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

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This study aims to evaluate the protective and autophagy induction properties of pterostilbene and sitagliptin on modulating the degree of atherosclerosis in rabbit models treated with an atherogenic diet. Methods 80 rabbits were randomly placed into one of four study groups (20 in each group): normal control diet (NC) fed normal diet for eight weeks, atherogenic control (AC) fed atherogenic diet for eight weeks, pterostilbene treated group (PT) fed atherogenic diet with pterostilbene (at 10 mg/kg/day) orally daily for eight weeks, and sitagliptin treated group (ST) fed atherogenic diet with sitagliptin (at 12 mg/kg/day) orally daily for eight weeks. Results While serum lipids and F2-isoprostane were elevated significantly in the AC study cohort compared to NC study cohort, (P < 0.001), both pterostilbene and sitagliptin supplementations provided significant improvements in serum lipid parameters and F2-isoprostane in the PT study cohort and ST study cohort, respectively, when compared to the AC study cohort, (P<0.001). Total cholesterol, triglycerides and LDL levels were significantly reduced among the PT and ST study cohorts as compared to the AC study cohort. This was coupled with a significant rise in LC3B levels (marker of tissue autophagy) among the PT study cohort and the ST study cohort, as compared to the AC study cohort, (P < 0.001). The RNA expression of mTORC1 was reduced significantly at both PT study cohort and ST study cohort, (P<0.001). Pterostilbene supplementation induced a significant reduction in tissue expression of PI3K and AKT, (P<0.01), while sitagliptin induced significant increase in 5’ adenosine monophosphate-activated protein kinase (AMPK) levels, (P<0.001). Conclusions The results indicate that pterostilbene and/or sitagliptin supplementation can significantly improve the outcome of atherosclerosis due to their effects on the inflammatory pathways which hinder the progression of atherosclerotic plaque formation. 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F1000Research 2025, 12 :339 ( https://doi.org/10.12688/f1000research.130682.4 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Revised Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] Hussam H Sahib https://orcid.org/0000-0001-8099-3425 1 , Bassim I Mohammad https://orcid.org/0000-0001-6732-5940 2 , Najah R Hadi 3 , [...] Azhar Al-Shaibany 4 , Anil K Philip 5 , Wisam J Mohammed 6 , Dina A Jamil https://orcid.org/0000-0002-0537-375X 7,8 , Hayder A Al-Aubaidy https://orcid.org/0000-0001-9564-0120 8 Hussam H Sahib https://orcid.org/0000-0001-8099-3425 1 , Bassim I Mohammad https://orcid.org/0000-0001-6732-5940 2 , [...] Najah R Hadi 3 , Azhar Al-Shaibany 4 , Anil K Philip 5 , Wisam J Mohammed 6 , Dina A Jamil https://orcid.org/0000-0002-0537-375X 7,8 , Hayder A Al-Aubaidy https://orcid.org/0000-0001-9564-0120 8 PUBLISHED 14 May 2025 Author details Author details 1 Department of Pharmacology and Therapeutics, College of Pharmacy, University of Al-Qadisiyah, Al-Qadisiyah, Iraq 2 Department of Pharmacology and Therapeutics, College of Medicine, University of Al-Qadisiyah, Al-Qadisiyah, Iraq 3 Department of Pharmacology and Therapeutics, College of Medicine, University of Kufa, Al-Najaf, Iraq 4 Health Service Executive, GIM Navan, Ireland 5 School of Pharmacy, University of Nizwa, Birkat AlMouz, Oman 6 Baghdad Medical Complex-Iraqi, Cardiology Center, Baghdad, Iraq 7 Oceania University of Medicine, Melbourne, Australia 8 Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, VIC, 3086, Australia Hussam H Sahib Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation Bassim I Mohammad Roles: Conceptualization, Investigation, Project Administration, Supervision, Writing – Original Draft Preparation Najah R Hadi Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Project Administration, Supervision Azhar Al-Shaibany Roles: Investigation, Resources, Validation, Writing – Review & Editing Anil K Philip Roles: Formal Analysis, Methodology, Resources, Writing – Review & Editing Wisam J Mohammed Roles: Formal Analysis, Methodology, Resources, Writing – Original Draft Preparation Dina A Jamil Roles: Methodology, Validation, Writing – Review & Editing Hayder A Al-Aubaidy Roles: Conceptualization, Project Administration, Resources, Validation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Background Inflammation is the key contributor to the development of atherosclerotic plague. This study aims to evaluate the protective and autophagy induction properties of pterostilbene and sitagliptin on modulating the degree of atherosclerosis in rabbit models treated with an atherogenic diet. Methods 80 rabbits were randomly placed into one of four study groups (20 in each group): normal control diet (NC) fed normal diet for eight weeks, atherogenic control (AC) fed atherogenic diet for eight weeks, pterostilbene treated group (PT) fed atherogenic diet with pterostilbene (at 10 mg/kg/day) orally daily for eight weeks, and sitagliptin treated group (ST) fed atherogenic diet with sitagliptin (at 12 mg/kg/day) orally daily for eight weeks. Results While serum lipids and F2-isoprostane were elevated significantly in the AC study cohort compared to NC study cohort, ( P < 0.001), both pterostilbene and sitagliptin supplementations provided significant improvements in serum lipid parameters and F2-isoprostane in the PT study cohort and ST study cohort, respectively, when compared to the AC study cohort, ( P <0.001). Total cholesterol, triglycerides and LDL levels were significantly reduced among the PT and ST study cohorts as compared to the AC study cohort. This was coupled with a significant rise in LC3B levels (marker of tissue autophagy) among the PT study cohort and the ST study cohort, as compared to the AC study cohort, ( P < 0.001). The RNA expression of mTORC1 was reduced significantly at both PT study cohort and ST study cohort, ( P <0.001). Pterostilbene supplementation induced a significant reduction in tissue expression of PI3K and AKT, ( P <0.01), while sitagliptin induced significant increase in 5’ adenosine monophosphate-activated protein kinase (AMPK) levels, ( P <0.001). Conclusions The results indicate that pterostilbene and/or sitagliptin supplementation can significantly improve the outcome of atherosclerosis due to their effects on the inflammatory pathways which hinder the progression of atherosclerotic plaque formation. READ ALL READ LESS Keywords Pterostilbene, Sitagliptin, Atherosclerosis, PI3K, AMPK, AKT, Rabbits. Corresponding Author(s) Hayder A Al-Aubaidy ( [email protected] ) Close Corresponding author: Hayder A Al-Aubaidy Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2025 Sahib HH et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Sahib HH, Mohammad BI, Hadi NR et al. Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.12688/f1000research.130682.4 ) First published: 27 Mar 2023, 12 :339 ( https://doi.org/10.12688/f1000research.130682.1 ) Latest published: 14 May 2025, 12 :339 ( https://doi.org/10.12688/f1000research.130682.4 ) Revised Amendments from Version 3 The revised version of the manuscript incorporates the corrections suggested by the reviewer. The abstract, results and the discussion sections were amended to address the reviewer’s comments. Please see the author’s detailed response to the reviewer (Dr. Xin-Fang Leong) for more details. The revised version of the manuscript incorporates the corrections suggested by the reviewer. The abstract, results and the discussion sections were amended to address the reviewer’s comments. Please see the author’s detailed response to the reviewer (Dr. Xin-Fang Leong) for more details. See the authors' detailed response to the review by Jelica Grujic Milanovic See the authors' detailed response to the review by Xin-Fang Leong READ REVIEWER RESPONSES Introduction 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. Methods 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: • Normal Control (NC): Rabbits received a traditional chow diet and water ad libitum for eight weeks. • Atherogenic Control (AC): Rabbits were fed an atherogenic diet containing 2% cholesterol and provided water ad libitum for eight weeks. • Pterostilbene Treated (PT): Rabbits received an atherogenic diet, water ad libitum, and pterostilbene supplements (purity 98%, Hangzhou Hyper Chemicals Limited, China; CAS No. 537-42-8) at a dose of 10 mg/kg orally daily for eight weeks. • Sitagliptin Treated (ST): Rabbits received an atherogenic diet, water ad libitum, and sitagliptin supplements (purity 98%, Hangzhou Hyper Chemicals Limited, China; Batch No. 20112301) at a dose of 12 mg/kg orally daily for eight weeks. 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: • Serum lipid profile (total cholesterol – TC, triglyceride – TG, low density lipoprotein cholesterol – LDL, high density lipoprotein cholesterol – HDL, and very low-density lipoprotein cholesterol – VLDL. Serum lipids were measured using enzymatic methods (Abbott, Alcyon 300 Chemistry Analyzer, USA). • Serum F2-isoprostane to assess lipid peroxidation. This has been assessed colormetrically via ELISA (Sunlong, China, SL0284Rb). • Tissue LC3B as a marker of tissue autophagy marker using the LC3 Antibody Kit for Autophagy (Thermo Fisher, USA, catalogue number: L10382). • Assessments of mTOR (PI3K, AKT, AMPK, and mTORC1) using RT-PCR (see details below). • Histopathological examination of the aorta looking for atherosclerotic changes. Extraction of total RNA from aorta and reverse transcriptase polymerase chain reaction RNA extraction Total RNA was extracted from aorta tissue samples using the TRIzol ® reagent kit (Thermo Fisher, Catalogue number 12183555). Approximately 100 mg of aorta tissue was homogenised in 750 μl of TRIzol ® reagent. To this, 200 μl of chloroform was added, mixed vigorously for 15 seconds, and then placed on ice for 5 minutes. The mixture was centrifuged, and the aqueous phase (500 μl) was transferred to a new tube. An equal volume (500 μl) of isopropanol was added, and the sample was incubated at 4°C for 10 minutes before centrifugation. The supernatant was discarded, and the RNA pellet was washed with 1 ml of ethanol, followed by centrifugation. After discarding the supernatant, the RNA pellet was air-dried and resuspended in 100 μl of nuclease-free H 2 O for RNA extraction. 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 (MgCl 2 ), 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: 1. Intimal Thickness: Scored from 0 (normal) to 3 (severe thickening). 2. Lipid Deposition: Scored from 0 (none) to 3 (extensive lipid accumulation). 3. Inflammatory Cell Infiltration: Scored from 0 (no infiltration) to 3 (dense infiltration). 4. Plaque Vulnerability Indicators: Including necrotic core formation and fibrous cap thinning, scored from 0 (absent) to 3 (prominent). 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. Results 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 ). Table 1. Levels of serum lipid profile among the four study groups. Normal control group Atherogenic control group Pterostilbene treated group Sitagliptin treated group TC (mg/dl) 68.1±5.1 810.3±78.2 634.8±19.1 * 478.5±56.1 # TG (mg/dl) 47.8±1.5 294.5±25.6 201.3±16.7 150.1±19.4 # LDL (mg/dl) 25.3±2.8 647.3±70.3 347.1±20.9 * 274.6±31.8 # HDL (mg/dl) 15.6±0.8 24.6±0.5 21.3±0.7 18.7±0.9 # F2-Isoprstane (pg/ml) 167.9±7.5 798.3±43.1 523.4±25.8 * 598.3±45.9 # * Significance ( P <0.001) among the pterostilbene & the atherogenic study cohorts. # Significance ( P <0.01) among the sitagliptin & the atherogenic study cohorts. 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. Table 2. The difference in median atherosclerotic lesion degree between the 4 study groups. Normal diet control group N (%) Atherogenic diet control group N (%) Sitagliptin treated group N (%) Pterostilbene treated group N (%) P (Kruskal-Wallis) Atherosclerotic lesion degree <0.001 Normal 7 (100) 0 (0) 1 (14.3) 0 (0) Initial 0 (0) 0 (0) 4 (57.1) 5 (71.4) Intermediate 0 (0) 1 (14.3) 2 (28.6) 2 (28.6) Advanced 0 (0) 3 (42.9) 0 (0) 0 (0) Complicated 0 (0) 3 (42.9) 0 (0) 0 (0) Total 7 (100) 7 (100) 7 (100) 7 (100) Median Normal Advanced Initial Initial Mean rank 4.5 24.86 15 13.64 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 ). Discussion The current study evaluated the effects of pterostilbene and sitagliptin supplementation on reducing inflammation and improving atherosclerosis outcomes in a rabbit model. Our findings demonstrate that an atherogenic diet significantly impairs lipid profiles, as evidenced by increased total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL) levels, along with decreased high-density lipoprotein (HDL) levels compared to the normal control diet. 17 Following 8 weeks of supplementation, both pterostilbene and sitagliptin significantly improved total cholesterol, triglycerides, and LDL levels; however, HDL levels were lower in the treatment groups than in the atherogenic control group. This unexpected finding warrants further investigation, as HDL reductions may reflect treatment-specific effects on lipid metabolism or reverse cholesterol transport mechanisms. 18 Pterostilbene’s lipid-lowering effects may mimic those of peroxisome proliferator-activated receptor alpha (PPAR-α) agonists, known to reduce lipogenesis and lipid peroxidation, which are key drivers of multiorgan diseases such as liver disease and heart failure. 18 However, while these molecular mechanisms are plausible, they are based on indirect evidence and warrant further experimental validation. Clinically, the observed improvements in lipid profiles are relevant, as dyslipidemia is a major risk factor for atherosclerosis. Compared to existing lipid-lowering therapies like statins, pterostilbene and sitagliptin may offer complementary benefits, particularly through their anti-inflammatory and autophagy-modulating effects. Future research should compare these agents directly with standard treatments to determine their relative efficacy and potential for clinical application. Our study also revealed a significant reduction in plasma F2-isoprostane levels following pterostilbene supplementation, suggesting decreased oxidative stress. Although direct evidence linking pterostilbene to F2-isoprostane modulation is limited, we hypothesise that this effect may be mediated by the inhibition of 8-iso-prostaglandin-α production. 19 Since F2-isoprostanes are markers of lipid peroxidation, their reduction aligns with decreased oxidised LDL levels, further supporting the antioxidant potential of pterostilbene. 20 At the molecular level, we observed increased mRNA expression of PI3K, AKT, and mTORC1 in cholesterol-fed rabbits compared to controls, consistent with the role of oxidised LDL in activating PI3K signalling pathways. This activation promotes inflammatory responses, monocyte chemotaxis, and foam cell formation, contributing to atherosclerosis progression. 21 , 22 Pterostilbene appears to inhibit the PI3K/Akt/mTOR pathway, potentially regulating macrophage autophagy and influencing plaque stability. Elevated LC3B levels following pterostilbene treatment suggest enhanced autophagosome formation, indicating that pterostilbene may counteract the autophagy suppression observed in atherosclerotic conditions. 23 , 24 However, while autophagy induction can be protective, excessive autophagy may lead to cell death and exacerbate tissue damage. This dual role underscores the need for a balanced autophagic response, as both insufficient and excessive autophagy can contribute to atherosclerosis progression. Future studies should investigate the threshold at which autophagy shifts from being protective to detrimental in vascular tissues. Sitagliptin supplementation was associated with increased AMPK expression in aortic tissue, an effect not observed with pterostilbene. AMPK activation is known to enhance plaque stability by promoting lipid metabolism, reducing inflammation, and improving endothelial function. 