Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits

Atherosclerosis and inflammation

Atherosclerosis is a chronic inflammatory condition that affects various tissues and organs, leading to serious complications such as cardiovascular disease, stroke, and diabetes mellites.¹ During disease progression, atherosclerotic plaques can detach from the vascular wall, resulting in major cardiovascular events.¹ While the exact mechanisms underlying plaque rupture remain unclear, several studies have highlighted the critical roles of inflammation and oxidative damage in the progression of atherogenic plaques.² For example, Libby et al. (2002) demonstrated that inflammatory cytokines, such as interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α), contribute to plaque instability and rupture by promoting matrix metalloproteinase (MMP) activity, which degrades the fibrous cap.² Additionally, Witold et al. (2017) identified oxidative stress as a key factor in endothelial dysfunction and foam cell formation, both of which accelerate atherogenesis.³ These findings underscore the close correlation between the degree of oxidation, inflammation, and atherosclerosis progression. Therefore, monitoring inflammatory markers is clinically valuable, as they may provide critical insights into the progression of atherosclerosis.

Role of autophagy in atherosclerosis

Autophagy is a normal physiological mechanism through which the body can recycle cytoplasmic components (such as damaged/aged organelles and other cellular proteins), which are phagocytosed, and flagged for destruction by lysosomes.⁴ Autophagy plays a key role in cellular homeostasis and can help get rid of damaged cells in disease conditions such as cancer and chronic illnesses.⁵ Therefore, induction of autophagy in blood vessels can protect endothelial cells and smooth muscle cells damage, which may occur due to atherosclerosis and reduce the vulnerability of the plaques.⁵ Macrophage autophagy can be useful in inhibiting atherosclerotic plaque rupture, which could reduce the severity of the condition.⁵ mTOR is Ser/Thr protein kinase, considered to be a key control in cellular nutrition and energy expenditure, thus playing a central role in regulation of autophagy.⁶ It can integrate multiple signals from upstream pathways and block the formation of autophagosomes.⁶ Accordingly, there are several signaling paths which monitor autophagy induction. These include PI3K-Akt-mTOR and AMPK-mTOR pathways.⁷ Previously, it was indicated that the (PI3K-Akt-mTOR) represent the primary pathway which is responsible for regulating a variety of cellular behaviors such as growth, apoptosis, and autophagy.⁷ The activation of (PI3K/Akt/mTOR) signaling pathway in atherosclerosis may provide insight on the therapeutic role of inhibiting this pathway to control the development of atherosclerosis. LC3B is defined as an RNA-binding protein, which can be used to initiate mRNA degradation during autophagy, and hence will be measured in the current study to indicate the degree of autophagy.⁵

Potential therapeutic agents: Pterostilbene and sitagliptin

Pterostilbene is a di-methylated analog of resveratrol, recognised for its potent anti-inflammatory properties,⁸ making it a promising candidate for reducing inflammation and potentially improving atherosclerosis outcomes. Previous studies have demonstrated that pterostilbene downregulates NF-κB and Toll-like receptor 5 expression,⁹ both of which are critical mediators of inflammatory responses in vascular diseases. Additionally, pterostilbene has been shown to inhibit smooth muscle activation through modulation of the adenosine-monophosphate-activated-protein-kinase (AMPK) pathway.⁷ This AMPK-STAT3 signalling axis is a key indicator of endothelial inflammation within the vascular wall, suggesting a mechanistic pathway through which pterostilbene could exert protective effects against atherosclerosis.⁷

Sitagliptin, an oral antidiabetic medication from the gliptin family, is commonly used as a second-line treatment to manage hyperglycaemia in patients with diabetes mellitus.¹⁰ Gliptins function by stimulating insulin production and secretion through the inhibition of the dipeptidyl peptidase-4 enzyme, thereby prolonging the half-life of incretin hormones in response to dietary intake.¹¹ By improving insulin sensitivity, sitagliptin may also mitigate the development of diabetic complications, including atherosclerosis, given the close link between metabolic dysregulation and vascular inflammation.

