The Synergistic Impact of Probiotic Supplementation and High-intensity Interval Training on Dyslipidemia and the Liver Longevity Genes in Ovariectomized Rats

The Synergistic Impact of Probiotic Supplementation and High-intensity Interval Training on Dyslipidemia and the Liver Longevity Genes in Ovariectomized Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Synergistic Impact of Probiotic Supplementation and High-intensity Interval Training on Dyslipidemia and the Liver Longevity Genes in Ovariectomized Rats Elahe Abbaspour, Sahand Karimzadhagh, Zeinab Bayat, Arsalan Damirchi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4110400/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: This study aimed to explore the synergistic impact of supplementing with local probiotic consumption and engaging in high-intensity interval training (HIIT) on liver health. The focus was particularly on targeting the revitalization genes Cisd2 and Hnf4α and improving dyslipidemia. Methods: In this study, forty ovariectomized (OVX) Wistar rats were divided into five groups: one control, sham surgery, and four ovariectomized (OVX) groups receiving either 1 ml of synthesized local probiotic or engaged in HIIT at 90-95% VO2 max for eight weeks and finally co-treatment of exercise and probiotics. Following the interventions, protein expressions were measured using Western blotting, and assessments of lipid profiles were conducted using colorimetric analysis and enzyme-linked immunosorbent assay. Results: Western blot analysis demonstrated a notable increase in Cisd2 and Hnf4a levels following the co-administration of HIIT and probiotics compared to the OVX + Vehicle group (P=0.001). Furthermore, the analysis revealed a significant decrease in weight, WC, visceral fat, BMI, TG, TC, and LDL-C concentrations, coupled with increased HDL-C (P = 0.001). Conclusion: Our study indicates the efficacy of combined HIIT and lactobacillus probiotic treatment in elevating liver longevity markers of Cisd2 and Hnf4a and mitigating factors associated with obesity and dyslipidemia. Post-Menopause Metabolic syndrome Probiotics Obesity High-intensity interval training Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction As women experience menopause, hormonal transition predisposes the likelihood of developing metabolic syndrome(MetS), cardiovascular disease, and type 2 diabetes 1 – 3 . It is estimated that one in three postmenopausal women is affected by MetS, experiencing a heightened risk 4 , 5 . MetS is characterized by factors such as abdominal obesity, insulin resistance, dyslipidemia, and hypertension in post-menopausal women, mostly related to the decline in estrogen but elevation in androgen levels 6 – 8 . These hormonal shifts lead to alterations in body fat distribution and the onset of abdominal obesity 9 . The liver is among the primary organs affected by MetS 10 , and it plays a crucial role in various metabolic functions, such as detoxification of harmful substances, producing proteins, generating bile, and maintaining energy balance. As the liver ages, it undergoes a range of morphological and physiological changes 11 . For example, accumulation of fat due to aging and dyslipidemia can impair the liver's normal functioning and result in the development of fatty liver disease along with associated histopathological damage 12 . While the exact reasons behind liver aging are not fully understood, numerous studies have suggested that some genes might be essential in controlling lifespan and upholding cell balance. 13 One of the genes that have recently been reported to act as an anti-aging agent is Cisd2, a CDGSH iron-sulfur domain-containing protein family member 14 . This gene is primarily situated within the endoplasmic reticulum (ER), the outer membrane of mitochondria, and the mitochondria-associated ER membrane (MAM). It plays a key role in preserving calcium (Ca2+) balance, regulating mitochondrial performance, and managing oxidative stress, leading to various forms of cellular protection by controlling both ER and mitochondrial functions 15 , 16 . Furthermore, Cisd2 appears to inhibit the age-related disruption of several transcription regulators, including interleukin-6 (IL-6) and nuclear factor erythroid 2-related factor 2 (NRF2). As a result, it protects the transcriptional network, leading to liver rejuvenation. 17 On the other hand, HNF4α plays a pivotal role as a primary controller of liver metabolism and the balance of lipids by interacting with various signaling pathways to effectively manage nutrient metabolism 18 . The expression of hepatic HNF4α is significantly reduced in conditions like diabetes and non-alcoholic fatty liver disease (NAFLD) 19 – 21 . Hence, increasing Hnf4a expression in insulin-resistant situations might help restore the normal function of the liver, suggesting that activating Hnf4a may be the key to achieving the benefits of Cisd2TG. 22 , 23 Acknowledging prevention's superiority over treatment in medicine, the spotlight has increasingly turned to non-pharmacological methods, such as exercise and diet, over recent years. Research shows that physical exercise, including both aerobic and resistance training, reduces liver fat by improving insulin sensitivity and fat metabolism and reducing inflammation. These findings strongly support the idea that regular exercise, with specific intensity, frequency, and duration, benefits NAFLD management and, consequently, MetS 24 – 26 . The European Association for the Study of Obesity has verified the ample evidence endorsing the effectiveness of aerobic, resistance, and high-intensity interval training (HIIT) 27 .HIIT has demonstrated its effectiveness in addressing cardiometabolic risk factors, especially among individuals who are overweight or obese 28 . Prior studies have shown that HIIT can be more effective than moderate-intensity exercise for managing fatty liver. It can improve aerobic capacity and lipid profiles, reduce insulin resistance, and lower liver enzyme levels while recovering liver stiffness and function. 29 On the other hand, the gut-liver axis has gotten attention in the last few years due to the important influence of microbiota and its metabolic byproducts on liver health 30 . In this context, probiotics, which are beneficial living microorganisms, play an essential role in activating the immune system, modulating gastrointestinal function, and contributing to overall well-being. On the contrary, alterations in the gut microbiota have been linked to various disturbances, including the aging process 31 , 32 , metabolic disorders, neurodegenerative, immune system dysregulation, and gastrointestinal disorders. Moreover, gut microbial imbalance, or dysbiosis, is believed to occur as part of the natural aging process, and its prevention has been associated with an increased potential for longevity 33 . Considering the lack of an appropriate solution for MetS and liver dysfunction, finding a new strategy to prevent the progression of MetS toward diabetes is of great importance. Therefore, this study aimed to investigate the effect of probiotic supplementation and HIIT on liver health in ovariectomized rats, pointing to revitalization genes. To achieve these goals, we employed ovariectomized rats as a model for both menopause and metabolic syndrome. 