25 Beyond its glucose-lowering effects, sitagliptin may exert vascular protective actions through AMPK-mediated pathways, highlighting its potential utility in atherosclerosis management. Additionally, sitagliptin increased LC3B expression, suggesting enhanced autophagic activity, which may contribute to its anti-inflammatory effects and plaque-stabilising properties 26 and suggests that the reduction in autophagosome formation in the atherogenic group was significantly prevented by sitagliptin treatment. Sitagliptin protects atherogenic rabbits against inflammation through reduction of macrophage accumulation, and prevention of the inflammatory pathway concurrent with improved autophagic processes via activation of the AMPK/mTORC1 pathway. Our study has several limitations. The assessment of autophagy was limited to the aorta, and additional vascular regions were not evaluated due to time constraints. Histopathological scoring relied on haematoxylin and eosin staining, which may lack the sensitivity of advanced imaging techniques such as electron microscopy. Moreover, the molecular mechanisms proposed are based on indirect evidence and require further validation through mechanistic studies. Future research should focus on elucidating the precise molecular pathways involved in pterostilbene and sitagliptin’s effects on atherosclerosis. Clinical trials are needed to confirm their efficacy and safety in humans, with an emphasis on optimising dosing regimens and identifying potential synergistic effects with existing therapies. Investigating the long-term impact of these compounds on vascular health and their potential role in combination therapies could pave the way for novel strategies in atherosclerosis management. Conclusion In conclusion, supplementation with pterostilbene or sitagliptin significantly reduced oxidative stress, lipid peroxidation, and inflammation in rabbits fed an atherogenic diet, which may ultimately help mitigate the severity of atherosclerotic lesions. These effects are potentially mediated through the modulation of inflammatory pathways, likely involving the PI3K/Akt/mTOR and AMPK/mTOR signalling cascades. However, it is important to interpret these findings with caution, as the mechanisms proposed are based on indirect evidence and require further experimental validation. This study was conducted in an animal model, which may not fully replicate human pathophysiology. Therefore, while these results are promising, additional research, including well-designed human clinical trials, is necessary to confirm the efficacy and safety of pterostilbene and sitagliptin in managing atherosclerosis. The potential clinical implications of these findings suggest that both pterostilbene and sitagliptin could serve as adjunctive therapies to existing atherosclerosis treatments. Furthermore, considering the distinct but complementary mechanisms of action observed, exploring combination therapy may offer synergistic benefits, enhancing therapeutic outcomes in atherosclerosis management. Future studies should investigate the optimal dosing strategies and the long-term effects of these compounds, both individually and in combination, to better understand their potential in clinical practice. Author contributions The authors responsibilities were as follows: Conceptualization, H.S., W.M., B.M., and N.H.; methodology, H.S., D.J., and N.H.; formal analysis, H.S., A.A., A.P., and H.A.; investigation, H.S., B.M., N.H., and H.A.; writing—original draft preparation, H.S., B.M., and N.H.; writing—review and editing, B.M., A.A., W.M., D.J., A.P., and H.A.; supervision, B.M., and N.H. All authors have read and agreed to the published version of the manuscript. Data availability Underlying data figshare: Pterostilbene versus sitagliptin study, https://doi.org/10.6084/m9.figshare.22028744.v4 . 27 This project contains the following data: - Pterostilbene vs sitagliptin study. Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Reporting guidelines ARRIVE checklist for ‘Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits’, https://doi.org/10.6084/m9.figshare.22028744.v4 . 27 Acknowledgments The authors thank members of the Pharmacology and Therapeutic Department of Al-Qadisiyah Medical College. References 1. Hassanpour M, Rahbarghazi R, Nouri M, et al. : Role of autophagy in atherosclerosis: foe or friend? J. Inflamm. 2019; 16 (1): 8. PubMed Abstract | Publisher Full Text | Free Full Text 2. Libby P, Ridker PM, Maseri A: Inflammation and Atherosclerosis. Circulation. 2002; 105 (9): 1135–1143. Publisher Full Text 3. Nowak WN, Deng J, Ruan XZ, et al. : Reactive Oxygen Species Generation and Atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2017; 335 (5):e41–e52. 4. Yang Z, Klionsky DJ: An overview of the molecular mechanism of autophagy. Curr. Top. Microbiol. 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Publisher Full Text Comments on this article Comments (0) Version 4 VERSION 4 PUBLISHED 27 Mar 2023 ADD YOUR COMMENT Comment Author details Author details 1 Department of Pharmacology and Therapeutics, College of Pharmacy, University of Al-Qadisiyah, Al-Qadisiyah, Iraq 2 Department of Pharmacology and Therapeutics, College of Medicine, University of Al-Qadisiyah, Al-Qadisiyah, Iraq 3 Department of Pharmacology and Therapeutics, College of Medicine, University of Kufa, Al-Najaf, Iraq 4 Health Service Executive, GIM Navan, Ireland 5 School of Pharmacy, University of Nizwa, Birkat AlMouz, Oman 6 Baghdad Medical Complex-Iraqi, Cardiology Center, Baghdad, Iraq 7 Oceania University of Medicine, Melbourne, Australia 8 Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, VIC, 3086, Australia Hussam H Sahib Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation Bassim I Mohammad Roles: Conceptualization, Investigation, Project Administration, Supervision, Writing – Original Draft Preparation Najah R Hadi Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Project Administration, Supervision Azhar Al-Shaibany Roles: Investigation, Resources, Validation, Writing – Review & Editing Anil K Philip Roles: Formal Analysis, Methodology, Resources, Writing – Review & Editing Wisam J Mohammed Roles: Formal Analysis, Methodology, Resources, Writing – Original Draft Preparation Dina A Jamil Roles: Methodology, Validation, Writing – Review & Editing Hayder A Al-Aubaidy Roles: Conceptualization, Project Administration, Resources, Validation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information The author(s) declared that no grants were involved in supporting this work. Article Versions (4) version 4 Revised Published: 14 May 2025, 12:339 https://doi.org/10.12688/f1000research.130682.4 version 3 Revised Published: 07 Apr 2025, 12:339 https://doi.org/10.12688/f1000research.130682.3 version 2 Revised Published: 20 Sep 2023, 12:339 https://doi.org/10.12688/f1000research.130682.2 version 1 Published: 27 Mar 2023, 12:339 https://doi.org/10.12688/f1000research.130682.1 Copyright © 2025 Sahib HH et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Sahib HH, Mohammad BI, Hadi NR et al. Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.12688/f1000research.130682.4 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 4 VERSION 4 PUBLISHED 14 May 2025 Revised Views 0 Cite How to cite this report: Orecchioni M. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.181862.r393700 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v4#referee-response-393700 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 21 Aug 2025 Marco Orecchioni , Immunology Center of Georgia, Augusta University, Augusta, Georgia, USA Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.181862.r393700 This revised manuscript by Sahib et al. highlights the role of pterostilbene and sitagliptin supplementation in mitigating the progression of atherosclerosis. Overall, the study is of interest; however, some points still require clarification. The scope ... Continue reading READ ALL This revised manuscript by Sahib et al. highlights the role of pterostilbene and sitagliptin supplementation in mitigating the progression of atherosclerosis. Overall, the study is of interest; however, some points still require clarification. The scope statement claims to investigate the combined effects of pterostilbene and sitagliptin, yet the data address only the individual compounds. To evaluate synergy, an additional group should be included in which rats receive both compounds simultaneously. Control rats should also receive each compound while on a standard (non-atherogenic) diet to determine the supplements’ effects under steady-state conditions (especially on lipid content). This should be commented. The authors did not examine the expression of key pro-inflammatory markers (e.g., TNF-α, IL-1β, IL-6, IL-10, TGF-β). Including these data would provide valuable mechanistic insight. Because pterostilbene and sitagliptin affect cholesterol levels, rat body weights after treatment should be reported. The observed reduction in atherosclerosis progression may stem from improved lipid profiles rather than a direct anti-inflammatory effect; this distinction should be discussed. Are complete blood counts comparable across groups? Please provide these data or clarify. A more thorough plaque analysis is warranted. For example, does supplementation alter macrophage content or plaque stability? As a minor, the word "regression" should be used carefully since the data presented do not show regression, but rather a slower progression of the disease. Please adjust. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Macrophage biology, atherosclerosis, GPCRs, Olfactory receptors I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Orecchioni M. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.181862.r393700 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v4#referee-response-393700 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Leong XF. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.181862.r384829 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v4#referee-response-384829 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 02 Jul 2025 Xin-Fang Leong , Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Approved VIEWS 0 https://doi.org/10.5256/f1000research.181862.r384829 No further comments. The manuscript ... Continue reading READ ALL No further comments. The manuscript is okay to be indexed. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Pharmacology, cardiovascular system, animal study I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Leong XF. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.181862.r384829 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v4#referee-response-384829 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Milanovic JG. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.181862.r384830 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v4#referee-response-384830 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 28 May 2025 Jelica Grujic Milanovic , University of Belgrade, Belgrade, Serbia Approved VIEWS 0 https://doi.org/10.5256/f1000research.181862.r384830 I have ... Continue reading READ ALL I have no comment Competing Interests: No competing interests were disclosed. Reviewer Expertise: polyphenols, resveratrol, oxidative stress, hyperlipidaemia, hypertension I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Milanovic JG. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.181862.r384830 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v4#referee-response-384830 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 3 VERSION 3 PUBLISHED 07 Apr 2025 Revised Views 0 Cite How to cite this report: Leong XF. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.178031.r376390 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v3#referee-response-376390 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 08 May 2025 Xin-Fang Leong , Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.178031.r376390 The revised manuscript has shown marked improvement in terms of clarity, coherence, and overall quality. It is now well-written and considerably easier to understand. The authors have clearly addressed the previous comments, and the manuscript is much more compelling as ... Continue reading READ ALL The revised manuscript has shown marked improvement in terms of clarity, coherence, and overall quality. It is now well-written and considerably easier to understand. The authors have clearly addressed the previous comments, and the manuscript is much more compelling as a result. However, there are still some issues that need to be addressed before final acceptance. Firstly, there is a discrepancy (reduction vs. increase) between the mRNA expression findings for sitagliptin and pterostilbene reported in the abstract and those presented in the results section, particularly in the Figure 4. Please ensure these are consistent throughout the manuscript. Secondly, the statement regarding HDL levels requires clarification, as the current data indicate that HDL levels were increased in both the supplemented groups and the normal control group compared to the atherogenic diet group—this should be clearly reflected in the results/Table 1 and discussion. Thirdly, while H&E staining images have been provided, it would strengthen the histological analysis if Oil Red O staining images were also included, if available. Finally, the methodology section mentions that "PCR products were then separated on a 1% agarose gel, visualized, and photographed under ultraviolet light," but this data appears to be missing from both the manuscript and the supplementary materials. Please ensure that this data is either included or the statement is revised accordingly. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Pharmacology, cardiovascular system, animal study I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Leong XF. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.178031.r376390 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v3#referee-response-376390 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 22 Jul 2025 Hayder Al-Aubaidy , Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, 3086, Australia 22 Jul 2025 Author Response Dear Dr Leong, Thank you very much for your review. Please see below our response: Reviewer (Dr. Xin-Fang Leong) Comments: The revised manuscript has shown marked improvement in ... Continue reading Dear Dr Leong, Thank you very much for your review. Please see below our response: Reviewer (Dr. Xin-Fang Leong) Comments: The revised manuscript has shown marked improvement in terms of clarity, coherence, and overall quality. It is now well-written and considerably easier to understand. The authors have clearly addressed the previous comments, and the manuscript is much more compelling as a result. However, there are still some issues that need to be addressed before final acceptance. Firstly , there is a discrepancy (reduction vs. increase) between the mRNA expression findings for sitagliptin and pterostilbene reported in the abstract and those presented in the results section, particularly in the Figure 4. Please ensure these are consistent throughout the manuscript. Response: We thank the reviewer for highlighting this inconsistency. We have carefully reviewed the data and confirmed that sitagliptin increased AMPK expression, as shown in Figure 4. Accordingly, we have revised the abstract to accurately reflect this finding. The sentence now reads: “Pterostilbene supplementation induced a significant reduction in tissue expression of PI3K and AKT, while sitagliptin induced a significant increase in 5’ adenosine monophosphate-activated protein kinase (AMPK) levels.” This correction ensures consistency across the abstract, results, and figures. Secondly , the statement regarding HDL levels requires clarification, as the current data indicate that HDL levels were increased in both the supplemented groups and the normal control group compared to the atherogenic diet group—this should be clearly reflected in the results/Table 1 and discussion. Response: We appreciate the reviewer’s careful examination of the lipid profile data. Upon reviewing Table 1, we confirm that HDL levels were actually higher in the atherogenic control group than in the pterostilbene and sitagliptin groups . To address this, we have revised the relevant sections as follows: In the Results , we state: “HDL levels were significantly reduced in the PT and ST groups compared to the AC group (P<0.01), indicating that neither treatment restored HDL to normal levels.” In the Discussion , the paragraph has been updated to acknowledge the lower HDL levels in the treatment groups, and a note is added that this unexpected result may be due to compound-specific effects on lipid metabolism. These adjustments ensure the data and narrative are aligned. Thirdly, while H&E staining images have been provided, it would strengthen the histological analysis if Oil Red O staining images were also included, if available. Response: We thank the reviewer for this suggestion. Although Oil Red O staining was initiated, the image analysis and quantification were not complete at the time of this revision. As a result, we have removed all references to Oil Red O staining from the manuscript to avoid presenting incomplete data. We plan to include these findings in a future update or follow-up publication once the analysis is finalised. Finally, the methodology section mentions that "PCR products were then separated on a 1% agarose gel, visualized, and photographed under ultraviolet light," but this data appears to be missing from both the manuscript and the supplementary materials. Please ensure that this data is either included or the statement is revised accordingly. Response: We appreciate the reviewer’s attention to this detail. As gel images were not prepared for inclusion in the current version, we have removed this statement to avoid confusion. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards Hayder Al-Aubaidy On behalf of all authors for this manuscript Dear Dr Leong, Thank you very much for your review. Please see below our response: Reviewer (Dr. Xin-Fang Leong) Comments: The revised manuscript has shown marked improvement in terms of clarity, coherence, and overall quality. It is now well-written and considerably easier to understand. The authors have clearly addressed the previous comments, and the manuscript is much more compelling as a result. However, there are still some issues that need to be addressed before final acceptance. Firstly , there is a discrepancy (reduction vs. increase) between the mRNA expression findings for sitagliptin and pterostilbene reported in the abstract and those presented in the results section, particularly in the Figure 4. Please ensure these are consistent throughout the manuscript. Response: We thank the reviewer for highlighting this inconsistency. We have carefully reviewed the data and confirmed that sitagliptin increased AMPK expression, as shown in Figure 4. Accordingly, we have revised the abstract to accurately reflect this finding. The sentence now reads: “Pterostilbene supplementation induced a significant reduction in tissue expression of PI3K and AKT, while sitagliptin induced a significant increase in 5’ adenosine monophosphate-activated protein kinase (AMPK) levels.” This correction ensures consistency across the abstract, results, and figures. Secondly , the statement regarding HDL levels requires clarification, as the current data indicate that HDL levels were increased in both the supplemented groups and the normal control group compared to the atherogenic diet group—this should be clearly reflected in the results/Table 1 and discussion. Response: We appreciate the reviewer’s careful examination of the lipid profile data. Upon reviewing Table 1, we confirm that HDL levels were actually higher in the atherogenic control group than in the pterostilbene and sitagliptin groups . To address this, we have revised the relevant sections as follows: In the Results , we state: “HDL levels were significantly reduced in the PT and ST groups compared to the AC group (P<0.01), indicating that neither treatment restored HDL to normal levels.” In the Discussion , the paragraph has been updated to acknowledge the lower HDL levels in the treatment groups, and a note is added that this unexpected result may be due to compound-specific effects on lipid metabolism. These adjustments ensure the data and narrative are aligned. Thirdly, while H&E staining images have been provided, it would strengthen the histological analysis if Oil Red O staining images were also included, if available. Response: We thank the reviewer for this suggestion. Although Oil Red O staining was initiated, the image analysis and quantification were not complete at the time of this revision. As a result, we have removed all references to Oil Red O staining from the manuscript to avoid presenting incomplete data. We plan to include these findings in a future update or follow-up publication once the analysis is finalised. Finally, the methodology section mentions that "PCR products were then separated on a 1% agarose gel, visualized, and photographed under ultraviolet light," but this data appears to be missing from both the manuscript and the supplementary materials. Please ensure that this data is either included or the statement is revised accordingly. Response: We appreciate the reviewer’s attention to this detail. As gel images were not prepared for inclusion in the current version, we have removed this statement to avoid confusion. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards Hayder Al-Aubaidy On behalf of all authors for this manuscript Competing Interests: Nothing to declare. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 22 Jul 2025 Hayder Al-Aubaidy , Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, 3086, Australia 22 Jul 2025 Author Response Dear Dr Leong, Thank you very much for your review. Please see below our response: Reviewer (Dr. Xin-Fang Leong) Comments: The revised manuscript has shown marked improvement in ... Continue reading Dear Dr Leong, Thank you very much for your review. Please see below our response: Reviewer (Dr. Xin-Fang Leong) Comments: The revised manuscript has shown marked improvement in terms of clarity, coherence, and overall quality. It is now well-written and considerably easier to understand. The authors have clearly addressed the previous comments, and the manuscript is much more compelling as a result. However, there are still some issues that need to be addressed before final acceptance. Firstly , there is a discrepancy (reduction vs. increase) between the mRNA expression findings for sitagliptin and pterostilbene reported in the abstract and those presented in the results section, particularly in the Figure 4. Please ensure these are consistent throughout the manuscript. Response: We thank the reviewer for highlighting this inconsistency. We have carefully reviewed the data and confirmed that sitagliptin increased AMPK expression, as shown in Figure 4. Accordingly, we have revised the abstract to accurately reflect this finding. The sentence now reads: “Pterostilbene supplementation induced a significant reduction in tissue expression of PI3K and AKT, while sitagliptin induced a significant increase in 5’ adenosine monophosphate-activated protein kinase (AMPK) levels.” This correction ensures consistency across the abstract, results, and figures. Secondly , the statement regarding HDL levels requires clarification, as the current data indicate that HDL levels were increased in both the supplemented groups and the normal control group compared to the atherogenic diet group—this should be clearly reflected in the results/Table 1 and discussion. Response: We appreciate the reviewer’s careful examination of the lipid profile data. Upon reviewing Table 1, we confirm that HDL levels were actually higher in the atherogenic control group than in the pterostilbene and sitagliptin groups . To address this, we have revised the relevant sections as follows: In the Results , we state: “HDL levels were significantly reduced in the PT and ST groups compared to the AC group (P<0.01), indicating that neither treatment restored HDL to normal levels.” In the Discussion , the paragraph has been updated to acknowledge the lower HDL levels in the treatment groups, and a note is added that this unexpected result may be due to compound-specific effects on lipid metabolism. These adjustments ensure the data and narrative are aligned. Thirdly, while H&E staining images have been provided, it would strengthen the histological analysis if Oil Red O staining images were also included, if available. Response: We thank the reviewer for this suggestion. Although Oil Red O staining was initiated, the image analysis and quantification were not complete at the time of this revision. As a result, we have removed all references to Oil Red O staining from the manuscript to avoid presenting incomplete data. We plan to include these findings in a future update or follow-up publication once the analysis is finalised. Finally, the methodology section mentions that "PCR products were then separated on a 1% agarose gel, visualized, and photographed under ultraviolet light," but this data appears to be missing from both the manuscript and the supplementary materials. Please ensure that this data is either included or the statement is revised accordingly. Response: We appreciate the reviewer’s attention to this detail. As gel images were not prepared for inclusion in the current version, we have removed this statement to avoid confusion. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards Hayder Al-Aubaidy On behalf of all authors for this manuscript Dear Dr Leong, Thank you very much for your review. Please see below our response: Reviewer (Dr. Xin-Fang Leong) Comments: The revised manuscript has shown marked improvement in terms of clarity, coherence, and overall quality. It is now well-written and considerably easier to understand. The authors have clearly addressed the previous comments, and the manuscript is much more compelling as a result. However, there are still some issues that need to be addressed before final acceptance. Firstly , there is a discrepancy (reduction vs. increase) between the mRNA expression findings for sitagliptin and pterostilbene reported in the abstract and those presented in the results section, particularly in the Figure 4. Please ensure these are consistent throughout the manuscript. Response: We thank the reviewer for highlighting this inconsistency. We have carefully reviewed the data and confirmed that sitagliptin increased AMPK expression, as shown in Figure 4. Accordingly, we have revised the abstract to accurately reflect this finding. The sentence now reads: “Pterostilbene supplementation induced a significant reduction in tissue expression of PI3K and AKT, while sitagliptin induced a significant increase in 5’ adenosine monophosphate-activated protein kinase (AMPK) levels.” This correction ensures consistency across the abstract, results, and figures. Secondly , the statement regarding HDL levels requires clarification, as the current data indicate that HDL levels were increased in both the supplemented groups and the normal control group compared to the atherogenic diet group—this should be clearly reflected in the results/Table 1 and discussion. Response: We appreciate the reviewer’s careful examination of the lipid profile data. Upon reviewing Table 1, we confirm that HDL levels were actually higher in the atherogenic control group than in the pterostilbene and sitagliptin groups . To address this, we have revised the relevant sections as follows: In the Results , we state: “HDL levels were significantly reduced in the PT and ST groups compared to the AC group (P<0.01), indicating that neither treatment restored HDL to normal levels.” In the Discussion , the paragraph has been updated to acknowledge the lower HDL levels in the treatment groups, and a note is added that this unexpected result may be due to compound-specific effects on lipid metabolism. These adjustments ensure the data and narrative are aligned. Thirdly, while H&E staining images have been provided, it would strengthen the histological analysis if Oil Red O staining images were also included, if available. Response: We thank the reviewer for this suggestion. Although Oil Red O staining was initiated, the image analysis and quantification were not complete at the time of this revision. As a result, we have removed all references to Oil Red O staining from the manuscript to avoid presenting incomplete data. We plan to include these findings in a future update or follow-up publication once the analysis is finalised. Finally, the methodology section mentions that "PCR products were then separated on a 1% agarose gel, visualized, and photographed under ultraviolet light," but this data appears to be missing from both the manuscript and the supplementary materials. Please ensure that this data is either included or the statement is revised accordingly. Response: We appreciate the reviewer’s attention to this detail. As gel images were not prepared for inclusion in the current version, we have removed this statement to avoid confusion. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards Hayder Al-Aubaidy On behalf of all authors for this manuscript Competing Interests: Nothing to declare. Close Report a concern COMMENT ON THIS REPORT Version 2 VERSION 2 PUBLISHED 20 Sep 2023 Revised Views 0 Cite How to cite this report: Leong XF. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.156125.r358416 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v2#referee-response-358416 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 05 Feb 2025 Xin-Fang Leong , Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.156125.r358416 Introduction: 1. The introduction mentions that "previous studies have illustrated the action of inflammation and oxidative damage in atherogenic plaques progression," but it does not specify any specific studies or key findings that have directly contributed to this knowledge. ... Continue reading READ ALL Introduction: 1. The introduction mentions that "previous studies have illustrated the action of inflammation and oxidative damage in atherogenic plaques progression," but it does not specify any specific studies or key findings that have directly contributed to this knowledge. 2. The introduction could better highlight how the current study fills a gap in the existing literature. What makes pterostilbene and sitagliptin particularly interesting for atherosclerosis? How does this research differ from prior studies on these compounds? 3. The writing sometimes shifts abruptly between concepts (e.g., from the role of mTOR in autophagy to the effects of pterostilbene). To improve coherence, consider dividing the introduction into clearly defined subsections, such as "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents," etc. Materials & Methods: 1. Provide more details on the composition of the atherogenic diet -The diet was a traditional chow with 2% cholesterol? 