INTRODUCTION:

Smoking is the most well-known risk factor, and inflammatory changes are a clear indication of pathological changes in cardiovascular disease¹. Smoking not only causes physiological harm to the respiratory lot but also adds to the advancement of cardiovascular disease².

Smoking is a significant factor in causing endothelial dysfunction, and understanding the mechanisms linking smoking to atherosclerosis can be challenging. The numerous chemicals presents in cigarettes, exceeding 5,000 in number, disrupt the balance between prooxidant and antioxidant levels, promotes inflammation over anti-inflammation, as well as disturb prothrombin and antithrombin, leading to endothelial dysfunction^3-6. Caffeine in cigarettes can increase breathing rate, diaphragm contractility, and sensitivity to carbon dioxide⁷.

The biggest cause of death worldwide is cardiovascular disease, which typically starts with endothelial dysfunction⁸.It is a critical indicator of the seriousness and mortality of atherosclerosis confusion as it adds to the improvement of atherosclerosis⁹. Since atherosclerosis is a main source of coronary vein infection (computer-aided design), endothelial brokenness becomes a critical indicator for CAD^10-11. The vast majority of deaths in developed countries are caused by cardiovascular disease, most of which are related to some form of irregular blood flow in the arteries¹². In problems related to the heart, the arteries are affected by hardening due to the accumulation of fatty substances in the lumen or plaque formation¹³. The pathogenesis of endothelial dysfunction is largely influenced by the role of endothelial nitric oxide synthase (eNOS), an enzyme present in endothelial cells responsible for nitric oxide (NO) production^14,15. Early indicators of atherosclerosis include reduced nitric oxide (NO) activity, increased production of reactive oxygen species, and augmented endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the arterial wall¹⁶. Adenosine triphosphate (ATP) concentration as a functionally high energy carrier occurs in all cells and in the extracellular space¹⁷. Reactive oxygen species (ROS) have been implicated in the initiation and progression of atherosclerosis¹⁸. The fluid dynamics of blood through arterial segments play an important role in cardiovascular disease and can lead to strokes, heart attacks, and various cardiovascular diseases¹⁹.

Ketone body, including β-hydroxybutyrate, have shown potential benefits for patients with cardiovascular disease and exhibit potential for clinical applications²⁰.β-hydroxybutyrate acts as a stress response molecule, regulating the antioxidant defense program to maintain redox homeostasis²¹. Development in the field of medicine with accelerated drug delivery systems to target organs can increase therapeutic efficacy²². In addition, therapies aimed at increasing the circulating ketones holds potential for improving endothelial dysfunction. In this study, the investigation was to determine the effects of β-hydroxybutyrate in animal models exposed to cigarette smoke as an initial step towards developing ketone body therapy. The essential goal of this study is to look at the effect of β-hydroxybutyrate on endothelial brokenness, zeroing in on eNOS levels and VCAM-1 articulation in male Wistar rodents presented to tobacco smoke.

MATERIALS AND METHODS:

Materials:

Animals:

In this study, 25 male Wistar rats (Rattus norvegicus) were eight weeks old and weighed between 150 and 200 grams on average. The Farma Veterinary Center, Directorate General of Livestock and Animal Health Services, Ministry of Agriculture in Surabaya, East Java, Indonesia, evaluated the rats' health after they were obtained from an experimental animal farm. The rats were housed in ventilated cages with a 12-hour light/dark cycle and the room temperature was maintained between 2 and 30 degrees Celsius. The rats were fed a standard rodent-balanced diet and provided with water ad libitum. This study was approved by the Institutional Animal Care and Use Committee of Universitas Airlangga, Surabaya, Indonesia with the Number of Approval Certificate 2.KE.113.08.2022. The study adhered in the guidelines outlined the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Experimental Design and Groups:

A total of 25 Wistar rats were randomly allocated to five experimental groups. The study comprised two control groups: one group (K-) received no intervention, while the other group (K+) was exposed to daily cigarette smoke. In addition, three treatment groups were included, receiving daily doses of β-hydroxybutyrate-(R)-1,3-butanediol monoester supplement (DeltaG; KE) at 1.5g/kg/day (P1), 3g/kg/day (P2), and 6g/kg/day (P3), respectively. All treatment groups were also exposed to daily cigarette smoke. After a duration of 28 days of exposure to cigarette smoke, samples of the aortic tissue were collected and subsequently analyzed to determine the levels of eNOS and the expression of VCAM-1. The collected data were subjected to analysis using the ANOVA test.