2. Materials and Methods 2.1. Animal Selection and Care Forty adult female Wistar rats, aged three months, with an approximate weight ranging between 200 -250g, were obtained for this study. The rats were housed in groups of four per cage in standard conditions, including a temperature of 22°C ± 2°C and a 12:12-hour light/dark cycle with the lights on at 07:00 34 . The animals were provided free access to standard pellet rat chow and water. Ethical considerations were meticulously observed throughout the study, adhering to the European Union's ethical guidelines for animal experiments (Directive 2010/63/EU). The study received ethics approval from the Sport Sciences Research Institute of Iran (IR.SSRI.REC.1400.1087) and is reported in accordance with ARRIVE guidelines 35 . 2.2. Ovariectomy Surgery With the exception of the sham group (n = 8), animals underwent ovariectomy (OVX) surgery following the technique elucidated by Babaei et al. 36 . Briefly, animals were anesthetized via an intraperitoneal (i.p) injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) at a 4:1 ratio. Following complete anesthesia, ovaries were accurately removed through a midline incision on the abdomen muscle, and then abdominal muscle and skin were sutured. The sham group underwent the same surgical procedure, excluding the removal of ovaries. A schematic illustration depicting the key steps and procedures is presented in Fig. 1 . 2.3. Group Allocation and Intervention Protocol One month after ovariectomy, rats displaying MetS criteria, including elevated serum cholesterol LDL, triglycerides (TG) glucose, weight gain, and waist circumference, were categorized into five groups (n = 8 rats per group): probiotic (OVX + prob), (OVX + exercise), a combination of exercise with probiotic (OVX + Exe + Prob), control (OVX + control), and the sham group. The probiotic groups consumed 1 ml lactobacillus cocktail (10 9 cells) three times weekly via gavage. The control group (OVX + control) received an equivalent volume of drinkable water via gavage. The exercised groups engaged in treadmill running (DSI-580; Danesh Salar Iranian) at an intensity of 90–95% of their VO2 max, on a zero-degree gradient, three times a week for eight weeks. __Figure1__ 2.4. Preparation of Lactobacillus Strains The probiotic source was derived from fecal samples collected during a prior cross-sectional study conducted in 2019 under the ethics approval code IR.GUMS.REC.1398.016 37 . It involved 83 volunteers from Rudbar, a nonindustrial, humid, rainy-climate area in northern Iran known for a vegetable-rich diet. Samples were gathered from volunteers at rural health centers using convenience sampling, with all participants providing written informed consent. Inclusion criteria ensured the absence of gastrointestinal diseases and a six-month antibiotic-free history. The Lactobacillus strains used exhibited several probiotic properties, including bacteriocin production, bactericidal effects, and resilience to acid and bile. Isolation consisted of spreading glycerol bacterial stocks onto An De Man, Rogosa, and Sharpe (MRS) agar plates and then incubating them at 37°C for 48 hours to ensure purity. Afterward, a solitary colony was cultivated in MRS broth, resulting in a concentration of 10^9 colony-forming units per milliliter (CFU/mL). Each rat was administered 1 ml of this solution through a gavage syringe three times weekly. 2.5. High-Intensity Interval Training Program Before initiating the HIIT program, animals underwent treadmill acclimatization, running at 10 meters per minute for 10 minutes with no incline for one week. Following this, two groups participated in an eight-week HIIT regimen three times per week. HIIT sessions comprised nine one-minute intervals at 90–95% of their VO2 max and 8 one-minute intervals at 50% of their VO2 max between them, interspersed with four-minute intervals at 55% of VO2 max for warm-up and cool-down. (Fig. 2 ) VO2 max was assessed by having rats run at 6 meters per minute on a flat treadmill for 5 minutes, with a 3-meter-per-minute speed increase every 3 minutes until exhaustion 4 , 38 . __Figure 2__ 2.6. Morphometric parameters measurement The study followed the methodology outlined by Bayat et al. 39 . After an overnight fast, rats were anesthetized and sacrificed for measurements, including weight and waist circumference (WC). WC was measured in the largest abdominal region. Subsequently, blood samples were collected from the inferior vena cava, centrifuged, and stored at − 80°C for later biochemical analysis. Intra-abdominal fat depots (mesenteric, urogenital, and retroperitoneal) were dissected, and their weights were promptly measured using a laboratory weighing scale (SKY-3000; Jadevar) to avoid evaporative weight loss. 2.7. Biochemical measurements The measurement of serum total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) concentrations was carried out using the enzymatic colorimetric method (GOD-PAP; Pars Azmoun), while triglyceride (TG) levels were assessed using the Triglyceride Quantification Kit, Catalog Number MAK266 (Pars Azmoun). Additionally, the estimation of low-density lipoprotein cholesterol (LDL-C) was conducted based on the methodology outlined in previous studies, specifically following the procedure described by Babaei et al. 40 . 2.8. Liver Extraction and Immunoblot Analysis After the blood sampling and visceral fat removal, the livers were carefully dissected and kept at -80 for further analysis. Subsequently, the frozen liver samples were prepared for western blotting analysis.500 mg of liver tissue (approximately 10 6 cells) was used for protein extraction. The tissue was lysed in 500 µl of lysis buffer, which consisted of 10 µl Triton X-100, 0.003 g EDTA, 500 µl Tris-HCl (pH 8), 0.08 g NaCl, 0.01 g sodium dodecyl sulfate (SDS), one tablet of Protease Inhibitor Cocktail, and 0.025 g Sodium Deoxycholate. The lysate was then incubated for 1 hour with primary antibodies at the following dilutions: β-Actin (sc-47778, 1:300), CISD2 (13318-1-AP, 1:300), and HNF-4a (sc-374229, 1:300). After incubation, the membrane was washed with PBS for 30 minutes at room temperature. Following the primary antibody incubation and wash steps, the membrane was probed with secondary antibodies, specifically HRP-conjugated mouse anti-rabbit IgG (sc-2357, 1:1000) and HRP-conjugated mouse IgG (sc-516102, 1:1000), with the incubation conducted at room temperature for 1 hour. Additionally, β-actin antibody was used as the loading control. After the final wash steps, the protein bands on the membrane were visualized using the Amersham ECL Prime Western Blotting Detection kit (GE Healthcare Life Sciences). Band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA). 2.9. Statistical analysis The normality of the data was assessed through the Shapiro-Wilk test using IBM SPSS Statistics software (version 22.0). Group comparisons were conducted using one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test. The results are presented as mean ± standard error (SE) of the mean, and statistical significance was set at P < 0.05. 