Aerobic Exercise and Nano-curcumin Supplementation Prevent cancer symptom development through MAPK/ERK pathway

Aerobic Exercise and Nano-curcumin Supplementation Prevent cancer symptom development through MAPK/ERK pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Aerobic Exercise and Nano-curcumin Supplementation Prevent cancer symptom development through MAPK/ERK pathway Zahra Vafaee Mastan Ababd, Nader Hamed Chaman, Masoumeh Hosseini, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3257588/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective/background: Impaired cell internal settings and its excessive proliferation causes the occurrence of various syndrom and diseases such as cancer. One of the cell signaling pathways that lead to these outcomes is the MAPK/ERK pathway. So, the goal of the present study is to investigate the effect of aerobic training and Nano-curcumin supplementation on RAS and ERK gene expression in rat muscle tissue. Materials and methods In this experimental study, 32 male wistar rats (aged 4–6 weeks, 130–150 g) were randomly assigned into four groups (n = 8/groups), including Control (C), Moderate Intensity Aerobic Exercise (MICT), Nano-Curcumin Supplementation (NCS) and moderate intensity aerobic exercise + Nano-curcumin (MICT + NCS). The training groups implemented the MICT for 4 weeks and 5 sessions per week. The Supplement groups received 80 mg/kg/day through oral gavage for 4 weeks. Genes expression of RAS and ERK were measured with real-time PCR method. Data were analyzed by one-way ANOVA and Bonferroni post-hoc tests (P < 0.05). Findings: Results showed that 4 weeks of moderate intensity aerobic exercise and Nano-curcumin supplementation significantly decreased the gene expression levels of RAS and ERK in muscle tissue of rats. Conclusion Implementation of 4 weeks of MICT combined with Nano-Curcumin supplementation, more efficiently alleviated the RAS/ERK gene expression as symptom development of cancer. Aerobic exercise Nano-curcumin RAS ERK Figures Figure 1 Figure 2 Introduction Today, cancer is one of the most important public hygiene problems all over the world ( 1 ). Cancer is one of the main causes of morbidity in the world ( 2 ). There is a deficiency in ERK/MAPK route in many cancers. This route can be a potentially proper target for cancer treatment. The RAS/RAF/MEK/REK cascade transfers signals to the transcription factors from cell surface receptors that cause gene expression regulation. In addition, it regulates the activity of the proteins involved in apoptosis as well ( 3 ). RAS is an ancho protein that is mainly active in many human cancer types. In over 90 percent of cancers, mutation in RAS gene has been observed. RAS proteins act as molecular switches and become active by bonding to GTP and the messaging cascade creates the cellular procedures control, including replication, differentiation, adhesion, apoptosis, migration and cell division ( 1 ). RAS protein plays an important role in MAPK cascade route. MAPK signaling route plays a critical role in signal transfer from kinases protein and phosphatase protein. The well-known kinase in this route is ERK. ERK signaling route includes important mediators that are proper for growth and replication ( 4 ). Kinase MAP cascade trend plays a role in setting up the cellular replication responses via agonists that stimulate C kinase protein, and through growth factors acting on tyrosine receptors and lead to RAS activation. In cascade trend, it causes the kinase RAF activation, also activating MEK whether mediated by C kinase protein or by RAS. The phosphorylated MEK activates and phosphorylases ERK that in turn phosphorylases transcription factors, protein substrates and other kinase proteins that are important in setting up the cellular replication and other cellular responses ( 5 ). The protective role of ERK happens by activating kinase tyrosine, BCL2, BAD and NF-Kβ imposition or inhibiting the C cytochrome release ( 6 ). Some studies have shown that physical activity increases the gene expression and ERK and RAS protein. In most of these studies, ERK response to one or more activity sessions has been examined and immediately after the activity, the activity level and ERK phosphorylation amount have been investigated. For instance, the in-situ studies ( 7 ), cycling protocols in the exercised and non-exercised people, resistive and power exercises ( 8 ) have shown that ERK is activated immediately after the activity. Nemati et al (2019) showed an increase in ERK after eight weeks of resistive exercise in the rats’ flexor muscles of the big toe ( 9 ). Xie et al (2007) also reported an increase in RAS protein levels in rats’ skin tissue in reaction to a 60-minutes activity session of high intensity over working wheel ( 10 ). On the contrary, Mirdar Harijani and Mousavi (2020) reported the invariability of RAS after 12 weeks of rats’ lung below- maximum swimming exercise exposed to NNK carcinogen ( 11 ). The application of curcumin Nanoparticles is an effective method in treatment of cancer. Curcumin is a natural substance existing in CURCUMA LONGA plant, a member of ginger family, and is predominantly known as turmeric. Given the fact that no special toxic side-effects have been reported of this natural by-product, the consumption of this product is considered in treatment regimes in cancer patients as a well-known compound factor and drug supplementation. Causing cellular replication suppression and apoptosis imposition in a wide range of tumoral cells via unknown mechanisms, performs its anti-cancer and pharmaceutical activity to a large extent by inhibiting defensive signaling routes of NF-ĸB, PPAR-Y, CYCLIN-D1 and PI3/AKT ( 12 ). By imposing apoptosis via releasing cytochrome C and by affecting P53 protein, curcumin prevents from cancer cells replication ( 13 ). SANDHIUTAMI et al (2021) concluded in a study that prescription of sys platin and Nano- Curcumin lead to a considerable reduction in ovarian tumor. They hence concluded that Nano-Curcumin, that is administered as a joint treatment with Sys Platin, has a treatment potential in ovarian cancer models via reducing signaling routes regulation of PI3K/AKT, JAK/STAT3 ( 14 ). As the pharmaceutical plants have robust anti-cancer