Investigation of the Effects of Different Dietary Treatment Methods on Liver Apoptosis in Mice With Pentylenetetrazole Induced Epilepsy Model

Investigation of the Effects of Different Dietary Treatment Methods on Liver Apoptosis in Mice With Pentylenetetrazole Induced Epilepsy Model | 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 Research Article Investigation of the Effects of Different Dietary Treatment Methods on Liver Apoptosis in Mice With Pentylenetetrazole Induced Epilepsy Model Sacide Sarıçiçek, Ravza Tosunbayraktar, Ali Tuğrul Akin, Ayça Kara, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6880346/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 Purpose: In this study, it was aimed to evaluate the effects of ketogenic diet, intermittent fasting and high-fat diet models on the liver in Balb-c mice in which epilepsy pentylenetetrazole (PTZ) kindling model was created. Materials and methods: 40 mice which is 8-week-old Balb/c were used in the study. To create a pentylenetetrazole kindling model, a total of 15 doses of 30mg/kg PTZl injection were administered every other day. Dietary practices were started simultaneously with the first PTZ injection. Mice; They were divided into 4 groups as standard rodent diet, high-fat diet, intermittent fasting and ketogenic diet. At the end of the three-month experimental period, all groups were sacrificed. Liver tissues were taken from mice and fixed in 4% formaldehyde solution and embedded in paraffin blocks. After deparaffinization procedures, 5 µm sections were taken on the microtome device. Immunohistochemistry with Cas3, Cas8, CHOP, GRP78 and TNF-alpha antibodies; Hematoxylin staining was performed to analyze the pathological status. Statistical analyzes were made in the Graphpad application by taking 20X images under the microscope. Graphs were created by comparing all groups with each other. Results: While the group with the highest expression of Cas3, Cas8 and TNF-a was the PTZ+KD group, the group with the highest expression of GRP78 was PTZ+HFD. When Cas3, Cas 8, TNF-a and GRP78 expressions were examined in the experimental groups, the lowest expression level was observed in the PTZ+IF group. Conclusion: We suggest that intermittent fasting diets can be used as an alternative, at least considering the benefits for the liver, but we emphasize the need for further studies. Nutrition & Dietetics Neurobiology of Disease epilepsy diet liverpathology intermittent fasting hexokinase liver ketogenic diet high-fat diet Figures Figure 1 Figure 2 Figure 3 INTRODUCTION The liver is a critical centre for numerous physiological processes. These include macronutrient metabolism, blood volume regulation, immune system maintenance, endocrine control of growth signalling pathways, lipid and cholesterol homeostasis, and the breakdown of xenobiotic compounds, including many available drugs. The processing, breakdown and metabolism of macronutrients provide the energy needed to carry out the processes specified above and are therefore among the most critical functions of the liver. Furthermore, the liver's capacities to store glucose in the form of glycogen with nutrition and to assemble glucose via the gluconeogenic pathway in response to fasting are critical. The liver oxidises lipids, but can also package excess lipid for secretion and storage in other tissues such as fat. Finally, the liver is the main processor of protein and amino acid metabolism, as it is responsible for most of the proteins released in the blood (depending on mass or unique protein range), the processing of amino acids for energy, and the excretion of nitrogenous waste (Trefts et al., 2017). Epilepsy is a common neurological disorder affecting more than 70 million people worldwide (Ngugi et al., 2011), with an increase of approximately 2 million cases each year (Rezaei et al., 2019). Today, the primary treatment method for these patients is antiepileptic drugs (Tang et al., 2017). Drugs used in the treatment of epilepsy are metabolised in the liver and lead to changes in the metabolism of bile acids, cholesterol, other lipids, bilirubin and many other endogenous molecules and exogenous drugs metabolised by these enzymes (Havel and Kane, 1995). In addition to epilepsy drug treatments, surgery, neuromodulation and dietary treatments are also applied (D'Andrea et al., 2019). The classical ketogenic diet is a biochemical fasting model applied in resistant cases, which enables the use of ketone bodies as an alternative glucose resource for the brain rich in lipids and low in carbohydrates and protein (Armeno et al., 2014) to induce ketosis in metabolism and simulate a fasting state (Ułamek-Kozioł et al., 2019). There are studies showing the preventive and suppressive effects of intermittent fasting on PTZ-induced seizures (Karimzadeh et al., 2013). The effects of these and similar metabolism-based dietary treatment methods on seizure control have been investigated (Rho and Boison., 2022). In order to better understand the pathogenesis of epilepsy, evaluation of systemic metabolism and inflammation, as well as system biology and whole organism approach is considered necessary (Kirchner et al., 2020). In this study, we aimed to investigate the effects of different ketogenic diets, intermittent fasting and high fat diet on apoptosis, inflammation and endoplasmic reticulum stress (ERS) in mouse liver tissues in PTZ-induced experimental kindling model. METHOD Animal modelling The experimental study was conducted with the ethical approval of ERU Animal Experiments Local Ethics Committee (HADYEK NUMBER: 24/158). All procedures performed in the study were carried out in accordance with the 1964 Declaration of Helsinki standards. A total of 40 8-week-old male Balb-c mice were randomly divided into 5 groups (n=40). Kindling model was established in all animals (n=32) except the control group (n=8). Briefly, 30 mg/kg