Melatonin Ameliorates High Fat Diet Induced Oxidative Stress-Mediated Insulin Resistance, Amyloid Burden and Memory Dysfunction in Female Mice and their offspring

Melatonin Ameliorates High Fat Diet Induced Oxidative Stress-Mediated Insulin Resistance, Amyloid Burden and Memory Dysfunction in Female Mice and their offspring | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Melatonin Ameliorates High Fat Diet Induced Oxidative Stress-Mediated Insulin Resistance, Amyloid Burden and Memory Dysfunction in Female Mice and their offspring Amin Jan, Mohsin Shah, Shahid Ali Shah, Hamid Habib Syed, Ehtesham Ehtesham This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3376865/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 High fat diet (HFD) is a prime factor, which contributes to the present epidemic of metabolic syndrome. HFD induces oxidative stress (OS) that in turn causes neuroinflammation, neurodegeneration, synaptic dysfunction, insulin resistance and amyloid burden. Pineal gland secretes a potent anti-apoptotic hormone, melatonin that has the potential to nullify the toxic effect of reactive oxygen species (ROS) and ameliorate various complications induced by HFD in rodent models. We aimed to find the therapeutic effects of melatonin against HFD-induced neuroinflammation and neurodegeneration mediated by OS in pregnant mice and their offspring. The neuroprotective effects of melatonin against HFD induced neurotoxicity were analyzed by Western blotting and immunofluorescence in the brain homogenates of pregnant albino mice and their offspring. Short and long term memory loss was evaluated. HFD significantly induced OS leading to neuroinflammation and apoptotic neurodegeneration in cortex and hippocampus of the pregnant mice and their offspring. However, administration of melatonin with HFD significantly improved synaptic dysfunction and inhibited both the neuroinflammation and neurodegeneration in albino mice. Here, we report that melatonin exerts these effects via SIRT1 mediated NRF-2/HO-1 signaling pathway to reduce HFD-induced OS and insulin resistance. This study revealed that melatonin could reverse HFD induced multiple complications leading to the memory dysfunction in pregnant female mice and their successive generation via activation of SIRT1/NRF2 signaling pathway. HFD Obesity Neuroinflammation Neurodegeneration Melatonin Treatment Synaptic Dysfunction ROS Oxidative Stress SIRT1/NRF2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Diet containing high amount of refined sugars and saturated fats is a prime factor contributing to the present epidemic of metabolic syndrome and insulin resistance [1]. HFD causes metabolic disorders which can have negative implication on cognition, behavior, and brain health in general [2]. In human studies, HFD, obesity, DM, and other metabolic disorders have shown a strong correlation with the development of Alzheimer’s disease (AD) and other cognitive impairments [3,4]. Inflammation in various parts of the brain is linked to abnormal behavioral, cognitive dysfunction and AD [5]. It is previously reported that feeding mice with HFD for 14 weeks affects brain insulin signaling and cognitive capabilities [6]. In addition, Amyloid β production in brain is increased after 16 weeks HFD which further exacerbated AD [7]. Furthermore, HFD accelerates the progression of AD-like pathology by increasing brain amyloid deposition and oxidative stress (OS) [8]. Consumption of HFDs has shown to develop obesity and peripheral insulin resistance as well as disruption of blood brain barrier (BBB) [9]. As a result, glucose transport to the brain decreases that lead to the reduction of brain insulin and peripheral hyper-insulinemia. Therefore, it is presumed that HFD-induced peripheral hyperinsulinemia might cause brain insulin deficit leading to reduction in insulin degrading enzyme [10]. In addition, in the cortex and hippocampus, HFD enhances the production of Aβ [9]. This Aβ can be found in a variety of forms in the brain, including monomers, oligomers and insoluble fibrils that form plaques outside the cells. The soluble form of oligomeric Aβ decreases synapses and hinders long-term potentiation (LTP) [11,12]. Melatonin is an important hormone secreted mainly by the pineal gland [13]. It acts as a potent anti-oxidant, anti-apoptotic and anti-inflammatory agent [14]. In addition, it upregulates several antioxidant enzymes and subsequently downregulate other pro-oxidants [15], in both pre-clinical and clinical studies by modifying pro and anti-inflammatory cytokines [16,17]. Melatonin also has anti-apoptotic properties mainly by inhibiting the reduction of pBad and pAkt levels [18]. Maternal circulating melatonin levels gradually increase after 24 weeks of pregnancy spiking at 32 weeks and then reverting to baseline after delivery in a normal pregnancy [19]. Human placental melatonin levels are 6 to 7 times higher in comparison to plasma levels, suggesting a role in placental development [20]. Previous reports have shown that melatonin activate SIRT1 which in turn reduce oxidative stress, inflammation and neurodegeneration in various models by stimulating Nrf2/HO-1 signaling pathway [21]. Here, we investigated the SIRT1 mediated NRF2/HO-1 signaling pathway of melatonin against HFD-induced OS and impaired insulin-signaling in the cortex and hippocampus of dams and their offspring. We also assessed the effects of melatonin against HFD-induced changes in Aβ and synaptic markers in the dams and their offspring providing a potential useful therapeutic index for preventing neurodegeneration and its consequences. MATERIALS AND METHOD Additional details for all procedures are given in the Supplementary Materials and Methods. Animals Healthy adult female albino mice (7-8 weeks old and 30 g body weight) were purchased from Riphah International University Islamabad, Pakistan. Sample size comprised of 60 albino mice (36 female adult mice and 24 offspring), calculated via resource equation approach. Female adult mice were randomly distributed into three groups, i.e.; a control group (n=13), a HFD group (n=13) and HFD + Melatonin group (n=10). Control group mice were fed with a normal standard diet. HFD group mice were fed ad libitum on commercially available obesogenic HFD, containing 45% fats, 35% carbohydrates, and 20% proteins per dry weight [22]. After successful conception, melatonin treatment was started to one HFD group while keeping the animals on HFD until the termination of pregnancy. Study design and approval This was an experimental study carried out at the Institute of Basic Medical Sciences (IBMS), Khyber Medical University (KMU), Peshawar, Pakistan, under institutional Ethical board approval number (Dir/KMU EB/EM/000806). The study consisted of three maternal and two offspring interventions, described below. Maternal interventions: The adult female mice were distributed into two groups i.e. normal standard diet fed animals (n=13) and HFD fed animals (n=23). After three weeks of HFD and standard diet feeding, one set (n=3) from each group was selected randomly and assessed for behavioral studies and then sacrificed for biochemical measurements. The remaining normal (n=10) and HFD fed mice (n=20) were allowed to mate for about one week time. After successful conception, the HFD fed mice were randomly divided into two groups. Melatonin (10 mg/kg BW) was administered intra-peritoneally to one of the HFD fed pregnant mice group (n=10) until the termination of pregnancy. While the remaining mice (n=10) were kept feeding on HFD without treating with melatonin. 3 mice from each category i.e. normal diet mice, HFD and HFD plus melatonin were subjected to behavioral studies and intraperitoneal glucose tolerance test (IPGTT) and then sacrificed for further biochemical analysis. While 2 mice from the same groups were subjected to immunofluorescence analysis. Remaining female mice (n=5/group) were allowed to complete the weaning period. After completion of the weaning period, 3 mice from each group were again subjected into all the above mentioned experiments, while 2 mice of each group were assessed by immunofluorescence analysis. Offspring intervention : First offspring intervention with three pups of each respective maternal group was carried out at the age of four weeks. Selected proteins were analyzed in the four weeks neonatal intervention. Remaining offspring from respective parental groups (n=5) were kept to grow to the adulthood until they reached 8 weeks. These adult offsprings were subjected to behavioral and other investigations followed by sacrificing to check the expression of different proteins through Western blotting and immunofluorescence techniques. Behavioral tests Therapeutic effects of melatonin on HFD induced memory impairment were evaluated by Y-Maze [23] and Morris Water Maze behavioral tests [24]. The investigator carrying out the behavioral tests was blinded to mice tags and treatment. Western blotting All animals were slaughtered at the end of each intervention after completion of behavioral studies as per method reported previously [21]. Different mouse derived primary antibodies such as anti-BCL-2 (SC-7382), anti-BAX (SC-7480), anti-CAS-3 (SC-7272), anti-PARP-1 (SC-8007), anti-SIRT1 (SC-74504), anti-NRF-2 (SC-365949), anti-HO-1 (SC-136960), anti-Aβ (SC-28365), anti-BACE1 (SC-33711), anti-PSD95 (SC-71933), anti-SYP (SC-17750), anti-p-IRS (SC-8038), anti-p-AKT (SC-514032), anti-p-GSK3B (SC-81496), anti-TNF-α (SC-52746), anti-COX2 (SC-376861), anti-IL-1β (SC-32294) and β-actin (SC-47778) of Santa Cruz Biotech, CA, USA were used to detect and localize protein of interest. ECL was used to detect cross-reacting proteins after incubation with horseradish peroxidase-conjugated secondary antibodies. ImageJ software was used to perform the densitometry analysis of the bands in arbitrary units (A.U.) relative to the untreated control. Detailed procedure can be seen in the supplementary methods. Tissue preparation and immunofluorescence Equal number of mice (n=2) were kept in each group for conducting morphological study. Primary anti-bodies such as anti-SIRT1, anti NF-κB and anti-Aβ (1:100 in PBS) were used to detect protein of interest. Secondary anti-bodies (FITC and TRITC conjugated, Santa Cruz, 1:50 in PBS) were used for cross reaction with the primary antibodies. Immunofluorescence staining was done as reported previously [25]. Images were captured with confocal microscopy (FluoView FV 1000 Olympus, Japan). Detailed procedure can be seen in the supplementary methods. Statistical Analysis The original X-ray films of the Western blot analysis were scanned. Image J software was used to perform densitometry analysis of the bands. Integral optical density (IOD) of proteins were expressed in arbitrary units (A.Us) as Mean±S.E.M. Significant difference in the first intervention was determined using t-test whereas significant difference in both the maternal and offspring interventions was determined using one-way analysis of variance (ANOVA), followed by