Antioxidant activity of pomegranate juice reduces emphysematous changes and injury secondary to cigarette smoke in an animal model and human alveolar cells

Figures & data

Figure 1 Wet-to-dry ratio (W/D) and expression of inflammatory mediators after acute exposure to CS.

Notes: Mean W/D (A), transcriptional expression of IL-1β (B), IL-6 (C), and TNF-α (D) of Control, acute CS exposure (CS), CS + PJ, and PJ. Error bars represent SE. Asterisks indicate statistically significant associations (P<0.05).

Abbreviations: CS, cigarette smoke; IL, interleukin; PJ, pomegranate juice; SE, standard error; TNF, tumor necrosis factor.

Figure 2 PJ attenuates CS-induced ROS formation and apoptosis.

Notes: All images captured using 20× objective. (A) ROS detection in lung tissues after acute CS exposure. 5 μm thick sections mounted on microscope slides were incubated with DHE. ROS levels were induced after CS and were attenuated in CS + PJ group. (B) Apoptosis assessment after acute CS exposure. TUNEL and Hoechst staining of lung demonstrated an increase in apoptotic activity in CS, which was significantly reduced in CS + PJ.
Abbreviations: CS, cigarette smoke; DHE, dihydroethidium; PJ, pomegranate juice; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Figure 3 H&E examination under light microscopy (original magnification: ×100).

Notes: (A) H&E of normal lung showing thin interstitial alveolar wall and capillary vessels. Rare inflammatory cells are noted. (B) PJ lung tissue demonstrating normal lung architecture similar to that in A. (C) After acute CS exposure, note thickening of the interstitial wall, capillary congestion, and infiltration by inflammatory cells (arrows). (D) When compared to Control, CS + PJ-treated animals demonstrated remarkable reduction in inflammatory cell migration, alveolar wall thickening, and capillary congestion.
Abbreviations: CS, cigarette smoke; H&E, hematoxylin and eosin; PJ, pomegranate juice.

Figure 4 Mean transcriptional expression of TNF-α after chronic exposure.

Notes: Significantly increased levels of TNF-α were observed in CS animals at 1 month (A) and 3 months (B) of CS. The increased levels of TNF-α were significantly attenuated with PJ supplementation. Error bars represent SE. Asterisks indicate statistically significant associations (P<0.05).

Abbreviations: CS, cigarette smoke; PJ, pomegranate juice; SE, standard error; TNF, tumor necrosis factor.

Figure 5 H&E examination under light microscopy at 1 month (original magnifications: ×20).

Notes: Normal lung architecture observed in Control (A) and PJ (B). (C) After 1 month of CS exposure, destruction of the alveolar architecture with distension of the alveolar sacs (arrows). (D) CS + PJ-treated animals demonstrate minimal damage to the lung parenchyma and preservation of pulmonary alveoli.
Abbreviations: CS, cigarette smoke; H&E, hematoxylin and eosin; PJ, pomegranate juice.

Figure 6 H&E examination under light microscopy at 3 months (original magnification: ×20).

Notes: Normal mouse lung architecture in Control (A) and PJ (B). (C) After 3 months of CS exposure, extensive destruction of the alveolar architecture with distension of the alveolar sacs (arrows). (D) PJ supplementation in CS + PJ limited the damage observed in the lungs of CS-exposed animals.
Abbreviations: CS, cigarette smoke; H&E, hematoxylin and eosin; PJ, pomegranate juice.

Figure 7 Cellular morphology examination under light microscopy (original magnification: ×20).

Notes: Changes in cellular morphology were observed with increasing concentrations of CS extract. Cell shrinkage, rounding, and vacuoles were observed with CSE of concentrations ≥1 mg/mL.

Abbreviations: CS, cigarette smoke; CSE, CS extract; PJ, pomegranate juice.

Figure 8 CSE-induced oxidative stress in A549 cells.

Notes: All mages captured using 20× objective. Fluorescent images of CS and CS + PJ-treated A549 cells stained with DHE dye. H₂O₂-treated cells were included as positive control. Oxidative stress in A549 cells treated with smoke extract increased with increased concentrations of CS extract. PJ attenuated CS-induced ROS status in A549 cells.
Abbreviations: CS, cigarette smoke; CSE, CS extract; DHE, dihydroethidium; PJ, pomegranate juice; ROS, reactive oxygen species.