Cigarette Smoke Exposure:

The rodents were presented to sidestream smoke utilizing a peristaltic siphon. They were set inside an acrylic box with aspects of 95 x 80 x 65cm, which would oblige five rodents. To expose the rats to the cigarette smoke, the cigarettes were lit simultaneously and placed in the box for 30minutes. The rats were exposed to the smoke from a daily dose of 40 cigarettes, divided into five cycles per day and eight cigarettes per cycle. This exposure regimen was maintained for a period of four weeks. The cigarettes utilized in the study were Indonesian unfiltered cigarettes from Dji Sam Soe® by HM Sampoerna according to the manufacturer's label, these cigarettes contain approximately 39mg of tar and 2.3mg of nicotine.

β-hydroxybutyrate:

We used β-hydroxybutyrate-(R)-1,3-butanediol monoester (DeltaG Performance) purchased from TDeltaS Global, Inc. (Orlando, Florida). The exogenous ketone was administered orally to the rats at a daily dose.

Measurement of eNOS Levels:

ELISA (Elabscience E-EL-R0367) was used to measure eNOS levels in the aorta in accordance with the manufacturer's instructions. In rundown, the examples were added to a well plate with the essential enemy of eNOS neutralizer and brooded for the time being at 4 degrees Celsius. After washing, the secondary anti-eNOS antibody was added and incubated for two hours. Following another wash, substrate solution was added, and after a 30-minute incubation, the reaction was stopped using a stop solution. The absorbance of the examples was then estimated at 450nm utilizing an ELISA peruser.

VCAM-1 Immunohistochemistry:

The aortic tissue was prepared, deparaffinized, and incubated with the anti-VCAM-1 primary antibody (Santacruz biotech SC-13160) for approximately 30 minutes. After washing, the tissue was incubated with the secondary antibody, followed by streptavidin-horseradish peroxidase (SA-HRP). Staining was performed using Chromogen DAB, and counterstaining was done with hematoxylin-eosin. The VCAM-1 expression in the tunica intima was observed under a light microscope at 400x magnification. A semi-quantitative measurement of VCAM-1 expression was conducted using the immunoreactivity scoring system (IRS) outlined in Table 1. IRS score by calculating the score of the proportion of positive cells multiplied by the intensity of staining.

Table 1: Immunoreactivity scoring system (IRS)

Score for percentage of cell staining	Score for intensity of staining
0 = no stained cells	0 = no reaction
1 = <10% cells are stained	1 = mild intensity of staining
2 = 10-50% cells are stained	2 = moderate intensity of staining
3 = 51-80% cells are stained	3 = heavy intensity of staining
4 = >80% cells are stained

Statistical Analysis:

The information examination included ascertaining mean and standard deviations (SD) for regularly dispersed information, and middle with higher and lower values for non-typically disseminated information. The Shapiro-Wilk test was used to determine whether the data were normal. For regularly circulated information, autonomous t-tests and one-way ANOVA tests were performed to look at mean qualities between factors. For non-ordinarily circulated information, the Mann-Whitney and Kruskal-Wallis tests were utilized all things being equal. Post hoc examination was directed utilizing Fisher's LSD test. SPSS adaptation 25.0 (IBM Corp., Chicago, USA) was utilized for the factual examination.

RESULT:

Impact of Cigarette Smoke Exposure and β-hydroxybutyrate Administration on eNOS Levels:

After 28 days of the experiment, we measured the eNOS levels in different groups. The results showed that the eNOS levels were 234.74±225.50 in the K(-) group, 21.19±12.50 in the K(+) group, 182.75±111.07 in the P1 group, 263.39±206.90 in the P2 group, and 21.40±10.08 in the P3 group. Our analysis indicated significant differences between the groups (p=0.036). Further examination using post-hoc testing revealed that the P2 group exhibited a significant decrease in aortic IMT compared to the K(-) group (p= 0.016). The summarized findings are presented in Table 2 and Figure 1 to provide a visual representation of the results.

