Crocin attenuates cigarette smoke-induced lung injury and cardiac dysfunction by anti-oxidative effects: the role of Nrf2 antioxidant system in preventing oxidative stress

Chronic obstructive pulmonary disease (COPD) as a global health problem is a major cause of morbidity and mortality that is set to become the third leading cause of death worldwide by 2020 [1].

There is overwhelming evidence that oxidative stress plays an essential role in the pathogenesis of COPD [2]. This oxidative damage to cellular organelles plays a central role in mediating a wide array of downstream processes that contribute to the development and progression of COPD. Oxidative stress also stimulates epithelial cells and alveolar macrophages to generate additional inflammatory cells into the lung which resulting in severe pulmonary injury [3, 4]. Increased oxidative stress in the respiratory compartment of COPD patients originates from the increased burden of oxidants from environmental exposures such as air pollutants, cigarette smoke (CS), and increased amounts of reactive oxygen species (ROS) released from inflammatory cells involved in the destructive inflammatory process in the lungs of COPD subjects [5]. Exposure of macrophages and lymphocytes to oxidative stress results in release various inflammatory substances including cytokines and chemokines, which could destroy collagen and elastin, stimulate mucosal secretions in lung tissues, and even lead to more destructive processes in lungs [6].

Cigarette smoking is the major risk factor for the development of COPD. CS contains more than 5000 different chemicals and generates more than 10¹⁵ oxidants per puff, directly or indirectly, through various processes such as the Haber-Weiss reaction [7]. Reactive oxygen species, in turn, can induce lipid peroxidation and yield products such as malondialdehyde (MDA), which have the ability to stimulate pulmonary inflammation [8].

Under normal physiological conditions oxidant challenge is normally neutralized by the antioxidants in the epithelial lining fluid. Oxidative stress occurs if antioxidant levels in the epithelial lining fluid are inadequate to neutralize the inhaled oxidants/free radicals [9]. Among various antioxidants the reduced glutathione (GSH), as the most abundant cellular thiol antioxidant, plays an essential role in the maintenance of intracellular redox balance in epithelial lining fluid and is involved in the detoxification reaction through enzyme-catalyzed reactions or by direct conjugation [10]. This critical antioxidant has been reported to be depleted in pulmonary disorders, such as cystic fibrosis, acute respiratory distress syndrome, and COPD, suggesting a role for oxidative stress in the pathogenesis of these chronic inflammatory lung diseases [11, 12].

The synthesis of GSH depends on the rate of synthesis of the GCL catalytic (GCLC) subunit through increased transcription and mRNA stability [13].

Nuclear erythroid-related factor 2 (Nrf2), a member of the cap-N-collar family, is the principal transcription factor that regulates antioxidant response element-mediated expression of antioxidant enzymes. Under basal conditions, Nrf2 is sequestered in the cytoplasm by actin-binding protein (Keap1); on exposure of lungs to oxidative stress, Nrf2 dissociates from its repressor protein (Keap1), translocates into the nucleus, binds to antioxidant response elements, and transactivates antioxidant genes [14]. Nrf2 phosphorylation is mediated by protein kinase C (PKC), phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinases (MAPK). The modifications of each of the kinases in Nrf2 activation depends on the stress stimuli [15]. Nrf2 play a critical role in induction of multiple cytoprotective genes. One of the genes regulated by Nrf2 is heme oxygenase-1 (HO-1). The induction of HO-1 was able to exert protective role against oxidative stress [16]. Several experiences demonstrated a reduced expression of HO-1 related to an altered expression of Nrf2 in cigarette smoke related lung disease such as COPD [17‐19]. This process has pathophysiological roles since it decreases endogenous antioxidant capacity [16]. Among the spectrum of antioxidant genes controlled by Nrf2 pathway, the gene encoding GCLc is of particular interest, especially in CS-induced oxidative stress [20]. Therefore, agents that modulate Nrf2 as Nrf2 activators would be expected to have significant beneficial health effects in CS-mediated oxidative stress by upregulation of antioxidant enzymes.

In addition to pathology in the lungs, COPD is now believed to have systemic features. An increase in the risk of cardiovascular disease is one such systemic feature. There is considerable evidence of an association between COPD and cardiac disease [21]. The range of cardiovascular disease includes coronary artery disease (CAD), right ventricular dysfunction and arrhythmias. Cardiac disorders associated with COPD increase morbidity and worsen survival. Patients with COPD also carry an increased risk of mortality due to myocardial infarction, arrhythmia or congestive heart failure compared with those who do not. Since the cardiac dysfunction and abnormalities obviously contribute to the overall morbidity associated with COPD [22], therefore, an understanding of their role and potential for treatment is necessary.

