CS: cigarette smoke; H&E: hematoxylin and eosin; PJ: pomegranate juice. Open in a separate window Figure 2 H&E examination under light microscopy of lung tissues from CS animals. Lung cancer constitutes more than a sixth of all cancer CaMKII-IN-1 deaths worldwide and is the leading cause of cancer death in the United States for both men and women [1, 2]. In 2014, chronic lower respiratory disease (COPD) was the third leading cause of death in the United States after heart disease and cancer [1]. Most cases of lung cancer and COPD are associated with combustible tobacco smoke, and cigarette smoke (CS) remains the basic vehicle for delivering combustible tobacco smoke leading to the development of lung cancer and COPD [3, 4]. COPD is also an independent risk factor for lung cancer among smokers, and the probability of lung cancer increases with the increasing severity of COPD [5, 6]. The pathophysiology of lung cancer and COPD shares similar pathways that lead to cellular injury, DNA damage, and mutations [7]. Oxidative stress (OS) is a common pathway in the pathogenesis of COPD and lung cancer. Previously, we reported higher OS in animals exposed to CaMKII-IN-1 CS. Emphysematous changes and destruction of lung architecture were observed with three-month CS exposure as well [8]. CS contains free radicals and reactive oxygen species (ROS) that deplete extracellular and intracellular antioxidants causing oxidative damage to DNA, proteins, and SLCO2A1 lipids [9, 10]. Antioxidant depletion, induced by CS, correlates with carcinogenesis of lungs and other organs as well [11]. DNA is particularly vulnerable to OS, and DNA repair may result in mutations leading to the development of malignant cells [12, 13]. Avoidance of CS exposure and quitting smoking remain the key and most effective strategy that will prevent OS and tissue injury. An alternative strategy, however, is to strengthen the defense mechanisms against carcinogenesis of CS by exogenous supplementation of pharmacological and dietary agents. This approach, referred to as chemoprevention, is currently utilized in the fight against cancer and other human disorders such as cardiovascular diseases [14, 15]. Chemoprevention supplementation that will result in significant reduction in OS may prevent tissue injury and eventually may reduce the incidence of lung cancer secondary to CS exposure. L. (Punicaceae) is very rich in polyphenols and other biologically active compounds like flavonoids, gallic acid, ellagic acid, and ellagitannins. The combination of polyphenols with other phytochemicals such as ellagic acid synergistically augments the superior antioxidant properties of PJ [16, 17]. Polyphenols act as oxygen radical and hydroxyl radical scavengers and increase antioxidant glutathione levels by inducing glutamate cysteine ligase expression. As an anti-inflammatory agent, polyphenols inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-Expression Hypoxia-inducible factor 1(HIF-1levels, tissue sections were incubated with rabbit polyclonal HIF-1antibodies at a dilution 1?:?50 in 1% normal goat serum (Santa Cruz Biotechnology Inc., CaMKII-IN-1 CA, USA). Hypoxic lung tissues from previous animal experiments were utilized for positive control, and fluorescent images were scanned for signal with the laser scanning confocal microscope (LSM 710, Zeiss, Germany) [19]. 3. Results 3.1. Histopathology and Lung Nodule Enumeration Control (Figures 1(a) and 1(b)) and PJ (Figures 1(c) and 1(d)) groups revealed normal alveolar structure with minimal infiltrates of inflammatory cells and thin alveolar walls. In contrast, lung tissues of the CS-exposed mice showed damaged alveolar architecture, emphysema, and higher levels of inflammatory cells (Figure 2, A). In addition, a significant number of lung nodules were identified in CS-exposed mice (< 0.05 when compared to control) (Table 1). Observed lung nodules in the CS group consisted of atypical round or oval epithelial cells, containing hyperchromatic nuclei and forming characteristic glands or acini (Figures 1(d) and 1(e)). PJ supplementation to CS animals attenuated the findings noted in CS animals; normal alveolar structure with minimal emphysematous changes and no pulmonary nodules were observed (Figures 1(g) and 1(f)). Open in a separate window Figure 1 H&E examination under light microscopy of lung tissues from the control (a, b), PJ (c, d), CS (e, f), and PJ?+?CS (g, h). Note: normal lung architecture observed in lung tissue from control showing, at lower magnification (original magnification: 20), thin interstitial alveolar wall and capillary vessels (a). At higher magnification (original magnification: 40), normal lung tissue is shown and rare inflammatory cells are noted (b). Similar findings were observed in the PJ-only group (c, d) and PJ (b). (e) A pulmonary nodule noted with CS exposure and at a higher magnification (original magnification: 40), the nodule consists of round epithelial cells with high nuclear-cytoplasm ratio grouping together and forming characteristic acini. The CS?+?PJ revealed no lung nodules and minimal injury and damage to the lung.