Study rationale and objectives

Despite extensive research on the individual roles of pterostilbene and sitagliptin, there remains a gap in understanding their combined effects on atherosclerosis, particularly in the context of diet-induced vascular inflammation. Most prior studies have focused on their isolated impacts within metabolic or inflammatory pathways without exploring potential synergistic effects. The current study aims to address this gap by investigating the benefits of pterostilbene and sitagliptin supplementation in improving atherosclerosis outcomes in rabbits fed an atherogenic diet. This research seeks to elucidate not only the individual contributions of these compounds but also their potential interactive mechanisms, providing new insights into combination therapies for atherosclerosis management.

Ethical approval

All the procedures were performed according to the guidelines approved by the National Institutes of Health. The research study received ethics approval from the Animal Research Ethics, College of Medicine, University of Kufa, Iraq (approval no. 15792, on 14th December 2020) where the research took place. All experimental procedures involving animals were carried out in keeping with guidelines from the National Institutes of Health Guide for the Care and Use of Laboratory Animals to ameliorate any suffering of animals.

Animal protocol

This study included 80 New Zealand White rabbits (26 males and 54 females), aged between 1 and 3 years and weighing between 1300 and 3000 grams. All rabbits were housed individually in polycarbonate cages (0.90 × 0.60 × 0.60 m) for two weeks to allow for acclimatization to the environment. The housing conditions were maintained on a 12-hour light/dark cycle at a constant temperature of 25°C with 50% humidity.

During the acclimatization period, the rabbits were fed a standard pellet diet and had access to tap water ad libitum. Food consumption and faecal characteristics were routinely monitored to assess health status. Rabbits were excluded from the study if they appeared unwell before enrolment. None of the animals had been previously used in research.

Following acclimatization, the rabbits were assigned to experimental groups and fed an atherogenic diet composed of a traditional chow supplemented with 2% cholesterol to induce atherosclerosis. This diet was designed to promote the development of atherosclerotic lesions, providing a suitable model for evaluating the effects of the tested interventions. The diet’s composition was chosen based on established protocols to ensure consistency and reproducibility of atherosclerotic outcomes.

Study design

Following a two-week acclimatization period, animals were randomly assigned to one of four study groups, with 20 animals per group. A sample size of 20 animals per group was determined through power analysis, providing more than 85% power to detect significant differences with an effect size of 0.45 at a significance level of α = 0.05. Randomisation was conducted using simple random sampling: each animal was assigned a tag number, and a blindfolded researcher (B.M.) randomly selected numbered tags from a hat to allocate animals to the groups.

Study groups:

Justification for dosage and feeding duration

The selected dosages for pterostilbene (10 mg/kg) and sitagliptin (12 mg/kg) were based on previously published studies that demonstrated their efficacy in modulating inflammatory and metabolic pathways relevant to atherosclerosis without causing adverse effects.^12,13 The pterostilbene dosage was chosen considering its bioavailability and anti-inflammatory potential observed in preclinical studies, while the sitagliptin dose reflects effective glucose-lowering and vascular protective outcomes in animal models.^12,13

An eight-week feeding duration was chosen to ensure sufficient time for the development of diet-induced atherosclerotic changes and to allow for the therapeutic effects of the interventions to manifest. This duration aligns with established protocols in similar rabbit models of atherosclerosis, providing a balance between the progression of the disease and the ethical considerations of prolonged animal experimentation.

Induction of atherosclerosis

To induce hyperlipidemia and subsequent development of atherosclerotic changes, animals were provided with 2% high cholesterol (BDH Chemicals Ltd, England), in their food to develop atherosclerotic changes in the aorta following 8 weeks supplementation.¹⁴ During the study, animals were monitored on a daily basis to check their vital signs. In addition, blood pressure, body weight and blood samples were collected fortnightly to measure blood glucose levels.

At the conclusion of the study, rabbits were kept fasted overnight, then they were euthanized using ketamine (HIKMA Pharmaceuticals, 3310), using (66 mg/kg), and xylazine (Alfasan, 1004111-07), sing (6 mg/kg), via intramuscular injection.^15,16 Following euthanasia, thoracotomy was performed to expose the heart and collect blood. Aortic arch was dissected, and samples collected as well as the following:

Extraction of total RNA from aorta and reverse transcriptase polymerase chain reaction

RT-PCR analysis

For reverse transcriptase polymerase chain reaction (RT-PCR), a reaction mixture was prepared containing 3 μg of total RNA, 2.5 μM oligo (dT) primers, 1.5 mM magnesium chloride (MgCl₂), 0.01 M dithiothreitol (DTT), and 200 units of SuperScript III reverse transcriptase in a total volume of 20 μl. The RT process included an initial reverse transcription step at 42°C for 60 minutes, followed by a denaturation step at 70°C for 5 minutes.