2. Expand on the rationale for sample size and the method of calculation for statistical power. Why 26 male and 54 female rabbits? 3. Justify the chosen single dosage for PT and ST and the feeding duration for 8 weeks. 4. Detail how autophagy and RT-PCR measurements will be quantified and analyzed. a) RT-PCR protocol is described, the specific genes of interest (e.g., PI3K, AKT, AMPK, mTOR) are listed but without any context about their relevance to atherosclerosis. Information about housekeeping genes used for normalization should be included. b) The use of LC3B as a marker of autophagy is appropriate, but a brief explanation of how this marker will be measured and explaining why LC3B was chosen as a representative marker would enhance the depth of rationale for the study. 5. Provide more information on the histopathological examination, including scoring criteria and staining methods (the scoring criteria should not be placed in the Results section). 6. Kruskal-Wallis or Mann-Whitney for histopathological grading? Any comparison done between ST and PT? Results: 1. To enhance clarity and avoid redundancy, do refrain from repeating the same data in the text, table, and figure within the Results section. Each element should serve a distinct purpose: the text can summarize key findings, while tables and figures provide visual or detailed data, ensuring they complement each other without overlap. For example: level of F2-isoprostane (data) can be found in body text as well as in the Table and Figure. Same goes to the results for lipid profile. For LC3B - Figure 3 can be removed to avoid redundancy. 2. Histopathological examination: The progression of the lesion should be demonstrated by the AC group, while the improvement of the lesion is more effectively represented by the PT or ST groups. Discussion: 1. Refine interpretations of molecular mechanisms: Emphasize that some of the proposed mechanisms are hypotheses based on indirect evidence and suggest future experimental validation. 2. Provide more detailed analysis of the clinical relevance of the lipid profile changes, including comparison to existing treatments. 3. The role of autophagy in atherosclerosis is discussed, but the potential dangers of excessive autophagy are not considered. In some contexts, autophagy may contribute to cell death, and an overactive autophagic response could potentially exacerbate tissue damage. 4. More thoroughly explain how sitagliptin activates AMPK and its role in atherosclerosis beyond glucose regulation. 5. Address study limitations in more detail. 6. Specific future research suggestions: Outline clear next steps for validating and translating these findings into clinical practice (e.g. how could pterostilbene or sitagliptin be optimized for clinical use). Conclusion: 1. The conclusion could be more cautious in attributing the observed effects solely to the PI3K/Akt/mTOR and AMPK/mTOR pathways. Incorporating phrases like "potentially through" or "likely due to" would make the conclusion more nuanced and scientifically precise. 2. Acknowledging the study’s limitations in the conclusion would be beneficial. For example, noting that the results are based on animal models and that further studies, particularly human clinical trials, are necessary would provide a more balanced perspective. 3. The conclusion does not address the potential implications of these findings for human clinical practice. 4. Considering the positive effects of both compounds, the conclusion could briefly explore the potential for combination therapy, highlighting its possible therapeutic advantages. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Pharmacology, cardiovascular system, animal study I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Leong XF. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.156125.r358416 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v2#referee-response-358416 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 07 Apr 2025 Hayder Al-Aubaidy , Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, 3086, Australia 07 Apr 2025 Author Response Dear Dr. Xin-Fang Leong (Reviewer #2): Thank you very much for your highly valuable input. Please see below our response to your comments, highlighting the main changes we ... Continue reading Dear Dr. Xin-Fang Leong (Reviewer #2): Thank you very much for your highly valuable input. Please see below our response to your comments, highlighting the main changes we implemented in the manuscript, version 3: Introduction: 1. The introduction mentions that "previous studies have illustrated the action of inflammation and oxidative damage in atherogenic plaques progression," but it does not specify any specific studies or key findings that have directly contributed to this knowledge. Thank you for pointing this out. We have revised the introduction to specify key studies that have directly contributed to the understanding of inflammation and oxidative damage in atherogenic plaque progression. For example, Libby et al. (2002) highlighted the role of IL-6 and TNF-α in promoting plaque instability, while Witold et al. (2017) demonstrated the involvement of oxidative stress in endothelial dysfunction and foam cell formation. 2. The introduction could better highlight how the current study fills a gap in the existing literature. What makes pterostilbene and sitagliptin particularly interesting for atherosclerosis? How does this research differ from prior studies on these compounds? We appreciate this suggestion. The revised introduction now explicitly outlines the gap in existing literature, emphasising the limited data on the combined effects of pterostilbene and sitagliptin on atherosclerosis. We have elaborated on their potential synergistic effects, which differ from prior studies focusing solely on their individual impacts. 3. The writing sometimes shifts abruptly between concepts (e.g., from the role of mTOR in autophagy to the effects of pterostilbene). To improve coherence, consider dividing the introduction into clearly defined subsections, such as "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents," etc. To enhance clarity, we have reorganised the introduction into clearly defined subsections: "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents." This restructuring ensures a smoother transition between concepts. Materials & Methods: 1. Provide more details on the composition of the atherogenic diet -The diet was a traditional chow with 2% cholesterol? The manuscript now specifies that the atherogenic diet consisted of traditional chow supplemented with 2% cholesterol, following established protocols for inducing atherosclerosis in animal models. 2. Expand on the rationale for sample size and the method of calculation for statistical power. Why 26 male and 54 female rabbits? We have provided details on the power analysis conducted to determine the sample size, which offers over 85% power to detect significant differences. The unequal male-to-female ratio was due to availability constraints while ensuring both sexes were represented. 3. Justify the chosen single dosage for PT and ST and the feeding duration for 8 weeks. The dosages for pterostilbene (10 mg/kg) and sitagliptin (12 mg/kg) were based on prior studies demonstrating their efficacy without adverse effects. The eight-week duration aligns with protocols sufficient for the development of atherosclerotic changes and therapeutic assessment. 4. Detail how autophagy and RT-PCR measurements will be quantified and analyzed. a) RT-PCR protocol is described, the specific genes of interest (e.g., PI3K, AKT, AMPK, mTOR) are listed but without any context about their relevance to atherosclerosis. Information about housekeeping genes used for normalization should be included. We have elaborated on the relevance of PI3K, AKT, AMPK, and mTOR to atherosclerosis and included GAPDH as the housekeeping gene for normalisation. b) The use of LC3B as a marker of autophagy is appropriate, but a brief explanation of how this marker will be measured and explaining why LC3B was chosen as a representative marker would enhance the depth of rationale for the study. The rationale for selecting LC3B as a marker of autophagy is now explained in detail, highlighting its role in autophagosome formation and relevance in vascular studies. 5. Provide more information on the histopathological examination, including scoring criteria and staining methods (the scoring criteria should not be placed in the Results section). We have included comprehensive information on the scoring criteria and staining methods, which were previously only partially described. 6. Kruskal-Wallis or Mann-Whitney for histopathological grading? Any comparison done between ST and PT? The Kruskal-Wallis test was used for overall comparisons, with Mann-Whitney U tests for pairwise comparisons, including between the PT and ST groups. Results: 1. To enhance clarity and avoid redundancy, do refrain from repeating the same data in the text, table, and figure within the Results section. Each element should serve a distinct purpose: the text can summarize key findings, while tables and figures provide visual or detailed data, ensuring they complement each other without overlap. For example: level of F2-isoprostane (data) can be found in body text as well as in the Table and Figure. Same goes to the results for lipid profile. For LC3B - Figure 3 can be removed to avoid redundancy. We have streamlined the Results section to avoid redundancy, summarising key findings in the text while using tables and figures to present detailed data. 2. Histopathological examination: The progression of the lesion should be demonstrated by the AC group, while the improvement of the lesion is more effectively represented by the PT or ST groups. The revised section now better illustrates lesion progression in the AC group and improvements in the PT and ST groups, with clear histological descriptions supported by representative images. Discussion: 1. Refine interpretations of molecular mechanisms: Emphasize that some of the proposed mechanisms are hypotheses based on indirect evidence and suggest future experimental validation. We have clarified that the proposed mechanisms are hypotheses based on indirect evidence, suggesting future studies for experimental validation. 2. Provide more detailed analysis of the clinical relevance of the lipid profile changes, including comparison to existing treatments. The discussion now includes a comparison to existing treatments, highlighting potential complementary roles for pterostilbene and sitagliptin. 3. The role of autophagy in atherosclerosis is discussed, but the potential dangers of excessive autophagy are not considered. In some contexts, autophagy may contribute to cell death, and an overactive autophagic response could potentially exacerbate tissue damage. We have acknowledged the potential risks of excessive autophagy, which could lead to cell death and exacerbate atherosclerosis in certain contexts. 4. More thoroughly explain how sitagliptin activates AMPK and its role in atherosclerosis beyond glucose regulation. The revised discussion provides a more thorough explanation of how sitagliptin activates AMPK and its broader role in vascular health beyond glucose regulation. 5. Address study limitations in more detail. Additional limitations are discussed, including the reliance on animal models and potential sensitivity issues with histological methods. 6. Specific future research suggestions: Outline clear next steps for validating and translating these findings into clinical practice (e.g. how could pterostilbene or sitagliptin be optimized for clinical use). We have outlined specific next steps for clinical translation, such as optimising dosing, exploring combination therapies, and conducting human trials. Conclusion: 1. The conclusion could be more cautious in attributing the observed effects solely to the PI3K/Akt/mTOR and AMPK/mTOR pathways. Incorporating phrases like "potentially through" or "likely due to" would make the conclusion more nuanced and scientifically precise. The conclusion now uses terms like "potentially" and "likely" to present a more nuanced interpretation of the mechanisms involved. 2. Acknowledging the study’s limitations in the conclusion would be beneficial. For example, noting that the results are based on animal models and that further studies, particularly human clinical trials, are necessary would provide a more balanced perspective. We have explicitly noted that the findings are based on animal models and highlighted the need for human clinical trials. 3. The conclusion does not address the potential implications of these findings for human clinical practice. The potential implications for human atherosclerosis management are discussed, considering both agents as adjunctive therapies. 4. Considering the positive effects of both compounds, the conclusion could briefly explore the potential for combination therapy, highlighting its possible therapeutic advantages. We have added a brief discussion on the potential benefits of combination therapy, considering the complementary mechanisms of pterostilbene and sitagliptin. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards, Hayder Al-Aubaidy On behalf of all authors for this manuscript Dear Dr. Xin-Fang Leong (Reviewer #2): Thank you very much for your highly valuable input. Please see below our response to your comments, highlighting the main changes we implemented in the manuscript, version 3: Introduction: 1. The introduction mentions that "previous studies have illustrated the action of inflammation and oxidative damage in atherogenic plaques progression," but it does not specify any specific studies or key findings that have directly contributed to this knowledge. Thank you for pointing this out. We have revised the introduction to specify key studies that have directly contributed to the understanding of inflammation and oxidative damage in atherogenic plaque progression. For example, Libby et al. (2002) highlighted the role of IL-6 and TNF-α in promoting plaque instability, while Witold et al. (2017) demonstrated the involvement of oxidative stress in endothelial dysfunction and foam cell formation. 2. The introduction could better highlight how the current study fills a gap in the existing literature. What makes pterostilbene and sitagliptin particularly interesting for atherosclerosis? How does this research differ from prior studies on these compounds? We appreciate this suggestion. The revised introduction now explicitly outlines the gap in existing literature, emphasising the limited data on the combined effects of pterostilbene and sitagliptin on atherosclerosis. We have elaborated on their potential synergistic effects, which differ from prior studies focusing solely on their individual impacts. 3. The writing sometimes shifts abruptly between concepts (e.g., from the role of mTOR in autophagy to the effects of pterostilbene). To improve coherence, consider dividing the introduction into clearly defined subsections, such as "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents," etc. To enhance clarity, we have reorganised the introduction into clearly defined subsections: "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents." This restructuring ensures a smoother transition between concepts. Materials & Methods: 1. Provide more details on the composition of the atherogenic diet -The diet was a traditional chow with 2% cholesterol? The manuscript now specifies that the atherogenic diet consisted of traditional chow supplemented with 2% cholesterol, following established protocols for inducing atherosclerosis in animal models. 