PTZ dissolved in saline was injected 14 times every other day. The reminder dose was repeated 10 days after the last injection. Throughout the study, all animals were kept at 23°-25°C in a 12 h light/12 h dark rhythm (Sarıçiçek et al., 2025). Experimental Design Mice were randomly divided into 5 groups; control group (C), high fat diet group (PTZ+HFD), intermittent fasting group (PTZ+IF), standard rodent diet group (PTZ+CD) and ketogenic diet group (PTZ+KD). No treatment other than standard care was applied to the control group. Dietary interventions were started simultaneously with the injection in all animals. C and PTZ+CD groups received standard rodent feed, PTZ+HFD group received rodent feed containing 25% fat, 22% protein, 53% carbohydrate (Altromin 1090-25), PTZ+KD group received rodent feed containing 84% fat, 11% protein, 5% carbohydrate (Altromin C1084). PTZ+IF group received 24 hours fasting and 24 hours standard rodent feed. Diets were administered for 3 months and at the end of the period, all groups were sacrificed. Liver tissues were taken from mice and fixed in 4% formaldehyde solution. The tissues were followed and embedded in paraffin blocks. After 5 μm sections were taken on microtome device, haematoxylin staining and immunohistochemical staining with Cas3, Cas8, GRP78, TNF-alpha, Hk1 and Hk2 antibodies were performed. 20X images were taken under a microscope. Statistical analysis IHC staining measurements were performed using ImageJ application. Statistical analysis and significance calculation were performed using Graphpad application. Graphs were created by comparing all groups with each other. RESULT Table 1: IHC staining results in mouse liver tissues Groups Control PTZ + HFD PTZ + IF PTZ + CD PTZ + KD P Cas-3 66.06±4.26 a 73.73±5.16 b 69.43±3.66 c 74.87±4.45 d 78.79±8.57 e 0.0001 Cas-8 49.67±10.04 a 74.22±4.67 b 63.81±13.85 c 73.74±5.72 d 90.82±7.01 e 0.0001 TNF-a 62.03±8.09 a 68.98±10.16 b 64.00±8.72 c 80.35±6.02 d 89.12±6.40 e 0.0001 GRP78 72.4±5.71 a 85.32±8.42 b 69.78±3.23 c 73.54±7.39 d 76.66±8.91 e 0.0001 Hk1 78.66±5.31 a 86.16±12.42 b 77.8±5.58 c 80.0±7.53 d 97.27±9.08 e 0.0001 Hk2 106.6±11.02 a 103.3±8.64 b 96.96±9.69 c 97.74±9.30 c 94.93±12.73 d 0.0001 P<0.05: considered statistically significant (C: control group, PTZ+HFD: high-fat diet group, PTZ+IF: intermittent fasting group, PTZ+CD: standard rodent diet group, PTZ+KD: ketogenic diet group). When Cas3 expression was evaluated, a statistically significant difference was found between the groups (p<0.05). It was found to be significantly higher in the PTZ+KD group compared to all other groups. The fact that the Cas3 expression level in the PTZ+KD group was higher than that in the PTZ+CD group indicates that the number of cells undergoing apoptosis in the liver was higher than in animals fed a normal diet. As expected, the group with the lowest Cas3 expression was the control group, followed closely by the PTZ+IF group. When Cas8 expression was evaluated, a statistically significant difference was found between the groups (p<0.05). It was found to be statistically significantly higher in the PTZ+KD group compared to all other groups. The higher Cas8 expression level in the PTZ+KD group compared to the PTZ+CD group indicates that the number of cells undergoing apoptosis in the liver is higher than in animals fed a normal diet. As expected, the group with the lowest Cas8 expression was the control group, while the group closest to it was the PTZ+IF group. When TNF-a expression was evaluated, a statistically significant difference was found between the groups (p<0.05). TNF-a expression was found to be significantly higher in the PTZ+KD group compared to all other groups. The higher TNF-α expression level in the PTZ+KD group compared to the PTZ+CD group indicates that inflammation in the liver is greater than in animals fed a normal diet. When the experimental groups were compared, the lowest expression was found to be significantly lower in the PTZ+IF group. When GRP78 expression was evaluated, a statistically significant difference was found between the groups (p<0.05). GRP78 expression was found to be significantly higher in the PTZ+HFD group compared to all other groups. The group with the lowest expression was the PTZ+IF group. When Hk1 expression was evaluated, a statistically significant difference was found between the groups (p<0.05). Hk1 expression was found to be significantly higher in the PTZ+KD group compared to all other groups. The lowest expression level was found to be significantly lower in the PTZ+IF group. When Hk2 expression was evaluated, a statistically significant difference was found between the groups (p<0.05). Hk2 expression was found to be significantly higher in the control group compared to all other groups. The lowest expression level was found to be significantly lower in the PTZ+KD group. DISCUSSION Anticonvulsant drugs are the first choice for treating epilepsy patients. However, in drug-resistant cases, non-pharmacological dietary treatments are often preferred (Ruan et al., 2022). The aim of the KD is to induce and maintain a state of nutritional ketosis through an increase in hepatic ketogenesis. The increased production of liver ketone bodies is the result of fatty acid oxidation, which mimics a state of starvation. Thus, ketogenesis increases as the use of acetyl CoA in the Krebs cycle decreases (Simeone et al., 2018). Although the effect of the ketogenic diet on preventing seizure frequency cannot be fully explained, various mechanisms, including inhibition of glycolysis, disruption of glutamatergic synaptic transmission, and activation of ATP-sensitive potassium channels, are thought to be involved (Lutas and Yellen, 2013). It is important to evaluate the effects of different dietary interventions in patients with epilepsy on peripheral tissues and organs and systemic inflammation in order to understand