property and sporting is also one of the life-style factors that reduces the risk of suffering cancer, there is the possibility that the consumption of Nano- Curcumin, along with sportive exercises, could have major impacts in treating such patients. Very limited studies have been conducted over the fact that sportive exercise, combined with Nano-Curcumin, have an infinite impact. The scholar of the present study sought to answer the question of whether or not the synergy of moderate intensity aerobic exercise and receiving Nano-Curcumin supplementation have an impact on RAS and ERK genes expression in rats’ muscular cell. Materials and methods Animals In this experimental study, Fifty-six male Wistar rats, weighing 130–150 g that were bred in the Center of Razi Animal Institute, Karaj Medical University was obtained. Animals were allowed food and water ad libitum before and during the experiment. Upon delivery to the our laboratory animal experiment location, rats were provided 1 week interval for acclimation to the environment (24 ± 1°C; humidity of 45–55%, 12:12 dark/light cycle), with free access to water and standard chow (comprising of 54% mixed carbohydrate, 19% protein and 3% lipid). After this period, the rats were devided into 4 groups of Control (C), Moderate Intensity Aerobic Exercise (MICT), Nano-Curcumin Supplementation (NCS) and moderate intensity aerobic exercise + Nano-curcumin (MICT + NCS). Animal care and ethical principles were based on the Guide for the Care and Use of Laboratory Animals [34] approved by the Ethics Committee of Islamic Azad University, eastern Tehran branch [IR.IAU.SRB.REC.1400.055]. Aerobic exercise training protocol and load measurments After a week of adaptation to treadmill running (3 times/week, 10-min/session), MICT groups performed 18 m/min for a total time of 20 min a day at 0% incline for 4 weeks, 5 sessions per week on a motorized treadmill. The exercise speed and duration were increased gradually, in a way that in the 4th week, the rats were able to run at a speed of 20m per minutes and 35 minutes a day ( 15 ) (Table 1 ). To simulate the conditions, in order to reduce the stress of the cages, the sedentary rats were kept out of the cages on a stationary treadmill for the same period of exercise as the training groups. Table 1 MICT training protocol Variable Week 1 Week 2 Week 3 Week 4 Intensity (meter) 18 m/min 18 m/min 20 m/min 20 m/min Duration (min) 20 min/day 25 min/day 30 min/day 35 min/day Nano-Curcumin supplementation The dose of Vanadium-Zinc supplementation was chosen in accordance with the weight of the rats (80 mg/kg/day) diluted in 1.0 mL of distilled water daily for eight weeks through oral gavage ( 16 ). Tissue extraction and blood collection After spending a 4-week period of treatment, the rats in the fasting state (12 hours) were anesthetized with intraperitoneal injection of a combination of ketamine and xylazine (80 and 12 mg/kg, respectively) 48 hours right after the last exercise session. Soleus tissue was excised and separated via the gap on the lateral dorsal region of the lower limb and immediately freezes with liquid nitrogen and stored at − 80 ◦C until processing for realtime (RT) polymerase chain reaction (PCR) assessment ( 35 ). RT-PCR analysis Briefly, total RNA was isolated from the soleus tissue by total RNA extraction mini kit (Favorgen Biotech, Taiwan) as described in the supplier’s protocol. The products were then treated with DNase I Kit (Sinaclon, Iran) to remove possible DNA. First-strand cDNA was synthesized from RNA samples with MMLV reverse transcriptase (YTA, Iran), utilizing oligo (dT) primer and dNTP mix according to the manufacturer’s protocol. In this study, specific primers (RAS, ERK and GAPDH) were designed with Primer premier 5 software. The primer sequences used in this study have been presented in Table 2 . Real-time Polymerase Chain Reaction (PCR) was performed using RealQ Plus 2x Master Mix Green (Ampliqon, Denmark) in Rotor Gene 6000 (Corbett Research, Australia). The thermal cycling conditions were followed with 95 ◦C for 15 min, 40 cycles at 95 ◦ C for 30s, 60 ◦ C for 30s and 72 ◦ C for 20s. The expression level of each sample was normalized against the expression of GAPDH as a suitable internal control ( 35 ). The relative expression level was also calculated using the comparative CT method 2 − ΔΔCT ( 36 ). Table 2 RT-PCR forward and reverse primer sequences Gene Reverse and forward primer NCBI accession number Amplicon Size Ras (Hras) F-5'-TGTTACCAACTGGGACGACA-3' R-5'-TCTCAGCTGTGGTGGTGAAG-3' NM_001130442.2 121 Ephb1 (ERK) F-5´-ATGCGCTTCACTGTGAGAGAC-3´ R-5´-ATTCCGAGTAAGAGGCCCAAA-3´ NM_001038663.1 160 GAPDH F-5'-ATCAGGTTACTTTCTTGTTCAGCG-3' R-5'-TGATGCCCTGGTCAGTCTTG-3' NM_012520.2 147 Statistical analysis All data were presented as means ± SEM. Groups comparisons were achieved using one-way ANOVA, followed by the Bonferoni post-hoc test. Statistical significance was considered at P < 0.05. The statistical analysis was performed using SPSS statistical software (Version 22). Results The results of ANOVA showed that 4 weeks of Nano-curcumin supplementation and moderate intensity aerobic exercise led to a significant difference in the RAS gene expression levels (p = 0.029) among the study groups; according to the results of Bonferroni test, NCS (p = 0.338) and MICT + NCS (p = 0.493) were able to down-regulate RAS gene expression levels but MICT (p = 1.56) increased RAS gene expression in relation to Control group. Also, Nano-curcumin supplementation showed the greatest effects on modulating RAS gene expression levels (Fig. 1). Additionally, the results of ANOVA revealed a significant difference inthe ERK gene expression levels in the muscle tissue of rats. The results of Bonferroni test showed that ERK variations in response to MICT, NCS and MICT + NCS groups was decreased relative to control group. However, this reduction was significant just in NCS groups in comparison to Control (Fig. 2). Discussion The results displayed that moderate intensity aerobic exercise by itself caused an increase in RAS and the combination of the moderate intensity aerobic exercise with reception of Nano-Curcumin supplementation caused a significant reduction of RAS gene in rats’ muscular cell. The interruption in ERK/MAPK