2. Expand on the rationale for sample size and the method of calculation for statistical power. Why 26 male and 54 female rabbits? We have provided details on the power analysis conducted to determine the sample size, which offers over 85% power to detect significant differences. The unequal male-to-female ratio was due to availability constraints while ensuring both sexes were represented. 3. Justify the chosen single dosage for PT and ST and the feeding duration for 8 weeks. The dosages for pterostilbene (10 mg/kg) and sitagliptin (12 mg/kg) were based on prior studies demonstrating their efficacy without adverse effects. The eight-week duration aligns with protocols sufficient for the development of atherosclerotic changes and therapeutic assessment. 4. Detail how autophagy and RT-PCR measurements will be quantified and analyzed. a) RT-PCR protocol is described, the specific genes of interest (e.g., PI3K, AKT, AMPK, mTOR) are listed but without any context about their relevance to atherosclerosis. Information about housekeeping genes used for normalization should be included. We have elaborated on the relevance of PI3K, AKT, AMPK, and mTOR to atherosclerosis and included GAPDH as the housekeeping gene for normalisation. b) The use of LC3B as a marker of autophagy is appropriate, but a brief explanation of how this marker will be measured and explaining why LC3B was chosen as a representative marker would enhance the depth of rationale for the study. The rationale for selecting LC3B as a marker of autophagy is now explained in detail, highlighting its role in autophagosome formation and relevance in vascular studies. 5. Provide more information on the histopathological examination, including scoring criteria and staining methods (the scoring criteria should not be placed in the Results section). We have included comprehensive information on the scoring criteria and staining methods, which were previously only partially described. 6. Kruskal-Wallis or Mann-Whitney for histopathological grading? Any comparison done between ST and PT? The Kruskal-Wallis test was used for overall comparisons, with Mann-Whitney U tests for pairwise comparisons, including between the PT and ST groups. Results: 1. To enhance clarity and avoid redundancy, do refrain from repeating the same data in the text, table, and figure within the Results section. Each element should serve a distinct purpose: the text can summarize key findings, while tables and figures provide visual or detailed data, ensuring they complement each other without overlap. For example: level of F2-isoprostane (data) can be found in body text as well as in the Table and Figure. Same goes to the results for lipid profile. For LC3B - Figure 3 can be removed to avoid redundancy. We have streamlined the Results section to avoid redundancy, summarising key findings in the text while using tables and figures to present detailed data. 2. Histopathological examination: The progression of the lesion should be demonstrated by the AC group, while the improvement of the lesion is more effectively represented by the PT or ST groups. The revised section now better illustrates lesion progression in the AC group and improvements in the PT and ST groups, with clear histological descriptions supported by representative images. Discussion: 1. Refine interpretations of molecular mechanisms: Emphasize that some of the proposed mechanisms are hypotheses based on indirect evidence and suggest future experimental validation. We have clarified that the proposed mechanisms are hypotheses based on indirect evidence, suggesting future studies for experimental validation. 2. Provide more detailed analysis of the clinical relevance of the lipid profile changes, including comparison to existing treatments. The discussion now includes a comparison to existing treatments, highlighting potential complementary roles for pterostilbene and sitagliptin. 3. The role of autophagy in atherosclerosis is discussed, but the potential dangers of excessive autophagy are not considered. In some contexts, autophagy may contribute to cell death, and an overactive autophagic response could potentially exacerbate tissue damage. We have acknowledged the potential risks of excessive autophagy, which could lead to cell death and exacerbate atherosclerosis in certain contexts. 4. More thoroughly explain how sitagliptin activates AMPK and its role in atherosclerosis beyond glucose regulation. The revised discussion provides a more thorough explanation of how sitagliptin activates AMPK and its broader role in vascular health beyond glucose regulation. 5. Address study limitations in more detail. Additional limitations are discussed, including the reliance on animal models and potential sensitivity issues with histological methods. 6. Specific future research suggestions: Outline clear next steps for validating and translating these findings into clinical practice (e.g. how could pterostilbene or sitagliptin be optimized for clinical use). We have outlined specific next steps for clinical translation, such as optimising dosing, exploring combination therapies, and conducting human trials. Conclusion: 1. The conclusion could be more cautious in attributing the observed effects solely to the PI3K/Akt/mTOR and AMPK/mTOR pathways. Incorporating phrases like "potentially through" or "likely due to" would make the conclusion more nuanced and scientifically precise. The conclusion now uses terms like "potentially" and "likely" to present a more nuanced interpretation of the mechanisms involved. 2. Acknowledging the study’s limitations in the conclusion would be beneficial. For example, noting that the results are based on animal models and that further studies, particularly human clinical trials, are necessary would provide a more balanced perspective. We have explicitly noted that the findings are based on animal models and highlighted the need for human clinical trials. 3. The conclusion does not address the potential implications of these findings for human clinical practice. The potential implications for human atherosclerosis management are discussed, considering both agents as adjunctive therapies. 4. Considering the positive effects of both compounds, the conclusion could briefly explore the potential for combination therapy, highlighting its possible therapeutic advantages. We have added a brief discussion on the potential benefits of combination therapy, considering the complementary mechanisms of pterostilbene and sitagliptin. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards, Hayder Al-Aubaidy On behalf of all authors for this manuscript Competing Interests: Nothing to disclose Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 07 Apr 2025 Hayder Al-Aubaidy , Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, 3086, Australia 07 Apr 2025 Author Response Dear Dr. Xin-Fang Leong (Reviewer #2): Thank you very much for your highly valuable input. Please see below our response to your comments, highlighting the main changes we ... Continue reading Dear Dr. Xin-Fang Leong (Reviewer #2): Thank you very much for your highly valuable input. Please see below our response to your comments, highlighting the main changes we implemented in the manuscript, version 3: Introduction: 1. The introduction mentions that "previous studies have illustrated the action of inflammation and oxidative damage in atherogenic plaques progression," but it does not specify any specific studies or key findings that have directly contributed to this knowledge. Thank you for pointing this out. We have revised the introduction to specify key studies that have directly contributed to the understanding of inflammation and oxidative damage in atherogenic plaque progression. For example, Libby et al. (2002) highlighted the role of IL-6 and TNF-α in promoting plaque instability, while Witold et al. (2017) demonstrated the involvement of oxidative stress in endothelial dysfunction and foam cell formation. 2. The introduction could better highlight how the current study fills a gap in the existing literature. What makes pterostilbene and sitagliptin particularly interesting for atherosclerosis? How does this research differ from prior studies on these compounds? We appreciate this suggestion. The revised introduction now explicitly outlines the gap in existing literature, emphasising the limited data on the combined effects of pterostilbene and sitagliptin on atherosclerosis. We have elaborated on their potential synergistic effects, which differ from prior studies focusing solely on their individual impacts. 3. The writing sometimes shifts abruptly between concepts (e.g., from the role of mTOR in autophagy to the effects of pterostilbene). To improve coherence, consider dividing the introduction into clearly defined subsections, such as "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents," etc. To enhance clarity, we have reorganised the introduction into clearly defined subsections: "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents." This restructuring ensures a smoother transition between concepts. Materials & Methods: 1. Provide more details on the composition of the atherogenic diet -The diet was a traditional chow with 2% cholesterol? The manuscript now specifies that the atherogenic diet consisted of traditional chow supplemented with 2% cholesterol, following established protocols for inducing atherosclerosis in animal models. 2. Expand on the rationale for sample size and the method of calculation for statistical power. Why 26 male and 54 female rabbits? We have provided details on the power analysis conducted to determine the sample size, which offers over 85% power to detect significant differences. The unequal male-to-female ratio was due to availability constraints while ensuring both sexes were represented. 3. Justify the chosen single dosage for PT and ST and the feeding duration for 8 weeks. The dosages for pterostilbene (10 mg/kg) and sitagliptin (12 mg/kg) were based on prior studies demonstrating their efficacy without adverse effects. The eight-week duration aligns with protocols sufficient for the development of atherosclerotic changes and therapeutic assessment. 4. Detail how autophagy and RT-PCR measurements will be quantified and analyzed. a) RT-PCR protocol is described, the specific genes of interest (e.g., PI3K, AKT, AMPK, mTOR) are listed but without any context about their relevance to atherosclerosis. Information about housekeeping genes used for normalization should be included. We have elaborated on the relevance of PI3K, AKT, AMPK, and mTOR to atherosclerosis and included GAPDH as the housekeeping gene for normalisation. b) The use of LC3B as a marker of autophagy is appropriate, but a brief explanation of how this marker will be measured and explaining why LC3B was chosen as a representative marker would enhance the depth of rationale for the study. The rationale for selecting LC3B as a marker of autophagy is now explained in detail, highlighting its role in autophagosome formation and relevance in vascular studies. 5. Provide more information on the histopathological examination, including scoring criteria and staining methods (the scoring criteria should not be placed in the Results section). We have included comprehensive information on the scoring criteria and staining methods, which were previously only partially described. 6. Kruskal-Wallis or Mann-Whitney for histopathological grading? Any comparison done between ST and PT? The Kruskal-Wallis test was used for overall comparisons, with Mann-Whitney U tests for pairwise comparisons, including between the PT and ST groups. Results: 1. To enhance clarity and avoid redundancy, do refrain from repeating the same data in the text, table, and figure within the Results section. Each element should serve a distinct purpose: the text can summarize key findings, while tables and figures provide visual or detailed data, ensuring they complement each other without overlap. For example: level of F2-isoprostane (data) can be found in body text as well as in the Table and Figure. Same goes to the results for lipid profile. For LC3B - Figure 3 can be removed to avoid redundancy. We have streamlined the Results section to avoid redundancy, summarising key findings in the text while using tables and figures to present detailed data. 2. Histopathological examination: The progression of the lesion should be demonstrated by the AC group, while the improvement of the lesion is more effectively represented by the PT or ST groups. The revised section now better illustrates lesion progression in the AC group and improvements in the PT and ST groups, with clear histological descriptions supported by representative images. Discussion: 1. Refine interpretations of molecular mechanisms: Emphasize that some of the proposed mechanisms are hypotheses based on indirect evidence and suggest future experimental validation. We have clarified that the proposed mechanisms are hypotheses based on indirect evidence, suggesting future studies for experimental validation. 2. Provide more detailed analysis of the clinical relevance of the lipid profile changes, including comparison to existing treatments. The discussion now includes a comparison to existing treatments, highlighting potential complementary roles for pterostilbene and sitagliptin. 3. The role of autophagy in atherosclerosis is discussed, but the potential dangers of excessive autophagy are not considered. In some contexts, autophagy may contribute to cell death, and an overactive autophagic response could potentially exacerbate tissue damage. We have acknowledged the potential risks of excessive autophagy, which could lead to cell death and exacerbate atherosclerosis in certain contexts. 4. More thoroughly explain how sitagliptin activates AMPK and its role in atherosclerosis beyond glucose regulation. The revised discussion provides a more thorough explanation of how sitagliptin activates AMPK and its broader role in vascular health beyond glucose regulation. 5. Address study limitations in more detail. Additional limitations are discussed, including the reliance on animal models and potential sensitivity issues with histological methods. 6. Specific future research suggestions: Outline clear next steps for validating and translating these findings into clinical practice (e.g. how could pterostilbene or sitagliptin be optimized for clinical use). We have outlined specific next steps for clinical translation, such as optimising dosing, exploring combination therapies, and conducting human trials. Conclusion: 1. The conclusion could be more cautious in attributing the observed effects solely to the PI3K/Akt/mTOR and AMPK/mTOR pathways. Incorporating phrases like "potentially through" or "likely due to" would make the conclusion more nuanced and scientifically precise. The conclusion now uses terms like "potentially" and "likely" to present a more nuanced interpretation of the mechanisms involved. 2. Acknowledging the study’s limitations in the conclusion would be beneficial. For example, noting that the results are based on animal models and that further studies, particularly human clinical trials, are necessary would provide a more balanced perspective. We have explicitly noted that the findings are based on animal models and highlighted the need for human clinical trials. 3. The conclusion does not address the potential implications of these findings for human clinical practice. The potential implications for human atherosclerosis management are discussed, considering both agents as adjunctive therapies. 4. Considering the positive effects of both compounds, the conclusion could briefly explore the potential for combination therapy, highlighting its possible therapeutic advantages. We have added a brief discussion on the potential benefits of combination therapy, considering the complementary mechanisms of pterostilbene and sitagliptin. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards, Hayder Al-Aubaidy On behalf of all authors for this manuscript Dear Dr. Xin-Fang Leong (Reviewer #2): Thank you very much for your highly valuable input. Please see below our response to your comments, highlighting the main changes we implemented in the manuscript, version 3: Introduction: 1. The introduction mentions that "previous studies have illustrated the action of inflammation and oxidative damage in atherogenic plaques progression," but it does not specify any specific studies or key findings that have directly contributed to this knowledge. Thank you for pointing this out. We have revised the introduction to specify key studies that have directly contributed to the understanding of inflammation and oxidative damage in atherogenic plaque progression. For example, Libby et al. (2002) highlighted the role of IL-6 and TNF-α in promoting plaque instability, while Witold et al. (2017) demonstrated the involvement of oxidative stress in endothelial dysfunction and foam cell formation. 2. The introduction could better highlight how the current study fills a gap in the existing literature. What makes pterostilbene and sitagliptin particularly interesting for atherosclerosis? How does this research differ from prior studies on these compounds? We appreciate this suggestion. The revised introduction now explicitly outlines the gap in existing literature, emphasising the limited data on the combined effects of pterostilbene and sitagliptin on atherosclerosis. We have elaborated on their potential synergistic effects, which differ from prior studies focusing solely on their individual impacts. 3. The writing sometimes shifts abruptly between concepts (e.g., from the role of mTOR in autophagy to the effects of pterostilbene). To improve coherence, consider dividing the introduction into clearly defined subsections, such as "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents," etc. To enhance clarity, we have reorganised the introduction into clearly defined subsections: "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents." This restructuring ensures a smoother transition between concepts. Materials & Methods: 1. Provide more details on the composition of the atherogenic diet -The diet was a traditional chow with 2% cholesterol? The manuscript now specifies that the atherogenic diet consisted of traditional chow supplemented with 2% cholesterol, following established protocols for inducing atherosclerosis in animal models. 2. Expand on the rationale for sample size and the method of calculation for statistical power. Why 26 male and 54 female rabbits? We have provided details on the power analysis conducted to determine the sample size, which offers over 85% power to detect significant differences. The unequal male-to-female ratio was due to availability constraints while ensuring both sexes were represented. 3. Justify the chosen single dosage for PT and ST and the feeding duration for 8 weeks. The dosages for pterostilbene (10 mg/kg) and sitagliptin (12 mg/kg) were based on prior studies demonstrating their efficacy without adverse effects. The eight-week duration aligns with protocols sufficient for the development of atherosclerotic changes and therapeutic assessment. 4. Detail how autophagy and RT-PCR measurements will be quantified and analyzed. a) RT-PCR protocol is described, the specific genes of interest (e.g., PI3K, AKT, AMPK, mTOR) are listed but without any context about their relevance to atherosclerosis. Information about housekeeping genes used for normalization should be included. We have elaborated on the relevance of PI3K, AKT, AMPK, and mTOR to atherosclerosis and included GAPDH as the housekeeping gene for normalisation. b) The use of LC3B as a marker of autophagy is appropriate, but a brief explanation of how this marker will be measured and explaining why LC3B was chosen as a representative marker would enhance the depth of rationale for the study. The rationale for selecting LC3B as a marker of autophagy is now explained in detail, highlighting its role in autophagosome formation and relevance in vascular studies. 5. Provide more information on the histopathological examination, including scoring criteria and staining methods (the scoring criteria should not be placed in the Results section). We have included comprehensive information on the scoring criteria and staining methods, which were previously only partially described. 6. Kruskal-Wallis or Mann-Whitney for histopathological grading? Any comparison done between ST and PT? The Kruskal-Wallis test was used for overall comparisons, with Mann-Whitney U tests for pairwise comparisons, including between the PT and ST groups. Results: 1. To enhance clarity and avoid redundancy, do refrain from repeating the same data in the text, table, and figure within the Results section. Each element should serve a distinct purpose: the text can summarize key findings, while tables and figures provide visual or detailed data, ensuring they complement each other without overlap. For example: level of F2-isoprostane (data) can be found in body text as well as in the Table and Figure. Same goes to the results for lipid profile. For LC3B - Figure 3 can be removed to avoid redundancy. We have streamlined the Results section to avoid redundancy, summarising key findings in the text while using tables and figures to present detailed data. 2. Histopathological examination: The progression of the lesion should be demonstrated by the AC group, while the improvement of the lesion is more effectively represented by the PT or ST groups. The revised section now better illustrates lesion progression in the AC group and improvements in the PT and ST groups, with clear histological descriptions supported by representative images. Discussion: 1. Refine interpretations of molecular mechanisms: Emphasize that some of the proposed mechanisms are hypotheses based on indirect evidence and suggest future experimental validation. We have clarified that the proposed mechanisms are hypotheses based on indirect evidence, suggesting future studies for experimental validation. 2. Provide more detailed analysis of the clinical relevance of the lipid profile changes, including comparison to existing treatments. The discussion now includes a comparison to existing treatments, highlighting potential complementary roles for pterostilbene and sitagliptin. 3. The role of autophagy in atherosclerosis is discussed, but the potential dangers of excessive autophagy are not considered. In some contexts, autophagy may contribute to cell death, and an overactive autophagic response could potentially exacerbate tissue damage. We have acknowledged the potential risks of excessive autophagy, which could lead to cell death and exacerbate atherosclerosis in certain contexts. 4. More thoroughly explain how sitagliptin activates AMPK and its role in atherosclerosis beyond glucose regulation. The revised discussion provides a more thorough explanation of how sitagliptin activates AMPK and its broader role in vascular health beyond glucose regulation. 5. Address study limitations in more detail. Additional limitations are discussed, including the reliance on animal models and potential sensitivity issues with histological methods. 6. Specific future research suggestions: Outline clear next steps for validating and translating these findings into clinical practice (e.g. how could pterostilbene or sitagliptin be optimized for clinical use). We have outlined specific next steps for clinical translation, such as optimising dosing, exploring combination therapies, and conducting human trials. Conclusion: 1. The conclusion could be more cautious in attributing the observed effects solely to the PI3K/Akt/mTOR and AMPK/mTOR pathways. Incorporating phrases like "potentially through" or "likely due to" would make the conclusion more nuanced and scientifically precise. The conclusion now uses terms like "potentially" and "likely" to present a more nuanced interpretation of the mechanisms involved. 2. Acknowledging the study’s limitations in the conclusion would be beneficial. For example, noting that the results are based on animal models and that further studies, particularly human clinical trials, are necessary would provide a more balanced perspective. We have explicitly noted that the findings are based on animal models and highlighted the need for human clinical trials. 3. The conclusion does not address the potential implications of these findings for human clinical practice. The potential implications for human atherosclerosis management are discussed, considering both agents as adjunctive therapies. 4. Considering the positive effects of both compounds, the conclusion could briefly explore the potential for combination therapy, highlighting its possible therapeutic advantages. We have added a brief discussion on the potential benefits of combination therapy, considering the complementary mechanisms of pterostilbene and sitagliptin. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards, Hayder Al-Aubaidy On behalf of all authors for this manuscript Competing Interests: Nothing to disclose Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Milanovic JG. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.156125.r208141 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v2#referee-response-208141 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 22 Sep 2023 Jelica Grujic Milanovic , University of Belgrade, Belgrade, Serbia Approved VIEWS 0 https://doi.org/10.5256/f1000research.156125.r208141 Authors well addressed my previous ... Continue reading READ ALL Authors well addressed my previous comments. The paper improved very much. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Oxidative stress, polyphenols, hypertension, cardiovascular diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Milanovic JG. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.156125.r208141 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v2#referee-response-208141 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 27 Mar 2023 Views 0 Cite How to cite this report: Milanovic JG. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.143454.r199143 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v1#referee-response-199143 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 14 Sep 2023 Jelica Grujic Milanovic , University of Belgrade, Belgrade, Serbia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.143454.r199143 The article by Sahib et al. has been clearly defined. The methodology applied is overall correct. The authors support their paper with recent references. However, the following changes to the main text are suggested: In ... Continue reading READ ALL The article by Sahib et al. has been clearly defined. The methodology applied is overall correct. The authors support their paper with recent references. However, the following changes to the main text are suggested: In the results, use the abbreviation TC instead of total cholesterol. In Figure 1 and Table 1 the authors presented the same results. The authors must improve this part of the results. In Figure 1, use the same colors for marking as a group as in Figure 1/4/5. Does the letter F in Figure 3 stand for the ST group? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: oxidative stress, polyphenols, hypertension, cardiovascular diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Milanovic JG. Reviewer Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.143454.r199143 ) The direct URL for this report is: https://f1000research.com/articles/12-339/v1#referee-response-199143 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 06 Oct 2023 Hayder Al-Aubaidy , Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, 3086, Australia 06 Oct 2023 Author Response Dear Professor Grujic, Many thanks for reviewing the manuscript and providing us with your comments. We confirm addressing all your comments in Version 2 of the manuscript. A summary of the ... Continue reading Dear Professor Grujic, Many thanks for reviewing the manuscript and providing us with your comments. We confirm addressing all your comments in Version 2 of the manuscript. A summary of the changes has been listed below: The article by Sahib et al. has been clearly defined. The methodology applied is overall correct. The authors support their paper with recent references. However, the following changes to the main text are suggested: In the results, use the abbreviation TC instead of total cholesterol. Response: Thank you for your comment. This has been fixed across the results section. In Figure 1 and Table 1 the authors presented the same results. The authors must improve this part of the results. Response: We have removed Figure 1 (serum lipid parameters) and kept Table 1 to avoid duplication of data. We have renamed the remaining figures accordingly. In Figure 1, use the same colors for marking as a group as in Figure 1/4/5. Response: Figure 1 – Serum lipid parameters has been removed from the manuscript as per the comment above. Does the letter F in Figure 3 stand for the ST group? Response: In this figure, letter F shows complicated atherosclerotic changes - hemorrhagic thrombus as seen in the atherogenic study cohort (no supplementations). The changes in the sitagliptin group are shown in letter B. Please refer to the figure legend for more details about the changes in the other study groups. Best Regards Prof. Hayder Al-Aubaidy On behalf of all authors for this manuscript Dear Professor Grujic, Many thanks for reviewing the manuscript and providing us with your comments. We confirm addressing all your comments in Version 2 of the manuscript. A summary of the changes has been listed below: The article by Sahib et al. has been clearly defined. The methodology applied is overall correct. The authors support their paper with recent references. However, the following changes to the main text are suggested: In the results, use the abbreviation TC instead of total cholesterol. Response: Thank you for your comment. This has been fixed across the results section. In Figure 1 and Table 1 the authors presented the same results. The authors must improve this part of the results. Response: We have removed Figure 1 (serum lipid parameters) and kept Table 1 to avoid duplication of data. We have renamed the remaining figures accordingly. In Figure 1, use the same colors for marking as a group as in Figure 1/4/5. Response: Figure 1 – Serum lipid parameters has been removed from the manuscript as per the comment above. Does the letter F in Figure 3 stand for the ST group? Response: In this figure, letter F shows complicated atherosclerotic changes - hemorrhagic thrombus as seen in the atherogenic study cohort (no supplementations). The changes in the sitagliptin group are shown in letter B. Please refer to the figure legend for more details about the changes in the other study groups. Best Regards Prof. Hayder Al-Aubaidy On behalf of all authors for this manuscript Competing Interests: Nothing to declare Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 06 Oct 2023 Hayder Al-Aubaidy , Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, 3086, Australia 06 Oct 2023 Author Response Dear Professor Grujic, Many thanks for reviewing the manuscript and providing us with your comments. We confirm addressing all your comments in Version 2 of the manuscript. A summary of the ... Continue reading Dear Professor Grujic, Many thanks for reviewing the manuscript and providing us with your comments. We confirm addressing all your comments in Version 2 of the manuscript. A summary of the changes has been listed below: The