the pathogenesis of the disease (Kirchner et al., 2020). In particular, the potential side effects of high-fat diets in individuals are a cause for concern (Zhang et al., 2016). Therefore, there is a need to investigate dietary models that may have fewer side effects on peripheral organs in addition to reducing seizure frequency. Proinflammatory cytokines such as TNF-α are one of the markers used to detect inflammation (Horiuchi et al., 2010). In the study conducted by Zhang et al., it was reported that TNF-α expression increased in the liver tissue of mice fed a KD for 3 months, indicating a proinflammatory state, and molecular signs of hepatic steatosis were observed (Zhang et al., 2016). In our study, TNF-α levels were significantly higher in mice fed a KD compared to other experimental groups. Cas3 is a lysosomal enzyme specific for the detection of apoptotic cells in the apoptotic pathway (Asadi et al., 2022). Cas8, on the other hand, is an initiator caspase specifically involved in the activation of apoptotic pathways. The high expression of Cas3 and Cas8 in the experimental group fed a ketogenic diet indicates that hepatocytes underwent more apoptosis compared to other experimental groups. When examining other experimental groups, the lowest expression level was observed in the group fed intermittent fasting. In a study by Mu et al. on rats with an infantile epilepsy model, probiotic supplementation was added to the ketogenic diet to reduce the hepatic steatosis induced by the diet, and the potential of its use in combination with KD to prevent liver steatosis was emphasised (Mu et al., 2022). Studies have shown that intermittent fasting diets can vary. In a study conducted by Ma et al. on mice, liver histopathology was evaluated after 12 hours of intermittent fasting was applied for one month, and it was stated that hepatocyte plaques developed well and sinusoids were clearly visible (Ma et al., 2021). In a study by Garbow et al., it was shown that the expression of CHOP/GADD153, a proapoptotic antibody, was significantly increased in the liver tissues of mice fed a KD compared to the control group. In our study, it was shown that it increased in mice fed a HFD, but there was no similar increase in the KD group (Garbow et al., 2011). Hexokinases are a family of four enzymes found in all tissues, primarily in the liver. In the first stage of glycolysis, they transfer a phosphate group from ATP to a glucose molecule. The phosphate group enables glucose to be retained within the cell. It is an irreversible enzyme used only in the glycolysis phase. In ketogenic diets, since the glycolysis phase is not actively used, the gluconeolytic pathway becomes active. In our study, it was shown that Hk2 was lower in the KD group compared to other groups, as expected. Declarations Funding This study was supported by Erciyes University Scientific Research Projects Unit with project number TYL-2020-10701. Thanks I would like to thank Erciyes University Betül Ziya Eren Genome and Stem Cell (GENKÖK) Center, where part of the study was carried out, Experimental Research Application and Research Center (DEKAM), GENKÖK staff. Ethic: In this study, approval was received from Erciyes University Animal Experiments Ethics Committee dated 04.09.2025 and numbered 24/158. References Armeno M, Caraballo R, Vaccarezza M, et al. Consenso nacional sobre dieta cetogénica [National consensus on the ketogenic diet]. Rev Neurol. 2014;59(5):213-223. Asadi M, Taghizadeh S, Kaviani E, et al. Caspase-3: Structure, function, and biotechnological aspects. Biotechnol Appl Biochem. 2022;69(4):1633-1645. doi:10.1002/bab.2233 D'Andrea Meira I, Romão TT, Pires do Prado HJ, Krüger LT, Pires MEP, da Conceição PO. Ketogenic Diet and Epilepsy: What We Know So Far. Front Neurosci. 2019;13:5. Published 2019 Jan 29. doi:10.3389/fnins.2019.00005 Garbow JR, Doherty JM, Schugar RC, et al. Hepatic steatosis, inflammation, and ER stress in mice maintained long term on a very low-carbohydrate ketogenic diet. Am J Physiol Gastrointest Liver Physiol. 2011;300(6):G956-G967. doi:10.1152/ajpgi.00539.2010 Horiuchi, T., Mitoma, H., Harashima, S. I., Tsukamoto, H., & Shimoda, T. Transmembrane TNF-α: structure, function and interaction with anti-TNF agents. Rheumatology, 2010, 49.7: 1215-1228. Kirchner A, Dachet F, Loeb JA. Identifying targets for preventing epilepsy using systems biology of the human brain. Neuropharmacology. 2020;168:107757. doi:10.1016/j.neuropharm.2019.107757 Lutas A, Yellen G. The ketogenic diet: metabolic influences on brain excitability and epilepsy. Trends Neurosci. 2013 Jan;36(1):32-40. doi: 10.1016/j.tins.2012.11.005. Epub 2012 Dec 8. PMID: 23228828; PMCID: PMC3534786. Ma J, Cheng Y, Su Q, et al. Effects of intermittent fasting on liver physiology and metabolism in mice. Exp Ther Med. 2021;22(3):950. doi:10.3892/etm.2021.10382 Ma J, Cheng Y, Su Q, et al. Effects of intermittent fasting on liver physiology and metabolism in mice. Exp Ther Med. 2021;22(3):950. doi:10.3892/etm.2021.10382 Martin-McGill KJ, Bresnahan R, Levy RG, Cooper PN. Ketogenic diets for drug-resistant epilepsy. Cochrane Database Syst Rev. 2020;6(6):CD001903. Published 2020 Jun 24. doi:10.1002/14651858.CD001903.pub5 Mu C, Nikpoor N, Tompkins TA, Rho JM, Scantlebury MH, Shearer J. Probiotics counteract hepatic steatosis caused by ketogenic diet and upregulate AMPK signaling in a model of infantile epilepsy. EBioMedicine. 2022;76:103838. doi:10.1016/j.ebiom.2022.103838 Ngugi AK, Kariuki SM, Bottomley C, Kleinschmidt I, Sander JW, Newton CR. Incidence of epilepsy: a systematic review and meta-analysis. Neurology. 2011;77(10):1005-1012. doi:10.1212/WNL.0b013e31822cfc90 Rezaei S, Abdurahman AA, Saghazadeh A, Badv RS, Mahmoudi M. Short-term and long-term efficacy of classical ketogenic diet and modified Atkins diet in children and adolescents with epilepsy: a systematic review and meta-analysis. Nutr Neurosci. 