route exists in the majority of cancers. This route can be as a proper potential target for the treatment of cancer. The RAS/RAF/MEK/ERK cascade of cell surface receptors, transfers signals into transcription factors that causes gene expression regulation, as well as regulating the activities of the proteins involved in apoptosis ( 3 ). When the RAS protein is activated, it activates a protein called BRAF that is the key member and the initiator of MAPK route ( 17 ). The kinase cascade that begins from BRAF, enables MEK and kinase MAP family proteins following the activation (phosphorylation) to both activate parallel routes including PI3K and to pass through core membrane and to cause the genes expression of the cellular division via activating the transcription factors. In line with the result of the present survey, Xie et al (2008) in a survey reported an increase in the RAS protein levels in rats’ skin tissue in response to a 60 minutes activity session of high intensity over the working wheel ( 10 ). On the contrary, Mir dar Harijani and Mousavi (2020) reported that 12 weeks of below- maximum swimming exercise created no significant variation in the rats’ RAS subjected to NNK carcinogen ( 11 ) that is not in line with the results of the present survey. Reviewing the findings of various surveys over cellular replication indices response to sportive exercises or activity, the result is obtained that the variations of such indices are likely to occur in response to the sportive activity under a state dependent on the intensity and duration. The results displayed that Nano-Curcumin and the compound of exercise plus Nano-Curcumin caused a significant reduction of RAS. In line with the results of the present survey, Cao et al (2015) displayed, in a survey, that via activating the RAS/ERK signaling route and reducing Caspase cascade, Curcumin prevents AGS cells from growing and it causes apoptosis. And Curcumin might be a potential target for the treatment of liver cancer ( 18 ). Gao et al (2011) concluded, in a study, that Curcumin, reduced intra-cellular signaling proteins of RAS, B-RAF, P-MEK, P-ERK, C-FOS, and EGR-1 ( 19 ). By inhibiting NF-KB route, Curcumin inhibits the anti-apoptosis genes expression and increases the BAX apoptosis protein expression ( 20 ). By inhibiting the WNT route, that plays a role in cellular differentiation and tumor generation, Curcumin causes the inhibition of apoptosis imposition and differentiation ( 20 ). Inflammation is one of the causes associated with cancer progression where Curcumin causes an increase in HMOX1 and GCLM genes expression and a reduction in EGR1, PTGS2/COX2 (SYNTHASE2) and chemokines of CXCL-1 and CXCL-2 ( 21 ). By inhibiting the route associated with α6β4 integrin, inhibiting the imposition of TGF-β1 and inhibiting the MMP-9 expression, Curcumin causes the inhibition of cancer cells movement, as well as their invasive power ( 22 ). Another result of the survey displayed that moderate intensity aerobic exercise and the reception of Nano-Curcumin supplementation caused a significant reduction of ERK gene in rats’ muscular cells. The ERK kinase protein is phosphorylated and activated via the residuals of threonine and tyrosine. This protein activates many transcription proteins and factors with various purposes. The activation of ERK causes the expression of over 600 genes and its impact on genes transcription expression is applied either through the activation of messaging routes in cytoplasm or via dimerization and displacement to the core and the phosphorylation of transcription factors ( 23 ). In cytosol, ERK applies its impacts as follows: by activating such proteins as P90RSK, that go into the core and activate the SRF transcription factors. In addition, it can also activate such factors as ELK1, ATF2, AP1, CREB, and MEF2C into the core ( 24 ). The activity of this transcription factor, helps with the regeneration of muscular cells and repairing muscular damages by expressing the genes involved in numerous procedures as mitochondrial biogenesis, vessel creation or various types of cellular replication of mitoses, muse and post-mitoses performance like differentiation, apoptosis and D cyclin expression ( 25 ). MAPK route, ERK, in particular, could be activated affected by many factors. In addition, via different mechanisms, such as growth factor secretion and muscular tensile, oxidants and PH reduction, the physical activity is also able to conduct such activation ( 26 ). In line with the results of the present survey, WENCKER et al (2012) reported that six weeks of exercise over the rotating tape caused a reduction of ERK in rats ( 27 ). On the contrary, Nemati et al (2018) reported that eight weeks of resistive exercise created no significant variation in ERK in rats’ flexor muscles of the big toe ( 9 ). In addition, Thompson et al (2003) displayed that after 48 hours of eccentric activity of biceps, ERK content maintains its significant increase. Justifying this matter, the type of activity can be addressed. The eccentric contraction creates more muscular damage than other contractions. It has been shown, in the studies, that muscular damage and the destruction of the damaged muscular cell, cause an increase of free radicals and releasing of inflammatory factors: factors that maintain their increase after the first activity session as well ( 28 ). Jiang et al (2015) have shown that muscular damage, activates lipin-1 protein, one of the upstream factors of ERK messaging route. The lipin-1 protein causes the phosphorylation if ERK, forming a complex together that enters into the cell core and expresses cyclin D protein. Eventually, cyclin D causes the renovation of the damaged muscular cell ( 29 ). The results illustrated that Nano-Curcumin and the exercise plus Nano-Curcumin compound, caused a significant reduction of ERK. In line with the results of the present survey, Mortazavi et al (2020) reported that Nano-Curcumin prescription or mediating the ERK1/2 and JNK routes, has the ability to prevent from thrombocytopenia in mice undergoing chemotherapy ( 30 ). Zhang et al (2019) reported that by under-regulating the signaling route activity of ERK/C-JUN, Curcumin prevents the endometrial cancer ( 31 ). Zhang et al (2016) reported