Post- hoc tukey's test. RESULTS HFD feeding induces multiple complications in female mice A statistically significant increase (p<0.001) in the expression of neuroinflammatory markers (TNF-α, COX2 and IL-1β) (Figure 1A), pro-apoptotic marker, PARP-1 (p<0.001) and significant decrease (p<0.001) was observed in the anti-apoptotic BCl-2 in the HFD fed mice. Likewise, significant increase (p<0.001) was observed in the Bax and Caspase-3 levels (Figure 1B). Similarly, significant decrease (p<0.001) in the expression of p-IRS-1, p-AKT and p-GSK3-β levels were observed in the brain homogenates of HFD group as compared to normal chow diet group (Figure 1C). Significant decrease (p<0.001) was also observed in the expression of SIRT-1, Nrf-2 and HO-1 (p<0.01) in HFD group as compared to normal chow diet group (Figure 1D). Maternal obesity/HFD induced an obvious decrease in the synaptic receptor proteins and increased amyloidogenic burden. Significant decrease (p<0.001) in the expression of synaptic proteins (SYP and PSD-95) (Figure 2A), and significant increase (p<0.001) was observed in the expression of BACE-1 and Aβ in HFD group as compared to normal diet group (Figure 2B). After feeding the female mice with HFD for three weeks, the animals showed a significant increase in body weight accompanied by, deranged lipid profiles, oxidative stress and altered behaviour shown in the Supplementary Figure S1. Melatonin exhibits reversal effect on HFD induced complications in the female mice after parturition and post weaning interventions We evaluated the neuroprotective potential of melatonin against HFD induced OS and associated neuroinflammation and neurodegeneration in the cortex and hippocampus of the mice brains. Statistically significant differences (p<0.001) were observed in the expression of neuroinflammatory and neurodegenerative markers. Post hoc comparison revealed that HFD induced significant increase (p<0.001) in the expression of neuroinflammatory markers (TNF-α, IL-1β and COX-2). However, melatonin significantly decreased (p<0.001) all the proinflammatory markers (TNF-α, IL-1β and COX-2) in both post-parturition intervention (Figure 3A) and post-weaning intervention (Figure 3B). Similarly, HFD markedly increased (p<0.001) Bax, Caspase-3 and PARP-1 and decreased (p<0.001) Bcl-2, while melatonin significantly reversed (p<0.001) Bax, BCl-2, Caspase-3 and PARP-1 expression in post-parturition (Figure 3C) and post-weaning interventions (Figure 3D). A significant differences (p<0.001) were also observed in the expression of insulin resistance markers (IRS-1, p-AKT and p-GSK3-β). Post hoc tukey’s analysis showed significant decrease (p<0.001) in the expression of IRS-1, p-AKT and p-GSK3-β proteins in the brain homogenates of HFD group as compared to control group in both maternal interventions. Melatonin, however showed significant increase (p<0.001) in the expression of almost all the assessed markers (IRS-1, p-AKT and p-GSK3-β) in post-parturition intervention (Figure 4A) both maternal interventions (Figure 4A and 4B). Melatonin activated the SIRT1/Nrf2 pathway to reduce HFD induced OS. A statistically significant differences (p<0.001) were observed in the expression of signaling proteins. Nrf-2 and HO-1 along with a significant decrement in SIRT-1 (p<0.001) in the in HFD group was observed as compared to control group. Melatonin, on the other hand markedly increased (p<0.05) the expression of the signaling pathway proteins (SIRT1/Nrf2/HO-1) in the post-parturition intervention (Figure 4C). However, highly significant increase (p<0.001) was observed in the expression levels of SIRT1, Nrf2 and HO-1 in the post-weaning intervention (Figure 4D). HFD significantly decreased (p<0.001) synaptic receptor proteins (SYP & PSD95) and significantly increased amyloid burden proteins (BACE-1 and Aβ) in both interventions. Melatonin, however markedly increased the expression of SYP (p<0.01) & PSD95 (p<0.001) (Figure 5A and 5B) and decreased (p<0.001) BACE-1 and Aβ in the mice brains in both post-parturition and post-weaning interventions (Figure 5C and 5D). Selected proteins Aβ, NF-kB and SIRT-1 expressions were analysed through immunofluorescence technique. Significant differences (p<0.001) were revealed in the expression of these proteins among various groups. Post hoc tukey test showed significant increase (p<0.001) in the expression of the NF-kB and Aβ proteins and significant decrease (p<0.001) in SIRT1 expression in HFD group as compared to normal chow diet group. Melatonin treatment significantly rectified (p<0.001) the effect of HFD in both post-parturition and post- weaning intervention (Figure 7). Maternal melatonin treatment protects mice offspring from the deteriorating effects of HFD Maternal HFD and HFD + melatonin effects were observed in two offspring’s interventions at the age of 4 and 8 weeks respectively. Post hoc tukey’s analysis showed significant increase (p<0.001) in the expression of neuro-inflammatory markers (TNF-α and IL-1β) in the HFD groups of neonatal mice as compared to control group (Figure 7A). Similarly, a significant increase (p<0.001) was observed in the expression of apoptotic markers (Caspase-3 and PARP-1) in HFD group neonatal mice. Melatonin meticulously reversed all the damages caused by HFD. A significant decrease of pro-inflammatory markers, TNF-α (p<0.01) and IL-1β (p<0.05) was observed (Figure 8A) along with a highly significant decrease (p<0.001) in the pro-apoptotic marker in the brain homogenates of young offsprings of HFD + Melatonin group (Figure 7B). Neuro-inflammatory and amyloidogenic markers were also investigated in the brain homogenates of the adult offsprings. Significant increase (p<0.001) in the neuro-inflammatory markers i.e.; TNF-α, IL-1β and COX-2 levels was observed in the HFD groups of adult mice as compared to control group. Similarly, a significant increase (p<0.001) was observed in the expression of BACE-1 and Aβ proteins in HFD group adult mice as compared to normal standard diet group. Melatonin successfully reversed all the damages caused by HFD. Post hoc tukey test showed highly significant decrease (p<0.001) in the pro-inflammatory markers (TNF-α, IL-1β and COX-2) levels (Figure 7C) along with significant decrease (p<0.001) in the expression of BACE-1 and Aβ proteins in the brain homogenates of the HFD + melatonin group adult mice (Figure 7D). Significant differences (p<0.001) were observed in the expression of insulin resistance markers (p-IRS-1, p-AKT and p-GSK3β) among various groups of adult offsprings. Significant decrease (p<0.001) in the expression of p-IRS-1, p-AKT and p-GSK3β was observed in the HFD group adult mice brain. Melatonin on the other hand significantly retrieved (p<0.001) these markers (Figure 8A). SIRT-1 expression and activity was analysed in both young and adult offsprings. Significant differences (p<0.001) were observed among the expression levels of SIRT-1 signaling proteins. Significant decrease (p<0.001) in the expression of NRF-2 and HO-1 along with SIRT-1 levels in HFD group neonatal and adult mice were observed as compared to the control group. Melatonin however, successfully rectified this decrease in both the offspring interventions. Significant increase (p<0.001) in SIRT-1, Nrf-2 and HO-1 expression levels were observed in HFD + melatonin group adult offsprings (Figure 8B). Similarly, melatonin significantly increased (p<0.001) the expression levels of the signalling protein in HFD + melatonin group neonatal mice (Figure 8C). Selected proteins NF-kB, SIRT-1 and Aβ proteins expressions were analysed through immunofluorescence technique. Post hoc comparison demonstrate significant increase (p<0.001) in the expression of the NF-kB and Aβ proteins and significant decrease (p<0.001) in SIRT1 expression in HFD group. Melatonin treatment significantly improved (p<0.001) the detrimental effects of HFD in the HFD + Melatonin group (Figure 9A and 9B). DISCUSSION Prolonged consumption of HFD causes obesity which in turn may lead to the development of diseases like cardiovascular disease, certain types of cancer and type 2 diabetes mellitus [26]. Adipocytes play a contributing role in inflammation during progression of obesity by secreting common cytokines like IL-1, IL-6, and TNFα etc. [27]. Proinflammatory cytokines (IL-1β, IL-6, and TNF-α) initiate inflammation in response to multiple factors as they can cross the blood brain barrier [28]. Our study revealed that brains homogenates of HFD fed mice showed increased neuroinflammatory (TNF-α, IL-1β and COX2) and neurodegenerative (Bax, BCl-2, Caspase-3 and PARP-1) proteins expression as compared to control diet fed mice. Melatonin’s ability to regulate immune system modulation along with reduction in acute and chronic inflammation has been reported previously [30]. Various studies suggest that melatonin modifies proinflammatory cytokines and exerts anti-inflammatory effects in various pathophysiological conditions [31,32]. According to Liu et al. (2013), melatonin through its action on inflammasome genes expression, reduces the LPS-induced inflammation in mouse adipose tissue [33]. Here, we observed that chronic consumption of HFD increases both neuroinflammation and neurodegeneration in the dams and their offsprings but subsequent treatment with melatonin was found effective in reducing these neuroinflammatory and neurodegenerative markers in both the maternal and offsprings’ interventions. Multiple studies reported that diet containing high amount of refined sugar and saturated fats can negatively affect cognitive functions [34,35]. HFD exacerbates cognitive decline along with other conditions such as chronic stress, sleep deprivation, and aging [36]. Consistent with these results, we observed, HFD induced oxidative stress mediated synaptic dysfunction and increased amyloidogenic burden with gross decline in the expression of synaptic proteins SYP and PSD95 and significant increase in BACE-1 and Aβ expression showing amyloidogenic burden in all the maternal and adult intervention. Matsubara et al. (2003) showed that melatonin administration to APP695 transgenic mice was associated with a decrease in brain Aβ levels [37]. The underlying mechanism of this decrease in the brain amyloid is still not clear, but it could be due to a direct action of melatonin on the metabolism of β-amyloid precursor protein [38]. In our study, administration of melatonin during pregnancy has long lasting effect on restoring memory, synaptotoxicity and amyloid burden by significantly increasing, synaptic proteins (SYP and PSD95) and decreasing amyloid protein (BACE1 and Aβ) in the brain homogenates. It has previously been shown that diets containing high amount of saturated fat are usually associated with obesity, IR, and T2DM [39]. McNeilly et al. (2015) reported raised fasting blood glucose and insulin levels in rats fed on HFD [40]. Similarly, Kothari et al. (2017) reported that HFD resulted in peripheral and central insulin resistance [41]. On the other hand Brydon et al. (2001) showed that melatonin