3. Results 3.1. HIIT and probiotics supplementation significantly increased Cisd2 and Hnf4-a expression Western blot analysis showed that the application of HIIT and probiotics individually significantly increased Cisd2 expression compared to the OVX + Vehicle group (P = 0.04 and P = 0.03, respectively). The co-treatment of HIIT and probiotics resulted in the highest Cisd2 expression compared to the OVX + Vehicle group (P = 0.01). ( Fig. 3 ) Additionally, HIIT and probiotics individual administration showed a significant increase in Hnf4-a expression (P = 0.005 and P = 0.007, respectively), while the co-treatment group showed a synergistic increase (p = 0.001). The full-length blots displaying the entire gel are provided in supplementary figures S1 -S3 . __Figure 3__ 3.2. Co-treatment of probiotics and HIIT modulated lipid profile The results from the one-way ANOVA test revealed notable differences between groups in TG, TC, HDL-C, and LDL-C levels (P = 0.001). TG levels significantly decreased in OVX + Exe + Prob, OVX + Exe, and OVX + Prob compared to OVX + Vehicle. TC also decreased in OVX + Exe + Prob, OVX + Exe (P = 0.001), and OVX + Prob (P = 0.01) compared to OVX + Vehicle. HDL levels demonstrated an increase in OVX + Exe + Prob, OVX + Exe, and OVX + Prob (P = 0.001). No significant differences were observed between these groups in terms of TG, TC, LDL, and HDL. ( Fig. 4 ) __Figure 4__ 4. Discussion Our study, in line with the previous studies, demonstrated the MetS model after ovariectomy surgery, confirming the crucial anti-obesity role of estrogen 41 – 43 . The liver also showed significant downregulation in the expression of both Cisd2 and Hnf4a after ovariectomy. Further intervention using co-treatment of HIIT protocol and probiotic consumption revealed the upregulation of these two longevity genes. In a related study, Huang et al. 44 investigated the impact of the pro-longevity gene Cisd2 on liver aging, revealing that maintaining high levels of Cisd2 in transgenic mice attenuates age-related dysregulation in lipid metabolism, pathological abnormalities, and oxidative stress. Furthermore, in separate studies conducted in elderly transgenic mice, Cisd2 shows a youthful gene expression profile, indicating its role in preventing age-related dysregulation of important transcription regulators 14 , 23 , 44 , 45 . These findings underscore Cisd2 as a promising therapeutic target to decelerate liver aging. In addition, studies have consistently indicated that reduced HNF4α expression is associated with diverse liver pathological conditions, underscoring its role in preserving liver homeostasis 46 , 47 . A study by Melis et al. 48 demonstrated HNF4α downregulation in chronic liver diseases, while Lu et al. 18 emphasized its crucial role in preventing the onset of fatty liver and maintaining lipid metabolism. According to our data, the elevation in Cisd2 and Hnf4a suggests modulatory effects of the liver on lipid profiles. Moreover, our study revealed a significant decrease in TG, TC, and LDL and an increase in HDL in response to HIIT and probiotic supplementation. In line with our results, previous research has shown that the combined use of HIIT and probiotics effectively addresses dyslipidemia and obesity-related factors, leading to reductions in weight and visceral fat 4 , 8 . Our findings confirm the study by Iqbal et al., suggesting positive impacts of probiotics on lipid profiles 48 , and Plaza-Diaz et al., indicating anti-inflammatory effects of probiotics in the liver 49 . Remarkably, the most favorable outcome in our study was evident with co-treatment, underscoring the potential synergy between HIIT and probiotics in effectively reducing hepatic steatosis linked to obesity. Clinical evidence suggests that exercise and probiotics impact gene expression through epigenetic mechanisms, resulting in heightened endogenous activity and subsequent changes in lipid metabolism 50 , 51 .HIIT has been known to emerge as a time-efficient and effective intervention for addressing metabolic dysfunction associated with menopause 52 , 53 by activating the AMP-activated protein kinase (AMPK) pathway and lipolysis 52 . Also, animal and human studies have demonstrated that exercise-induced activation of AMPK leads to decreased liver fat accumulation by suppressing de novo lipogenesis, enhancing fatty acid oxidation, and supporting mitochondrial function and integrity in adipose tissue. This activation is driven by the increased demand for ATP during intense exercise, resulting in a rise in the AMP/ADP: ATP ratio, which in turn activates AMPK 54 – 57 . Furthermore, the intensity and duration of exercise play a crucial role, with higher-intensity workouts leading to increased ATP usage and subsequent AMPK activation 58 . Meanwhile, probiotics modulate gut microbiota and produce metabolites such as short-chain fatty acids (SCFAs), which inhibit histone deacetylase activity, an epigenetic mechanism regulating gene expression 59 . SCFAs also play a role in immunomodulation, activating liver cAMP signaling to regulate lipid metabolism and alleviating inflammatory conditions. These actions can directly affect gene expression, shape gut microbiota, improve lipid profiles, and ultimately promote metabolic health 59 – 62 . Consequently, the combination of HIIT and probiotics may synergistically enhance these effects, providing a promising approach for addressing dyslipidemia and obesity-related factors while preserving liver homeostasis and potentially slowing age-related metabolic decline 63 , 64 . From a clinical importance view, these findings highlight the promising role of HIIT and probiotics as effective interventions for dyslipidemia in postmenopausal women. However, further research is needed to understand the specific mechanisms behind these effects and optimize the integration of HIIT and probiotics for enhanced liver health and potential age-related deceleration. Conclusion Our study suggests promising combined effects of simultaneous treatment with HIIT and lactobacillus probiotics consumption on mitigating lipid profiles through upregulation of Cisd2 and Hnf4a, indicating improved liver function and mitochondrial activity. However, further research is necessary to refine this approach for enhancing liver health and addressing age-related factors. Declarations Ethics approval The institutional board review at Guilan University of Medical Sciences granted approval for the study is available upon request from the corresponding author. (Ethics approval code: IR.SSRI.REC.1400.1087) Conflict of interest disclosure The authors declare that they have no competing interests. Acknowledgment The authors created the graphical abstract using www.biorender.com Data availability statement Data is provided within the manuscript. Any additional data used to support the findings of this study are available from the corresponding author upon request. Funding None Author Contributions: We acknowledge the co-first authorship of E.A. and S.K. for their equal contributions to this work. E.A. and S.K. designed the study and prepared the manuscript. E.A. and Z.B. were responsible for the methodology and data collection. 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Cisd2 slows down liver aging and attenuates age-related metabolic dysfunction in male mice. Aging Cell . 2021;20(12):e13523. doi:10.1111/acel.13523 Liao HY, Liao B, Zhang HH. CISD2 plays a role in age-related diseases and cancer. Biomed Pharmacother . 2021;138:111472. doi:10.1016/j.biopha.2021.111472 Deans JR, Deol P, Titova N, et al. HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver. Front Endocrinol . 