that the combination of two drugs of Sorafenib and Curcumin, significantly causes apoptosis imposition and a reduction of AKT and ERK proteins level in the FTC133 cancer cell ranking in comparison with mono-drug treatment ( 32 ). Mousavi et al (2018) reported that Curcumin could partially prevent from cellular death caused by OHDA-6 and the reduction of ERK. In addition to the inhibition of various intra-cellular anti-oxidant mechanisms, the increased ROS production could cause some chronic diseases such as the cancer via irreversible oxidative damage in nucleic acid, DNA, protein and membrane lipid, creation of interruption in cellular procedures such as cellular metabolism, message transfer, gene expression, cell replication, and planned cellular death. The impact of Curcumin in the ERK activity reconstruction refers to its inhibitory impact on ROS production ( 33 ). Conclusion The results displayed that moderate intensity aerobic exercise caused a significant increase in RAS gene expression and a significant reduction of ERK in rats’ soleus muscle. In addition, the consumption of Nano-Curcumin also caused a significant reduction of RAS and ERK genes. 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Curcumin prevents 6 - OHDA induced cell death and ERK disruption in Human neuroblastoma cells. Journal of Knowledge & Health in Basic Medical Sciences 2018;13(3):1-7 . Council NR. Textbook of guide for the care and use of laboratory animals. Fathi R, Nasiri K, Akbari A, Ahmadi-KaniGolzar F, Farajtabar Z. Exercise protects against ethanol-induced damage in rat heart and liver through the inhibition of apoptosis and activation of Nrf2/Keap-1/HO-1 pathway. Life Sciences. 2020 Sep 1;256:117958. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(−Delta Delta C(T)) method, Methods 25 (2001) 402–408. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3257588","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":226651675,"identity":"05bc5d7e-06c2-4024-ba65-727dc0565333","order_by":0,"name":"Zahra Vafaee Mastan Ababd","email":"","orcid":"","institution":"Azad Islamic University of Tehran East","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"Vafaee Mastan","lastName":"Ababd","suffix":""},{"id":226651676,"identity":"77247bf4-8998-4cff-ae9d-f27306f5b669","order_by":1,"name":"Nader Hamed Chaman","email":"","orcid":"","institution":"University of Mazandaran","correspondingAuthor":false,"prefix":"","firstName":"Nader","middleName":"Hamed","lastName":"Chaman","suffix":""},{"id":226651678,"identity":"4246edec-9e3b-4f9e-9c22-3a2dea771b8e","order_by":2,"name":"Masoumeh Hosseini","email":"","orcid":"","institution":"Azad Islamic University of Tehran East","correspondingAuthor":false,"prefix":"","firstName":"Masoumeh","middleName":"","lastName":"Hosseini","suffix":""},{"id":226651680,"identity":"475eb73e-6785-4119-90ee-615c6bf5449c","order_by":3,"name":"Amir Maleki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYFAC5gbGBgMbOfvjzQcYGBuI0sII0pJmzHDmWAIpWhgOJTLcyDEgTotu+8HGhzMKDiQwNuR8k/i5w0aOgf3w0Q34tJidSWw23GBwJ4+Z4ew2yd4zQBfypKXdwKvlQGKb5AODZ8VsjL3bJHjbDic2SPCY4ddy/mH7zwcGhxN7mHmeSf4lSsuNxDbGDUAtM9h42KSJs+XGw2bJGcBANuBhM7aWbUszZiPol/PJBz/2/LGRM5B//PDm2zYbOX72w8fwakEGLBIgko1Y5SDA/IEU1aNgFIyCUTByAADXmlMrUgauRAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Mazandaran","correspondingAuthor":true,"prefix":"","firstName":"Amir","middleName":"","lastName":"Maleki","suffix":""}],"badges":[],"createdAt":"2023-08-12 08:14:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3257588/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3257588/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":41875561,"identity":"596228a5-1fed-4267-a591-05c3c31e3132","added_by":"auto","created_at":"2023-08-21 13:56:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65199,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3257588/v1/333d4c3d08f5df2a486ae8fa.png"},{"id":41875562,"identity":"f2e7e5d4-639d-42c2-9761-0e3055fa9d04","added_by":"auto","created_at":"2023-08-21 13:56:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69151,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3257588/v1/54ce08e8949bba0984a78990.png"},{"id":44391723,"identity":"e8d539b3-c31b-4133-87ea-b16964848b38","added_by":"auto","created_at":"2023-10-10 20:37:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":390295,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3257588/v1/837fb145-d29b-47a3-8ee0-b1b47e1e15a2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Aerobic Exercise and Nano-curcumin Supplementation Prevent cancer symptom development through MAPK/ERK pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eToday, cancer is one of the most important public hygiene problems all over the world (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Cancer is one of the main causes of morbidity in the world (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). There is a deficiency in ERK/MAPK route in many cancers. This route can be a potentially proper target for cancer treatment. The RAS/RAF/MEK/REK cascade transfers signals to the transcription factors from cell surface receptors that cause gene expression regulation. In addition, it regulates the activity of the proteins involved in apoptosis as well (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). RAS is an ancho protein that is mainly active in many human cancer types. In over 90 percent of cancers, mutation in RAS gene has been observed. RAS proteins act as molecular switches and become active by bonding to GTP and the messaging cascade creates the cellular procedures control, including replication, differentiation, adhesion, apoptosis, migration and cell division (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). RAS protein plays an important role in MAPK cascade route. MAPK signaling route plays a critical role in signal transfer from kinases protein and phosphatase protein. The well-known kinase in this route is ERK. ERK signaling route includes important mediators that are proper for growth and replication (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Kinase MAP cascade trend plays a role in setting up the cellular replication responses via agonists that stimulate C kinase protein, and through growth factors acting on tyrosine