activates MT2 receptors in human adipocytes which modulates glucose uptake [42]. The present study is in agreement with the above mentioned studies, where we found insulin signaling impairment in HFD fed female mice and their offsprings. Similarly, after the melatonin treatment during pregnancy significant restoration was observed in the expression of p-IRS, p-Akt and p-GSK3β proteins in both maternal and offspring intervention. Several studies have revealed that silent information regulator transcript-1 (SIRT1) reduces oxidative stress level and the extent of inflammation [43,44]. Nuclear factor erythroid-2 related factor 2 (Nrf2) is an important antioxidant protein essential for cellular defense [45]. Once activated, Nrf2 is translocated to the nucleus from the cytoplasm and interacts with the antioxidant defense system to facilitate transcription of hemeoxygenase 1 (HO-1). HO-1 increases resistance to OS and exhibit protective role against inflammation [46]. Shahid et al. (2017) confirmed the stimulation of SIRT1/Nrf2 signaling pathway by melatonin against LPS-induced oxidative stress in of the hippocampus of PND7 rats [21]. Here, we found that HFD significantly decreased SIRT1 expression and activity in female mice and their offsprings. Melatonin administration on the other hand reinstated brain SIRT1 expression with antagonistic effect to HFD induced OS in all the interventions of the mothers fed on HFD and their neonates and adults mice. The present study is in agreement with previous studies which report that melatonin is an important antioxidant and has a strong tendency to mitigate HFD-induced decreased expression of Nrf-2 and HO-1 in both maternal and offspring interventions. This study has some limitations. Here, we observed the therapeutic effects of melatonin only in the offspring groups of their own respective maternal groups. Cross-fostering of the neonates was not possible because mothers used to eat pups from the other groups despite having free access to food and water. Moreover, this study is conducted on four weeks and eight weeks adolescent offspring probably because of the lack of expertise to deal the neonatal brain samples. CONCLUSION We conclude that HFD induced neuro-inflammation and neuro-degeneration in pregnant mice and their offsprings via oxidative stress. Oxidative stress further triggered insulin resistance and amyloid-β generation in the brains of pregnant mice and their offsprings. Melatonin stabilized the damages caused by HFD by activating SIRT1/NRF-2/HO-1 signaling pathway, thus rectified the HFD induced toxic effects. Since there are limited remedies to effectively treat HFD induced brain damages, this study could be helpful in the development of potential treatment strategies in obese pregnant mothers. Declarations Ethical approval: The institutional Ethical board granted ethical approval to the study (Dir/KMU EB/EM/000806). Competing interest: None declared. Author’s contributions: AJ was involved in the execution of the project and writing of the manuscript. MS supervised, designed, executed the study and wrote the manuscript. SAS co-supervised the study and provided clinical expertise. SHH and E did critical editing of the manuscript. All the authors have read and approved the final version of the manuscript. Funding information: This research was supported by the Organization of Research Innovation and Commercialization of Khyber Medical University. Acknowledgment: The authors acknowledge with thanks Organization of Research Innovation and commercialization of Khyber Medical University for their financial and technical support. Data availability: Available from the corresponding author on reasonable request. References Grundy SM. Metabolic syndrome update. Trends Cardiovasc Med. 2016;26:364–73. Pasinetti GM, Eberstein JA. Metabolic syndrome and the role of dietary lifestyles in Alzheimer’s disease. J Neurochem. 2008;106:1503–14. Profenno LA, Porsteinsson AP, Faraone SV. Meta-analysis of Alzheimer’s disease risk with obesity, diabetes, and related disorders. Biol Psychiatry. 2010;67:505–12. Arshad N, Lin TS, Yahaya MF. Metabolic syndrome and its effect on the brain: possible mechanism. CNS Neurol Disord-Drug Targets Former Curr Drug Targets-CNS Neurol Disord. 2018;17:595–603. Almeida-Suhett CP, Graham A, Chen Y, Deuster P. Behavioral changes in male mice fed a high-fat diet are associated with IL-1β expression in specific brain regions. Physiol Behav. 2017;169:130–40. Kothari V, Luo Y, Tornabene T, O’Neill AM, Greene MW, Geetha T, et al. High fat diet induces brain insulin resistance and cognitive impairment in mice. Biochim Biophys Acta BBA-Mol Basis Dis. 2017;1863:499–508. Busquets O, Ettcheto M, Pallàs M, Beas-Zarate C, Verdaguer E, Auladell C, et al. Long-term exposition to a high fat diet favors the appearance of β-amyloid depositions in the brain of C57BL/6J mice. A potential model of sporadic Alzheimer’s disease. Mech Ageing Dev. 2017;162:38–45. Lin B, Hasegawa Y, Takane K, Koibuchi N, Cao C, Kim-Mitsuyama S. High-fat-diet intake enhances cerebral amyloid angiopathy and cognitive impairment in a mouse model of Alzheimer’s disease, independently of metabolic disorders. J Am Heart Assoc. 2016;5:e003154. Saiyasit N, Chunchai T, Prus D, Suparan K, Pittayapong P, Apaijai N, et al. Gut dysbiosis develops before metabolic disturbance and cognitive decline in high-fat diet–induced obese condition. Nutrition. 2020;69:110576. Petrov D, Pedrós I, Artiach G, Sureda FX, Barroso E, Pallàs M, et al. High-fat diet-induced deregulation of hippocampal insulin signaling and mitochondrial homeostasis deficiences contribute to Alzheimer disease pathology in rodents. Biochim Biophys Acta BBA-Mol Basis Dis. 2015;1852:1687–99. Butterfield DA, Boyd-Kimball D. Mitochondrial oxidative and nitrosative stress and Alzheimer disease. Antioxidants. 2020;9:818. Mastrocola R, Restivo F, Vercellinatto I, Danni O, Brignardello E, Aragno M, et al. Oxidative and nitrosative stress in brain mitochondria of diabetic rats. J Endocrinol. 2005;187:37–44. Amaral FG do, Cipolla-Neto J. A brief review about melatonin, a pineal hormone. Arch Endocrinol Metab. 2018;62:472–9. Muñoz-Jurado A, Escribano BM, Caballero-Villarraso J, Galván A, Agüera E, Santamaría A, et al. Melatonin and multiple sclerosis: antioxidant, anti-inflammatory and immunomodulator mechanism of action. Inflammopharmacology. 2022;30:1569–96. García-Navarro A, González-Puga C, Escames G, López LC, López A, López-Cantarero M, et al. Cellular mechanisms involved in the melatonin inhibition of HT-29 human colon cancer cell proliferation in culture. J Pineal Res. 2007;43:195–205. Favero G, Franceschetti L, Bonomini F, Rodella LF, Rezzani R. Melatonin as an anti-inflammatory agent modulating inflammasome activation. Int J Endocrinol. 2017;2017. Mauriz JL, Collado PS, Veneroso C, Reiter RJ, González-Gallego J. A review of the molecular aspects of melatonin’s anti-inflammatory actions: recent insights and new perspectives. J Pineal Res. 2013;54:1–14. Koh P-O. Melatonin attenuates the focal cerebral ischemic injury by inhibiting the dissociation of pBad from 14-3-3. J Pineal Res. 2008;44:101–6. Nakamura Y, Tamura H, Kashida S, Takayama H, Yamagata Y, Karube A, et al. Changes of serum melatonin level and its relationship to feto-placental unit during pregnancy. J Pineal Res. 2001;30:29–33. Iwasaki S, Nakazawa K, Sakai J, Kometani K, Iwashita M, Yoshimura Y, et al. Melatonin as a local regulator of human placental function. J Pineal Res. 2005;39:261–5. Shah SA, Khan M, Jo M-H, Jo MG, Amin FU, Kim MO. Melatonin stimulates the SIRT 1/Nrf2 signaling pathway counteracting lipopolysaccharide (LPS)-induced oxidative stress to rescue postnatal rat brain. CNS Neurosci Ther. 2017;23:33–44. Heinonen I, Rinne P, Ruohonen ST, Ruohonen S, Ahotupa M, Savontaus E. The effects of equal caloric high fat and western diet on metabolic syndrome, oxidative stress and vascular endothelial function in mice. Acta Physiol. 2014;211:515–27. Prieur EA, Jadavji NM. Assessing spatial working memory using the spontaneous alternation Y-maze test in aged male mice. Bio-Protoc. 2019;9:e3162–e3162. Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc. 2006;1:848–58. Donaldson JG. Immunofluorescence staining. Curr Protoc Cell Biol. 2015;69:4–3. Kesh SB, Sarkar D, Manna K. High-fat diet-induced oxidative stress and its impact on metabolic syndrome: a review. Asian J Pharm Clin Res. 2016;9:47–52. Kohlgruber A, Lynch L. Adipose tissue inflammation in the pathogenesis of type 2 diabetes. Curr Diab Rep. 2015;15:1–11. Skelly DT, Hennessy E, Dansereau M-A, Cunningham C. A systematic analysis of the peripheral and CNS effects of systemic LPS, IL-1β, TNF-α and IL-6 challenges in C57BL/6 mice. PloS One. 2013;8:e69123. Skelly DT, Hennessy E, Dansereau M-A, Cunningham C. A systematic analysis of the peripheral and CNS effects of systemic LPS, IL-1β, TNF-α and IL-6 challenges in C57BL/6 mice. PloS One. 2013;8:e69123. Carrillo-Vico A, Lardone PJ, Álvarez-Sánchez N, Rodríguez-Rodríguez A, Guerrero JM. Melatonin: buffering the immune system. Int J Mol Sci. 2013;14:8638–83. Nabavi SM, Nabavi SF, Sureda A, Xiao J, Dehpour AR, Shirooie S, et al. Anti-inflammatory effects of Melatonin: A mechanistic review. Crit Rev Food Sci Nutr. 2019;59:S4–16. Favero G, Franceschetti L, Bonomini F, Rodella LF, Rezzani R. Melatonin as an anti-inflammatory agent modulating inflammasome activation. Int J Endocrinol. 2017;2017. Liu X, Gong Y, Xiong K, Ye Y, Xiong Y, Zhuang Z, et al. Melatonin mediates protective effects on inflammatory response induced by interleukin-1 beta in human mesenchymal stem cells. J Pineal Res. 2013;55:14–25. Taylor ZB, Stevenson RJ, Ehrenfeld L, Francis HM. The impact of saturated fat, added sugar and their combination on human hippocampal integrity and function: A systematic review and meta-analysis. Neurosci Biobehav Rev. 2021;130:91–106. Kanoski SE, Davidson TL. Western diet consumption and cognitive impairment: links to hippocampal dysfunction and obesity. Physiol Behav. 2011;103:59–68. Askarova S, Umbayev B, Masoud A-R, Kaiyrlykyzy A, Safarova Y, Tsoy A, et al. The links between the gut microbiome, aging, modern lifestyle and Alzheimer’s disease. Front Cell Infect Microbiol. 2020;10:104. Matsubara E, Bryant-Thomas T, Pacheco Quinto J, Henry TL, Poeggeler B, Herbert D, et al. Melatonin increases survival and inhibits oxidative and amyloid pathology in a transgenic model of Alzheimer’s disease. J Neurochem. 2003;85:1101–8. Lahiri DK. Melatonin affects the metabolism of the β-amyloid precursor protein in different cell types. J Pineal Res. 1999;26:137–46. Parrott MD, Greenwood CE. Dietary influences on cognitive function with aging: from high-fat diets to healthful eating. Ann N Y Acad Sci. 2007;1114:389–97. McNeilly AD, Stewart CA, Sutherland C, Balfour DJ. High fat feeding is associated with stimulation of the hypothalamic-pituitary-adrenal axis and reduced anxiety in the rat. Psychoneuroendocrinology. 