Neurotrophic effect magnolol on MPTP/p induced parkinsons disease by regulation of PI3K-AKT-GSK3β via MAPK/mTOR signalling pathway

Neurotrophic effect magnolol on MPTP/p induced parkinsons disease by regulation of PI3K-AKT-GSK3β via MAPK/mTOR signalling 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 Neurotrophic effect magnolol on MPTP/p induced parkinsons disease by regulation of PI3K-AKT-GSK3β via MAPK/mTOR signalling pathway Ruili Chen, Dongdong Zhang, Annamalai Vijayalakshmi, Periyannan Velu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5906100/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 Introduction Parkinson's disease (PD) dopaminergic loss may be slowed down by neurotrophic factors (NTFs) activating at a higher level by regulating several signalling pathways, including PI3K, AKT, and Ras-MAPK. Therefore, the goal of the current investigation is to ascertain how neurotrophic magnolol (ML) is about the neurotoxicity of MPTP/p in Parkinson's disease. Methods Five weeks of ML treatment markedly reduced the motor impairments, decrease in tyrosine kinase receptor expression (TrKB) and dopamine deficiency and NTFs caused by MPTP/p. Results Additionally discovered that ML treatment markedly reduced the activation of MAPK/P38/JNK-related proteins. Additionally, ML therapy enhanced phosphorylation of PI3K, Akt, GSK-3β, and mTOR, indicating ML regulated the PI3K/Akt/mTOR signaling pathway, this reason ML has protected the Brain system. In a chronic parkinson's disease, the current study provides more comprehensive in vivo evidence supporting the neuroprotective action of ML on dopaminergic neurons. Conclusion It also raises the idea of employing ML as a novel chemotherapeutic medication. ML MPTP/p NTFs cell proliferation Apoptosis Parkinson disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Parkinson's disease (PD), deteriorating degenerative motor disease over time [ 1 , 2 ]. The current approaches to treating Parkinson's disease (PD) focus on raising dopamine levels by giving levodopa (L-Dopa), a dopamine counterpart, and/or dopamine agonists to contribute to get the motor circuitry functioning normally again [ 3 ]. This method does not protect the remaining dopaminergic neurons from injury and is unable to restore the injured neurons [ 4 ]. Rather of treating the clinical symptoms, researchers are increasingly spending more time on treatment techniques that slow reduction or terminate the neurotoxic process [ 5 ]. In PD animal models, VEGF, produced through the circulatory path, has been shown to support the proliferation of DA nerve cells [ 6 ]. Brain-derived neurotrophic factor (BDNF) stimulates brain generation process by regulating transcriptional proteins [ 7 ]. Many therapeutic experiments using the direct supply of trophic elements to the PD brain have inconsistent outcomes [ 8 ]. This could be because different injection or infusion procedures were used, as well as the site and manner of delivery. Approaches to raise endogenous trophic factor expression or augment their transmission function are receiving greater attention recently as potential PD therapy choices. Plant extract is mostly utilized in Chinese medicine, magnolil is a binaphthalene polyphenolic substance that was extracted from Magnolia officinalis . The structural isomer of magnolol is honokiol.Which was first isolated from the magnolia root in 1930 by Sugii, a Japanese scientist [ 10 – 12 ]. According to Ho et al. [ 13 ], by promoting apoptosis, a smooth muscle relaxant, ML inhibits the control, accommodation, and production of thrombus in vascular smooth muscle cells. This allows ML to reach the therapeutic level for cardiovascular diseases, protecting high-risk patients from cardiovascular diseases. ML significantly decreased the cytotoxicity and ROS production caused by MPP+ [ 14 ]. ML's neurotrophic qualities have not yet been established, nevertheless. Therefore, the goal of this work is to assess ML potential for neuroprotection against MPTP/p-caused neurodegeneration in a Parkinson's by examining motor impairments, neurochemical modifications, the expression of different NTFs, and its downstream pathway. Materials and methods Reagents and antibodies We bought MPTP, ML, and Probenecid from Sigma Aldrich in the United States. The antibodies and secondary antibodies listed below were acquired from Cell Signaling in the United States. Analytical-grade chemicals were employed for all other purposes. Animals In this work, adult male C57BL/6 mice (25–30 g of weight) were purchased. They were kept in an atmosphere that was 22°C, had a 12-hour light and dark cycle, and had limitless access to water. The Institution's local Institutional Animal Ethical Committee approved each experiment. Experimental design for dose fixation Twelve mice per experimental group were used for the concentration-dependent investigation (phase I) and 6 rats/group were used for the brain-protective study (phase II) to analyze the effective dose. Phase I The animals had prior training in both narrow beam and open field walking tests after a seven-day acclimatization period, and this process continued and then achieved. As a control, the Group-1 received saline treatment. Experimental design 1 (Phase I) is diagrammatically explained in Fig. 1 A. Two days following the experimental phase, animals were put to death and their brains were removed for additional research on neurochemical and immunoblot analysis investigations. Behavior assessments were carried out, including the dose-dependent study's narrow beam walking test and the open field test. Phase II Based on the DA level we determined the effective dose of ML to be 100 mg/kg bw., and we selected this dosage for additional trials. To put it