; (f) hyper inflated alveolar spaces and mild septal inflammatory cell infiltrate. Scale bar = 50 μm.

Figure 2

Effects of curcumin treatment on lung tissue: (a) control group: lung tissue structure was normal and no deposition of collagen fibre; (b) BaP -induced group showed extensive depositions of collagen fibre; (c) benzopyrene plus curcumin treated group: showed less depositions of collagen; (d) curcumin (50 mg/kg bw) group: there was no deposition of collagen fibre. Scale bar = 100 μm.

Figure 3

The anti-inflammatory effects of curcumin were evaluated through measurement of Tumour Necrosis Factor alpha (TNF-α), Interleukin 6 (IL-6), and C-reactive protein (CRP): BaP -induced group showed the levels of TNF-α and IL-6 were found to be substantial increased as compared with the control group (p <  0.05). However, production of TNF-α and IL-6 and were significantly decreased in the BaP plus curcumin treated group (p < 0.05). The level of CRP was increased in the BaP-treated group as compared to control group as well as co-administration of curcumin (50 mg/kg) with BaP (50 mg/kg), but difference was statically insignificant (p > 0.05).

Figure 4

The antioxidant effects of curcumin were measured in the serum of different groups. Benzopyrene treated groups exhibited a decreased level of Glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity enzymes as compared to the control group (p < 0.05). Treatment of curcumin (50 mg/kg b.w.) in BaP administered rats increased the levels of SOD, CAT, GPx, and total antioxidant capacity significantly compared to the BaP treated group (p < 0.05).

Figure 5

Effects of curcumin treatment on ultrastructural changes in BaP -induced lung tissue: (a) control group: lung tissue structure was normal and Normal alveolar space (AS), presence of vacuole (V). There is intact capillary endothelium submerged with alveolar epithelium i.e., normal condition, normal cell membrane of (Type I and Type-II) alveolar epithelial cell (PnI and PnII) and vacuole (V); (b) BaP -induced group showed marked irregular and degraded arrangement of microvilli(arrow), the cytoplasmic contents(arrow) escapes into the alveolar lumen, necrotic degenerative changes and ruptured cell membrane of (Type-II) alveolar epithelial cell (PnII). There is a condensed nucleus and dilated vesicles of rough endoplasmic reticulum showing compression of the interalveolar spaces that appears as clefts (green arrow); (c) benzopyrene plus curcumin treated group: Less necrotic degenerative changes and normal cell membrane of (Type-II) alveolar epithelial cell (PnII). Normal gas barrier facing alveolar lumen (A); red blood cells are seen in the capillary lumen (RBC). Decreased vacuolization, lung showing compression of the interalveolar spaces which appear as clefts (green arrow); (d) curcumin (50 mg/kg bw) group: the alveolar lumen (AS), contains normal red blood cells (RBC), a granulated cytoplasm normal. Regular arrangement of microvilli normalization and Normal Alveolar spaces with intact Blood gas barriers (arrow) and presence of less RBCs.

Figure 6

Effect of curcumin treatment on apoptosis in BaP -induced lung tissue: (a) control group: did not show apoptotic nuclei; (b) TUNEL-positive cells were detected in the BZ group treated cells; (c) benzopyrene plus curcumin treated group: curcumin significantly decreased the number of TUNEL-positive cells compared with the number in the BaP group. Scale bar = 50 μm.

Figure 7

Immunohistochemical analysis. P53 protein expression was evaluated in different treatment groups. (a) control group did not show P53 expression; (b) expression of p53 was detected in BaP treated group; (c) curcumin plus BaP decreased the protein expression as compared with the BaP group and intensity of positivity was low. Scale bar = 50 μm.

Figure 8

Effect of curcumin on cell cycle phase distribution by flow cytometry (a) control group 30.6% cell were arrested in G1; (b) high shift in G2/M phase in BaP-only treated group; (c) significant change in curcumin + BaP treated group; (d) marginal shift was observed from G2/M to G1 in the curcumin only treated group. The cell cycle distributions were calculated and expressed as means ± SDs of three independent experiments.

Figure 9

Effect of curcumin on apoptosis by flow cytometry. (a) control group 97.4% cells were viable as PI^- & Annexin V⁻; (b) viable cells decreased to 80 in BaP only group as 12.7% Annexin V⁺ (early apoptosis) and 5.6% PI⁺ and Annexin V+ (late apoptosis); (c) curcumin plus BaP group reduced the apoptosis; (d) curcumin only group showed no significant difference in comparison to the control group. The cell cycle distributions were calculated and expressed as means ± SDs of three independent experiments.

Figure 9

Effect of curcumin on apoptosis by flow cytometry. (a) control group 97.4% cells were viable as PI^- & Annexin V⁻; (b) viable cells decreased to 80 in BaP only group as 12.7% Annexin V⁺ (early apoptosis) and 5.6% PI⁺ and Annexin V+ (late apoptosis); (c) curcumin plus BaP group reduced the apoptosis; (d) curcumin only group showed no significant difference in comparison to the control group. The cell cycle distributions were calculated and expressed as means ± SDs of three independent experiments.