2023;14:1266527. doi:10.3389/fendo.2023.1266527 Qu N, Luan T, Liu N, et al. Hepatocyte nuclear factor 4 a (HNF4α): A perspective in cancer. Biomed Pharmacother . 2023;169:115923. doi:10.1016/j.biopha.2023.115923 Melis M, Marino R, Tian J, et al. Mechanism and Effect of HNF4α Decrease in a Rat Model of Cirrhosis and Liver Failure. Cell Mol Gastroenterol Hepatol . 2024;17(3):453-479. doi:10.1016/j.jcmgh.2023.11.009 Plaza-Diaz J, Gomez-Llorente C, Abadia-Molina F, et al. Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on hepatic steatosis in Zucker rats. PloS One . 2014;9(5):e98401. doi:10.1371/journal.pone.0098401 Santibañez-Gutierrez A, Fernández-Landa J, Calleja-González J, Delextrat A, Mielgo-Ayuso J. Effects of Probiotic Supplementation on Exercise with Predominance of Aerobic Metabolism in Trained Population: A Systematic Review, Meta-Analysis and Meta-Regression. Nutrients . 2022;14(3):622. doi:10.3390/nu14030622 Wang K, Liu H, Hu Q, et al. Epigenetic regulation of aging: implications for interventions of aging and diseases. Signal Transduct Target Ther . 2022;7(1):1-22. doi:10.1038/s41392-022-01211-8 Maillard F, Pereira B, Boisseau N. Effect of High-Intensity Interval Training on Total, Abdominal and Visceral Fat Mass: A Meta-Analysis. Sports Med Auckl NZ . 2018;48(2):269-288. doi:10.1007/s40279-017-0807-y Mahatme S, K V, Kumar N, Rao V, Kovela RK, Sinha MK. Impact of high-intensity interval training on cardio-metabolic health outcomes and mitochondrial function in older adults: a review. Med Pharm Rep . 2022;95(2):115-130. doi:10.15386/mpr-2201 Wu L, Zhang L, Li B, et al. AMP-Activated Protein Kinase (AMPK) Regulates Energy Metabolism through Modulating Thermogenesis in Adipose Tissue. Front Physiol . 2018;9. Accessed February 23, 2024. https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2018.00122 Jessen N, Sundelin EIO, Møller AB. AMP kinase in exercise adaptation of skeletal muscle. Drug Discov Today . 2014;19(7):999-1002. doi:10.1016/j.drudis.2014.03.009 Marcinko K, Sikkema SR, Samaan MC, Kemp BE, Fullerton MD, Steinberg GR. High intensity interval training improves liver and adipose tissue insulin sensitivity. Mol Metab . 2015;4(12):903-915. doi:10.1016/j.molmet.2015.09.006 Smith BK, Marcinko K, Desjardins EM, Lally JS, Ford RJ, Steinberg GR. Treatment of nonalcoholic fatty liver disease: role of AMPK. Am J Physiol-Endocrinol Metab . 2016;311(4):E730-E740. doi:10.1152/ajpendo.00225.2016 Richter EA, Ruderman NB. AMPK and the biochemistry of exercise: Implications for human health and disease. Biochem J . 2009;418(2):261-275. doi:10.1042/BJ20082055 Monda V, Villano I, Messina A, et al. Exercise Modifies the Gut Microbiota with Positive Health Effects. Oxid Med Cell Longev . 2017;2017:3831972. doi:10.1155/2017/3831972 Imierska M, Kurianiuk A, Błachnio-Zabielska A. The Influence of Physical Activity on the Bioactive Lipids Metabolism in Obesity-Induced Muscle Insulin Resistance. Biomolecules . 2020;10(12):1665. doi:10.3390/biom10121665 Zhang L, Zhang R, Li L. Effects of Probiotic Supplementation on Exercise and the Underlying Mechanisms. Foods . 2023;12(9):1787. doi:10.3390/foods12091787 Begum N, Mandhare A, Tryphena KP, et al. Epigenetics in depression and gut-brain axis: A molecular crosstalk. Front Aging Neurosci . 2022;14. Accessed February 26, 2024. https://www.frontiersin.org/articles/10.3389/fnagi.2022.1048333 Boisseau N, Barnich N, Koechlin-Ramonatxo C. The Nutrition-Microbiota-Physical Activity Triad: An Inspiring New Concept for Health and Sports Performance. Nutrients . 2022;14(5):924. doi:10.3390/nu14050924 Meroni M, Longo M, Dongiovanni P. The Role of Probiotics in Nonalcoholic Fatty Liver Disease: A New Insight into Therapeutic Strategies. Nutrients . 2019;11(11):2642. doi:10.3390/nu11112642 Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx GraphicalAbstract.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4110400","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":286264978,"identity":"32cb5f92-08c1-4ec3-8505-80eb13611056","order_by":0,"name":"Elahe Abbaspour","email":"","orcid":"","institution":"Guilan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Elahe","middleName":"","lastName":"Abbaspour","suffix":""},{"id":286264979,"identity":"12d895ba-18f5-4fe8-b2f6-b2382aebb302","order_by":1,"name":"Sahand Karimzadhagh","email":"","orcid":"","institution":"Guilan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sahand","middleName":"","lastName":"Karimzadhagh","suffix":""},{"id":286264980,"identity":"ebe85ca6-90c3-4d1f-b02b-736ec9ed2554","order_by":2,"name":"Zeinab Bayat","email":"","orcid":"","institution":"The University of Guilan","correspondingAuthor":false,"prefix":"","firstName":"Zeinab","middleName":"","lastName":"Bayat","suffix":""},{"id":286264981,"identity":"f871c4d6-ca89-4d39-ac43-f72a7784df21","order_by":3,"name":"Arsalan Damirchi","email":"","orcid":"","institution":"The University of Guilan","correspondingAuthor":false,"prefix":"","firstName":"Arsalan","middleName":"","lastName":"Damirchi","suffix":""},{"id":286264983,"identity":"69ebb061-e085-4440-89cd-ac333fe2c746","order_by":4,"name":"Meysam Hasannejad-Bibalan","email":"","orcid":"","institution":"Guilan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Meysam","middleName":"","lastName":"Hasannejad-Bibalan","suffix":""},{"id":286264984,"identity":"6ebdf1a9-51f8-4acf-a3eb-3ad6b0ea07bb","order_by":5,"name":"Parvin Babaei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYFACHgZmEMkPF2AmVotkA6laGAwOEOsscwbeg58LamxkjM+vMd34g8FOnoGd9wFeLZYNfMnSM46l8ZjdeGN2m4ch2bCBmd0ArxaDAzwG0jxsh4FazpjdBnokgYGZDb/DgFqMf/P8+89jPOOM2c0fDPVEaTGT5m0D2sXfY3aDh+EwYS1Av6RZz+xL5pG4wVZ2m8fguGEbIS3AEDt8u+CbnT1//+FtN39UVMvz8x8j4DD5B1CWRAKIy8BAwA6wGijgP0BI7SgYBaNgFIxUAAABqTj/jhsMUQAAAABJRU5ErkJggg==","orcid":"","institution":"Guilan University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Parvin","middleName":"","lastName":"Babaei","suffix":""}],"badges":[],"createdAt":"2024-03-15 21:44:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4110400/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4110400/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53960932,"identity":"61579b0b-5639-49a1-87c4-ab46ec31ad40","added_by":"auto","created_at":"2024-04-02 18:00:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":367650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSchematic Presentation of In Vivo Experiment Methodology. A visual guide illustrating key steps and procedures in the study's workflow.\u003c/em\u003e \u003cem\u003eHIIT: high-intensity interval training, MetS: metabolic syndrome.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4110400/v1/ff1335ccaa8883b9e48706f7.png"},{"id":53960934,"identity":"cf4d666b-55ad-4c97-9720-faefe3819a87","added_by":"auto","created_at":"2024-04-02 18:00:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":255942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDescription of the HIIT Protocol. The approach includes warming up and cooling down, incorporating a 4-minute segment at 55% of VO2max. The central HIIT session involves 9 one-minute intervals at 90-95% VO2 max, with one-minute breaks at 50% VO2 max on a zero-degree incline. This protocol is followed three times per week for a duration of 8 weeks. HIIT: High-Intensity Interval Training, VO2max: Maximal Oxygen Consumption.