receptors and lead to RAS activation. In cascade trend, it causes the kinase RAF activation, also activating MEK whether mediated by C kinase protein or by RAS. The phosphorylated MEK activates and phosphorylases ERK that in turn phosphorylases transcription factors, protein substrates and other kinase proteins that are important in setting up the cellular replication and other cellular responses (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The protective role of ERK happens by activating kinase tyrosine, BCL2, BAD and NF-Kβ imposition or inhibiting the C cytochrome release (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome studies have shown that physical activity increases the gene expression and ERK and RAS protein. In most of these studies, ERK response to one or more activity sessions has been examined and immediately after the activity, the activity level and ERK phosphorylation amount have been investigated. For instance, the in-situ studies (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), cycling protocols in the exercised and non-exercised people, resistive and power exercises (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) have shown that ERK is activated immediately after the activity. Nemati et al (2019) showed an increase in ERK after eight weeks of resistive exercise in the rats\u0026rsquo; flexor muscles of the big toe (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Xie et al (2007) also reported an increase in RAS protein levels in rats\u0026rsquo; skin tissue in reaction to a 60-minutes activity session of high intensity over working wheel (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). On the contrary, Mirdar Harijani and Mousavi (2020) reported the invariability of RAS after 12 weeks of rats\u0026rsquo; lung below- maximum swimming exercise exposed to NNK carcinogen (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe application of curcumin Nanoparticles is an effective method in treatment of cancer. Curcumin is a natural substance existing in CURCUMA LONGA plant, a member of ginger family, and is predominantly known as turmeric. Given the fact that no special toxic side-effects have been reported of this natural by-product, the consumption of this product is considered in treatment regimes in cancer patients as a well-known compound factor and drug supplementation. Causing cellular replication suppression and apoptosis imposition in a wide range of tumoral cells via unknown mechanisms, performs its anti-cancer and pharmaceutical activity to a large extent by inhibiting defensive signaling routes of NF-ĸB, PPAR-Y, CYCLIN-D1 and PI3/AKT (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). By imposing apoptosis via releasing cytochrome C and by affecting P53 protein, curcumin prevents from cancer cells replication (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). SANDHIUTAMI et al (2021) concluded in a study that prescription of sys platin and Nano- Curcumin lead to a considerable reduction in ovarian tumor. They hence concluded that Nano-Curcumin, that is administered as a joint treatment with Sys Platin, has a treatment potential in ovarian cancer models via reducing signaling routes regulation of PI3K/AKT, JAK/STAT3 (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs the pharmaceutical plants have robust anti-cancer property and sporting is also one of the life-style factors that reduces the risk of suffering cancer, there is the possibility that the consumption of Nano- Curcumin, along with sportive exercises, could have major impacts in treating such patients. Very limited studies have been conducted over the fact that sportive exercise, combined with Nano-Curcumin, have an infinite impact. The scholar of the present study sought to answer the question of whether or not the synergy of moderate intensity aerobic exercise and receiving Nano-Curcumin supplementation have an impact on RAS and ERK genes expression in rats\u0026rsquo; muscular cell.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eIn this experimental study, Fifty-six male Wistar rats, weighing 130\u0026ndash;150 g that were bred in the Center of Razi Animal Institute, Karaj Medical University was obtained. Animals were allowed food and water ad libitum before and during the experiment. Upon delivery to the our laboratory animal experiment location, rats were provided 1 week interval for acclimation to the environment (24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C; humidity of 45\u0026ndash;55%, 12:12 dark/light cycle), with free access to water and standard chow (comprising of 54% mixed carbohydrate, 19% protein and 3% lipid). After this period, the rats were devided into 4 groups of Control (C), Moderate Intensity Aerobic Exercise (MICT), Nano-Curcumin Supplementation (NCS) and moderate intensity aerobic exercise\u0026thinsp;+\u0026thinsp;Nano-curcumin (MICT\u0026thinsp;+\u0026thinsp;NCS). Animal care and ethical principles were based on the Guide for the Care and Use of Laboratory Animals [34] approved by the Ethics Committee of Islamic Azad University, eastern Tehran branch [IR.IAU.SRB.REC.1400.055].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAerobic exercise training protocol and load measurments\u003c/h2\u003e \u003cp\u003eAfter a week of adaptation to treadmill running (3 times/week, 10-min/session), MICT groups performed 18 m/min for a total time of 20 min a day at 0% incline for 4 weeks, 5 sessions per week on a motorized treadmill. The exercise speed and duration were increased gradually, in a way that in the 4th week, the rats were able to run at a speed of 20m per minutes and 35 minutes a day (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To simulate the conditions, in order to reduce the stress of the cages, the sedentary rats were kept out of the cages on a stationary treadmill for the same period of exercise as the training groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMICT training protocol\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeek 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeek 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWeek 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWeek 4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntensity (meter)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18 m/min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 m/min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20 m/min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 m/min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration (min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 min/day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25 min/day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 min/day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35 min/day\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNano-Curcumin supplementation\u003c/h2\u003e \u003cp\u003eThe dose of Vanadium-Zinc supplementation was chosen in accordance with the weight of the rats (80 mg/kg/day) diluted in 1.0 mL of distilled water daily for eight weeks through oral gavage (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTissue extraction and blood collection\u003c/h2\u003e \u003cp\u003eAfter spending a 4-week period of treatment, the rats in the fasting state (12 hours) were anesthetized with intraperitoneal injection of a combination of ketamine and xylazine (80 and 12 mg/kg, respectively) 48 hours right after the last exercise session. Soleus tissue was excised and separated via the gap on the lateral dorsal region of the lower limb and immediately freezes with liquid nitrogen and stored at \u0026minus;\u0026thinsp;80 ◦C until processing for realtime (RT) polymerase chain reaction (PCR) assessment (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRT-PCR analysis\u003c/h2\u003e \u003cp\u003eBriefly, total RNA was isolated from the soleus tissue by total RNA extraction mini kit (Favorgen Biotech, Taiwan) as described in the supplier\u0026rsquo;s protocol. The products were then treated with DNase I Kit (Sinaclon, Iran) to remove possible DNA. First-strand cDNA was synthesized from RNA samples with MMLV reverse transcriptase (YTA, Iran), utilizing oligo (dT) primer and dNTP mix according to the manufacturer\u0026rsquo;s protocol. In this study, specific primers (RAS, ERK and GAPDH) were designed with Primer premier 5 software. The primer sequences used in this study have been presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Real-time Polymerase Chain Reaction (PCR) was performed using RealQ Plus 2x Master Mix Green (Ampliqon, Denmark) in Rotor Gene 6000 (Corbett Research, Australia). The thermal cycling conditions were followed with 95 ◦C for 15 min, 40 cycles at 95\u003csup\u003e◦\u003c/sup\u003eC for 30s, 60\u003csup\u003e◦\u003c/sup\u003eC for 30s and 72\u003csup\u003e◦\u003c/sup\u003eC for 20s. The expression level of each sample was normalized against the expression of GAPDH as a suitable internal control (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The relative expression level was also calculated using the comparative CT method 2\u003csup\u003e\u0026minus; ΔΔCT\u003c/sup\u003e (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRT-PCR forward and reverse primer sequences\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse and forward primer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNCBI accession number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmplicon Size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRas (Hras)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5'-TGTTACCAACTGGGACGACA-3'\u003c/p\u003e \u003cp\u003eR-5'-TCTCAGCTGTGGTGGTGAAG-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_001130442.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEphb1 (ERK)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026acute;-ATGCGCTTCACTGTGAGAGAC-3\u0026acute;\u003c/p\u003e \u003cp\u003eR-5\u0026acute;-ATTCCGAGTAAGAGGCCCAAA-3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_001038663.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGAPDH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5'-ATCAGGTTACTTTCTTGTTCAGCG-3'\u003c/p\u003e \u003cp\u003eR-5'-TGATGCCCTGGTCAGTCTTG-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_012520.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e147\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Groups comparisons were achieved using one-way ANOVA, followed by the Bonferoni post-hoc test. Statistical significance was considered at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The statistical analysis was performed using SPSS statistical software (Version 22).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe results of ANOVA showed that 4 weeks of Nano-curcumin supplementation and moderate intensity aerobic exercise led to a significant difference in the RAS gene expression levels (p\u0026thinsp;=\u0026thinsp;0.029) among the study groups; according to the results of Bonferroni test, NCS (p\u0026thinsp;=\u0026thinsp;0.338) and MICT\u0026thinsp;+\u0026thinsp;NCS (p\u0026thinsp;=\u0026thinsp;0.493) were able to down-regulate RAS gene expression levels but MICT (p\u0026thinsp;=\u0026thinsp;1.56) increased RAS gene expression in relation to Control group. Also, Nano-curcumin supplementation showed the greatest effects on modulating RAS gene expression levels (Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAdditionally, the results of ANOVA revealed a significant difference inthe ERK gene expression levels in the muscle tissue of rats. The results of Bonferroni test showed that ERK variations in response to MICT, NCS and MICT\u0026thinsp;+\u0026thinsp;NCS groups was decreased relative to control group. However, this reduction was significant just in NCS groups in comparison to Control (Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results displayed that moderate intensity aerobic exercise by itself caused an increase in RAS and the combination of the moderate intensity aerobic exercise with reception of Nano-Curcumin supplementation caused a significant reduction of RAS gene in rats\u0026rsquo; muscular cell. The interruption in ERK/MAPK route exists in the majority of cancers. This route can be as a proper potential target for the treatment of cancer. The RAS/RAF/MEK/ERK cascade of cell surface receptors, transfers signals into transcription factors that causes gene expression regulation, as well as regulating the activities of the proteins involved in apoptosis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). When