2019;22:317–34. Rho JM, Boison D. The metabolic basis of epilepsy. Nat Rev Neurol. 2022;18(6):333-347. doi:10.1038/s41582-022-00651-8 Ruan Y, Chen L, She D, Chung Y, Ge L, Han L. Ketogenic diet for epilepsy: an overview of systematic review and meta-analysis. Eur J Clin Nutr. 2022;76(9):1234-1244. doi:10.1038/s41430-021-01060-8 Sarıçiçek S, Tosunbayraktar R, Yaşar Fırat Y, Kara A, Erdoğan FF. Assessment of ketogenic diet, intermittent fasting and high fat diet effect on BDNF and behaviour in epilepsy mice models. Research Square. Published June 2025. doi:10.21203/rs.3.rs-6801463/v1 Simeone TA, Simeone KA, Stafstrom CE, Rho JM. Do ketone bodies mediate the anti-seizure effects of the ketogenic diet? Neuropharmacology. 2018;133:233–41. Tang F, Hartz AMS, Bauer B. Drug-Resistant Epilepsy: Multiple Hypotheses, Few Answers. Front Neurol. 2017;8:301. Published 2017 Jul 6. doi:10.3389/fneur.2017.00301 Ułamek-Kozioł M, Czuczwar SJ, Pluta R, Januszewski S. Ketogenic diet and epilepsy. Nutrients. MDPI AG. 2019;11. Zhang X, Qin J, Zhao Y, et al. Long-term ketogenic diet contributes to glycemic control but promotes lipid accumulation and hepatic steatosis in type 2 diabetic mice. Nutr Res. 2016;36(4):349-358. doi:10.1016/j.nutres.2015.12.002 Graph Graph 1 is available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files graph1.png Graph 1: IHC staining evaluation in mouse liver tissues 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. We do this by developing innovative software and high quality services for the global research community. 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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-6880346","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":470373411,"identity":"220891ad-c560-4961-b9c9-e5828cb44fb0","order_by":0,"name":"Sacide 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design\u003c/p\u003e\n\u003cp\u003eC: control group, PTZ+HFD: high fat diet group, PTZ+ IF: intermittent fasting group, PTZ+CD: standard rodent diet group PTZ+KD: ketogenic diet group\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6880346/v1/8faec69636542cdcb1d708c9.png"},{"id":84678329,"identity":"d3f8a373-b2f8-4fd4-aa77-76f9f8119e3e","added_by":"auto","created_at":"2025-06-16 07:55:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1561914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1: \u003c/strong\u003eLiver IHC staining analysis (20X)\u003c/p\u003e\n\u003cp\u003e(C: control group, PTZ+HFD: high-fat diet group, PTZ+IF: intermittent fasting group, PTZ+CD: standard rodent diet group, PTZ+KD: ketogenic diet group)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6880346/v1/9b379e0d27d1b60fbdb64e07.png"},{"id":84678328,"identity":"38569556-f6d8-4a15-80f3-37ec2ac8d44c","added_by":"auto","created_at":"2025-06-16 07:55:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":311112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2\u003c/strong\u003e: Histological analysis of liver morphology (H\u0026amp;E, 20X).\u003c/p\u003e\n\u003cp\u003e(C: control group, PTZ+HFD: high-fat diet group, PTZ+IF: intermittent fasting group, PTZ+CD: standard rodent diet group, PTZ+KD: ketogenic diet group)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6880346/v1/3b09667f57839e0a08c8c2fb.png"},{"id":84678366,"identity":"bb275432-e96e-4af4-8698-1f411fcadbae","added_by":"auto","created_at":"2025-06-16 07:55:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2815808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6880346/v1/c25919c2-a051-4d14-8942-71c67c04f8b0.pdf"},{"id":84678300,"identity":"5b6e9c12-726a-471e-9696-93fa5ed77f90","added_by":"auto","created_at":"2025-06-16 07:54:56","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":178118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraph 1: IHC staining evaluation in mouse liver tissues\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"graph1.png","url":"https://assets-eu.researchsquare.com/files/rs-6880346/v1/186010d36fb02e22b0e5c90e.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eInvestigation of the Effects of Different Dietary Treatment Methods on Liver Apoptosis in Mice With Pentylenetetrazole Induced Epilepsy Model\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe liver is a critical centre for numerous physiological processes. These include macronutrient metabolism, blood volume regulation, immune system maintenance, endocrine control of growth signalling pathways, lipid and cholesterol homeostasis, and the breakdown of xenobiotic compounds, including many available drugs. The processing, breakdown and metabolism of macronutrients provide the energy needed to carry out the processes specified above and are therefore among the most critical functions of the liver. Furthermore, the liver's capacities to store glucose in the form of glycogen with nutrition and to assemble glucose via the gluconeogenic pathway in response to fasting are critical. The liver oxidises lipids, but can also package excess lipid for secretion and storage in other tissues such as fat. Finally, the liver is the main processor of protein and amino acid metabolism, as it is responsible for most of the proteins released in the blood (depending on mass or unique protein range), the processing of amino acids for energy, and the excretion of nitrogenous waste (Trefts et al., 2017).