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4110400/v1/f8560e549b534505803f91fd.png"},{"id":53960964,"identity":"3cf1acbd-4f70-4c0a-a660-64a9f2f05a57","added_by":"auto","created_at":"2024-04-02 18:00:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":470572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eProtein Expression Analysis of Cisd2 and Hnf4-a in Rat Liver Tissue(\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Protein expression of Cisd2 and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Hnf4-a. Experimental groups include OVX + Vehicle (ovariectomized only), OVX + Exe (HIIT), OVX + Prob (Probiotic consumption), and OVX + Exe + Prob (probiotic consumption with HIIT). Statistical analysis employed one-way ANOVA followed by Tukey’s post hoc test. Results presented as mean ± SE of the mean, n = 8 rats per group.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e (C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Representative Western blot images of Cisd2 and Hnf4a expression in rat liver tissue. Densitometric analysis of all protein bands was performed, and the data are presented as relative density normalized to β-actin, illustrating the modulation of protein expression in the experimental groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4110400/v1/e327b116f2be7b4fe355501e.png"},{"id":53960946,"identity":"c80167a4-2eff-40dd-9ee8-7713fb0831c3","added_by":"auto","created_at":"2024-04-02 18:00:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":323606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eComparative Effects of Exercise, Probiotic, and their Combination on Lipid Profiles. The study assesses the impact on (A) LDL-C, (B) TG, (C) HDL-C, and (D) TC. Experimental groups include OVX + Vehicle (ovariectomized only), OVX + Exe (HIIT), OVX + Prob (Probiotic consumption), and OVX + Exe + Prob (probiotic consumption with HIIT). Statistical analysis employed one-way ANOVA followed by Tukey’s post hoc test. Results presented as mean ± SE of the mean, n = 8 rats per group. HIIT: High-Intensity Interval Training, LDL-C: Low-Density Lipoprotein Cholesterol, TG: Triacylglyceride, HDL-C: High-Density Lipoprotein Cholesterol, TC: Total Cholesterol.\u003c/em\u003e \u003cem\u003e*P ≤ 0.05, ***P ≤ 0.001 vs OVX + Vehicle group. # P ≤ 0.05, ### P ≤ 0.001 vs sham group.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4110400/v1/c3cf9f4e7cfddbed6fc25f32.png"},{"id":59725862,"identity":"3a302b7d-95e3-47ea-a1d3-20a704b60f00","added_by":"auto","created_at":"2024-07-05 10:50:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2080028,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4110400/v1/f56f769a-a7d9-408e-ab63-5c040733454f.pdf"},{"id":53960933,"identity":"f7a74b8d-bfd0-42e3-993c-971ea952a6a7","added_by":"auto","created_at":"2024-04-02 18:00:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4479423,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4110400/v1/00c5ccb7b94f999244149473.docx"},{"id":53960930,"identity":"775c2ba3-3224-4e07-8d3e-b2750dd0834c","added_by":"auto","created_at":"2024-04-02 18:00:47","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":593957,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-4110400/v1/850ccce07875b0b0502197ec.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Synergistic Impact of Probiotic Supplementation and High-intensity Interval Training on Dyslipidemia and the Liver Longevity Genes in Ovariectomized Rats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs women experience menopause, hormonal transition predisposes the likelihood of developing metabolic syndrome(MetS), cardiovascular disease, and type 2 diabetes \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It is estimated that one in three postmenopausal women is affected by MetS, experiencing a heightened risk \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. MetS is characterized by factors such as abdominal obesity, insulin resistance, dyslipidemia, and hypertension in post-menopausal women, mostly related to the decline in estrogen but elevation in androgen levels \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These hormonal shifts lead to alterations in body fat distribution and the onset of abdominal obesity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe liver is among the primary organs affected by MetS\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and it plays a crucial role in various metabolic functions, such as detoxification of harmful substances, producing proteins, generating bile, and maintaining energy balance. As the liver ages, it undergoes a range of morphological and physiological changes \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. For example, accumulation of fat due to aging and dyslipidemia can impair the liver's normal functioning and result in the development of fatty liver disease along with associated histopathological damage \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. While the exact reasons behind liver aging are not fully understood, numerous studies have suggested that some genes might be essential in controlling lifespan and upholding cell balance. \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOne of the genes that have recently been reported to act as an anti-aging agent is Cisd2, a CDGSH iron-sulfur domain-containing protein family member \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This gene is primarily situated within the endoplasmic reticulum (ER), the outer membrane of mitochondria, and the mitochondria-associated ER membrane (MAM). It plays a key role in preserving calcium (Ca2+) balance, regulating mitochondrial performance, and managing oxidative stress, leading to various forms of cellular protection by controlling both ER and mitochondrial functions \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Furthermore, Cisd2 appears to inhibit the age-related disruption of several transcription regulators, including interleukin-6 (IL-6) and nuclear factor erythroid 2-related factor 2 (NRF2). As a result, it protects the transcriptional network, leading to liver rejuvenation. \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOn the other hand, HNF4α plays a pivotal role as a primary controller of liver metabolism and the balance of lipids by interacting with various signaling pathways to effectively manage nutrient metabolism \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The expression of hepatic HNF4α is significantly reduced in conditions like diabetes and non-alcoholic fatty liver disease (NAFLD) \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Hence, increasing Hnf4a expression in insulin-resistant situations might help restore the normal function of the liver, suggesting that activating Hnf4a may be the key to achieving the benefits of Cisd2TG.