2015;52:272–80. Kothari V, Luo Y, Tornabene T, O’Neill AM, Greene MW, Geetha T, et al. High fat diet induces brain insulin resistance and cognitive impairment in mice. Biochim Biophys Acta BBA-Mol Basis Dis. 2017;1863:499–508. Brydon L, Petit L, Delagrange P, Strosberg AD, Jockers R. Functional expression of MT2 (Mel1b) melatonin receptors in human PAZ6 adipocytes. Endocrinology. 2001;142:4264–71. Salminen A, Kaarniranta K, Kauppinen A. Crosstalk between oxidative stress and SIRT1: impact on the aging process. Int J Mol Sci. 2013;14:3834–59. Chan S-H, Hung C-H, Shih J-Y, Chu P-M, Cheng Y-H, Lin H-C, et al. SIRT1 inhibition causes oxidative stress and inflammation in patients with coronary artery disease. Redox Biol. 2017;13:301–9. Kim J, Cha Y-N, Surh Y-J. A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in inflammatory disorders. Mutat Res Mol Mech Mutagen. 2010;690:12–23. Xiang Q, Zhao Y, Lin J, Jiang S, Li W. The Nrf2 antioxidant defense system in intervertebral disc degeneration: Molecular insights. Exp Mol Med. 2022;54:1067–75. Additional Declarations No competing interests reported. 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Also discoverable on Platform About In Review Editorial Policies 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-3376865","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":236231106,"identity":"34931604-f417-4831-bcc3-7c1697450e56","order_by":0,"name":"Amin Jan","email":"","orcid":"","institution":"Khyber Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amin","middleName":"","lastName":"Jan","suffix":""},{"id":236231107,"identity":"19c80bad-c6fb-4196-ae22-d68a51e1622c","order_by":1,"name":"Mohsin Shah","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYFACHgaGBCBpwMDA+LABLALkMhwgTguzIfFaQACohU2SKC267b2HPzzcwSBjLnb8WeXMnMMM/Ow5BswFZ3BrMTtzLk0i8QwDj+XsHLObG7cdZpDseWPAPOMGHi03cswYEtuAfrmdw3bzIVCLwQ2gLTwf8Gox/gDRkv6sEKTFnggtBhIQLQlmjCCHGUiAtOBz2JkzZkAtEiC/GEvO3JbOI3HmWcHhGfi8f7zH+OPPNht7c+n0hx97t1nL8bcnb3xccAy3FiiQgLPA0XSYoAYMwEy6llEwCkbBKBjGAABxcVOj6YL1+AAAAABJRU5ErkJggg==","orcid":"","institution":"Khyber Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mohsin","middleName":"","lastName":"Shah","suffix":""},{"id":236231108,"identity":"a5313309-805e-480e-9a6c-9a1644c1a208","order_by":2,"name":"Shahid Ali Shah","email":"","orcid":"","institution":"Haripur University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shahid","middleName":"Ali","lastName":"Shah","suffix":""},{"id":236231109,"identity":"759fcef7-2413-437a-8dc2-54839e2f4a21","order_by":3,"name":"Hamid Habib Syed","email":"","orcid":"","institution":"Khyber Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hamid","middleName":"Habib","lastName":"Syed","suffix":""},{"id":236231110,"identity":"5812424d-4b9a-451e-b2ce-3dae78e6ae9d","order_by":4,"name":"Ehtesham Ehtesham","email":"","orcid":"","institution":"Khyber Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ehtesham","middleName":"","lastName":"Ehtesham","suffix":""}],"badges":[],"createdAt":"2023-09-22 08:44:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3376865/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3376865/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44081966,"identity":"541ede62-0ab2-48c4-9fac-8f1c695ce17a","added_by":"auto","created_at":"2023-10-04 14:30:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":143571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHFD induced significant changes in the expression levels various markers in the brains of the adult female mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShown are the Western blot results of maternal (A) neuroinflammatory markers (COX2, TNF-α and IL-1β), (B) neurodegeneration markers (BAX, Bcl-2, caspase-3 and PARP-1), (C) insulin resistance markers (p-IRS, p-Akt and p-GSK3β) and (D) signaling proteins (SIRT1, Nrf-2 and HO-1) in pre-pregnancy stage, along with their bar-chart representation respectively. β-Actin was used as a loading control. The results were determined using Image J software and bar-chart indicate mean in A.U ± SEM. Significance of control vs HFD is expressed as #. Significance: ##p\u0026lt;0.01 and ###p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/7605ce9b61dfc9582bc9b8cf.png"},{"id":44081967,"identity":"d928dc66-d94b-4e9d-8e40-5d69ab5225d5","added_by":"auto","created_at":"2023-10-04 14:30:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHFD induced significant\u003c/strong\u003e \u003cstrong\u003esynaptotoxicity and amyloid burden in the brains of the adult female mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShown are the Western blot results of maternal (A) synapse receptor proteins (SYP and PSD-95) and (B) amyloidogenic burdon proteins (BACE-1 and Aβ) in pre-pregnancy stage, along with their bar-chart representation respectively. β-Actin was used as a loading control. The results were determined using Image J software and bar-chart indicate mean in A.U ± SEM. Significance of control vs HFD is expressed as #. Significance: ##p\u0026lt;0.01 and ###p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/451b1ff2d2610eb14f1de3c0.png"},{"id":44082808,"identity":"85235156-f64f-4974-8026-3c8ce8b15b0b","added_by":"auto","created_at":"2023-10-04 14:38:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMelatonin showed significant improvement in the expression levels of neuro-inflammatory and neuro-degeneration markers in HFD fed female mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShown are the Western blot results of neuro-inflammatory markers (COX2, TNF-α and IL-1β) in (A) post parturition, and (B) post weaning stage, and neurodegeneration markers (BAX, Bcl-2, caspase-3 and PARP-1) in (C) post parturition, and (D) post weaning stage, along with their bar-chart representation respectively. β-Actin was used as a loading control. The results were determined using Image J software and bar-chart indicate mean in A.U ± SEM. Significance of one way ANOVA is expressed as α, significance of control vs HFD is expressed as #, while significance of HFD vs HFD+Melationin is expressed as*. Significance: \u003csup\u003eαα, **, ##\u003c/sup\u003ep\u0026lt;0.01 and \u003csup\u003eααα, ***, ###\u003c/sup\u003ep\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/f21276f41d6ba9f5bd0c7b8a.png"},{"id":44084298,"identity":"f8bc20a9-94c0-4454-8c96-2beafbd37f37","added_by":"auto","created_at":"2023-10-04 14:46:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":168549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMelatonin demonstrated substantial amelioration in the central insulin resistance signaling pathway proteins expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShown are the Western blot results of insulin resistance markers (p-IRS, p-Akt and p-GSK3β) in (A) post parturition, and (B) post weaning stage, and signaling proteins (SIRT1, Nrf-2 and HO-1) in (C) post parturition, and (D) post weaning stage, along with their bar-chart representation respectively. β-Actin was used as a loading control. The results were determined using Image J software and bar-chart indicate mean in A.U ± SEM. Significance of one way ANOVA is expressed as α, significance of control vs HFD is expressed as #, while significance of HFD vs HFD+Melationin is expressed as*. Significance: \u003csup\u003eαα, **, ##\u003c/sup\u003ep\u0026lt;0.01 and \u003csup\u003eααα, ***, ###\u003c/sup\u003ep\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/c736f94ec6a99ede6512e570.png"},{"id":44081968,"identity":"179c510c-ef60-4b14-8de0-1e307f5b019a","added_by":"auto","created_at":"2023-10-04 14:30:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":147755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMelatonin exhibited significant retrieval in the HFD-induced synaptotoxicity and amyloid burden in the female mice brain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShown are the Western blot results of synapse receptor proteins (SYP and PSD-95) in (A) post parturition, and (B) post weaning stage, and amyloidogenic burdon proteins (BACE-1 and Aβ) in (C) post parturition, and (D) post weaning stage, along with their bar-chart representation respectively. β-Actin was used as a loading control. The results were determined using Image J software and bar-chart indicate mean in A.U ± SEM. Significance of one way ANOVA is expressed as α, significance of control vs HFD is expressed as #, while significance of HFD vs HFD+Melationin is expressed as*. Significance: \u003csup\u003eαα, **, ##\u003c/sup\u003ep\u0026lt;0.01 and \u003csup\u003eααα, ***, ###\u003c/sup\u003ep\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/2e817f3278f31b34ceacf5f2.png"},{"id":44081969,"identity":"96a209dd-58c1-40a8-98bf-a8b03531bdb5","added_by":"auto","created_at":"2023-10-04 14:30:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1019338,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunofluorescence analysis of melatonin showing immense amelioration of selected proteins in post parturition and Post weaning stage of female mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShown are the (A) The immunofluorescence images of Aβ (green) along with the relative integral optical density (IOD) bar chart, in the CA1 region of the experimental groups in post parturition stage,\u003cstrong\u003e \u003c/strong\u003e(B) the immunofluorescence images (pink) of Aβ along with the relative integral optical density (IOD) bar chart, in the CA1 region in the experimental groups in post weaning stage. Also shown are the (C) the double immunofluorescence images of NF-kB (green) and SIRT1 (red) along with the relative integral optical density (IOD) bar chart, in the CA1 region of the experimental groups in post parturition stage,\u003cstrong\u003e \u003c/strong\u003eand (D) the double immunofluorescence images of NF-kB (green) and SIRT1 (red) along with the relative integral optical density (IOD)\u0026nbsp; bar chart, in the CA1 region of the experimental groups in post weaning stage. DAPI was used to stain the nucleus.\u003cstrong\u003e \u003c/strong\u003eThe complete procedures are depicted in the material and methods section.\u003cstrong\u003e \u003c/strong\u003eSignificance of one way ANOVA is expressed as α, significance of control vs HFD is expressed as #, while significance of HFD vs HFD+Melationin is expressed as*. Significance:\u0026nbsp; \u003csup\u003eααα\u003c/sup\u003eP \u0026lt; 0.001, \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/29a63bc93a97208f29ca9a1e.png"},{"id":44081977,"identity":"73d87504-815e-4cb5-85f0-a63bff39bde0","added_by":"auto","created_at":"2023-10-04 14:30:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":148721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMelatonin successfully ameliorated the expression levels of various proteins in the neonatal and adolescent mice brains of the HFD group.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShown are the Western blot results of (A) neuroinflammatory markers (TNF-α and IL-1β) and (B) neurodegeneration markers (caspase-3 and PARP-1) along with their bar chart respectively in the neonatal brains of the HFD group. Also shown are the Western blot results of (C) neuroinflammatory markers (COX2 and TNF-α) and (D) amyloid burdon (BACE-1 and Aβ) along with their bar chart respectively in the HFD group adolescents.