\u003c/p\u003e \u003cp\u003eEpilepsy is a common neurological disorder affecting more than 70\u0026nbsp;million people worldwide (Ngugi et al., 2011), with an increase of approximately 2\u0026nbsp;million cases each year (Rezaei et al., 2019). Today, the primary treatment method for these patients is antiepileptic drugs (Tang et al., 2017). Drugs used in the treatment of epilepsy are metabolised in the liver and lead to changes in the metabolism of bile acids, cholesterol, other lipids, bilirubin and many other endogenous molecules and exogenous drugs metabolised by these enzymes (Havel and Kane, 1995). In addition to epilepsy drug treatments, surgery, neuromodulation and dietary treatments are also applied (D'Andrea et al., 2019). The classical ketogenic diet is a biochemical fasting model applied in resistant cases, which enables the use of ketone bodies as an alternative glucose resource for the brain rich in lipids and low in carbohydrates and protein (Armeno et al., 2014) to induce ketosis in metabolism and simulate a fasting state (Ułamek-Kozioł et al., 2019). There are studies showing the preventive and suppressive effects of intermittent fasting on PTZ-induced seizures (Karimzadeh et al., 2013). The effects of these and similar metabolism-based dietary treatment methods on seizure control have been investigated (Rho and Boison., 2022). In order to better understand the pathogenesis of epilepsy, evaluation of systemic metabolism and inflammation, as well as system biology and whole organism approach is considered necessary (Kirchner et al., 2020).\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate the effects of different ketogenic diets, intermittent fasting and high fat diet on apoptosis, inflammation and endoplasmic reticulum stress (ERS) in mouse liver tissues in PTZ-induced experimental kindling model.\u003c/p\u003e"},{"header":"METHOD","content":"\u003cp\u003e\u003cstrong\u003eAnimal modelling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental study was conducted with the ethical approval of ERU Animal Experiments Local Ethics Committee (HADYEK NUMBER: 24/158). All procedures performed in the study were carried out in accordance with the 1964 Declaration of Helsinki standards.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA total of 40 8-week-old male Balb-c mice were randomly divided into 5 groups (n=40). Kindling model was established in all animals (n=32) except the control group (n=8). Briefly, 30 mg/kg PTZ dissolved in saline was injected 14 times every other day. The reminder dose was repeated 10 days after the last injection. Throughout the study, all animals were kept at 23\u0026deg;-25\u0026deg;C in a 12 h light/12 h dark rhythm (Sarı\u0026ccedil;i\u0026ccedil;ek et al., 2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were randomly divided into 5 groups; control group (C), high fat diet group (PTZ+HFD), intermittent fasting group (PTZ+IF), standard rodent diet group (PTZ+CD) and ketogenic diet group (PTZ+KD). No treatment other than standard care was applied to the control group. Dietary interventions were started simultaneously with the injection in all animals. C and PTZ+CD groups received standard rodent feed, PTZ+HFD group received rodent feed containing 25% fat, 22% protein, 53% carbohydrate (Altromin 1090-25), PTZ+KD group received rodent feed containing 84% fat, 11% protein, 5% carbohydrate (Altromin C1084). PTZ+IF group received 24 hours fasting and 24 hours standard rodent feed. Diets were administered for 3 months and at the end of the period, all groups were sacrificed. Liver tissues were taken from mice and fixed in 4% formaldehyde solution. The tissues were followed and embedded in paraffin blocks. After 5 \u0026mu;m sections were taken on microtome device, haematoxylin staining and immunohistochemical staining with Cas3, Cas8, GRP78, TNF-alpha, Hk1 and Hk2 antibodies were performed. 20X images were taken under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIHC staining measurements were performed using ImageJ application. Statistical analysis and significance calculation were performed using Graphpad application. Graphs were created by comparing all groups with each other.\u003c/p\u003e"},{"header":"RESULT","content":"\u003cp\u003e\u003cstrong\u003eTable 1: IHC staining results in mouse liver tissues\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"623\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5769%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePTZ + HFD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePTZ + IF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15.0641%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePTZ + CD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePTZ + KD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.29487%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5769%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCas-3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e66.06\u0026plusmn;4.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e73.73\u0026plusmn;5.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e69.43\u0026plusmn;3.66\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.0641%;\"\u003e\n \u003cp\u003e74.87\u0026plusmn;4.45\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e78.79\u0026plusmn;8.57\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.29487%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5769%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCas-8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e49.67\u0026plusmn;10.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e74.22\u0026plusmn;4.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e63.81\u0026plusmn;13.85\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.0641%;\"\u003e\n \u003cp\u003e73.74\u0026plusmn;5.72\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e90.82\u0026plusmn;7.01\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.29487%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5769%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTNF-a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e62.03\u0026plusmn;8.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e68.98\u0026plusmn;10.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e64.00\u0026plusmn;8.72\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.0641%;\"\u003e\n \u003cp\u003e80.35\u0026plusmn;6.