PCR amplification was carried out with the following thermal cycling conditions: initial denaturation at 95°C for 30 seconds, primer annealing at 60°C for 30 seconds, and elongation at 72°C for 30 seconds for 30 cycles. A final elongation step was performed at 72°C for 7 minutes.

Genes of interest and normalisation

The specific genes analysed in this study include PI3K, AKT, AMPK, and mTOR, which are key regulators in the autophagy signalling pathway and are highly relevant to atherosclerosis. PI3K and AKT are involved in cell growth and survival pathways, AMPK regulates cellular energy homeostasis, and mTOR plays a central role in inhibiting autophagy. These genes were selected to provide insights into the molecular mechanisms underlying atherosclerotic progression and the therapeutic effects of the tested compounds.

Gene expression levels were normalised against the housekeeping gene GAPDH, selected for its stable expression across experimental conditions. Relative gene expression was quantified using the 2^(-ΔΔCt) method, ensuring accurate comparison of target gene expression levels across different samples.

Measurement of Autophagy Marker (LC3B)

LC3B (microtubule-associated protein 1 light chain 3 beta) was chosen as a representative marker of autophagy due to its critical role in autophagosome formation. During autophagy, LC3B-I (cytosolic form) is lipidated to form LC3B-II, which associates with autophagosome membranes, serving as a reliable indicator of autophagic activity.

LC3B expression was quantified through RT-PCR using specific primers targeting LC3B mRNA. Additionally, the LC3B-II/LC3B-I ratio will be assessed to determine autophagy flux. The rationale for selecting LC3B as a marker is its well-established correlation with autophagic activity and its use as a standard autophagy biomarker in atherosclerosis-related studies.

Histopathological examination

Aorta tissue samples were collected from all animals for histopathological examination. The tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm thickness. Sections were stained using Hematoxylin and Eosin (H&E) to evaluate general histological architecture.

Scoring criteria

Histopathological grading of atherosclerotic lesions was performed based on a semi-quantitative scoring system evaluating the following parameters:

Each slide was evaluated independently by two blinded pathologists to ensure consistency and reduce observer bias. The final score for each parameter was averaged between the two observers.

Statistical analysis

The sample size of 80 rabbits (26 males and 54 females) was determined based on a power analysis conducted prior to the study, aiming to achieve a statistical power of 80% with an alpha level of 0.05. This calculation was performed using G*Power software, considering the expected effect size derived from preliminary data and previous studies on atherosclerosis interventions. The unequal distribution of males and females was due to availability constraints and to ensure representation of both sexes, allowing for potential assessment of sex-based differences in response to the interventions.

Means and standard error of the mean (SEM) were calculated and analysed using the Statistical Package for Social Sciences (SPSS, version 26, IBM, USA). For multiple group comparisons, one-way ANOVA was performed, followed by the Least Significant Difference (LSD) post-hoc test to identify specific group differences. A P-value of <0.05 was considered statistically significant.

For histopathological grading, the non-parametric Kruskal-Wallis test was used to compare histopathological scores among all four groups. When significant differences were identified, pairwise comparisons were conducted using the Mann-Whitney U test to assess differences between specific groups, including direct comparisons between the pterostilbene-treated (PT) and sitagliptin-treated (ST) groups. A P-value of ≤ 0.001 was considered statistically significant for these analyses to control for Type I errors due to multiple comparisons.

Atherogenic diet effects on lipid profile and atherogenic index

The atherogenic diet significantly elevated serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL) compared to the NC cohort (P < 0.001). Pterostilbene supplementation notably reduced TC and LDL levels, while sitagliptin supplementation resulted in significant reductions in TC, TG, and LDL levels (P < 0.001) (Table 1).