β-Actin was used as a loading control. The results were determined using Image J software and bar chart indicate mean in A.U ± SEM. Significance of one way ANOVA is expressed as \u003csup\u003eα\u003c/sup\u003e, significance of control vs HFD is expressed as #, while significance of HFD vs HFD+Melationin is expressed as*. Significance: \u003csup\u003eαα, **, ##\u003c/sup\u003ep\u0026lt;0.01 and \u003csup\u003eααα,***, ###\u003c/sup\u003ep\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/612b8d60fa82d1b89f049e7e.png"},{"id":44081971,"identity":"3351934e-580e-47d3-9885-95b964e66163","added_by":"auto","created_at":"2023-10-04 14:30:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":141199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMelatonin successfully rectified insulin resistance and enhance signaling proteins in in the neonatal and adolescent mice brain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShown are the Western blot results of (A) insulin resistance makers (p-IRS, p-Akt and p-GSK3β) and (B) signaling proteins (SIRT1, Nrf-2 and HO-1) along with their bar chart respectively in eight weeks adolescents. Also Shown are the Western blot results of signaling proteins (SIRT1, Nrf-2 and HO-1) along with their bar chart in the neonatal brains of the HFD group.\u0026nbsp; β-Actin was used as a loading control. The results were determined using Image J software and bar chart indicate mean in A.U ± SEM. Significance of one way ANOVA is expressed as \u003csup\u003eα\u003c/sup\u003e, significance of control vs HFD is expressed as #, while significance of HFD vs HFD+Melationin is expressed as*. Significance: \u003csup\u003eα,*,#\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003eαα,**, ##\u003c/sup\u003ep\u0026lt;0.01 and \u003csup\u003eααα,***, ###\u003c/sup\u003ep\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/aa7137211391e320231cc36c.png"},{"id":44081974,"identity":"18b4e0b9-9385-487b-a8ca-ec21a99236fa","added_by":"auto","created_at":"2023-10-04 14:30:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":993145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunofluorescence analysis of melatonin showing immense amelioration of selected proteins in the CA1 region of hippocampus of the adolescents.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShown are the (A) The immunofluorescence images of Aβ (green) along with the relative integral optical density (IOD) bar chart, in the CA1 region of the 8 weeks mice and (B) the double immunofluorescence images of NF-kB (green) and SIRT1 (red) along with the relative integral optical density (IOD) bar charts, in the CA1 region of the 8 weeks mice. DAPI was used to stain the nucleus. The complete procedures are depicted in the material and methods section. Significance of one way ANOVA is expressed as α, significance of control vs HFD is expressed as #, while significance of HFD vs HFD+Melationin is expressed as*. Significance: αα, **, ##p\u0026lt;0.01 and ααα, ***, ###p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/9565fbd9b91e61719bec1c52.png"},{"id":44094446,"identity":"63b4d885-649a-4eab-b22a-3519c6a2b01f","added_by":"auto","created_at":"2023-10-04 16:52:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3257093,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/dcffe626-31b2-42f4-a839-7d981c2c5e0e.pdf"},{"id":44081978,"identity":"f4a8b5f1-10ce-4e31-9169-921d9047c1bd","added_by":"auto","created_at":"2023-10-04 14:30:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2940477,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryArticle05062023.docx","url":"https://assets-eu.researchsquare.com/files/rs-3376865/v1/cfe406cc5dfd5abfa5cb140a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Melatonin Ameliorates High Fat Diet Induced Oxidative Stress-Mediated Insulin Resistance, Amyloid Burden and Memory Dysfunction in Female Mice and their offspring","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiet containing high amount of refined sugars and saturated fats is a prime factor contributing to the present epidemic of metabolic syndrome and insulin resistance\u0026nbsp;[1]. HFD causes metabolic disorders which can have negative implication on cognition, behavior, and brain health in general\u0026nbsp;[2]. In human studies, HFD, obesity, DM, and other metabolic disorders have shown a strong correlation with the development of Alzheimer\u0026rsquo;s disease (AD) and other cognitive impairments\u0026nbsp;[3,4].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInflammation in various parts of the brain is linked to abnormal behavioral, cognitive dysfunction and AD\u0026nbsp;[5]. It is previously reported that feeding mice with HFD for 14 weeks affects brain insulin signaling and cognitive capabilities\u0026nbsp;[6]. In addition, Amyloid \u0026beta; production in brain is increased after 16 weeks HFD which further exacerbated AD\u0026nbsp;[7]. Furthermore, HFD accelerates the progression of AD-like pathology by increasing brain amyloid deposition and oxidative stress (OS)\u0026nbsp;[8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsumption of HFDs has shown to develop obesity and peripheral insulin resistance as well as disruption of blood brain barrier (BBB)\u0026nbsp;[9]. As a result, glucose transport to the brain decreases that lead to the reduction of brain insulin and peripheral hyper-insulinemia. Therefore, it is presumed that HFD-induced peripheral hyperinsulinemia might cause brain insulin deficit leading to reduction in insulin degrading enzyme\u0026nbsp;[10]. In addition, in the cortex and hippocampus, HFD\u0026nbsp;enhances the production of A\u0026beta;\u0026nbsp;[9]. This A\u0026beta; can be found in a variety of forms in the brain, including monomers, oligomers and insoluble fibrils that form plaques outside the cells. The soluble form of oligomeric A\u0026beta; decreases synapses and hinders long-term potentiation (LTP)\u0026nbsp;[11,12].\u003c/p\u003e\n\u003cp\u003eMelatonin is an important hormone secreted mainly by the pineal gland [13]. \u0026nbsp;It acts as a potent anti-oxidant, anti-apoptotic and anti-inflammatory agent [14]. \u0026nbsp;In addition, it upregulates several antioxidant enzymes and subsequently downregulate other pro-oxidants [15], in both pre-clinical and clinical studies by modifying pro and anti-inflammatory cytokines [16,17]. \u0026nbsp;Melatonin also has anti-apoptotic properties mainly by inhibiting the reduction of pBad and pAkt levels [18]. Maternal circulating melatonin levels gradually increase after 24 weeks of pregnancy spiking at 32 weeks and then reverting to baseline after delivery in a normal pregnancy [19]. Human placental melatonin levels are 6 to 7 times higher in comparison to plasma levels, suggesting a role in placental development [20]. Previous reports have shown that melatonin activate SIRT1 which in turn reduce oxidative stress, inflammation and neurodegeneration in various models by stimulating Nrf2/HO-1 signaling pathway [21]. Here, we investigated the SIRT1 mediated NRF2/HO-1 signaling pathway of melatonin against HFD-induced OS and impaired insulin-signaling in the cortex and hippocampus of dams and their offspring. We also assessed the effects of melatonin against HFD-induced changes in A\u0026beta; and synaptic markers in the dams and their offspring providing a potential useful therapeutic index for preventing neurodegeneration and its consequences.\u003c/p\u003e"},{"header":"MATERIALS AND METHOD","content":"\u003cp\u003eAdditional details for all procedures are given in the Supplementary Materials and Methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHealthy adult female albino mice (7-8 weeks old and 30 g body weight) were purchased from Riphah International University Islamabad, Pakistan. Sample size comprised of 60 albino mice (36 female adult mice and 24 offspring), calculated via resource equation approach. Female adult mice were randomly distributed into three groups, i.e.; a control group (n=13), a HFD group (n=13) and HFD + Melatonin group (n=10). Control group mice were fed with a normal standard diet. HFD group mice were fed ad libitum on commercially available obesogenic HFD, containing 45% fats, 35% carbohydrates, and 20% proteins per dry weight\u0026nbsp;[22]. After successful conception, melatonin treatment was started to one HFD group while keeping the animals on HFD until the termination of pregnancy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy design and approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was an experimental study carried out at the Institute of Basic Medical Sciences (IBMS), Khyber Medical University (KMU), Peshawar, Pakistan, under institutional Ethical board approval number (Dir/KMU EB/EM/000806). The study consisted of three maternal and two offspring interventions, described below.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaternal interventions:\u003c/strong\u003e The adult female mice were distributed into two groups i.e. normal standard diet fed animals (n=13) and HFD fed animals (n=23). After three weeks of HFD and standard diet feeding, one set (n=3) from each group was selected randomly and assessed for behavioral studies and then sacrificed for biochemical measurements. The remaining normal (n=10) and HFD fed mice (n=20) were allowed to mate for about one week time. After successful conception, the HFD fed mice were randomly divided into two groups. Melatonin (10 mg/kg BW) was administered intra-peritoneally to one of the HFD fed pregnant mice group (n=10) until the termination of pregnancy. While the remaining mice (n=10) were kept feeding on HFD without treating with melatonin. 3 mice from each category i.e. normal diet mice, HFD and HFD plus melatonin were subjected to behavioral studies and intraperitoneal glucose tolerance test (IPGTT) and then sacrificed for further biochemical analysis. While 2 mice from the same groups were subjected to immunofluorescence analysis. Remaining female mice (n=5/group) were allowed to complete the weaning period. After completion of the weaning period, 3 mice from each group were again subjected into all the above mentioned experiments, while 2 mice of each group were assessed by immunofluorescence analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOffspring intervention\u003c/strong\u003e: First offspring intervention with three pups of each respective maternal group was carried out at the age of four weeks. Selected proteins were analyzed in the four weeks neonatal intervention. Remaining offspring from respective parental groups (n=5) were kept to grow to the adulthood until they reached 8 weeks. These adult offsprings were subjected to behavioral and other investigations followed by sacrificing to check the expression of different proteins through Western blotting and immunofluorescence techniques.