article by Sahib et al. has been clearly defined. The methodology applied is overall correct. The authors support their paper with recent references. However, the following changes to the main text are suggested: In the results, use the abbreviation TC instead of total cholesterol. Response: Thank you for your comment. This has been fixed across the results section. In Figure 1 and Table 1 the authors presented the same results. The authors must improve this part of the results. Response: We have removed Figure 1 (serum lipid parameters) and kept Table 1 to avoid duplication of data. We have renamed the remaining figures accordingly. In Figure 1, use the same colors for marking as a group as in Figure 1/4/5. Response: Figure 1 – Serum lipid parameters has been removed from the manuscript as per the comment above. Does the letter F in Figure 3 stand for the ST group? Response: In this figure, letter F shows complicated atherosclerotic changes - hemorrhagic thrombus as seen in the atherogenic study cohort (no supplementations). The changes in the sitagliptin group are shown in letter B. Please refer to the figure legend for more details about the changes in the other study groups. Best Regards Prof. Hayder Al-Aubaidy On behalf of all authors for this manuscript Dear Professor Grujic, Many thanks for reviewing the manuscript and providing us with your comments. We confirm addressing all your comments in Version 2 of the manuscript. A summary of the changes has been listed below: The article by Sahib et al. has been clearly defined. The methodology applied is overall correct. The authors support their paper with recent references. However, the following changes to the main text are suggested: In the results, use the abbreviation TC instead of total cholesterol. Response: Thank you for your comment. This has been fixed across the results section. In Figure 1 and Table 1 the authors presented the same results. The authors must improve this part of the results. Response: We have removed Figure 1 (serum lipid parameters) and kept Table 1 to avoid duplication of data. We have renamed the remaining figures accordingly. In Figure 1, use the same colors for marking as a group as in Figure 1/4/5. Response: Figure 1 – Serum lipid parameters has been removed from the manuscript as per the comment above. Does the letter F in Figure 3 stand for the ST group? Response: In this figure, letter F shows complicated atherosclerotic changes - hemorrhagic thrombus as seen in the atherogenic study cohort (no supplementations). The changes in the sitagliptin group are shown in letter B. Please refer to the figure legend for more details about the changes in the other study groups. Best Regards Prof. Hayder Al-Aubaidy On behalf of all authors for this manuscript Competing Interests: Nothing to declare Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 4 VERSION 4 PUBLISHED 27 Mar 2023 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 4 (revision) 14 May 25 read read read Version 3 (revision) 07 Apr 25 read Version 2 (revision) 20 Sep 23 read read Version 1 27 Mar 23 read Jelica Grujic Milanovic , University of Belgrade, Belgrade, Serbia Xin-Fang Leong , Universiti Kebangsaan Malaysia, Bangi, Malaysia Marco Orecchioni , Augusta University, Augusta, USA Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Orecchioni M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 21 Aug 2025 | for Version 4 Marco Orecchioni , Immunology Center of Georgia, Augusta University, Augusta, Georgia, USA 0 Views copyright © 2025 Orecchioni M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This revised manuscript by Sahib et al. highlights the role of pterostilbene and sitagliptin supplementation in mitigating the progression of atherosclerosis. Overall, the study is of interest; however, some points still require clarification. The scope statement claims to investigate the combined effects of pterostilbene and sitagliptin, yet the data address only the individual compounds. To evaluate synergy, an additional group should be included in which rats receive both compounds simultaneously. Control rats should also receive each compound while on a standard (non-atherogenic) diet to determine the supplements’ effects under steady-state conditions (especially on lipid content). This should be commented. The authors did not examine the expression of key pro-inflammatory markers (e.g., TNF-α, IL-1β, IL-6, IL-10, TGF-β). Including these data would provide valuable mechanistic insight. Because pterostilbene and sitagliptin affect cholesterol levels, rat body weights after treatment should be reported. The observed reduction in atherosclerosis progression may stem from improved lipid profiles rather than a direct anti-inflammatory effect; this distinction should be discussed. Are complete blood counts comparable across groups? Please provide these data or clarify. A more thorough plaque analysis is warranted. For example, does supplementation alter macrophage content or plaque stability? As a minor, the word "regression" should be used carefully since the data presented do not show regression, but rather a slower progression of the disease. Please adjust. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Macrophage biology, atherosclerosis, GPCRs, Olfactory receptors I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Orecchioni M. Peer Review Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.181862.r393700) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-339/v4#referee-response-393700 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Leong X. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 02 Jul 2025 | for Version 4 Xin-Fang Leong , Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia 0 Views copyright © 2025 Leong X. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions No further comments. The manuscript is okay to be indexed. Competing Interests No competing interests were disclosed. Reviewer Expertise Pharmacology, cardiovascular system, animal study I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Leong XF. Peer Review Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.181862.r384829) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-339/v4#referee-response-384829 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Milanovic J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 28 May 2025 | for Version 4 Jelica Grujic Milanovic , University of Belgrade, Belgrade, Serbia 0 Views copyright © 2025 Milanovic J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I have no comment Competing Interests No competing interests were disclosed. Reviewer Expertise polyphenols, resveratrol, oxidative stress, hyperlipidaemia, hypertension I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Milanovic JG. Peer Review Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.181862.r384830) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-339/v4#referee-response-384830 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Leong X. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 08 May 2025 | for Version 3 Xin-Fang Leong , Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia 0 Views copyright © 2025 Leong X. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The revised manuscript has shown marked improvement in terms of clarity, coherence, and overall quality. It is now well-written and considerably easier to understand. The authors have clearly addressed the previous comments, and the manuscript is much more compelling as a result. However, there are still some issues that need to be addressed before final acceptance. Firstly, there is a discrepancy (reduction vs. increase) between the mRNA expression findings for sitagliptin and pterostilbene reported in the abstract and those presented in the results section, particularly in the Figure 4. Please ensure these are consistent throughout the manuscript. Secondly, the statement regarding HDL levels requires clarification, as the current data indicate that HDL levels were increased in both the supplemented groups and the normal control group compared to the atherogenic diet group—this should be clearly reflected in the results/Table 1 and discussion. Thirdly, while H&E staining images have been provided, it would strengthen the histological analysis if Oil Red O staining images were also included, if available. Finally, the methodology section mentions that "PCR products were then separated on a 1% agarose gel, visualized, and photographed under ultraviolet light," but this data appears to be missing from both the manuscript and the supplementary materials. Please ensure that this data is either included or the statement is revised accordingly. Competing Interests No competing interests were disclosed. Reviewer Expertise Pharmacology, cardiovascular system, animal study I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 22 Jul 2025 Hayder Al-Aubaidy, Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, 3086, Australia Dear Dr Leong, Thank you very much for your review. Please see below our response: Reviewer (Dr. Xin-Fang Leong) Comments: The revised manuscript has shown marked improvement in terms of clarity, coherence, and overall quality. It is now well-written and considerably easier to understand. The authors have clearly addressed the previous comments, and the manuscript is much more compelling as a result. However, there are still some issues that need to be addressed before final acceptance. Firstly , there is a discrepancy (reduction vs. increase) between the mRNA expression findings for sitagliptin and pterostilbene reported in the abstract and those presented in the results section, particularly in the Figure 4. Please ensure these are consistent throughout the manuscript. Response: We thank the reviewer for highlighting this inconsistency. We have carefully reviewed the data and confirmed that sitagliptin increased AMPK expression, as shown in Figure 4. Accordingly, we have revised the abstract to accurately reflect this finding. The sentence now reads: “Pterostilbene supplementation induced a significant reduction in tissue expression of PI3K and AKT, while sitagliptin induced a significant increase in 5’ adenosine monophosphate-activated protein kinase (AMPK) levels.” This correction ensures consistency across the abstract, results, and figures. Secondly , the statement regarding HDL levels requires clarification, as the current data indicate that HDL levels were increased in both the supplemented groups and the normal control group compared to the atherogenic diet group—this should be clearly reflected in the results/Table 1 and discussion. Response: We appreciate the reviewer’s careful examination of the lipid profile data. Upon reviewing Table 1, we confirm that HDL levels were actually higher in the atherogenic control group than in the pterostilbene and sitagliptin groups . To address this, we have revised the relevant sections as follows: In the Results , we state: “HDL levels were significantly reduced in the PT and ST groups compared to the AC group (P<0.01), indicating that neither treatment restored HDL to normal levels.” In the Discussion , the paragraph has been updated to acknowledge the lower HDL levels in the treatment groups, and a note is added that this unexpected result may be due to compound-specific effects on lipid metabolism. These adjustments ensure the data and narrative are aligned. Thirdly, while H&E staining images have been provided, it would strengthen the histological analysis if Oil Red O staining images were also included, if available. Response: We thank the reviewer for this suggestion. Although Oil Red O staining was initiated, the image analysis and quantification were not complete at the time of this revision. As a result, we have removed all references to Oil Red O staining from the manuscript to avoid presenting incomplete data. We plan to include these findings in a future update or follow-up publication once the analysis is finalised. Finally, the methodology section mentions that "PCR products were then separated on a 1% agarose gel, visualized, and photographed under ultraviolet light," but this data appears to be missing from both the manuscript and the supplementary materials. Please ensure that this data is either included or the statement is revised accordingly. Response: We appreciate the reviewer’s attention to this detail. As gel images were not prepared for inclusion in the current version, we have removed this statement to avoid confusion. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards Hayder Al-Aubaidy On behalf of all authors for this manuscript View more View less Competing Interests Nothing to declare. reply Respond Report a concern Leong XF. Peer Review Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.178031.r376390) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-339/v3#referee-response-376390 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Leong X. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 05 Feb 2025 | for Version 2 Xin-Fang Leong , Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia 0 Views copyright © 2025 Leong X. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Introduction: 1. The introduction mentions that "previous studies have illustrated the action of inflammation and oxidative damage in atherogenic plaques progression," but it does not specify any specific studies or key findings that have directly contributed to this knowledge. 2. The introduction could better highlight how the current study fills a gap in the existing literature. What makes pterostilbene and sitagliptin particularly interesting for atherosclerosis? How does this research differ from prior studies on these compounds? 3. The writing sometimes shifts abruptly between concepts (e.g., from the role of mTOR in autophagy to the effects of pterostilbene). To improve coherence, consider dividing the introduction into clearly defined subsections, such as "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents," etc. Materials & Methods: 1. Provide more details on the composition of the atherogenic diet -The diet was a traditional chow with 2% cholesterol? 2. Expand on the rationale for sample size and the method of calculation for statistical power. Why 26 male and 54 female rabbits? 3. Justify the chosen single dosage for PT and ST and the feeding duration for 8 weeks. 4. Detail how autophagy and RT-PCR measurements will be quantified and analyzed. a) RT-PCR protocol is described, the specific genes of interest (e.g., PI3K, AKT, AMPK, mTOR) are listed but without any context about their relevance to atherosclerosis. Information about housekeeping genes used for normalization should be included. b) The use of LC3B as a marker of autophagy is appropriate, but a brief explanation of how this marker will be measured and explaining why LC3B was chosen as a representative marker would enhance the depth of rationale for the study. 5. Provide more information on the histopathological examination, including scoring criteria and staining methods (the scoring criteria should not be placed in the Results section). 6. Kruskal-Wallis or Mann-Whitney for histopathological grading? Any comparison done between ST and PT? Results: 1. To enhance clarity and avoid redundancy, do refrain from repeating the same data in the text, table, and figure within the Results section. Each element should serve a distinct purpose: the text can summarize key findings, while tables and figures provide visual or detailed data, ensuring they complement each other without overlap. For example: level of F2-isoprostane (data) can be found in body text as well as in the Table and Figure. Same goes to the results for lipid profile. For LC3B - Figure 3 can be removed to avoid redundancy. 