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAcknowledging prevention's superiority over treatment in medicine, the spotlight has increasingly turned to non-pharmacological methods, such as exercise and diet, over recent years. Research shows that physical exercise, including both aerobic and resistance training, reduces liver fat by improving insulin sensitivity and fat metabolism and reducing inflammation. These findings strongly support the idea that regular exercise, with specific intensity, frequency, and duration, benefits NAFLD management and, consequently, MetS \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe European Association for the Study of Obesity has verified the ample evidence endorsing the effectiveness of aerobic, resistance, and high-intensity interval training (HIIT)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.HIIT has demonstrated its effectiveness in addressing cardiometabolic risk factors, especially among individuals who are overweight or obese\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Prior studies have shown that HIIT can be more effective than moderate-intensity exercise for managing fatty liver. It can improve aerobic capacity and lipid profiles, reduce insulin resistance, and lower liver enzyme levels while recovering liver stiffness and function. \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOn the other hand, the gut-liver axis has gotten attention in the last few years due to the important influence of microbiota and its metabolic byproducts on liver health \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In this context, probiotics, which are beneficial living microorganisms, play an essential role in activating the immune system, modulating gastrointestinal function, and contributing to overall well-being. On the contrary, alterations in the gut microbiota have been linked to various disturbances, including the aging process \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, metabolic disorders, neurodegenerative, immune system dysregulation, and gastrointestinal disorders. Moreover, gut microbial imbalance, or dysbiosis, is believed to occur as part of the natural aging process, and its prevention has been associated with an increased potential for longevity\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsidering the lack of an appropriate solution for MetS and liver dysfunction, finding a new strategy to prevent the progression of MetS toward diabetes is of great importance. Therefore, this study aimed to investigate the effect of probiotic supplementation and HIIT on liver health in ovariectomized rats, pointing to revitalization genes. To achieve these goals, we employed ovariectomized rats as a model for both menopause and metabolic syndrome.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animal Selection and Care\u003c/h2\u003e \u003cp\u003eForty adult female Wistar rats, aged three months, with an approximate weight ranging between 200 -250g, were obtained for this study. The rats were housed in groups of four per cage in standard conditions, including a temperature of 22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and a 12:12-hour light/dark cycle with the lights on at 07:00\u003csup\u003e34\u003c/sup\u003e. The animals were provided free access to standard pellet rat chow and water. Ethical considerations were meticulously observed throughout the study, adhering to the European Union's ethical guidelines for animal experiments (Directive 2010/63/EU). The study received ethics approval from the Sport Sciences Research Institute of Iran (IR.SSRI.REC.1400.1087) and is reported in accordance with ARRIVE guidelines\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Ovariectomy Surgery\u003c/h2\u003e \u003cp\u003eWith the exception of the sham group (n\u0026thinsp;=\u0026thinsp;8), animals underwent ovariectomy (OVX) surgery following the technique elucidated by Babaei et al. \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Briefly, animals were anesthetized via an intraperitoneal (i.p) injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) at a 4:1 ratio. Following complete anesthesia, ovaries were accurately removed through a midline incision on the abdomen muscle, and then abdominal muscle and skin were sutured. The sham group underwent the same surgical procedure, excluding the removal of ovaries. A schematic illustration depicting the key steps and procedures is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Group Allocation and Intervention Protocol\u003c/h2\u003e \u003cp\u003eOne month after ovariectomy, rats displaying MetS criteria, including elevated serum cholesterol LDL, triglycerides (TG) glucose, weight gain, and waist circumference, were categorized into five groups (n\u0026thinsp;=\u0026thinsp;8 rats per group): probiotic (OVX\u0026thinsp;+\u0026thinsp;prob), (OVX\u0026thinsp;+\u0026thinsp;exercise), a combination of exercise with probiotic (OVX\u0026thinsp;+\u0026thinsp;Exe\u0026thinsp;+\u0026thinsp;Prob), control (OVX\u0026thinsp;+\u0026thinsp;control), and the sham group. The probiotic groups consumed 1 ml lactobacillus cocktail (10\u003csup\u003e9\u003c/sup\u003e cells) three times weekly via gavage. The control group (OVX\u0026thinsp;+\u0026thinsp;control) received an equivalent volume of drinkable water via gavage. The exercised groups engaged in treadmill running (DSI-580; Danesh Salar Iranian) at an intensity of 90\u0026ndash;95% of their VO2 max, on a zero-degree gradient, three times a week for eight weeks.\u003c/p\u003e \u003cp\u003e \u003cb\u003e__Figure1__\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Preparation of Lactobacillus Strains\u003c/h2\u003e \u003cp\u003eThe probiotic source was derived from fecal samples collected during a prior cross-sectional study conducted in 2019 under the ethics approval code IR.GUMS.REC.1398.016\u003csup\u003e37\u003c/sup\u003e. It involved 83 volunteers from Rudbar, a nonindustrial, humid, rainy-climate area in northern Iran known for a vegetable-rich diet. Samples were gathered from volunteers at rural health centers using convenience sampling, with all participants providing written informed consent. Inclusion criteria ensured the absence of gastrointestinal diseases and a six-month antibiotic-free history. The Lactobacillus strains used exhibited several probiotic properties, including bacteriocin production, bactericidal effects, and resilience to acid and bile. Isolation consisted of spreading glycerol bacterial stocks onto An De Man, Rogosa, and Sharpe (MRS) agar plates and then incubating them at 37\u0026deg;C for 48 hours to ensure purity. Afterward, a solitary colony was cultivated in MRS broth, resulting in a concentration of 10^9 colony-forming units per milliliter (CFU/mL). Each rat was administered 1 ml of this solution through a gavage syringe three times weekly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. High-Intensity Interval Training Program\u003c/h2\u003e \u003cp\u003eBefore initiating the HIIT program, animals underwent treadmill acclimatization, running at 10 meters per minute for 10 minutes with no incline for one week. Following this, two groups participated in an eight-week HIIT regimen three times per week. HIIT sessions comprised nine one-minute intervals at 90\u0026ndash;95% of their VO2 max and 8 one-minute intervals at 50% of their VO2 max between them, interspersed with four-minute intervals at 55% of VO2 max for warm-up and cool-down. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e VO2 max was assessed by having rats run at 6 meters per minute on a flat treadmill for 5 minutes, with a 3-meter-per-minute speed increase every 3 minutes until exhaustion \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e__Figure 2__\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Morphometric parameters measurement\u003c/h2\u003e \u003cp\u003eThe study followed the methodology outlined by Bayat et al. \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. After an overnight fast, rats were anesthetized and sacrificed for measurements, including weight and waist circumference (WC). WC was measured in the largest abdominal region. Subsequently, blood samples were collected from the inferior vena cava, centrifuged, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for later biochemical analysis. Intra-abdominal fat depots (mesenteric, urogenital, and retroperitoneal) were dissected, and their weights were promptly measured using a laboratory weighing scale (SKY-3000; Jadevar) to avoid evaporative weight loss.