Cigarette Smoking-Induced Cardiac Hypertrophy, Vascular Inflammation and Injury Are Attenuated by Antioxidant Supplementation in an Animal Model

Abstract

Background: Cardiovascular diseases are the leading causes of morbidity and mortality worldwide. Cigarette smoking remains a global health epidemic with associated detrimental effects on the cardiovascular system. In this work, we investigated the effects of cigarette smoke exposure on cardiovascular system in an animal model. The study then evaluated the effects of antioxidants (AO), represented by pomegranate juice, on cigarette smoke induced cardiovascular injury. This study aims at evaluating the effect of pomegranate juice supplementation on the cardiovascular system of an experimental rat model of smoke exposure. Methods: Adult rats were divided into four different groups: Control, Cigarette smoking (CS), AO, and CS + AO. Cigarette smoke exposure was for 4 weeks (5 days of exposure/week) and AO group received pomegranate juice while other groups received placebo. Assessment of cardiovascular injury was documented by assessing different parameters of cardiovascular injury mediators including: (1) cardiac hypertrophy, (2) oxidative stress, (3) expression of inflammatory markers, (4) expression of Bradykinin receptor 1 (Bdkrb1), Bradykinin receptor 2 (Bdkrb2), and (5) altered expression of fibrotic/atherogenic markers [(Fibronectin (Fn1) and leptin receptor (ObR))]. Results: Data from this work demonstrated that cigarette smoke exposure induced cardiac hypertrophy, which was reduced upon administration of pomegranate in CS + AO group. Cigarette smoke exposure was associated with elevation in oxidative stress, significant increase in the expression of IL-1β, TNFα, Fn1, and ObR in rat's aorta. In addition, an increase in aortic calcification was observed after 1 month of cigarette smoke exposure. Furthermore, cigarette smoke induced a significant up regulation in Bdkrb1 expression level. Finally, pomegranate supplementation exhibited cardiovascular protection assessed by the above findings and partly contributed to ameliorating cardiac hypertrophy in cigarette smoke exposed animals. Conclusion: Findings from this work showed that cigarette smoking exposure is associated with significant cardiovascular pathology such as cardiac hypertrophy, inflammation, pro-fibrotic, and atherogenic markers and aortic calcification in an animal model as assessed 1 month post exposure. Antioxidant supplementation prevented cardiac hypertrophy and attenuated indicators of atherosclerosis markers associated with cigarette smoke exposure.

Keywords: calcification; cardiovascular diseases; cigarette smoking; hypertrophy; inflammation; pomegranate juice; reactive oxygen species.

Figure 1

Reactive oxygen species assessment post experimental smoke. (A) AO attenuates CS induced ROS formation. Notes: 5 μm thick sections mounted on microscope slides were incubated with DHE. ROS levels were induced after CS and were attenuated in CS + AO. Images captured using 40X magnification. (B) Quantification of the intensity of ROS formation. Intensity of ROS staining was determined from ZEN software and normalized to the Hoechst level relative to the Control samples. CS group had a significantly higher ROS to Hoechst ratio compared to control. ROS was significantly reduced in CS + AO in comparison with CS. One Way Anova test was used to check for significance between the groups. Error bars represent SE. Asterisks indicate statistically significant associations (P < 0.05). CS, cigarette smoke; ROS, reactive oxygen species; AO, antioxidant.

Figure 2

(A) Heart to Body weight ratio (H/B) after 4 weeks of CS. Mean Heart to Body weight ratio (H/B) compared to control. CS group had a significantly higher H/B ratio compared to control. H/B ratio was significantly reduced in CS + AO in comparison with CS. Error bars represent SE. One Way Anova test was used to check for significance between the groups. Asterisks indicate statistically significant associations (P < 0.05). (B,C) AO reduces transcriptional expression of fibrotic (ObR and Fn1) markers in rat aortas. Transcriptional expression of (B) ObR and (C) Fn1 were induced in the aortas of CS group. AO reduced the smoking-induced gene expression of ObR and Fn1. Data on each target mRNA was normalized to GAPDH. One Way Anova test was used to check for significance between the groups. Error bars represent SE. Asterisks indicate statistically significant associations (P < 0.05). (D,E) AO reduces protein expression of fibrotic (ObR and Fn1) markers in rat aortas. Protein expression of (D) ObR and (E) Fn1 were induced in the aortas of CS group. Data on each protein was normalized to β-actin. Error bars represent SE. One Way Anova test was used to check for significance between the groups. Asterisks indicate statistically significant associations (P < 0.05).

Figure 3

Smoking-induced aorta calcification is stopped by AO. The effect of CS on calcification in rat aortas, was assessed using von Kossa staining. CS induced calcium deposition in the aortas, which was significantly reduced with AO supplementation in (CS +AO) group. All pictures were taken at 40X magnification. For interpretation of Von Kossa stain, calcium salt in mass deposits appeared black, calcium in dispersed deposits appeared gray, nuclei appeared red, and cytoplasm appeared light pink.

Figure 4

Assessment of Bdkrb1 and Bdkrb2 in the aortas of rat tissues. (A) Immunohistochemistry staining for Bdkrb1 and Bdkrb2 in the aortas of rat tissues. Bdkrb1 protein expression is induced in the aortas of smoking (CS) group. CS-induced expression was abolished in the presence of AO in the (CS + AO). All pictures were taken at 40X magnification. (B) Quantification of the intensity of the immunofluorescence of Bdkrb1. Intensity of Bdkrb1 staining was determined from ZEN software and normalized to the Hoechst level relative to the Control samples. CS group had a significantly higher Bdkrb1 to Hoechst ratio compared to control. Bdkrb1 staining was significantly reduced in CS + AO in comparison with CS. Error bars represent SE. One Way Anova test was used to check for significance between the groups. Asterisks indicate statistically significant associations (P < 0.05). (C) Quantification of the intensity of the immunofluorescence of Bdkrb2. Intensity of Bdkrb2 staining was determined from ZEN software and normalized to the Hoechst level relative to the Control samples. Bdkrb2 staining was significantly reduced in CS + AO in comparison with CS. Error bars represent SE. One Way Anova test was used to check for significance between the groups. Asterisks indicate statistically significant associations (P < 0.05). D and E: protein expression assessment of the Bdkrb1 and Bdkrb2 in rat aortas. Protein expression of (D) Bdkrb1, but not (E) Bdkrb2, was induced in the aortas of CS group. AO significantly reduced the expression of Bdkrb1 in the CS + AO group. Data on each protein was normalized to β-actin. Error bars represent SE. One Way Anova test was used to check for significance between the groups. Asterisks indicate statistically significant associations (P < 0.05).

Figure 5

AO reduces transcriptional expression of inflammatory (IL1β, TNF-α) in rat aortas. Transcriptional expression of (A) IL-1β, and (B) TNF-α were induced in the aortas of CS group. AO reduced the smoking-induced gene expression of IL-1β. Data on each target mRNA was normalized to GAPDH. Error bars represent SE. One Way Anova test was used to check for significance between the groups. Asterisks indicate statistically significant associations (P < 0.05).