2. Histopathological examination: The progression of the lesion should be demonstrated by the AC group, while the improvement of the lesion is more effectively represented by the PT or ST groups. Discussion: 1. Refine interpretations of molecular mechanisms: Emphasize that some of the proposed mechanisms are hypotheses based on indirect evidence and suggest future experimental validation. 2. Provide more detailed analysis of the clinical relevance of the lipid profile changes, including comparison to existing treatments. 3. The role of autophagy in atherosclerosis is discussed, but the potential dangers of excessive autophagy are not considered. In some contexts, autophagy may contribute to cell death, and an overactive autophagic response could potentially exacerbate tissue damage. 4. More thoroughly explain how sitagliptin activates AMPK and its role in atherosclerosis beyond glucose regulation. 5. Address study limitations in more detail. 6. Specific future research suggestions: Outline clear next steps for validating and translating these findings into clinical practice (e.g. how could pterostilbene or sitagliptin be optimized for clinical use). Conclusion: 1. The conclusion could be more cautious in attributing the observed effects solely to the PI3K/Akt/mTOR and AMPK/mTOR pathways. Incorporating phrases like "potentially through" or "likely due to" would make the conclusion more nuanced and scientifically precise. 2. Acknowledging the study’s limitations in the conclusion would be beneficial. For example, noting that the results are based on animal models and that further studies, particularly human clinical trials, are necessary would provide a more balanced perspective. 3. The conclusion does not address the potential implications of these findings for human clinical practice. 4. Considering the positive effects of both compounds, the conclusion could briefly explore the potential for combination therapy, highlighting its possible therapeutic advantages. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Pharmacology, cardiovascular system, animal study I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 07 Apr 2025 Hayder Al-Aubaidy, Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, 3086, Australia Dear Dr. Xin-Fang Leong (Reviewer #2): Thank you very much for your highly valuable input. Please see below our response to your comments, highlighting the main changes we implemented in the manuscript, version 3: Introduction: 1. The introduction mentions that "previous studies have illustrated the action of inflammation and oxidative damage in atherogenic plaques progression," but it does not specify any specific studies or key findings that have directly contributed to this knowledge. Thank you for pointing this out. We have revised the introduction to specify key studies that have directly contributed to the understanding of inflammation and oxidative damage in atherogenic plaque progression. For example, Libby et al. (2002) highlighted the role of IL-6 and TNF-α in promoting plaque instability, while Witold et al. (2017) demonstrated the involvement of oxidative stress in endothelial dysfunction and foam cell formation. 2. The introduction could better highlight how the current study fills a gap in the existing literature. What makes pterostilbene and sitagliptin particularly interesting for atherosclerosis? How does this research differ from prior studies on these compounds? We appreciate this suggestion. The revised introduction now explicitly outlines the gap in existing literature, emphasising the limited data on the combined effects of pterostilbene and sitagliptin on atherosclerosis. We have elaborated on their potential synergistic effects, which differ from prior studies focusing solely on their individual impacts. 3. The writing sometimes shifts abruptly between concepts (e.g., from the role of mTOR in autophagy to the effects of pterostilbene). To improve coherence, consider dividing the introduction into clearly defined subsections, such as "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents," etc. To enhance clarity, we have reorganised the introduction into clearly defined subsections: "Atherosclerosis and Inflammation," "Role of Autophagy in Atherosclerosis," and "Potential Therapeutic Agents." This restructuring ensures a smoother transition between concepts. Materials & Methods: 1. Provide more details on the composition of the atherogenic diet -The diet was a traditional chow with 2% cholesterol? The manuscript now specifies that the atherogenic diet consisted of traditional chow supplemented with 2% cholesterol, following established protocols for inducing atherosclerosis in animal models. 2. Expand on the rationale for sample size and the method of calculation for statistical power. Why 26 male and 54 female rabbits? We have provided details on the power analysis conducted to determine the sample size, which offers over 85% power to detect significant differences. The unequal male-to-female ratio was due to availability constraints while ensuring both sexes were represented. 3. Justify the chosen single dosage for PT and ST and the feeding duration for 8 weeks. The dosages for pterostilbene (10 mg/kg) and sitagliptin (12 mg/kg) were based on prior studies demonstrating their efficacy without adverse effects. The eight-week duration aligns with protocols sufficient for the development of atherosclerotic changes and therapeutic assessment. 4. Detail how autophagy and RT-PCR measurements will be quantified and analyzed. a) RT-PCR protocol is described, the specific genes of interest (e.g., PI3K, AKT, AMPK, mTOR) are listed but without any context about their relevance to atherosclerosis. Information about housekeeping genes used for normalization should be included. We have elaborated on the relevance of PI3K, AKT, AMPK, and mTOR to atherosclerosis and included GAPDH as the housekeeping gene for normalisation. b) The use of LC3B as a marker of autophagy is appropriate, but a brief explanation of how this marker will be measured and explaining why LC3B was chosen as a representative marker would enhance the depth of rationale for the study. The rationale for selecting LC3B as a marker of autophagy is now explained in detail, highlighting its role in autophagosome formation and relevance in vascular studies. 5. Provide more information on the histopathological examination, including scoring criteria and staining methods (the scoring criteria should not be placed in the Results section). We have included comprehensive information on the scoring criteria and staining methods, which were previously only partially described. 6. Kruskal-Wallis or Mann-Whitney for histopathological grading? Any comparison done between ST and PT? The Kruskal-Wallis test was used for overall comparisons, with Mann-Whitney U tests for pairwise comparisons, including between the PT and ST groups. Results: 1. To enhance clarity and avoid redundancy, do refrain from repeating the same data in the text, table, and figure within the Results section. Each element should serve a distinct purpose: the text can summarize key findings, while tables and figures provide visual or detailed data, ensuring they complement each other without overlap. For example: level of F2-isoprostane (data) can be found in body text as well as in the Table and Figure. Same goes to the results for lipid profile. For LC3B - Figure 3 can be removed to avoid redundancy. We have streamlined the Results section to avoid redundancy, summarising key findings in the text while using tables and figures to present detailed data. 2. Histopathological examination: The progression of the lesion should be demonstrated by the AC group, while the improvement of the lesion is more effectively represented by the PT or ST groups. The revised section now better illustrates lesion progression in the AC group and improvements in the PT and ST groups, with clear histological descriptions supported by representative images. Discussion: 1. Refine interpretations of molecular mechanisms: Emphasize that some of the proposed mechanisms are hypotheses based on indirect evidence and suggest future experimental validation. We have clarified that the proposed mechanisms are hypotheses based on indirect evidence, suggesting future studies for experimental validation. 2. Provide more detailed analysis of the clinical relevance of the lipid profile changes, including comparison to existing treatments. The discussion now includes a comparison to existing treatments, highlighting potential complementary roles for pterostilbene and sitagliptin. 3. The role of autophagy in atherosclerosis is discussed, but the potential dangers of excessive autophagy are not considered. In some contexts, autophagy may contribute to cell death, and an overactive autophagic response could potentially exacerbate tissue damage. We have acknowledged the potential risks of excessive autophagy, which could lead to cell death and exacerbate atherosclerosis in certain contexts. 4. More thoroughly explain how sitagliptin activates AMPK and its role in atherosclerosis beyond glucose regulation. The revised discussion provides a more thorough explanation of how sitagliptin activates AMPK and its broader role in vascular health beyond glucose regulation. 5. Address study limitations in more detail. Additional limitations are discussed, including the reliance on animal models and potential sensitivity issues with histological methods. 6. Specific future research suggestions: Outline clear next steps for validating and translating these findings into clinical practice (e.g. how could pterostilbene or sitagliptin be optimized for clinical use). We have outlined specific next steps for clinical translation, such as optimising dosing, exploring combination therapies, and conducting human trials. Conclusion: 1. The conclusion could be more cautious in attributing the observed effects solely to the PI3K/Akt/mTOR and AMPK/mTOR pathways. Incorporating phrases like "potentially through" or "likely due to" would make the conclusion more nuanced and scientifically precise. The conclusion now uses terms like "potentially" and "likely" to present a more nuanced interpretation of the mechanisms involved. 2. Acknowledging the study’s limitations in the conclusion would be beneficial. For example, noting that the results are based on animal models and that further studies, particularly human clinical trials, are necessary would provide a more balanced perspective. We have explicitly noted that the findings are based on animal models and highlighted the need for human clinical trials. 3. The conclusion does not address the potential implications of these findings for human clinical practice. The potential implications for human atherosclerosis management are discussed, considering both agents as adjunctive therapies. 4. Considering the positive effects of both compounds, the conclusion could briefly explore the potential for combination therapy, highlighting its possible therapeutic advantages. We have added a brief discussion on the potential benefits of combination therapy, considering the complementary mechanisms of pterostilbene and sitagliptin. Thank you again for your constructive feedback, which has significantly improved the clarity and quality of our manuscript. Best Regards, Hayder Al-Aubaidy On behalf of all authors for this manuscript View more View less Competing Interests Nothing to disclose reply Respond Report a concern Leong XF. Peer Review Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.156125.r358416) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-339/v2#referee-response-358416 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 Milanovic J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Sep 2023 | for Version 2 Jelica Grujic Milanovic , University of Belgrade, Belgrade, Serbia 0 Views copyright © 2023 Milanovic J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Authors well addressed my previous comments. The paper improved very much. Competing Interests No competing interests were disclosed. Reviewer Expertise Oxidative stress, polyphenols, hypertension, cardiovascular diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Milanovic JG. Peer Review Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.156125.r208141) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-339/v2#referee-response-208141 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 Milanovic J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 14 Sep 2023 | for Version 1 Jelica Grujic Milanovic , University of Belgrade, Belgrade, Serbia 0 Views copyright © 2023 Milanovic J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The article by Sahib et al. has been clearly defined. The methodology applied is overall correct. The authors support their paper with recent references. However, the following changes to the main text are suggested: In the results, use the abbreviation TC instead of total cholesterol. In Figure 1 and Table 1 the authors presented the same results. The authors must improve this part of the results. In Figure 1, use the same colors for marking as a group as in Figure 1/4/5. Does the letter F in Figure 3 stand for the ST group? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise oxidative stress, polyphenols, hypertension, cardiovascular diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 06 Oct 2023 Hayder Al-Aubaidy, Department of Microbiology, Anatomy, Physiology and Pharmacology & Centre for Cardiovascular Biology and Disease Research, School of Agriculture, Biomedicine & Environment, La Trobe University, Melbourne, 3086, Australia Dear Professor Grujic, Many thanks for reviewing the manuscript and providing us with your comments. We confirm addressing all your comments in Version 2 of the manuscript. A summary of the changes has been listed below: The article by Sahib et al. has been clearly defined. The methodology applied is overall correct. The authors support their paper with recent references. However, the following changes to the main text are suggested: In the results, use the abbreviation TC instead of total cholesterol. Response: Thank you for your comment. This has been fixed across the results section. In Figure 1 and Table 1 the authors presented the same results. The authors must improve this part of the results. Response: We have removed Figure 1 (serum lipid parameters) and kept Table 1 to avoid duplication of data. We have renamed the remaining figures accordingly. In Figure 1, use the same colors for marking as a group as in Figure 1/4/5. Response: Figure 1 – Serum lipid parameters has been removed from the manuscript as per the comment above. Does the letter F in Figure 3 stand for the ST group? Response: In this figure, letter F shows complicated atherosclerotic changes - hemorrhagic thrombus as seen in the atherogenic study cohort (no supplementations). The changes in the sitagliptin group are shown in letter B. Please refer to the figure legend for more details about the changes in the other study groups. Best Regards Prof. Hayder Al-Aubaidy On behalf of all authors for this manuscript View more View less Competing Interests Nothing to declare reply Respond Report a concern Milanovic JG. Peer Review Report For: Comparing the effectiveness of pterostilbene and sitagliptin on modulating inflammatory levels and inducing autophagy to improve atherosclerosis outcome: A preclinical study in rabbits [version 4; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 12 :339 ( https://doi.org/10.5256/f1000research.143454.r199143) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-339/v1#referee-response-199143 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. 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