High-density lipoprotein (HDL) levels were reduced in the PT and ST groups compared to the AC group (P<0.01), indicating that neither treatment restored HDL to normal levels (Table 1).

Effect of atherogenic diet and treatment on oxidative stress maker F2-Isoprostane

Plasma F2-Isoprostane levels, a marker of lipid peroxidation, were significantly elevated in rabbits fed an atherogenic diet for 8 weeks (AC cohort) compared to the normal control group (NC cohort) (P < 0.001). Supplementation with pterostilbene and sitagliptin for 8 weeks resulted in a significant reduction in plasma F2-Isoprostane levels compared to the AC cohort (P < 0.05).

Key trends observed include a notable decrease in oxidative stress markers in response to both treatments, highlighting their potential antioxidative effects. Detailed numerical data are presented in Table 1, and the comparative trends are visually depicted in Figure 1, ensuring a clear representation without redundancy.

Figure 1. Plasma F2-Isoprostane levels among the four study groups (P<0.005).

Effect of atherogenic diet and treatment options on aortic atherosclerotic lesion degree

Histopathological analysis revealed pronounced progression of atherosclerotic lesions in the AC cohort, characterised by advanced lesions with extracellular lipid cores and complicated lesions marked by hemorrhagic thrombus formation. These changes were significantly more severe compared to the NC cohort, which exhibited normal arterial architecture (P < 0.001).

In contrast, the PT and ST treated groups demonstrated marked improvements in aortic histopathology. The majority of rabbits in these groups exhibited initial or intermediate lesions with significantly reduced lipid accumulation and less pronounced intimal thickening. Pterostilbene treatment was associated with improved vascular integrity, while sitagliptin treatment resulted in reduced foam cell formation and inflammatory infiltration.

The differences in lesion severity among the groups were statistically significant (P < 0.001, Kruskal-Wallis test), with pairwise comparisons confirming significant improvements in both PT and ST groups compared to the AC cohort. These findings are summarised in Table 2, with representative histological images provided in Figure 2 to illustrate the progression and regression of atherosclerotic lesions.

Figure 2. Atherosclerotic changes; cross-section in aortic arch; high fat diet rabbit; hematoxylin and eosin stain, (×40) for figures A, B, C, D, F, (x10) for Figure E.

A: Normal arterial appearance in the control group; B: Initial atherosclerotic changes – lipid-laden foam cells in the sitagliptin study cohort; C: fatty streak and D: Intermediate atherosclerotic changes – extracellular lipid pool in the Pterostilbene study cohort; E: Advance atherosclerotic changes – a core of extracellular lipid and F: Complicated atherosclerotic changes – hemorrhagic thrombus as seen in the atherogenic study cohort (no supplementations).

We have used the scoring methodology to interpret the lesions ( Table 2).

In addition, the Tissue levels of LC3B, a marker of autophagy, were significantly reduced in the AC cohort compared to the NC cohort (P < 0.001). Both pterostilbene and sitagliptin supplementation significantly increased LC3B levels compared to the AC cohort (P < 0.001), indicating enhanced autophagic activity (Figure 3).

Figure 3. Marker of tissue autophagy (LC3B) in aortic tissue following the study conclusion (P<0.001).

Effect of pterostilbene and sitagliptin on mRNA expression levels of PI3K, AKT, AMPK and mTORC1 in aortic tissues

The atherogenic diet caused a significant increase in the degree of inflammation as manifested by the significant rise in the expression of mTORC1, PI3K and AKT (P < 0.001). Following the 8 weeks of supplementations, the expression of mTORC1 was significantly reduced in response to pterostilbene and sitagliptin supplementations as compared to the atherogenic study cohort (P < 0.001) ( Figure 4).

Figure 4. Markers of inflammatory changes (mTORC1, AKT, PI3K and AMPK) in aortic tissue following the end of the study (P<0.001).

Pterostilbene supplementation significantly improved the expression of AKT and PI3K in aortic tissue compared to the AC cohort (P < 0.001), an effect not observed with sitagliptin treatment. Additionally, the AMPK index was significantly reduced in the AC cohort compared to the NC cohort (P < 0.05). Sitagliptin supplementation resulted in a significant increase in AMPK expression compared to the AC cohort (P < 0.001) (Figure 4).