Saffron (Crocus sativus L.) has four major pharmacologically active constituents, namely crocetin, picrocrocin, safranal and crocin. Crocin is a water soluble carotenoid and the most important active constituent of saffron. In pharmacological studies, crocin has demonstrated anti-inflammatory, anticonvulsant and anti tumour activities. Radical scavenger effects as well as learning and memory improving properties [23], and it is reported to promote the diffusivity of oxygen in different tissues [24]. Crocin is also chemopreventive and has shown protective effects on genotoxins-induced oxidative stress in Swiss albino mice [25]. Current studies have shown that crocin exhibits significant radical scavenging activity and thus antioxidant activity. Moreover, the cardio protective effects of crocin have been documented in some studies in relation to modulating endogenous antioxidant enzymatic activities [26, 27].

In light of the findings described above, we hypothesize that crocin induces GSH Synthesis via Nrf2 dependent mechanisms and attenuates cigarette smoke-induced COPD mediated oxidative stress in lung tissue that leads to cardiac dysfunctions. Therefore, we investigated the protective role of crocin against cigarette smoke exposure (CSE)-mediated oxidative stress and the inflammatory process in COPD, modifications of Nrf2 and its upstream regulator genes (PKC, PI3K AND MAPK) and also GCLc and GSH as downstream enzymes controlled by Nrf2 and impairment of cardiac hemodynamics and remodeling in rats with COPD.

While smoking-induced cardiovascular and pulmonary disorders are widely used in many experimental models, but any studies have been reported on models of COPD- induced cardiovascular dysfunctions. Indeed, there is no prior rat model that demonstrates a composite picture of COPD and cardiac dysfunction after chronic CS exposure. This is the first study to evaluation of cardiac electrocardiogram and function changes mediated by cigarette smoke induced- COPD in rat model.

Rodents are the most commonly used models in experimental studies. The rat is a favorable model, since measurable COPD changes which further progress can be detected after only 2 months of smoke exposure [38].

Pathological changes of COPD often occurred in the airway wall and lung parenchyma. In this experimental study, in cigarette smoke exposure rats, significant inflammation of the lung parenchyma was observed by chest x-ray, including diffuse patchy alveolar infiltration. In line with previous studies, it has been documented that cigarette smoke exposure for only two months was enough to induce alveolar wall destruction and airspace enlargement in rats [39]. Also, rats exposed to CS for 2 months exhibited significantly diffuse patchy alveolar infiltration, hyperinflation and disordered lung markings in chest x-ray, suggesting that CS-induced lung injuries mdel was successfully established. To further assess the pathological changes in the lung tissue, H&E staining was performed on all groups. While asthma affects the large and small airways, with COPD smaller airways and especially the alveoli are most involved. In fact, COPD is characterized by destruction of alveolar walls, leading to abnormally large air spaces within the lung. Therefore, Better recognition of small airway disease as a distinct pathological process holds the promise of a more site specific and targeted therapy [40]. Accordingly, in current study we assayed H&E staining of lung tissue and measured Mean linear intercept (MLI) as an index of alveolar airspace enlargement in response to CS and crocin. The results showed significant increases in MLI after 2 month cigarette smoke exposure. The lung tissue was significantly damaged in the rat model of CS induced- lung injuries, in which there were necrosis and degeneration of bronchial mucosal epithelium cells, serous fluid exudation from alveolar space, and inflammatory cell infiltration in bronchial wall and pulmonary consolidation. On the other hand, measurement of arterial blood gas exchanges were an excellent index of destruction in alveolar wall and gas exchange that confirmed establishment of lung injuries model in rats.

Cigarette smoke is a complex mixture of an estimated 5000 toxic chemical compounds; each puff of CS contains over 10¹⁵ free radicals, which include H₂O₂, reactive aldehydes, benzo pyrene and quinines [41]. CS plays a major etiological role in the pathogenesis of inflammatory airway disease, such as COPD.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a regulator of cellular resistance to oxidative stress. The function of Nrf2 and its downstream target antioxidant genes have been shown to be important for cytoprotection against oxidant damage [42, 43]. Among the various antioxidant enzymes, the HO-1 is a major antioxidative and anti-inflammatory enzyme that is mainly regulated by Nrf2 activation [44]. Several studies demonstrated that up-regulation of this enzyme associated with its anti-inflammatory actions [45‐47]. In this study, cigarette smoke down-regulated the mRNA expression of HO-1 in CS rats and co-treatment with crocin markedly induced HO-1 expression. These findings suggest that crocin may be a potential therapeutic candidate as an anti-inflammatory or an antioxidant agent. This finding was supported by modification of Nrf2 gene expression level.