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e89.12\u0026plusmn;6.40\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.29487%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5769%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGRP78\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e72.4\u0026plusmn;5.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e85.32\u0026plusmn;8.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e69.78\u0026plusmn;3.23\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.0641%;\"\u003e\n \u003cp\u003e73.54\u0026plusmn;7.39\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e76.66\u0026plusmn;8.91\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.29487%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5769%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHk1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e78.66\u0026plusmn;5.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e86.16\u0026plusmn;12.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e77.8\u0026plusmn;5.58\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.0641%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 80.0\u0026plusmn;7.53\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e97.27\u0026plusmn;9.08\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.29487%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5769%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHk2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e106.6\u0026plusmn;11.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e103.3\u0026plusmn;8.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1859%;\"\u003e\n \u003cp\u003e96.96\u0026plusmn;9.69\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.0641%;\"\u003e\n \u003cp\u003e97.74\u0026plusmn;9.30\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3462%;\"\u003e\n \u003cp\u003e94.93\u0026plusmn;12.73\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.29487%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eP\u0026lt;0.05: considered statistically significant (C: control group, PTZ+HFD: high-fat diet group, PTZ+IF: intermittent fasting group, PTZ+CD: standard rodent diet group, PTZ+KD: ketogenic diet group).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen Cas3 expression was evaluated, a statistically significant difference was found between the groups (p\u0026lt;0.05). It was found to be significantly higher in the PTZ+KD group compared to all other groups. The fact that the Cas3 expression level in the PTZ+KD group was higher than that in the PTZ+CD group indicates that the number of cells undergoing apoptosis in the liver was higher than in animals fed a normal diet. As expected, the group with the lowest Cas3 expression was the control group, followed closely by the PTZ+IF group.\u003c/p\u003e\n\u003cp\u003eWhen Cas8 expression was evaluated, a statistically significant difference was found between the groups (p\u0026lt;0.05). It was found to be statistically significantly higher in the PTZ+KD group compared to all other groups. The higher Cas8 expression level in the PTZ+KD group compared to the PTZ+CD group indicates that the number of cells undergoing apoptosis in the liver is higher than in animals fed a normal diet. As expected, the group with the lowest Cas8 expression was the control group, while the group closest to it was the PTZ+IF group.\u003c/p\u003e\n\u003cp\u003eWhen TNF-a expression was evaluated, a statistically significant difference was found between the groups (p\u0026lt;0.05). TNF-a expression was found to be significantly higher in the PTZ+KD group compared to all other groups. The higher TNF-\u0026alpha; expression level in the PTZ+KD group compared to the PTZ+CD group indicates that inflammation in the liver is greater than in animals fed a normal diet. When the experimental groups were compared, the lowest expression was found to be significantly lower in the PTZ+IF group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen GRP78 expression was evaluated, a statistically significant difference was found between the groups (p\u0026lt;0.05). GRP78 expression was found to be significantly higher in the PTZ+HFD group compared to all other groups. The group with the lowest expression was the PTZ+IF group.\u003c/p\u003e\n\u003cp\u003eWhen Hk1 expression was evaluated, a statistically significant difference was found between the groups (p\u0026lt;0.05). Hk1 expression was found to be significantly higher in the PTZ+KD group compared to all other groups. The lowest expression level was found to be significantly lower in the PTZ+IF group.\u003c/p\u003e\n\u003cp\u003eWhen Hk2 expression was evaluated, a statistically significant difference was found between the groups (p\u0026lt;0.05). Hk2 expression was found to be significantly higher in the control group compared to all other groups. The lowest expression level was found to be significantly lower in the PTZ+KD group.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAnticonvulsant drugs are the first choice for treating epilepsy patients. However, in drug-resistant cases, non-pharmacological dietary treatments are often preferred (Ruan et al., 2022). The aim of the KD is to induce and maintain a state of nutritional ketosis through an increase in hepatic ketogenesis. The increased production of liver ketone bodies is the result of fatty acid oxidation, which mimics a state of starvation. Thus, ketogenesis increases as the use of acetyl CoA in the Krebs cycle decreases (Simeone et al., 2018). Although the effect of the ketogenic diet on preventing seizure frequency cannot be fully explained, various mechanisms, including inhibition of glycolysis, disruption of glutamatergic synaptic transmission, and activation of ATP-sensitive potassium channels, are thought to be involved (Lutas and Yellen, 2013).