the RAS protein is activated, it activates a protein called BRAF that is the key member and the initiator of MAPK route (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The kinase cascade that begins from BRAF, enables MEK and kinase MAP family proteins following the activation (phosphorylation) to both activate parallel routes including PI3K and to pass through core membrane and to cause the genes expression of the cellular division via activating the transcription factors. In line with the result of the present survey, Xie et al (2008) in a survey reported an increase in the RAS protein levels in rats\u0026rsquo; skin tissue in response to a 60 minutes activity session of high intensity over the working wheel (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). On the contrary, Mir dar Harijani and Mousavi (2020) reported that 12 weeks of below- maximum swimming exercise created no significant variation in the rats\u0026rsquo; RAS subjected to NNK carcinogen (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) that is not in line with the results of the present survey. Reviewing the findings of various surveys over cellular replication indices response to sportive exercises or activity, the result is obtained that the variations of such indices are likely to occur in response to the sportive activity under a state dependent on the intensity and duration. The results displayed that Nano-Curcumin and the compound of exercise plus Nano-Curcumin caused a significant reduction of RAS. In line with the results of the present survey, Cao et al (2015) displayed, in a survey, that via activating the RAS/ERK signaling route and reducing Caspase cascade, Curcumin prevents AGS cells from growing and it causes apoptosis. And Curcumin might be a potential target for the treatment of liver cancer (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Gao et al (2011) concluded, in a study, that Curcumin, reduced intra-cellular signaling proteins of RAS, B-RAF, P-MEK, P-ERK, C-FOS, and EGR-1 (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). By inhibiting NF-KB route, Curcumin inhibits the anti-apoptosis genes expression and increases the BAX apoptosis protein expression (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). By inhibiting the WNT route, that plays a role in cellular differentiation and tumor generation, Curcumin causes the inhibition of apoptosis imposition and differentiation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Inflammation is one of the causes associated with cancer progression where Curcumin causes an increase in HMOX1 and GCLM genes expression and a reduction in EGR1, PTGS2/COX2 (SYNTHASE2) and chemokines of CXCL-1 and CXCL-2 (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). By inhibiting the route associated with α6β4 integrin, inhibiting the imposition of TGF-β1 and inhibiting the MMP-9 expression, Curcumin causes the inhibition of cancer cells movement, as well as their invasive power (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother result of the survey displayed that moderate intensity aerobic exercise and the reception of Nano-Curcumin supplementation caused a significant reduction of ERK gene in rats\u0026rsquo; muscular cells. The ERK kinase protein is phosphorylated and activated via the residuals of threonine and tyrosine. This protein activates many transcription proteins and factors with various purposes. The activation of ERK causes the expression of over 600 genes and its impact on genes transcription expression is applied either through the activation of messaging routes in cytoplasm or via dimerization and displacement to the core and the phosphorylation of transcription factors (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In cytosol, ERK applies its impacts as follows: by activating such proteins as P90RSK, that go into the core and activate the SRF transcription factors. In addition, it can also activate such factors as ELK1, ATF2, AP1, CREB, and MEF2C into the core (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The activity of this transcription factor, helps with the regeneration of muscular cells and repairing muscular damages by expressing the genes involved in numerous procedures as mitochondrial biogenesis, vessel creation or various types of cellular replication of mitoses, muse and post-mitoses performance like differentiation, apoptosis and D cyclin expression (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). MAPK route, ERK, in particular, could be activated affected by many factors. In addition, via different mechanisms, such as growth factor secretion and muscular tensile, oxidants and PH reduction, the physical activity is also able to conduct such activation (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In line with the results of the present survey, WENCKER et al (2012) reported that six weeks of exercise over the rotating tape caused a reduction of ERK in rats (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). On the contrary, Nemati et al (2018) reported that eight weeks of resistive exercise created no significant variation in ERK in rats\u0026rsquo; flexor muscles of the big toe (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In addition, Thompson et al (2003) displayed that after 48 hours of eccentric activity of biceps, ERK content maintains its significant increase. Justifying this matter, the type of activity can be addressed. The eccentric contraction creates more muscular damage than other contractions. It has been shown, in the studies, that muscular damage and the destruction of the damaged muscular cell, cause an increase of free radicals and releasing of inflammatory factors: factors that maintain their increase after the first activity session as well (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Jiang et al (2015) have shown that muscular damage, activates lipin-1 protein, one of the upstream factors of ERK messaging route. The lipin-1 protein causes the phosphorylation if ERK, forming a complex together that enters into the cell core and expresses cyclin D protein. Eventually, cyclin D causes the renovation of the damaged muscular cell (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The results