Table 2: eNOS values (pg/ml) in each group

Group	n	*eNOS* (pg/ml)		p-value
Group	n	x ± SD	Min-Max
K(-)	5	234.74± 225.50^a	17.25-520.82	0.036*
K(+)	5	21.19 ± 12.50^b	5.61- 39.39
P1	5	182.75 ± 111.07^a	74.04 - 357.25
P2	5	263.39± 206.90^a	53.32- 553.32
P3	5	21.40 ± 10.08^b	12.25- 34.54

* Significant at p = 0.05 (one-way ANOVA)

ab Different superscripts shows significant differences between groups (post-hoc analysis: Fisher’s LSD test for eNOS)

Figure 1: The mean value of eNOS levels after administration of β-hydroxybutyrate

Effects of Cigarette Smoke Exposure and β-hydroxybutyrate Administration on VCAM-1 Expression:

The administration of β-hydroxybutyrate and exposure to cigarette smoke did not result in any significant differences in aortic VCAM-1 expression (p = 0.426; p > 0.05). The measured VCAM-1 expression levels were as follows: 8.20 ± 2.48 in the K(-) group, 9.00 ± 2.12 in the K(+) group, 7.20 ± 3.03 in the P1 group, 6.60 ± 1.94 in the P2 group, and 6.40 ± 2.50 in the P3 group. The results are presented in the following Table 3 and Figure 2.

Table 3: VCAM-1 expression values in each group

Group	n	VCAM-1 Expression (IRS)		p
Group	n	x ± SD	Min-Max
K(-)	5	8.20± 2.48	6.00-12.00	0.426*
K(+)	5	9.00± 2.12	6.00- 12.00
P1	5	7.20± 3.03	4.00- 12.00
P2	5	6.60± 1.94	4.00- 9.00
P3	5	6.40± 2.50	4.00- 9.00

* Significant at p = 0.05 (one-way ANOVA)

Figure 2: The mean value of VCAM-1 expression after administration of β-hydroxybutyrate

We examined the VCAM-1 expression through immunohistochemical analysis by counting the endothelial cells in the aortic tissue. Using a light microscope at 400x magnification, we observed ten different spots within the tunica intima of the aortic tissue. The findings indicated that after exposure to cigarette smoke, the K(-) group exhibited weak VCAM-1 expression in the intima (Figure 3A), while the K(+) group demonstrated strong expression (Figure 3B). Similarly, the P1, P2, and P3 groups, which received different doses of β-hydroxybutyrate along with exposure to cigarette smoke, displayed weak VCAM-1 expression in the intima. Notably, the P3 group showed the weakest VCAM-1 expression among these groups (Figure 3C, 3D, and 3E).

Figure 3. Observation results in the tunica intima of aortic tissue on the histopathological preparate with a light microscope at 400x magnification. (a) Control groups without intervention (K-). (b) Control groups with daily exposure to 40 cigarette smoke (K+). (c) Treatment groups with daily exposure to 40 cigarette smoke and daily doses of β-hydroxybutyrate 1.5 g/kg/day (P1). (d) Treatment groups with daily exposure to 40 cigarette smoke and daily doses of β-hydroxybutyrate 3 g/kg/day (P2). (e) Treatment groups with daily exposure to 40 cigarette smoke and daily doses of β-hydroxybutyrate 6 g/kg/day (P3).

DISCUSSION:

This study explored the impact of cigarette smoke exposure and the administration of β-hydroxybutyrate on eNOS levels and VCAM-1 expression, key factors in endothelial dysfunction, in the aorta. Our findings revealed that openness to cigarette smoke resulted in reduced eNOS levels, a ley player implicated in endothelial dysfunction²³. Endothelial nitric oxide synthase (eNOS) is a fundamental compound associated with the development of nitric oxide (NO) inside endothelial cells.NO plays a crucial role in maintaining vascular homeostasis by regulating various physiological processes, including vasodilation, platelet aggregation, and inflammation. As eNOS is responsible for synthesizing NO, its levels can serve as an indicator of NO bioavailability within endothelial cells, reflecting their ability to maintain proper vascular function^24-25. This lines up with past examinations showing diminished eNOS levels in endothelial cell societies presented to tobacco smoke, prompting cell brokenness and disabled multiplication, grip, and migration²⁶.

However, our study also demonstrated the potential of β-hydroxybutyrate, an active component of ketone body, to mitigate the effects of cigarette smoke on eNOS levels. β-hydroxybutyrate acts as a stress response molecule and regulates antioxidant defense mechanisms, thereby maintaining redox homeostasis²⁷. By reducing free radicals through its radical scavenging activity, β-hydroxybutyrate may help preserve eNOS activity and prevent endothelial dysfunction induced by cigarette smoke²⁸.The decrease in eNOS observed in our study can be attributed to the free radicals present in cigarette smoke, which can react with NO and form peroxynitrite, a highly reactive species with prooxidant properties, thereby reducing the availability of active NO²⁹. Endothelial dysfunction, characterized by impaired vascular tone and increased expression of adhesion molecules such as VCAM-1, can lead to coagulation and inflammation when NO levels are diminished³⁰.Notably, Soejima et al. demonstrated that ketone administration prevented a decrease in eNOS levels associated with vascular endothelial cell damage induced by low glucose exposure³¹.Additionally, Li et al. reported that medium doses of β-hydroxybutyrate increased the ratio of eNOS, resulting in a neuroprotective effect in ischemic stroke models³².