\u003c/p\u003e \u003cp\u003eIt is important to evaluate the effects of different dietary interventions in patients with epilepsy on peripheral tissues and organs and systemic inflammation in order to understand the pathogenesis of the disease (Kirchner et al., 2020). In particular, the potential side effects of high-fat diets in individuals are a cause for concern (Zhang et al., 2016). Therefore, there is a need to investigate dietary models that may have fewer side effects on peripheral organs in addition to reducing seizure frequency. Proinflammatory cytokines such as TNF-α are one of the markers used to detect inflammation (Horiuchi et al., 2010). In the study conducted by Zhang et al., it was reported that TNF-α expression increased in the liver tissue of mice fed a KD for 3 months, indicating a proinflammatory state, and molecular signs of hepatic steatosis were observed (Zhang et al., 2016). In our study, TNF-α levels were significantly higher in mice fed a KD compared to other experimental groups.\u003c/p\u003e \u003cp\u003eCas3 is a lysosomal enzyme specific for the detection of apoptotic cells in the apoptotic pathway (Asadi et al., 2022). Cas8, on the other hand, is an initiator caspase specifically involved in the activation of apoptotic pathways. The high expression of Cas3 and Cas8 in the experimental group fed a ketogenic diet indicates that hepatocytes underwent more apoptosis compared to other experimental groups. When examining other experimental groups, the lowest expression level was observed in the group fed intermittent fasting. In a study by Mu et al. on rats with an infantile epilepsy model, probiotic supplementation was added to the ketogenic diet to reduce the hepatic steatosis induced by the diet, and the potential of its use in combination with KD to prevent liver steatosis was emphasised (Mu et al., 2022).\u003c/p\u003e \u003cp\u003eStudies have shown that intermittent fasting diets can vary. In a study conducted by Ma et al. on mice, liver histopathology was evaluated after 12 hours of intermittent fasting was applied for one month, and it was stated that hepatocyte plaques developed well and sinusoids were clearly visible (Ma et al., 2021). In a study by Garbow et al., it was shown that the expression of CHOP/GADD153, a proapoptotic antibody, was significantly increased in the liver tissues of mice fed a KD compared to the control group. In our study, it was shown that it increased in mice fed a HFD, but there was no similar increase in the KD group (Garbow et al., 2011).\u003c/p\u003e \u003cp\u003eHexokinases are a family of four enzymes found in all tissues, primarily in the liver. In the first stage of glycolysis, they transfer a phosphate group from ATP to a glucose molecule. The phosphate group enables glucose to be retained within the cell. It is an irreversible enzyme used only in the glycolysis phase. In ketogenic diets, since the glycolysis phase is not actively used, the gluconeolytic pathway becomes active. In our study, it was shown that Hk2 was lower in the KD group compared to other groups, as expected.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was supported by Erciyes University Scientific Research Projects Unit with project number TYL-2020-10701.\u003c/p\u003e\n\u003ch2\u003eThanks\u003c/h2\u003e\n\u003cp\u003eI would like to thank Erciyes University Bet\u0026uuml;l Ziya Eren Genome and Stem Cell (GENK\u0026Ouml;K) Center, where part of the study was carried out, Experimental Research Application and Research Center (DEKAM), GENK\u0026Ouml;K staff.\u003c/p\u003e\n\u003ch2\u003eEthic:\u003c/h2\u003e\n\u003cp\u003eIn this study, approval was received from Erciyes University Animal Experiments Ethics Committee dated 04.09.2025 and numbered 24/158.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArmeno M, Caraballo R, Vaccarezza M, et al. Consenso nacional sobre dieta cetog\u0026eacute;nica [National consensus on the ketogenic diet]. Rev Neurol. 2014;59(5):213-223.\u003c/li\u003e\n \u003cli\u003eAsadi M, Taghizadeh S, Kaviani E, et al. Caspase-3: Structure, function, and biotechnological aspects.\u0026nbsp;Biotechnol Appl Biochem. 2022;69(4):1633-1645. doi:10.1002/bab.2233\u003c/li\u003e\n \u003cli\u003eD\u0026apos;Andrea Meira I, Rom\u0026atilde;o TT, Pires do Prado HJ, Kr\u0026uuml;ger LT, Pires MEP, da Concei\u0026ccedil;\u0026atilde;o PO. Ketogenic Diet and Epilepsy: What We Know So Far.\u0026nbsp;Front Neurosci. 2019;13:5. Published 2019 Jan 29. doi:10.3389/fnins.2019.00005\u003c/li\u003e\n \u003cli\u003eGarbow JR, Doherty JM, Schugar RC, et al. Hepatic steatosis, inflammation, and ER stress in mice maintained long term on a very low-carbohydrate ketogenic diet.\u0026nbsp;Am J Physiol Gastrointest Liver Physiol. 2011;300(6):G956-G967. doi:10.1152/ajpgi.00539.2010\u003c/li\u003e\n \u003cli\u003eHoriuchi, T., Mitoma, H., Harashima, S. I., Tsukamoto, H., \u0026amp; Shimoda, T. Transmembrane TNF-\u0026alpha;: structure, function and interaction with anti-TNF agents.\u0026nbsp;Rheumatology, 2010, 49.7: 1215-1228.\u003c/li\u003e\n \u003cli\u003eKirchner A, Dachet F, Loeb JA. Identifying targets for preventing epilepsy using systems biology of the human brain.\u0026nbsp;Neuropharmacology. 2020;168:107757. doi:10.1016/j.neuropharm.2019.107757\u003c/li\u003e\n \u003cli\u003eLutas A, Yellen G. The ketogenic diet: metabolic influences on brain excitability and epilepsy. Trends Neurosci. 2013 Jan;36(1):32-40. doi: 10.1016/j.tins.2012.11.005. Epub 2012 Dec 8. PMID: 23228828; PMCID: PMC3534786.\u003c/li\u003e\n \u003cli\u003eMa J, Cheng Y, Su Q, et al. Effects of intermittent fasting on liver physiology and metabolism in mice.\u0026nbsp;Exp Ther Med. 2021;22(3):950. doi:10.3892/etm.2021.10382\u003c/li\u003e\n \u003cli\u003eMa J, Cheng Y, Su Q, et al. Effects of intermittent fasting on liver physiology and metabolism in mice.\u0026nbsp;Exp Ther Med. 2021;22(3):950. doi:10.3892/etm.2021.10382\u003c/li\u003e\n \u003cli\u003eMartin-McGill KJ, Bresnahan R, Levy RG, Cooper PN. Ketogenic diets for drug-resistant epilepsy.