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Biochemical measurements\u003c/h2\u003e \u003cp\u003eThe measurement of serum total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) concentrations was carried out using the enzymatic colorimetric method (GOD-PAP; Pars Azmoun), while triglyceride (TG) levels were assessed using the Triglyceride Quantification Kit, Catalog Number MAK266 (Pars Azmoun). Additionally, the estimation of low-density lipoprotein cholesterol (LDL-C) was conducted based on the methodology outlined in previous studies, specifically following the procedure described by Babaei et al. \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Liver Extraction and Immunoblot Analysis\u003c/h2\u003e \u003cp\u003eAfter the blood sampling and visceral fat removal, the livers were carefully dissected and kept at -80 for further analysis. Subsequently, the frozen liver samples were prepared for western blotting analysis.500 mg of liver tissue (approximately 10 \u003csup\u003e6\u003c/sup\u003e cells) was used for protein extraction. The tissue was lysed in 500 \u0026micro;l of lysis buffer, which consisted of 10 \u0026micro;l Triton X-100, 0.003 g EDTA, 500 \u0026micro;l Tris-HCl (pH 8), 0.08 g NaCl, 0.01 g sodium dodecyl sulfate (SDS), one tablet of Protease Inhibitor Cocktail, and 0.025 g Sodium Deoxycholate. The lysate was then incubated for 1 hour with primary antibodies at the following dilutions: β-Actin (sc-47778, 1:300), CISD2 (13318-1-AP, 1:300), and HNF-4a (sc-374229, 1:300). After incubation, the membrane was washed with PBS for 30 minutes at room temperature. Following the primary antibody incubation and wash steps, the membrane was probed with secondary antibodies, specifically HRP-conjugated mouse anti-rabbit IgG (sc-2357, 1:1000) and HRP-conjugated mouse IgG (sc-516102, 1:1000), with the incubation conducted at room temperature for 1 hour. Additionally, β-actin antibody was used as the loading control. After the final wash steps, the protein bands on the membrane were visualized using the Amersham ECL Prime Western Blotting Detection kit (GE Healthcare Life Sciences). Band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe normality of the data was assessed through the Shapiro-Wilk test using IBM SPSS Statistics software (version 22.0). Group comparisons were conducted using one-way analysis of variance (ANOVA) followed by post hoc Tukey\u0026rsquo;s test. The results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE) of the mean, and statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. HIIT and probiotics supplementation significantly increased Cisd2 and Hnf4-a expression\u003c/h2\u003e \u003cp\u003eWestern blot analysis showed that the application of HIIT and probiotics individually significantly increased Cisd2 expression compared to the OVX\u0026thinsp;+\u0026thinsp;Vehicle group (P\u0026thinsp;=\u0026thinsp;0.04 and P\u0026thinsp;=\u0026thinsp;0.03, respectively). The co-treatment of HIIT and probiotics resulted in the highest Cisd2 expression compared to the OVX\u0026thinsp;+\u0026thinsp;Vehicle group (P\u0026thinsp;=\u0026thinsp;0.01). \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e Additionally, HIIT and probiotics individual administration showed a significant increase in Hnf4-a expression (P\u0026thinsp;=\u0026thinsp;0.005 and P\u0026thinsp;=\u0026thinsp;0.007, respectively), while the co-treatment group showed a synergistic increase (p\u0026thinsp;=\u0026thinsp;0.001). The full-length blots displaying the entire gel are provided in \u003cb\u003esupplementary figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e__Figure 3__\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Co-treatment of probiotics and HIIT modulated lipid profile\u003c/h2\u003e \u003cp\u003eThe results from the one-way ANOVA test revealed notable differences between groups in TG, TC, HDL-C, and LDL-C levels (P\u0026thinsp;=\u0026thinsp;0.001). TG levels significantly decreased in OVX\u0026thinsp;+\u0026thinsp;Exe\u0026thinsp;+\u0026thinsp;Prob, OVX\u0026thinsp;+\u0026thinsp;Exe, and OVX\u0026thinsp;+\u0026thinsp;Prob compared to OVX\u0026thinsp;+\u0026thinsp;Vehicle.\u003c/p\u003e \u003cp\u003eTC also decreased in OVX\u0026thinsp;+\u0026thinsp;Exe\u0026thinsp;+\u0026thinsp;Prob, OVX\u0026thinsp;+\u0026thinsp;Exe (P\u0026thinsp;=\u0026thinsp;0.001), and OVX\u0026thinsp;+\u0026thinsp;Prob (P\u0026thinsp;=\u0026thinsp;0.01) compared to OVX\u0026thinsp;+\u0026thinsp;Vehicle. HDL levels demonstrated an increase in OVX\u0026thinsp;+\u0026thinsp;Exe\u0026thinsp;+\u0026thinsp;Prob, OVX\u0026thinsp;+\u0026thinsp;Exe, and OVX\u0026thinsp;+\u0026thinsp;Prob (P\u0026thinsp;=\u0026thinsp;0.001). No significant differences were observed between these groups in terms of TG, TC, LDL, and HDL. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e__Figure 4__\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur study, in line with the previous studies, demonstrated the MetS model after ovariectomy surgery, confirming the crucial anti-obesity role of estrogen \u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e–\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The liver also showed significant downregulation in the expression of both Cisd2 and Hnf4a after ovariectomy. Further intervention using co-treatment of HIIT protocol and probiotic consumption revealed the upregulation of these two longevity genes. In a related study, Huang et al.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e investigated the impact of the pro-longevity gene Cisd2 on liver aging, revealing that maintaining high levels of Cisd2 in transgenic mice attenuates age-related dysregulation in lipid metabolism, pathological abnormalities, and oxidative stress. Furthermore, in separate studies conducted in elderly transgenic mice, Cisd2 shows a youthful gene expression profile, indicating its role in preventing age-related dysregulation of important transcription regulators\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. These findings underscore Cisd2 as a promising therapeutic target to decelerate liver aging. In addition, studies have consistently indicated that reduced HNF4α expression is associated with diverse liver pathological conditions, underscoring its role in preserving liver homeostasis\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. A study by Melis et al.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003edemonstrated HNF4α downregulation in chronic liver diseases, while Lu et al. \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e emphasized its crucial role in preventing the onset of fatty liver and maintaining lipid metabolism. According to our data, the elevation in Cisd2 and Hnf4a suggests modulatory effects of the liver on lipid profiles.