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioral tests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTherapeutic effects of melatonin on HFD induced memory impairment were evaluated by Y-Maze\u0026nbsp;[23]\u0026nbsp;and Morris Water Maze behavioral tests\u0026nbsp;[24]. The investigator carrying out the behavioral tests was blinded to mice tags and treatment. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animals were slaughtered at the end of each intervention after completion of behavioral studies as per method reported previously\u0026nbsp;[21]. Different mouse derived primary antibodies such as anti-BCL-2 (SC-7382), anti-BAX (SC-7480), anti-CAS-3 (SC-7272), anti-PARP-1 (SC-8007), anti-SIRT1 (SC-74504), anti-NRF-2 (SC-365949), anti-HO-1 (SC-136960), anti-A\u0026beta; (SC-28365), anti-BACE1 (SC-33711), anti-PSD95 (SC-71933), anti-SYP (SC-17750), anti-p-IRS (SC-8038), anti-p-AKT (SC-514032), anti-p-GSK3B (SC-81496), anti-TNF-\u0026alpha; (SC-52746), anti-COX2 (SC-376861), anti-IL-1\u0026beta; (SC-32294) and \u0026beta;-actin (SC-47778) \u0026nbsp;of Santa Cruz Biotech, CA, USA were used to detect and localize protein of interest. ECL was used to detect cross-reacting proteins after incubation with horseradish peroxidase-conjugated secondary antibodies. ImageJ software was used to perform the densitometry analysis of the bands in arbitrary units (A.U.) relative to the untreated control. Detailed procedure can be seen in the supplementary methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue preparation and immunofluorescence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEqual number of mice (n=2) were kept in each group for conducting morphological study. Primary anti-bodies such as anti-SIRT1, anti NF-\u0026kappa;B and anti-A\u0026beta; (1:100 in PBS) were used to detect protein of interest. \u0026nbsp;Secondary anti-bodies (FITC and TRITC conjugated, Santa Cruz, 1:50 in PBS) were used for cross reaction with the primary antibodies. Immunofluorescence staining was done as reported previously\u0026nbsp;[25]. \u0026nbsp;Images were captured with confocal microscopy (FluoView FV 1000 Olympus, Japan). Detailed procedure can be seen in the supplementary methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original X-ray films of the Western blot analysis were scanned. Image J software was used to perform densitometry analysis of the bands. Integral optical density (IOD) of proteins were expressed in arbitrary units (A.Us) as Mean\u0026plusmn;S.E.M. Significant difference in the first intervention was determined using t-test whereas significant difference in both the maternal and offspring \u0026nbsp;interventions was determined using one-way analysis of variance (ANOVA), followed by Post- hoc tukey\u0026apos;s test.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eHFD feeding induces multiple complications in female mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA statistically significant increase (p\u0026lt;0.001) in the expression of neuroinflammatory markers (TNF-\u0026alpha;, COX2 and IL-1\u0026beta;) (Figure 1A), pro-apoptotic marker, PARP-1 (p\u0026lt;0.001) and significant decrease (p\u0026lt;0.001) was observed in the anti-apoptotic BCl-2 in the HFD fed mice. Likewise, significant increase (p\u0026lt;0.001) was observed in the Bax and Caspase-3 levels (Figure 1B). Similarly, significant decrease (p\u0026lt;0.001) in the expression of p-IRS-1, p-AKT and p-GSK3-\u0026beta; levels were observed in the brain homogenates of HFD group as compared to normal chow diet group (Figure 1C). Significant decrease (p\u0026lt;0.001) was also observed in the expression of SIRT-1, Nrf-2 and HO-1 (p\u0026lt;0.01) in HFD group as compared to normal chow diet group (Figure 1D).\u003c/p\u003e\n\u003cp\u003eMaternal obesity/HFD induced an obvious decrease in the synaptic receptor proteins and increased amyloidogenic burden. Significant decrease (p\u0026lt;0.001) in the expression of synaptic proteins (SYP and PSD-95) (Figure 2A), and significant increase (p\u0026lt;0.001) was observed in the expression of BACE-1 and A\u0026beta; in HFD group as compared to normal diet group (Figure 2B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter feeding the female mice with HFD for three weeks, the animals showed a significant increase in body weight accompanied by, deranged lipid profiles, oxidative stress and altered behaviour shown in the Supplementary Figure S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMelatonin exhibits reversal effect on HFD induced complications in the female mice after parturition and post weaning interventions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe evaluated the neuroprotective potential of melatonin against HFD induced OS and associated neuroinflammation and neurodegeneration in the cortex and hippocampus of the mice brains. Statistically significant differences (p\u0026lt;0.001) were observed in the expression of neuroinflammatory and neurodegenerative markers. Post hoc comparison revealed that HFD induced significant increase (p\u0026lt;0.001) in the expression of neuroinflammatory markers (TNF-\u0026alpha;, IL-1\u0026beta; and COX-2). However, melatonin significantly decreased (p\u0026lt;0.001) all the proinflammatory markers (TNF-\u0026alpha;, IL-1\u0026beta; and COX-2) in both post-parturition intervention (Figure 3A) and post-weaning intervention (Figure 3B). Similarly, HFD markedly increased (p\u0026lt;0.001) Bax, Caspase-3 and PARP-1 and decreased (p\u0026lt;0.001) Bcl-2, while melatonin significantly reversed (p\u0026lt;0.001) Bax, BCl-2, Caspase-3 and PARP-1 expression in post-parturition (Figure 3C) and post-weaning interventions (Figure 3D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA significant differences (p\u0026lt;0.001) were also observed in the expression of insulin resistance markers (IRS-1, p-AKT and p-GSK3-\u0026beta;). Post hoc tukey\u0026rsquo;s analysis showed significant decrease (p\u0026lt;0.001) in the expression of IRS-1, p-AKT and p-GSK3-\u0026beta; proteins in the brain homogenates of HFD group as compared to control group in both maternal interventions. Melatonin, however showed significant increase (p\u0026lt;0.001) in the expression of almost all the assessed markers (IRS-1, p-AKT and p-GSK3-\u0026beta;) in post-parturition intervention (Figure 4A) both maternal interventions (Figure 4A and 4B).\u003c/p\u003e\n\u003cp\u003eMelatonin activated the SIRT1/Nrf2 pathway to reduce HFD induced OS. A statistically significant differences (p\u0026lt;0.001) were observed in the expression of signaling proteins. Nrf-2 and HO-1 along with a significant decrement in SIRT-1 (p\u0026lt;0.001) in the in HFD group was observed as compared to control group. Melatonin, on the other hand markedly increased (p\u0026lt;0.05) the expression of the signaling pathway proteins (SIRT1/Nrf2/HO-1) in the post-parturition intervention (Figure 4C). However, highly significant increase (p\u0026lt;0.001) was observed in the expression levels of SIRT1, Nrf2 and HO-1 in the post-weaning intervention (Figure 4D).\u003c/p\u003e\n\u003cp\u003eHFD significantly decreased (p\u0026lt;0.001) synaptic receptor proteins (SYP \u0026amp; PSD95) and significantly increased amyloid burden proteins (BACE-1 and A\u0026beta;) in both interventions. Melatonin, however markedly increased the expression of SYP (p\u0026lt;0.01) \u0026amp; PSD95 (p\u0026lt;0.001) \u0026nbsp;(Figure 5A and 5B) and decreased (p\u0026lt;0.001) BACE-1 and A\u0026beta; in the mice brains in both post-parturition and post-weaning interventions (Figure 5C and 5D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSelected proteins A\u0026beta;, NF-kB and SIRT-1 expressions were analysed through immunofluorescence technique. Significant differences (p\u0026lt;0.001) were revealed in the expression of these proteins among various groups. Post hoc tukey test showed significant increase (p\u0026lt;0.001) in the expression of the NF-kB and A\u0026beta; proteins and significant decrease (p\u0026lt;0.001) in SIRT1 expression in HFD group as compared to normal chow diet group. Melatonin treatment significantly rectified (p\u0026lt;0.001) the effect of HFD in both post-parturition and post- weaning intervention (Figure 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaternal melatonin treatment protects mice offspring from the deteriorating effects of HFD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaternal HFD and HFD + melatonin effects were observed in two offspring\u0026rsquo;s interventions at the age of 4 and 8 weeks respectively. Post hoc tukey\u0026rsquo;s analysis showed significant increase (p\u0026lt;0.001) in the expression of neuro-inflammatory markers (TNF-\u0026alpha; and IL-1\u0026beta;) in the HFD groups of neonatal mice as compared to control group (Figure 7A). Similarly, a significant increase (p\u0026lt;0.001) was observed in the expression of apoptotic markers (Caspase-3 and PARP-1) in HFD group neonatal mice. \u0026nbsp;Melatonin meticulously reversed all the damages caused by HFD. A significant decrease of pro-inflammatory markers, TNF-\u0026alpha; (p\u0026lt;0.01) and IL-1\u0026beta; (p\u0026lt;0.05) was observed (Figure 8A) along with a highly significant decrease (p\u0026lt;0.001) in the pro-apoptotic marker in the brain homogenates of young offsprings of HFD + Melatonin group (Figure 7B). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNeuro-inflammatory and amyloidogenic markers were also investigated in the brain homogenates of the adult offsprings. Significant increase (p\u0026lt;0.001) in the neuro-inflammatory markers i.e.; TNF-\u0026alpha;, IL-1\u0026beta; and COX-2 levels was observed in the HFD groups of adult mice as compared to control group. Similarly, a significant increase (p\u0026lt;0.001) was observed in the expression of BACE-1 and A\u0026beta; proteins in HFD group adult mice as compared to normal standard diet group. \u0026nbsp;Melatonin successfully reversed all the damages caused by HFD. Post hoc tukey test showed highly significant decrease (p\u0026lt;0.001) in the pro-inflammatory markers (TNF-\u0026alpha;, IL-1\u0026beta; and COX-2) levels (Figure 7C) along with significant decrease (p\u0026lt;0.001) in the expression of BACE-1 and A\u0026beta; proteins in the brain homogenates of the HFD + melatonin group adult mice (Figure 7D). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSignificant differences (p\u0026lt;0.001) were observed in the expression of insulin resistance markers (p-IRS-1, p-AKT and p-GSK3\u0026beta;) among various groups of adult offsprings. Significant decrease (p\u0026lt;0.001) in the expression of p-IRS-1, p-AKT and p-GSK3\u0026beta; was observed in the HFD group adult mice brain. Melatonin on the other hand significantly retrieved (p\u0026lt;0.001) these markers (Figure 8A). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSIRT-1 expression and activity was analysed in both young and adult offsprings. Significant differences (p\u0026lt;0.001) were observed among the expression levels of SIRT-1 signaling proteins. Significant decrease (p\u0026lt;0.001) in the expression of NRF-2 and HO-1 along with SIRT-1 levels in HFD group neonatal and adult mice were observed as compared to the control group. Melatonin however, successfully rectified this decrease in both the offspring interventions. Significant increase (p\u0026lt;0.001) in SIRT-1, Nrf-2 and HO-1 expression levels were observed in HFD + melatonin group adult offsprings (Figure 8B). Similarly, melatonin significantly increased (p\u0026lt;0.001) the expression levels of the signalling protein in HFD + melatonin group neonatal mice (Figure 8C).\u003c/p\u003e\n\u003cp\u003eSelected proteins NF-kB, SIRT-1 and A\u0026beta; proteins expressions were analysed through immunofluorescence technique. Post hoc comparison demonstrate significant increase (p\u0026lt;0.001) in the expression of the NF-kB and A\u0026beta; proteins and significant decrease (p\u0026lt;0.001) in SIRT1 expression in HFD group. Melatonin treatment significantly improved (p\u0026lt;0.001) the detrimental effects of HFD in the HFD + Melatonin group (Figure 9A and 9B).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eProlonged consumption of HFD causes obesity which in turn may lead to the development of diseases like cardiovascular disease, certain types of cancer and type 2 diabetes mellitus\u0026nbsp;[26]. Adipocytes play a contributing role in inflammation during progression of obesity by secreting common cytokines like IL-1, IL-6, and TNF\u0026alpha; etc.\u0026nbsp;[27]. Proinflammatory cytokines (IL-1\u0026beta;, IL-6, and TNF-\u0026alpha;) initiate inflammation in response to multiple factors as they can cross the blood brain barrier\u0026nbsp;[28]. Our study revealed that brains homogenates of HFD fed mice showed increased neuroinflammatory (TNF-\u0026alpha;, IL-1\u0026beta; and COX2) and neurodegenerative (Bax, BCl-2, Caspase-3 and PARP-1) proteins expression as compared to control diet fed mice. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMelatonin\u0026rsquo;s ability to regulate immune system modulation along with reduction in acute and chronic inflammation has been reported previously\u0026nbsp;[30]. Various studies suggest that melatonin modifies proinflammatory cytokines and exerts anti-inflammatory effects in various pathophysiological conditions\u0026nbsp;[31,32]. According to Liu et al. (2013), melatonin through its action on inflammasome genes expression, reduces the LPS-induced inflammation in mouse adipose tissue\u0026nbsp;[33]. Here, we observed that chronic consumption of HFD increases both neuroinflammation and neurodegeneration in the dams and their offsprings but subsequent treatment with melatonin was found effective in reducing these neuroinflammatory and neurodegenerative markers in both the maternal and offsprings\u0026rsquo; interventions.\u003c/p\u003e\n\u003cp\u003eMultiple studies reported that diet containing high amount of refined sugar and saturated fats can negatively affect cognitive functions\u0026nbsp;[34,35]. HFD exacerbates cognitive decline along with other conditions such as chronic stress, sleep deprivation, and aging\u0026nbsp;[36]. Consistent with these results, we observed, HFD induced oxidative stress mediated synaptic dysfunction and increased amyloidogenic burden with gross decline in the expression of synaptic proteins SYP and PSD95 and significant increase in BACE-1 and A\u0026beta; expression showing amyloidogenic burden in all the maternal and adult intervention.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMatsubara et al. (2003) showed that melatonin administration to APP695 transgenic mice was associated with a decrease in brain A\u0026beta; levels\u0026nbsp;[37]. The underlying mechanism of this decrease in the brain amyloid is still not clear, but it could be due to a direct action of melatonin on the metabolism of \u0026beta;-amyloid precursor protein\u0026nbsp;[38]. In our study, administration of melatonin during pregnancy has long lasting effect on restoring memory, synaptotoxicity and amyloid burden by significantly increasing, synaptic proteins (SYP and PSD95) and decreasing amyloid protein (BACE1 and A\u0026beta;) \u0026nbsp;in the brain homogenates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt has previously been shown that diets containing high amount of saturated fat are usually associated with obesity, IR, and T2DM\u0026nbsp;[39]. McNeilly et al. (2015) reported raised fasting blood glucose and insulin levels in rats fed on HFD\u0026nbsp;[40]. Similarly, Kothari et al. (2017) reported that HFD resulted in peripheral and central insulin resistance\u0026nbsp;[41]. On the other hand Brydon et al. (2001) showed that melatonin activates MT2 receptors in human adipocytes which modulates glucose uptake\u0026nbsp;[42]. The present study is in agreement with the above mentioned studies, where we found insulin signaling impairment in HFD fed female mice and their offsprings. \u0026nbsp;Similarly, after the melatonin treatment during pregnancy significant restoration was observed in the expression of p-IRS, p-Akt and p-GSK3\u0026beta; proteins in both maternal and offspring intervention.\u003c/p\u003e\n\u003cp\u003eSeveral studies have revealed that silent information regulator transcript-1 (SIRT1) reduces oxidative stress level and the extent of inflammation\u0026nbsp;[43,44]. Nuclear factor erythroid-2 related factor 2 (Nrf2) is an important antioxidant protein essential for cellular defense\u0026nbsp;[45]. Once activated, Nrf2 is translocated to the nucleus from the cytoplasm and interacts with the antioxidant defense system to facilitate transcription of hemeoxygenase 1 (HO-1). HO-1 increases resistance to OS and exhibit protective role against inflammation\u0026nbsp;[46]. Shahid et al. (2017) confirmed the stimulation of SIRT1/Nrf2 signaling pathway by melatonin against LPS-induced oxidative stress in of the hippocampus of PND7 rats\u0026nbsp;[21]. Here, we found that HFD significantly decreased SIRT1 expression and activity in female mice and their offsprings. Melatonin administration on the other hand reinstated brain SIRT1 expression with antagonistic effect to HFD induced OS in all the interventions of the mothers fed on HFD and their neonates and adults mice. The present study is in agreement with previous studies which report that melatonin is an important antioxidant and has a strong tendency to mitigate HFD-induced decreased expression of Nrf-2 and HO-1 in both maternal and offspring interventions.\u003c/p\u003e\n\u003cp\u003eThis study has some limitations. Here, we observed the therapeutic effects of melatonin only in the offspring groups of their own respective maternal groups. Cross-fostering of the neonates was not possible because mothers used to eat pups from the other groups despite having free access to food and water. Moreover, this study is conducted on four weeks and eight weeks adolescent offspring probably because of the lack of expertise to deal the neonatal brain samples.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eWe conclude that HFD induced neuro-inflammation and neuro-degeneration in pregnant mice and their offsprings via oxidative stress. Oxidative stress further triggered insulin resistance and amyloid-\u0026beta; generation in the brains of pregnant mice and their offsprings. Melatonin stabilized the damages caused by HFD by activating SIRT1/NRF-2/HO-1 signaling pathway, thus rectified the HFD induced toxic effects. Since there are limited remedies to effectively treat HFD induced brain damages, this study could be helpful in the development of potential treatment strategies in obese pregnant mothers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval:\u0026nbsp;\u003c/strong\u003eThe institutional Ethical board granted ethical approval to the study (Dir/KMU EB/EM/000806).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u0026nbsp;\u003c/strong\u003eNone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions:\u0026nbsp;\u003c/strong\u003eAJ was involved in the execution of the project and writing of the manuscript. MS supervised, designed, executed the study and wrote the manuscript. SAS co-supervised the study and provided clinical expertise. SHH and E did critical editing of the manuscript. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information:\u0026nbsp;\u003c/strong\u003eThis research was supported by\u0026nbsp;the Organization of Research Innovation and Commercialization of Khyber Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u0026nbsp;\u003c/strong\u003eThe authors acknowledge with thanks Organization of Research Innovation and commercialization of Khyber Medical University for their financial and technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eAvailable from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGrundy SM. Metabolic syndrome update. Trends Cardiovasc Med. 2016;26:364\u0026ndash;73.\u003c/li\u003e\n \u003cli\u003ePasinetti GM, Eberstein JA. Metabolic syndrome and the role of dietary lifestyles in Alzheimer\u0026rsquo;s disease. J Neurochem. 2008;106:1503\u0026ndash;14.\u003c/li\u003e\n \u003cli\u003eProfenno LA, Porsteinsson AP, Faraone SV. Meta-analysis of Alzheimer\u0026rsquo;s disease risk with obesity, diabetes, and related disorders. Biol Psychiatry. 2010;67:505\u0026ndash;12.\u003c/li\u003e\n \u003cli\u003eArshad N, Lin TS, Yahaya MF. Metabolic syndrome and its effect on the brain: possible mechanism. CNS Neurol Disord-Drug Targets Former Curr Drug Targets-CNS Neurol Disord. 2018;17:595\u0026ndash;603.\u003c/li\u003e\n \u003cli\u003eAlmeida-Suhett CP, Graham A, Chen Y, Deuster P. Behavioral changes in male mice fed a high-fat diet are associated with IL-1\u0026beta; expression in specific brain regions. Physiol Behav. 2017;169:130\u0026ndash;40.\u003c/li\u003e\n \u003cli\u003eKothari V, Luo Y, Tornabene T, O\u0026rsquo;Neill AM, Greene MW, Geetha T, et al. High fat diet induces brain insulin resistance and cognitive impairment in mice. Biochim Biophys Acta BBA-Mol Basis Dis. 2017;1863:499\u0026ndash;508.\u003c/li\u003e\n \u003cli\u003eBusquets O, Ettcheto M, Pall\u0026agrave;s M, Beas-Zarate C, Verdaguer E, Auladell C, et al. Long-term exposition to a high fat diet favors the appearance of \u0026beta;-amyloid depositions in the brain of C57BL/6J mice. A potential model of sporadic Alzheimer\u0026rsquo;s disease. Mech Ageing Dev. 2017;162:38\u0026ndash;45.\u003c/li\u003e\n \u003cli\u003eLin B, Hasegawa Y, Takane K, Koibuchi N, Cao C, Kim-Mitsuyama S. High-fat-diet intake enhances cerebral amyloid angiopathy and cognitive impairment in a mouse model of Alzheimer\u0026rsquo;s disease, independently of metabolic disorders. J Am Heart Assoc. 2016;5:e003154.\u003c/li\u003e\n \u003cli\u003eSaiyasit N, Chunchai T, Prus D, Suparan K, Pittayapong P, Apaijai N, et al. Gut dysbiosis develops before metabolic disturbance and cognitive decline in high-fat diet\u0026ndash;induced obese condition. Nutrition. 2020;69:110576.