illustrated that Nano-Curcumin and the exercise plus Nano-Curcumin compound, caused a significant reduction of ERK. In line with the results of the present survey, Mortazavi et al (2020) reported that Nano-Curcumin prescription or mediating the ERK1/2 and JNK routes, has the ability to prevent from thrombocytopenia in mice undergoing chemotherapy (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Zhang et al (2019) reported that by under-regulating the signaling route activity of ERK/C-JUN, Curcumin prevents the endometrial cancer (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Zhang et al (2016) reported that the combination of two drugs of Sorafenib and Curcumin, significantly causes apoptosis imposition and a reduction of AKT and ERK proteins level in the FTC133 cancer cell ranking in comparison with mono-drug treatment (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Mousavi et al (2018) reported that Curcumin could partially prevent from cellular death caused by OHDA-6 and the reduction of ERK. In addition to the inhibition of various intra-cellular anti-oxidant mechanisms, the increased ROS production could cause some chronic diseases such as the cancer via irreversible oxidative damage in nucleic acid, DNA, protein and membrane lipid, creation of interruption in cellular procedures such as cellular metabolism, message transfer, gene expression, cell replication, and planned cellular death. The impact of Curcumin in the ERK activity reconstruction refers to its inhibitory impact on ROS production (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results displayed that moderate intensity aerobic exercise caused a significant increase in RAS gene expression and a significant reduction of ERK in rats\u0026rsquo; soleus muscle. In addition, the consumption of Nano-Curcumin also caused a significant reduction of RAS and ERK genes. It can, thus, be stated that the consumption of Nano-Curcumin supplementation leads to the reductive trend of RAS and ERK genes that might lead to reduction of the cancer growth and development trend. However, the combination of moderate intensity aerobic exercise, associated with Nano-Curcumin supplementation, had a more effective role in reducing RAS and ERK and the down-regulating the cancer cells growth relative to any exclusive intervention. On this basis, it seems that, these two interventions could be used in pre-clinical studies as a complementary strategy with an effective potential for RAS and ERK suppression and cancer growth.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKheirandish Zarandi P, Zinatizadeh ZM, Yousefi MH, Mirzaei HR, Akbari M E. 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Curcumin prevents 6\u003cspan dir=\"RTL\"\u003e-\u003c/span\u003eOHDA induced\u003cspan dir=\"RTL\"\u003e \u003c/span\u003ecell death and ERK disruption in Human\u003cspan dir=\"RTL\"\u003e \u003c/span\u003eneuroblastoma cells.\u003cem\u003e Journal of Knowledge \u0026amp; Health\u003cspan dir=\"RTL\"\u003e \u003c/span\u003ein Basic Medical Sciences\u003c/em\u003e 2018;13(3):1-7\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eCouncil NR. Textbook of guide for the care and use of laboratory animals.\u003c/li\u003e\n\u003cli\u003eFathi R, Nasiri K, Akbari A, Ahmadi-KaniGolzar F, Farajtabar Z. Exercise protects against ethanol-induced damage in rat heart and liver through the inhibition of apoptosis and activation of Nrf2/Keap-1/HO-1 pathway. Life Sciences. 2020 Sep 1;256:117958. \u003c/li\u003e\n\u003cli\u003eK.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(\u0026minus;Delta Delta C(T)) method, Methods 25 (2001) 402\u0026ndash;408.\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":"Aerobic exercise, Nano-curcumin, RAS, ERK","lastPublishedDoi":"10.21203/rs.3.rs-3257588/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3257588/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective/background:\u003c/h2\u003e \u003cp\u003eImpaired cell internal settings and its excessive proliferation causes the occurrence of various syndrom and diseases such as cancer. One of the cell signaling pathways that lead to these outcomes is the MAPK/ERK pathway. So, the goal of the present study is to investigate the effect of aerobic training and Nano-curcumin supplementation on RAS and ERK gene expression in rat muscle tissue.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eIn this experimental study, 32 male wistar rats (aged 4\u0026ndash;6 weeks, 130\u0026ndash;150 g) were randomly assigned into four groups (n\u0026thinsp;=\u0026thinsp;8/groups), including Control (C), Moderate Intensity Aerobic Exercise (MICT), Nano-Curcumin Supplementation (NCS) and moderate intensity aerobic exercise\u0026thinsp;+\u0026thinsp;Nano-curcumin (MICT\u0026thinsp;+\u0026thinsp;NCS). The training groups implemented the MICT for 4 weeks and 5 sessions per week. The Supplement groups received 80 mg/kg/day through oral gavage for 4 weeks. Genes expression of RAS and ERK were measured with real-time PCR method. Data were analyzed by one-way ANOVA and Bonferroni post-hoc tests (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eFindings:\u003c/h2\u003e \u003cp\u003eResults showed that 4 weeks of moderate intensity aerobic exercise and Nano-curcumin supplementation significantly decreased the gene expression levels of RAS and ERK in muscle tissue of rats.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eImplementation of 4 weeks of MICT combined with Nano-Curcumin supplementation, more efficiently alleviated the RAS/ERK gene expression as symptom development of cancer.\u003c/p\u003e","manuscriptTitle":"Aerobic Exercise and Nano-curcumin Supplementation Prevent cancer symptom development through MAPK/ERK pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-21 13:56:52","doi":"10.21203/rs.3.rs-3257588/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":"89d58b0c-8552-457a-95bb-59e28ceb863b","owner":[],"postedDate":"August 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-10-10T20:29:22+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-21 13:56:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3257588","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3257588","identity":"rs-3257588","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}