Nrf2 has a pivotal role in the transcriptional regulation of phase II genes, including GCLc, thereby regulating GSH levels. GSH is an important lung antioxidant involved in maintenance of epithelial integrity, and its deficiency leads to airway injury and alveolar lung damage [48‐51]. Previous studies have shown that Nrf2-deficient mice were more susceptible to CS-induced emphysema and oxidative stress [52], suggesting a protective role of Nrf2 in lung injury. In the light of this finding, we hypothesized that CS impairs Nrf2-mediated transcriptional regulation of GCLc and biosynthesis of GSH (ROS-Nrf2-GCLc-GSH pathway), whereas an agent that modulate Nrf2, attenuates CS induced oxidative stress and restores CS-depleted GSH via an Nrf2-GCLc pathway-dependent mechanism. In the current study, we tested our hypothesis by investigating the mechanism of CS-mediated downregulation of antioxidant genes and the protective role of crocin in rat lungs tissue. Since a variety of oxidants and free radicals are implicated in the pathogenesis of chronic obstructive pulmonary disease, it is possible that induction of antioxidant genes is an effective approach for protection against environmental factor-induced oxidative stress.

Crocin has been well known for its possible antioxidant and anti-inflammatory role and protective effects against oxidant damage [53]. In the present study, we also examined the ability of crocin to attenuate the CS-mediated oxidative stress and GSH depletion probably via an Nrf2- pathway, in cigarette smoke exposure rats. Our results demonstrate that 2-month CS exposure resulted in increased oxidative stress via increasing lipid proxidation (MDA), which was associated with decreased levels of Nrf2, GCLc gene expression, GSH amount and other antioxidant enzymes activity in the lung tissue. Recent studies have shown that acute cigarette smoke exposure increased [54] the levels of antioxidant enzymes in both rat lung and epithelial cell, but chronic exposure decreased these levels [55]. Researchers believe that with repeated exposures, Nrf2 becomes less sensitive to the smoke-derived oxidants, although no mechanism for this decreased sensitivity has been demonstrated [56]. On the other hand, Aldehydes formed by lipid peroxidation form protein carbonyl or present in CSE adduct with sulfhydryl groups of Nrf2/Keap1, thereby leading to modulation of these sulfhydryl groups. This could be the reason for failure to localize Nrf2 into the nucleus or retention of Nrf2 in the cytoplasm of epithelial cells at a later time point which leaded to decreased GCLc gene expression [9]. Our results are in agreement with earlier studies suggesting that CS induces oxidative stress by ROS generation in various organ tissues. The decrease in the levels of GSH could be due to the formation of GSH conjugates with electrophilic compounds present in CS and inhibitory action of CS on Nrf2 or its upstream regulator genes expression by interaction of electrophilic components of CSE with the cysteine group in the active site [57]^. Co-treatment with crocin, significantly decreased CSE-induced ROS production, which was associated with increased levels of GSH activity and GCLc expression, compared with treatment with CSE alone. It is possible that crocin attenuates CSE-mediated depletion of GSH levels by increasing the biosynthesis of GSH and also by scavenging CSE-induced ROS. Indeed, in this possible mechanism, Nrf2 is phosphorylated through upstream signaling kinases. When these pathways are activated, phosphorylated Nrf2 can be liberated from Keap1. In fact, crocin might activate upstream signaling kinases, such as protein kinase C, phosphatidylinositol 3-kinase, and mitogen-activated kinase to modify Nrf2 pathway. PKC, PI3K and MAPK as the strongest intracellular pro-survival signaling systems are the target of many antioxidant agents for providing survival signaling [58]. Thus, to elucidate the upstream signaling pathway involved in crocin-mediated Nrf2 activation and GCLc and HO-1 induction, we studied the effects of crocin on the expression of these kinases in cigarette smoke induced CS rats. We found that after 2-months cigarette smoke exposure, the expression of PKC, PI3K and MAPK suppressed and then activated in crocin plus CS rats. The suppressed of Nrf2 pathway appears to be a common property of cigarette smoke exposure in chronic conditions [10] as we demonstrated in CS rats. Our data specify that this suppressed in chronic oxidative stress may arise from the changing in expression of PKC, PI3K and MAPK. Post-translational modification affects Nrf2 differently by altering Nrf2’s interaction with Keap1 and Nrf2 localization. These alterations of Nrf2 protein are essential for this protein to transactivate its downstream genes [59]. PKC, PI3K and MAPK are responsible for posphorylation and regulation of Nrf2 pathway in several cells and organs including lungs. Previous studies showed that PKC and PI3K strongly activate the Nrf2 pathway but, the effects of MAPKs on Nrf2 signaling appear to depend on the specific MAPK [60]. In this study, we report the first evidence that Nrf2 is upregulated by PKC, PI3K and MAPK (ERK2) modifications in crocin co-treatment CS rats. In this context, some studies demonstrated that some kinases such as PKC and MAPK have been implicated in playing a role in pro-inflammatory pathways [61, 62]. In contrast, some other studies provided evidence to suggest that there is a differential PKC regulation of production of inflammatory factors. For example, Conventional PKCs regulate the pro-inflammatory cytokine in a MAPK-dependent manner whereas anti-inflammatory IL-10 is regulated by a PKCζ-dependent pathway. In addition, cross-regulation between these pathways exists [63‐65]. Taken together, these observations suggest that anti-inflammatory and pro-inflammatory cytokines are differentially regulated by these kinases isoform.