\u003c/li\u003e\n \u003cli\u003ePetrov D, Pedr\u0026oacute;s I, Artiach G, Sureda FX, Barroso E, Pall\u0026agrave;s M, et al. High-fat diet-induced deregulation of hippocampal insulin signaling and mitochondrial homeostasis deficiences contribute to Alzheimer disease pathology in rodents. Biochim Biophys Acta BBA-Mol Basis Dis. 2015;1852:1687\u0026ndash;99.\u003c/li\u003e\n \u003cli\u003eButterfield DA, Boyd-Kimball D. Mitochondrial oxidative and nitrosative stress and Alzheimer disease. Antioxidants. 2020;9:818.\u003c/li\u003e\n \u003cli\u003eMastrocola R, Restivo F, Vercellinatto I, Danni O, Brignardello E, Aragno M, et al. Oxidative and nitrosative stress in brain mitochondria of diabetic rats. J Endocrinol. 2005;187:37\u0026ndash;44.\u003c/li\u003e\n \u003cli\u003eAmaral FG do, Cipolla-Neto J. A brief review about melatonin, a pineal hormone. Arch Endocrinol Metab. 2018;62:472\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eMu\u0026ntilde;oz-Jurado A, Escribano BM, Caballero-Villarraso J, Galv\u0026aacute;n A, Ag\u0026uuml;era E, Santamar\u0026iacute;a A, et al. Melatonin and multiple sclerosis: antioxidant, anti-inflammatory and immunomodulator mechanism of action. Inflammopharmacology. 2022;30:1569\u0026ndash;96.\u003c/li\u003e\n \u003cli\u003eGarc\u0026iacute;a-Navarro A, Gonz\u0026aacute;lez-Puga C, Escames G, L\u0026oacute;pez LC, L\u0026oacute;pez A, L\u0026oacute;pez-Cantarero M, et al. Cellular mechanisms involved in the melatonin inhibition of HT-29 human colon cancer cell proliferation in culture. J Pineal Res. 2007;43:195\u0026ndash;205.\u003c/li\u003e\n \u003cli\u003eFavero G, Franceschetti L, Bonomini F, Rodella LF, Rezzani R. Melatonin as an anti-inflammatory agent modulating inflammasome activation. Int J Endocrinol. 2017;2017.\u003c/li\u003e\n \u003cli\u003eMauriz JL, Collado PS, Veneroso C, Reiter RJ, Gonz\u0026aacute;lez-Gallego J. A review of the molecular aspects of melatonin\u0026rsquo;s anti-inflammatory actions: recent insights and new perspectives. J Pineal Res. 2013;54:1\u0026ndash;14.\u003c/li\u003e\n \u003cli\u003eKoh P-O. Melatonin attenuates the focal cerebral ischemic injury by inhibiting the dissociation of pBad from 14-3-3. J Pineal Res. 2008;44:101\u0026ndash;6.\u003c/li\u003e\n \u003cli\u003eNakamura Y, Tamura H, Kashida S, Takayama H, Yamagata Y, Karube A, et al. Changes of serum melatonin level and its relationship to feto-placental unit during pregnancy. J Pineal Res. 2001;30:29\u0026ndash;33.\u003c/li\u003e\n \u003cli\u003eIwasaki S, Nakazawa K, Sakai J, Kometani K, Iwashita M, Yoshimura Y, et al. Melatonin as a local regulator of human placental function. J Pineal Res. 2005;39:261\u0026ndash;5.\u003c/li\u003e\n \u003cli\u003eShah SA, Khan M, Jo M-H, Jo MG, Amin FU, Kim MO. Melatonin stimulates the SIRT 1/Nrf2 signaling pathway counteracting lipopolysaccharide (LPS)-induced oxidative stress to rescue postnatal rat brain. CNS Neurosci Ther. 2017;23:33\u0026ndash;44.\u003c/li\u003e\n \u003cli\u003eHeinonen I, Rinne P, Ruohonen ST, Ruohonen S, Ahotupa M, Savontaus E. The effects of equal caloric high fat and western diet on metabolic syndrome, oxidative stress and vascular endothelial function in mice. Acta Physiol. 2014;211:515\u0026ndash;27.\u003c/li\u003e\n \u003cli\u003ePrieur EA, Jadavji NM. Assessing spatial working memory using the spontaneous alternation Y-maze test in aged male mice. Bio-Protoc. 2019;9:e3162\u0026ndash;e3162.\u003c/li\u003e\n \u003cli\u003eVorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc. 2006;1:848\u0026ndash;58.\u003c/li\u003e\n \u003cli\u003eDonaldson JG. Immunofluorescence staining. Curr Protoc Cell Biol. 2015;69:4\u0026ndash;3.\u003c/li\u003e\n \u003cli\u003eKesh SB, Sarkar D, Manna K. High-fat diet-induced oxidative stress and its impact on metabolic syndrome: a review. Asian J Pharm Clin Res. 2016;9:47\u0026ndash;52.\u003c/li\u003e\n \u003cli\u003eKohlgruber A, Lynch L. Adipose tissue inflammation in the pathogenesis of type 2 diabetes. Curr Diab Rep. 2015;15:1\u0026ndash;11.\u003c/li\u003e\n \u003cli\u003eSkelly DT, Hennessy E, Dansereau M-A, Cunningham C. A systematic analysis of the peripheral and CNS effects of systemic LPS, IL-1\u0026beta;, TNF-\u0026alpha; and IL-6 challenges in C57BL/6 mice. PloS One. 2013;8:e69123.\u003c/li\u003e\n \u003cli\u003eSkelly DT, Hennessy E, Dansereau M-A, Cunningham C. A systematic analysis of the peripheral and CNS effects of systemic LPS, IL-1\u0026beta;, TNF-\u0026alpha; and IL-6 challenges in C57BL/6 mice. PloS One. 2013;8:e69123.\u003c/li\u003e\n \u003cli\u003eCarrillo-Vico A, Lardone PJ, \u0026Aacute;lvarez-S\u0026aacute;nchez N, Rodr\u0026iacute;guez-Rodr\u0026iacute;guez A, Guerrero JM. Melatonin: buffering the immune system. Int J Mol Sci. 2013;14:8638\u0026ndash;83.\u003c/li\u003e\n \u003cli\u003eNabavi SM, Nabavi SF, Sureda A, Xiao J, Dehpour AR, Shirooie S, et al. Anti-inflammatory effects of Melatonin: A mechanistic review. Crit Rev Food Sci Nutr. 2019;59:S4\u0026ndash;16.\u003c/li\u003e\n \u003cli\u003eFavero G, Franceschetti L, Bonomini F, Rodella LF, Rezzani R. Melatonin as an anti-inflammatory agent modulating inflammasome activation. Int J Endocrinol. 2017;2017.\u003c/li\u003e\n \u003cli\u003eLiu X, Gong Y, Xiong K, Ye Y, Xiong Y, Zhuang Z, et al. Melatonin mediates protective effects on inflammatory response induced by interleukin-1 beta in human mesenchymal stem cells. J Pineal Res. 2013;55:14\u0026ndash;25.\u003c/li\u003e\n \u003cli\u003eTaylor ZB, Stevenson RJ, Ehrenfeld L, Francis HM. The impact of saturated fat, added sugar and their combination on human hippocampal integrity and function: A systematic review and meta-analysis. Neurosci Biobehav Rev. 2021;130:91\u0026ndash;106.\u003c/li\u003e\n \u003cli\u003eKanoski SE, Davidson TL. Western diet consumption and cognitive impairment: links to hippocampal dysfunction and obesity. Physiol Behav. 2011;103:59\u0026ndash;68.\u003c/li\u003e\n \u003cli\u003eAskarova S, Umbayev B, Masoud A-R, Kaiyrlykyzy A, Safarova Y, Tsoy A, et al. The links between the gut microbiome, aging, modern lifestyle and Alzheimer\u0026rsquo;s disease. Front Cell Infect Microbiol. 2020;10:104.\u003c/li\u003e\n \u003cli\u003eMatsubara E, Bryant-Thomas T, Pacheco Quinto J, Henry TL, Poeggeler B, Herbert D, et al. Melatonin increases survival and inhibits oxidative and amyloid pathology in a transgenic model of Alzheimer\u0026rsquo;s disease. J Neurochem. 2003;85:1101\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eLahiri DK. Melatonin affects the metabolism of the \u0026beta;-amyloid precursor protein in different cell types. J Pineal Res. 1999;26:137\u0026ndash;46.\u003c/li\u003e\n \u003cli\u003eParrott MD, Greenwood CE. Dietary influences on cognitive function with aging: from high-fat diets to healthful eating. Ann N Y Acad Sci. 2007;1114:389\u0026ndash;97.\u003c/li\u003e\n \u003cli\u003eMcNeilly AD, Stewart CA, Sutherland C, Balfour DJ. High fat feeding is associated with stimulation of the hypothalamic-pituitary-adrenal axis and reduced anxiety in the rat. Psychoneuroendocrinology. 2015;52:272\u0026ndash;80.\u003c/li\u003e\n \u003cli\u003eKothari V, Luo Y, Tornabene T, O\u0026rsquo;Neill AM, Greene MW, Geetha T, et al. High fat diet induces brain insulin resistance and cognitive impairment in mice. Biochim Biophys Acta BBA-Mol Basis Dis. 2017;1863:499\u0026ndash;508.\u003c/li\u003e\n \u003cli\u003eBrydon L, Petit L, Delagrange P, Strosberg AD, Jockers R. Functional expression of MT2 (Mel1b) melatonin receptors in human PAZ6 adipocytes. Endocrinology. 2001;142:4264\u0026ndash;71.\u003c/li\u003e\n \u003cli\u003eSalminen A, Kaarniranta K, Kauppinen A. Crosstalk between oxidative stress and SIRT1: impact on the aging process. Int J Mol Sci. 2013;14:3834\u0026ndash;59.\u003c/li\u003e\n \u003cli\u003eChan S-H, Hung C-H, Shih J-Y, Chu P-M, Cheng Y-H, Lin H-C, et al. SIRT1 inhibition causes oxidative stress and inflammation in patients with coronary artery disease. Redox Biol. 2017;13:301\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eKim J, Cha Y-N, Surh Y-J. A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in inflammatory disorders. Mutat Res Mol Mech Mutagen. 2010;690:12\u0026ndash;23.\u003c/li\u003e\n \u003cli\u003eXiang Q, Zhao Y, Lin J, Jiang S, Li W. The Nrf2 antioxidant defense system in intervertebral disc degeneration: Molecular insights. Exp Mol Med. 2022;54:1067\u0026ndash;75.\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","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":"HFD, Obesity, Neuroinflammation, Neurodegeneration, Melatonin Treatment, Synaptic Dysfunction, ROS, Oxidative Stress, SIRT1/NRF2","lastPublishedDoi":"10.21203/rs.3.rs-3376865/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3376865/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"High fat diet (HFD) is a prime factor, which contributes to the present epidemic of metabolic syndrome. HFD induces oxidative stress (OS) that in turn causes neuroinflammation, neurodegeneration, synaptic dysfunction, insulin resistance and amyloid burden. Pineal gland secretes a potent anti-apoptotic hormone, melatonin that has the potential to nullify the toxic effect of reactive oxygen species (ROS) and ameliorate various complications induced by HFD in rodent models. We aimed to find the therapeutic effects of melatonin against HFD-induced neuroinflammation and neurodegeneration mediated by OS in pregnant mice and their offspring. The neuroprotective effects of melatonin against HFD induced neurotoxicity were analyzed by Western blotting and immunofluorescence in the brain homogenates of pregnant albino mice and their offspring. Short and long term memory loss was evaluated. HFD significantly induced OS leading to neuroinflammation and apoptotic neurodegeneration in cortex and hippocampus of the pregnant mice and their offspring. However, administration of melatonin with HFD significantly improved synaptic dysfunction and inhibited both the neuroinflammation and neurodegeneration in albino mice. Here, we report that melatonin exerts these effects via SIRT1 mediated NRF-2/HO-1 signaling pathway to reduce HFD-induced OS and insulin resistance. This study revealed that melatonin could reverse HFD induced multiple complications leading to the memory dysfunction in pregnant female mice and their successive generation via activation of SIRT1/NRF2 signaling pathway.","manuscriptTitle":"Melatonin Ameliorates High Fat Diet Induced Oxidative Stress-Mediated Insulin Resistance, Amyloid Burden and Memory Dysfunction in Female Mice and their offspring","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-04 14:30:53","doi":"10.21203/rs.3.rs-3376865/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":"cc69d46e-538c-47d1-bbcb-ac878d231af4","owner":[],"postedDate":"October 4th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-10-04T16:44:24+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-04 14:30:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3376865","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3376865","identity":"rs-3376865","version":["v1"]},"buildId":"uzvnyPIH0yfUW73ClG4p1","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}