\u0026nbsp;Cochrane Database Syst Rev. 2020;6(6):CD001903. Published 2020 Jun 24. doi:10.1002/14651858.CD001903.pub5\u003c/li\u003e\n \u003cli\u003eMu C, Nikpoor N, Tompkins TA, Rho JM, Scantlebury MH, Shearer J. Probiotics counteract hepatic steatosis caused by ketogenic diet and upregulate AMPK signaling in a model of infantile epilepsy.\u0026nbsp;EBioMedicine. 2022;76:103838. doi:10.1016/j.ebiom.2022.103838\u003c/li\u003e\n \u003cli\u003eNgugi AK, Kariuki SM, Bottomley C, Kleinschmidt I, Sander JW, Newton CR. Incidence of epilepsy: a systematic review and meta-analysis.\u0026nbsp;Neurology. 2011;77(10):1005-1012. doi:10.1212/WNL.0b013e31822cfc90 Rezaei S, Abdurahman AA, Saghazadeh A, Badv RS, Mahmoudi M. Short-term and long-term efficacy of classical ketogenic diet and modified Atkins diet in children and adolescents with epilepsy: a systematic review and meta-analysis. Nutr Neurosci. 2019;22:317\u0026ndash;34.\u003c/li\u003e\n \u003cli\u003eRho JM, Boison D. The metabolic basis of epilepsy.\u0026nbsp;Nat Rev Neurol. 2022;18(6):333-347. doi:10.1038/s41582-022-00651-8\u003c/li\u003e\n \u003cli\u003eRuan Y, Chen L, She D, Chung Y, Ge L, Han L. Ketogenic diet for epilepsy: an overview of systematic review and meta-analysis.\u0026nbsp;Eur J Clin Nutr. 2022;76(9):1234-1244. doi:10.1038/s41430-021-01060-8\u003c/li\u003e\n \u003cli\u003eSarı\u0026ccedil;i\u0026ccedil;ek S, Tosunbayraktar R, Yaşar Fırat Y, Kara A, Erdoğan FF. Assessment of ketogenic diet, intermittent fasting and high fat diet effect on BDNF and behaviour in epilepsy mice models. Research Square. Published June 2025. doi:10.21203/rs.3.rs-6801463/v1\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSimeone TA, Simeone KA, Stafstrom CE, Rho JM. Do ketone bodies mediate the anti-seizure effects of the ketogenic diet? Neuropharmacology. 2018;133:233\u0026ndash;41.\u003c/li\u003e\n \u003cli\u003eTang F, Hartz AMS, Bauer B. Drug-Resistant\u0026nbsp;Epilepsy: Multiple Hypotheses, Few Answers. Front Neurol. 2017;8:301. Published 2017 Jul 6. doi:10.3389/fneur.2017.00301\u003c/li\u003e\n \u003cli\u003eUłamek-Kozioł\u0026nbsp;M,\u0026nbsp;Czuczwar\u0026nbsp;SJ,\u0026nbsp;Pluta\u0026nbsp;R,\u0026nbsp;Januszewski\u0026nbsp;S.\u0026nbsp;Ketogenic diet and epilepsy. Nutrients. MDPI AG.\u0026nbsp;2019;11.\u003c/li\u003e\n \u003cli\u003eZhang X, Qin J, Zhao Y, et al. Long-term ketogenic diet contributes to glycemic control but promotes lipid accumulation and hepatic steatosis in type 2 diabetic mice. Nutr Res. 2016;36(4):349-358. doi:10.1016/j.nutres.2015.12.002\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Graph","content":"\u003cp\u003eGraph 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"9247b06b-afa4-4aa3-9662-5c4d498a3656","identifier":"10.13039/501100016209","name":"Bilimsel Araştırma Projeleri, Erciyes Üniversitesi","awardNumber":"10701","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Erciyes University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"epilepsy, diet, liverpathology, intermittent fasting, hexokinase, liver, ketogenic diet, high-fat diet","lastPublishedDoi":"10.21203/rs.3.rs-6880346/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6880346/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e In this study, it was aimed to evaluate the effects of ketogenic diet, intermittent fasting and high-fat diet models on the liver in Balb-c mice in which epilepsy pentylenetetrazole (PTZ) kindling model was created.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and methods:\u003c/strong\u003e 40 mice which is 8-week-old Balb/c were used in the study. To create a pentylenetetrazole kindling model, a total of 15 doses of 30mg/kg PTZl injection were administered every other day. Dietary practices were started simultaneously with the first PTZ injection. Mice; They were divided into 4 groups as standard rodent diet, high-fat diet, intermittent fasting and ketogenic diet. At the end of the three-month experimental period, all groups were sacrificed. Liver tissues were taken from mice and fixed in 4% formaldehyde solution and embedded in paraffin blocks. After deparaffinization procedures, 5 µm sections were taken on the microtome device. Immunohistochemistry with Cas3, Cas8, CHOP, GRP78 and TNF-alpha antibodies; Hematoxylin staining was performed to analyze the pathological status. Statistical analyzes were made in the Graphpad application by taking 20X images under the microscope. Graphs were created by comparing all groups with each other.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWhile the group with the highest expression of Cas3, Cas8 and TNF-a was the PTZ+KD group, the group with the highest expression of GRP78 was PTZ+HFD. When Cas3, Cas 8, TNF-a and GRP78 expressions were examined in the experimental groups, the lowest expression level was observed in the PTZ+IF group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e We suggest that intermittent fasting diets can be used as an alternative, at least considering the benefits for the liver, but we emphasize the need for further studies.\u003c/p\u003e","manuscriptTitle":"Investigation of the Effects of Different Dietary Treatment Methods on Liver Apoptosis in Mice With Pentylenetetrazole Induced Epilepsy Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 07:53:50","doi":"10.21203/rs.3.rs-6880346/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":"ff434f25-63d8-402e-8a38-96eacdc64a51","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49982435,"name":"Nutrition \u0026 Dietetics"},{"id":49982436,"name":"Neurobiology of Disease"}],"tags":[],"updatedAt":"2025-06-16T07:53:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-16 07:53:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6880346","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6880346","identity":"rs-6880346","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}