In our study, we also evaluated the impact of β-hydroxybutyrate on VCAM-1 expression in response to cigarette smoke exposure. Although the differences were not statistically significant, the administration of β-hydroxybutyrate resulted in reduced VCAM-1 expression in the treatment groups. Ji et al. demonstrated that β-hydroxybutyrate can attenuate mitochondrial superoxide production and mitigate mitochondrial oxidative stress in vascular endothelial cells by activating antioxidant pathways, thus potentially contributing to the observed decrease in VCAM-1 expression³³. Although the difference did not reach statistical significance, there was an observed trend of dose-dependent reduction in VCAM-1 levels in our study.

Numerous previous studies have highlighted the pleiotropic effects of ketone body and their potential cardiovascular benefits²⁰. Treatment with 1,3-butanediol, for instance, has been shown to enhance nitric oxide synthase activity in rat arteries³⁴. Following transaortic constriction (TAC), the presence of beta-hydroxybutyrate dehydrogenase 1 (BDH1) in rats was associated with increased oxidation of cardiac ketone body, upregulation of antioxidant superoxide dismutase expression, and suppression of oxidative stress³⁵. β-OHB, when present in mitochondria, serves as an alternative fuel and can mitigate the effects of oxidative stress³⁶.Moreover, higher concentrations of ketone body (β-OHB) have been found to improve cardiac remodeling pathology and enhance cardiac function in small and large animal models³⁷.

Ketone body have also demonstrated immunoregulatory properties in animals and humans, attenuating pathological inflammation through various mechanisms³⁸. β-hydroxybutyrate has been extensively studied and shown to decrease the production of reactive oxygen species (ROS), enhance mitochondrial respiration, and stimulate cellular endogenous antioxidant systems³⁹. Oxidative stress is caused by an imbalance between free radicals and antioxidant defense mechanisms in the vascular system. It plays a crucial role in mediating the production of cytokines and linking reactive oxygen species (ROS) to endothelial dysfunction⁴⁰. ROS-induced oxidative damage can exacerbate endothelial cell injury, triggering the activation of transcription factor Nrf2 in endothelial cells, which, in turn, protects against endothelial damage⁴¹. Huang et al. demonstrated that Nrf2 improves endothelial function by combating oxidative stress and mitigating mitochondrial damage, thus delaying atherosclerosis⁴². β-hydroxybutyrate, functioning as a stress response molecule, regulates antioxidant defense programs to keep redox homeostasis in response to environmental challenges, and its metabolism is key to its positive effects on stress response²⁰. Ketone body induce oxidative stress, activating the transcription factor Nrf2, which leads to the transcription of target genes involved in cellular antioxidant defense systems⁴³. The initiation of the Nrf2 pathway safeguards cells from cell demise incited by oxidative pressure through expanded articulation of cancer prevention agent proteins, for example, cytoplasmic superoxide dismutase (SOD1) and mitochondrial superoxide dismutase (SOD2)⁴⁴. Based on these findings, β-hydroxybutyrate emerges as a potent therapeutic agent with vasodilatory effects on the endothelium.

CONCLUSION:

In summary, our study provides evidence that exposure to cigarette smoke reduces eNOS levels, while the administration of β-hydroxybutyrate shows potential in mitigating the detrimental effects of cigarette smoke on eNOS levels. These findings suggest that β-hydroxybutyrate may have beneficial effects on endothelial function and oxidative stress management. Further exploration is justified to completely investigate the remedial capability of ketone body treatment as a vasodilator on the endothelium and its suggestions for cardiovascular health.

CONFLICT OF INTEREST:

There were no competing interests declared by the authors.

ACKNOWLEDGMENTS:

This study was funded by the Institute for Community Service and Development (LPPM), Universitas Airlangga, Surabaya, Indonesia, under grant No. 251/UN3/2022.

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Received on 14.06.2023 Modified on 25.10.2023

Research J. Pharm. and Tech 2024; 17(5):2235-2240.

DOI: 10.52711/0974-360X.2024.00352