In summary, in this part we have specified the molecular mechanisms of regulation of Nrf2 pathway by crocin during the chronic cigarette smoke exposure induced lung injuries. The mechanism of changing in these kinases is still unclear. However, it has been reported that the activity of this kinases can be affected by several factors including alteration of calcium homeostasis [66].

Many of the intracellular signaling pathways triggering and/or driving the release of these inflammatory mediators is sensitive to oxidative stress. Elevated levels of ROS have been further implicated in initiating various inflammatory processes in the lungs through the activation of transcription factors, signal transduction and gene expression of pro-inflammatory mediators which correlates with pathological changes in airway wall and lung parenchyma [67]. In this study, the alteration of the inflammatory cells and total protein in BALF and also, gene expression and levels of TNF-α and IL-6, as important pro-inflammatory cytokines involved in immunoregulation and inflammation in lung tissue and BALF, was investigated for an in-depth evaluation of the inflammatory changes of lung in cigarette smoke induced COPD rat model. These findings suggest that total protein and inflammatory cytokines plays a pivotal role in cigarette smoke induced lung injury. In accordance with previous studies, exposure to cigarette smoke caused elevated in TNF-α and IL-6 gene expression in lung tissue and enhanced levels of these cytokines in the BALF of rats compared to the control group which was correlated with the increase in inflammatory cells in bronchoalveolar lavage fluid, suggesting that increased inflammatory factors production increases COPD severity. However, the co-administration of crocin to the CS rats decreased cytokines gene expression and levels (IL-6 and TNF-α) and the number of inflammatory cells (macrophages and lymphocyte cells), ameliorated lung tissue injury. These effects may be related to the inhibition of releasing IL-6 and TNF-α by crocin, resulting in a decrease in the inflammatory cells recruitment responsible for lung injury in cigarette smoke exposure rats.

Chronic obstructive pulmonary disease is a chronic inflammatory condition of the lungs whose manifestations are linked to other systemic diseases, among which an increased risk of cardiovascular disorder is that, contributes significantly to both morbidity and mortality in COPD [68]. Smoking is the causative factor in the majority of individuals with COPD and in the development of coronary artery disease [69]. It is, therefore, difficult to show in a COPD population that the increased risk of cardiovascular disease is due to COPD alone, because COPD and smoking are inextricably linked [70]. However, to determine if CS-induced lung injuries established model has any in vivo and in vitro effect on the heart, ECG recording and isolated-perfused rat heart were performed to compare cardiac electrocardiogram and function between COPD and control rats.

To our knowledge, this is the first study that demonstrates significant deleterious effects of CS- induced COPD on the general cardiac hemodynamic, function and electrocardiogram in a rat model. We observed that CS-induced lung injuries resulted in significant shortening of the QRS complex, whereas the other waves did not show much change. Our results showed shortening of the QTc interval in CS rats, which was not statistically significant. These results were in accordance with the Karjalainen et al.’s study [71] which explained that a shortened QT interval and ST segment were as a risk factor for smokers and could cause death. The shortened ST segments and QT interval warn that there may be shortening in the ventricular filling phase, during which the coronary supply occurs. This may lead to an insufficient myocardial perfusion, which may lead to ischemic episodes. Such altered ventricular electrical activities, like a shortened ST segment and QRS complex also predisposes COPD patients to episodes of arrhythmia. The increased heart rate which was shown in our study probably was due to an increased catecholamine secretion from the adrenal medulla [72]. There are studies whose results demonstrate that cigarette smoke also may significantly increase levels of epinephrine and norepinephrine [73]; however, we did not assess plasma and urinary catecholamines in rats. All the above changes in our study were the result of the 2 months cigarette smoke exposure, which could lead to cardiovascular disorders easily identified by the wave duration in electrocardiography. COPD can increase the risk of sudden cardiac deaths. In the long term, the mortality which is caused by COPD is due to either coronary artery disease or electrophysiological disturbances, which lead to arrhythmias.