\u003c/p\u003e \u003cp\u003eMoreover, our study revealed a significant decrease in TG, TC, and LDL and an increase in HDL in response to HIIT and probiotic supplementation. In line with our results, previous research has shown that the combined use of HIIT and probiotics effectively addresses dyslipidemia and obesity-related factors, leading to reductions in weight and visceral fat\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Our findings confirm the study by Iqbal et al., suggesting positive impacts of probiotics on lipid profiles \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and Plaza-Diaz et al., indicating anti-inflammatory effects of probiotics in the liver \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Remarkably, the most favorable outcome in our study was evident with co-treatment, underscoring the potential synergy between HIIT and probiotics in effectively reducing hepatic steatosis linked to obesity.\u003c/p\u003e \u003cp\u003eClinical evidence suggests that exercise and probiotics impact gene expression through epigenetic mechanisms, resulting in heightened endogenous activity and subsequent changes in lipid metabolism\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.HIIT has been known to emerge as a time-efficient and effective intervention for addressing metabolic dysfunction associated with menopause\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e by activating the AMP-activated protein kinase (AMPK) pathway and lipolysis\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Also, animal and human studies have demonstrated that exercise-induced activation of AMPK leads to decreased liver fat accumulation by suppressing de novo lipogenesis, enhancing fatty acid oxidation, and supporting mitochondrial function and integrity in adipose tissue. This activation is driven by the increased demand for ATP during intense exercise, resulting in a rise in the AMP/ADP: ATP ratio, which in turn activates AMPK\u003csup\u003e\u003cspan additionalcitationids=\"CR55 CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e–\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Furthermore, the intensity and duration of exercise play a crucial role, with higher-intensity workouts leading to increased ATP usage and subsequent AMPK activation\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMeanwhile, probiotics modulate gut microbiota and produce metabolites such as short-chain fatty acids (SCFAs), which inhibit histone deacetylase activity, an epigenetic mechanism regulating gene expression \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. SCFAs also play a role in immunomodulation, activating liver cAMP signaling to regulate lipid metabolism and alleviating inflammatory conditions. These actions can directly affect gene expression, shape gut microbiota, improve lipid profiles, and ultimately promote metabolic health \u003csup\u003e\u003cspan additionalcitationids=\"CR60 CR61\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e–\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Consequently, the combination of HIIT and probiotics may synergistically enhance these effects, providing a promising approach for addressing dyslipidemia and obesity-related factors while preserving liver homeostasis and potentially slowing age-related metabolic decline \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. From a clinical importance view, these findings highlight the promising role of HIIT and probiotics as effective interventions for dyslipidemia in postmenopausal women. However, further research is needed to understand the specific mechanisms behind these effects and optimize the integration of HIIT and probiotics for enhanced liver health and potential age-related deceleration.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eOur study suggests promising combined effects of simultaneous treatment with HIIT and lactobacillus probiotics consumption on mitigating lipid profiles through upregulation of Cisd2 and Hnf4a, indicating improved liver function and mitochondrial activity. However, further research is necessary to refine this approach for enhancing liver health and addressing age-related factors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe institutional board review at Guilan University of Medical Sciences granted approval for the\u003c/p\u003e\n\u003cp\u003estudy is available upon request from the corresponding author.\u0026nbsp;(Ethics approval code:\u0026nbsp;IR.SSRI.REC.1400.1087)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of interest disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors created the graphical abstract using www.biorender.com\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript. Any additional data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the co-first authorship of E.A. and S.K. for their equal contributions to this work. E.A. and S.K. designed the study and prepared the manuscript. E.A. and Z.B. were responsible for the methodology and data collection. A.D. and M.H. contributed to concept development. P.B. supervised the project and provided oversight in the organization and writing of the manuscript. All authors participated in drafting the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJeong HG, Park H. 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The focus was particularly on targeting the revitalization genes Cisd2 and Hnf4α and improving dyslipidemia.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods: \u003c/strong\u003e\u003c/em\u003eIn this study, forty ovariectomized (OVX) Wistar rats were divided into five groups: one control, sham surgery, and four ovariectomized (OVX) groups receiving either 1 ml of synthesized local probiotic or engaged in HIIT at 90-95% VO2 max for eight weeks and finally co-treatment of exercise and probiotics. Following the interventions, protein expressions were measured using Western blotting, and assessments of lipid profiles were conducted using colorimetric analysis and enzyme-linked immunosorbent assay.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults: \u003c/strong\u003e\u003c/em\u003eWestern blot analysis demonstrated a notable increase in Cisd2 and Hnf4a levels following the co-administration of HIIT and probiotics compared to the OVX + Vehicle group (P=0.001). Furthermore, the analysis revealed a significant decrease in weight, WC, visceral fat, BMI, TG, TC, and LDL-C concentrations, coupled with increased HDL-C (P = 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003e\u003c/em\u003eOur study indicates the efficacy of combined HIIT and lactobacillus probiotic treatment in elevating liver longevity markers of Cisd2 and Hnf4a and mitigating factors associated with obesity and dyslipidemia.\u003c/p\u003e","manuscriptTitle":"The Synergistic Impact of Probiotic Supplementation and High-intensity Interval Training on Dyslipidemia and the Liver Longevity Genes in Ovariectomized Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-02 18:00:40","doi":"10.21203/rs.3.rs-4110400/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3a30d5de-2221-46c7-9708-cfcb4291e879","owner":[],"postedDate":"April 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-05T10:42:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-02 18:00:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4110400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4110400","identity":"rs-4110400","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}