COPD also can impair cardiac hemodynamic and function. We observed an adverse effect of cigarette smoke exposure on the in vitro baseline cardiac contractile function at the organ level. It is important to note that hypoxemia can influence cardiac function [74]. We performed ABG analysis as mainly used to evaluate and monitor gas exchange in the lungs. Two months of CS exposure showed a significant decrease in PO₂ in CS rats as opposed to the control group. Hypoxia is a potent stimulus for the generation of ROS and also induces hemodynamic stress [70]. Under physiological conditions, ROS are important mediators of a wide variety of cell functions. However, excessive generation of ROS and the fall in antioxidant defenses can lead to cellular oxidative stress. In our study, isolated hearts from CS rats showed significantly enhanced values for dP/dt_max and for the rate-pressure product. While co-treatment with crocin in CS-induced lung injuries rats restored these cardiac responses towards values measured in CS group. Several studies have also shown that hypoxia can lead to cardiac dysfunction in rats. Some adverse effects include altered metabolism and myocardial structure, a change in cardiac performance and heightened cardiac susceptibility to adult ischemic injury [75]. These experimental studies are of substantial clinical relevance, as it has now also been reported that, hypoxia modulated by CS induced-COPD show changes in cardiac morphology (RV hypertrophy) and function [52]. Here, we show that COPD has permanently altered the mechanical properties of the myocardium. An increase in the rate-pressure product is associated with increased myocardial work load and oxygen consumption and an increase in dP/dt_max is an established index of increased myocardial contractility [76]. It is possible that the increase in myocardial contractility occurs in response to the increased afterload derived from the increased peripheral vascular impedance. Sustained increases in myocardial contractility can strongly be associated with cardiovascular disease, and this cardiac phenotype is a known predictor of eventual heart failure in humans [76].

Our results also suggested that the effects of CS-induced lung injuries on the heart of CS rats can be prevented by crocin co-treatment during CS exposure, providing new evidence for the mechanism driving the developmental process of heart dysfunction by COPD to be also secondary to oxidative stress. Co-treatment with crocin may prevent hypoxia influences on the heart triggered by COPD-associated lung dysfunction by ameliorating oxidative stress via Nrf2 pathway. Further studies are needed to investigate the therapeutic effects of crocin. In this regard, using novel approaches such as Q3D-compartment multiscale model can be used to modeling the drug transport across the layers and along the lung and optimizing the target-specific drug delivery and increasing the localized bio-availability [77, 78].

In summary, our study was designed to investigate the effects of a 2-months cigarette smoke exposure in establishment of rat lung injuries model and provide substantial data regarding the pathogenic oxidative stress processes in this model. We sought to demonstrate whether the CS exposure-dependent alterations in lung morphology and parameters and cardiac disorder in COPD are concurrent with oxidative stress, particularly, in Nrf2 pathway (both upstream and downstream genes) (Fig. 16). Although our findings provide convincing insights into a versatile rat model of chronic smoking, the exposure time was moderate, and by designing only 2 months CS exposure was tested. Further studies with different levels of CS exposure and longer periods would be useful in further determining the relationship between duration and dose of exposure in the process of chronic cigarette smoking induced COPD flowed by cardiac disorders. Along with the future studies, this rat model of smoking-induced COPD should be useful to determine the mechanisms of COPD-induced disease and approaches for early detection and prevention.

The present in vivo rat model provides convincing evidence that chronic CS exposure induces significant alterations in pulmonary system that mediated cardiac function, structure, and electrocardiogram with cellular oxidative stress and decreased PKC, PI3K, MAPK, Nrf2 and GCLc genes expression decreasing antioxidant enzymes level. On the other hand, our results provided the evidence that crocin co-administration is protective against lung injury caused by CS cigarette smoke exposure and related cardiac dysfunction. Our data provided the evidence that these protective effects are probably related to Nrf2 pathway (both upstream and downstream genes). Further molecular and cellular studies in this model should enable the determination of important mechanistic insights in CS-induced diseases.