briefly, twenty-four male C57BL/6 mice were sacrificed, split up into 4 groups (n = 6). Experimental design 2 (Phase II) explained diagrammatically in Fig. 1 B. Open field test A wooden box with dimensions of W100 × D100 × H4 cm was utilized in an open field test. A rexin cloth covered the floor, which was then divided into 16 smaller squares. After being carefully placed in the open field's right corner, the animals were given five minutes to investigate. When the animal used both of its forelimbs to enter a square, a count was made. The animal's assessed motions have been separated into two main groups: periphery (outer central 16 squares) and core (inner central 9 squares). The quantity of raising (i.e., resting on the wall with the forelegs while standing on hind limbs, sniffing, and looking around) and preparation for activities was also counted [ 16 ]. Narrow beam walking The beam walk test was carried out using Rajasankar et al. methodology [ 16 ]. Before the experiment, mice were taught by being placed in an L100 cm × W1 cm narrow beam and having to crawl to a pitch-black goal box. The narrow beam apparatus was illuminated with a strong light. 48 hours after the final MPTP/p injection, every mouse was put in its spot and given the freedom to move. The mice's trip time from the start region to the dark box as well as the quantity of foot slip faults were noted. The treatment groups were hidden from the observer who graded the mice's behavior. The striatum (ST) and SN Franklin and Paxinos [ 17 ] were tested using a mouse brain atlas after behavioural changes. Analysis of striatal DA and its metabolites To measure the amount of DA and its metabolites by following the procedure [ 18 ]. The Beckman Gold 118 integrator was used to record and quantify the signals. By comparing peak levels with external norms, DA levels were determined. The moist weight of the brain tissue was reported as ng/mg for the results. Western blot analysis Following the completion of experiments protein expression levels in the kidney tissues were examined by western blotting [ 18 ]. The nuclear extraction kit was used to extract the protein according to the company’s guidelines. Nuclear Fyn and Nrf2 were determined using Western blot according to guidelines. Statistical analysis IBM SPSS Statistics for Windows, version 25 (IBM Corp., Armonk, N.Y., USA) and GraphPad Prism v. 8.0.2 (GraphPad Software, San Diego, USA) were used to evaluate the data from each group statistically. The data were deemed statistically significant when p < 0.05. RESULTS ML on long-term behavioural deficits Prolonged MPTP/p therapy impeded movement in the open field test and diminished balance in the narrow beam walking test. Open field test observed that MPTP/p-injected mice drastically lowered their peripheral, central, rearing and grooming, and other activities (Fig. 2 A). Furthermore, compared to control mice, they took longer to reach the goal box due to better foot slip errors (Fig. 2 B). The behavioral issues caused by MPTP/p were substantially alleviated by ML co-administration at doses of 50 and 100 mg/kg; ML (25 mg/kg) had no impact. Nevertheless, no appreciable variations were discovered between the ML and control animals. Effect of ML reduces the persistent MPTP/p-induced depletion of DA In ST-tissue, chronic MPTP/p-injected animals significantly decrease in DA, DOPAC, and HVA. When compared to PD mice treated with high doses of ML, the amounts of striatal DA and its by products in mice receiving MPTP/p were not significantly increased by ML used orally at low or medium doses. Thus, the effective dose of ML was determined to be 100 mg/kg, and more research was done (Fig. 2 C). ML effect on DAT and VMAT-2 expressions In comparison to the control group, MPTP/p dramatically reduced the expression of DAT and VMAT2 in both SN and ST. In the meantime, ML therapy clearly restored these protein expressions in contrast to the MPTP/p group. The control and ML-only treatment groups did not vary in any noticeable ways (Figs. 3 A&B). Impact of ML on NTF depletion induced by MPTP/p In comparison to matched control mice, the SN region's expressions of BDNF, GDNF, VEGF, and TrkB were dramatically reduced following a prolonged course of MPTP/p treatment. Treatment of mice with MPTP/p alone. The SN region of the injected animals showed significantly higher levels of BDNF, GDNF, VEGF, and TrkB expressions when ML was co-administered. Research on BDNF, GDNF, VEGF, and TrkB protein expression in mice given with saline as a control group and with ML alone did not reveal any significant differences (Fig. 4 ). Impact of ML on expressions of the p38 MAPK signaling cascade Comparing the MPTP/p treatment group to the control group, Western blot analysis showed that p-P38, MAPK, p-ERK, and p-JNK all had noticeably elevated expressions. MPTP/p group vs treatment with ML resulted in a significant decrease in p-P38 MAPK, p-ERK, and p-JNK. These findings imply that JNK, ERK, and P38 MAPK-mediated signalling pathways were inhibited in order for ML to have neuroprotective effects on MPTP/p-induced neurotoxicity (Fig. 5A). Figure 5A ) Shows a western blot examination of p-JNK/JNK, p-ERK/ERK, and p-P38 MAPK/P38 MAPK in the supernatant of experimental animals. B) PI3K, p-PI3K, Akt, p-Akt, GSK-3β, and p-GSK-3β in the supernatants of experimental animals were analyzed using Western blots. Scanning densitometry was used to measure the band density. Comparisons between groups are displayed in a bar graph. β-actin served as an internal control for measuring the expression of proteins. Values not sharing common superscript letter differ statistically significant at *, # p < 0.05. Activation of ML treatment GSK3β/mTOR/PI3K/AKT signaling Mice treated with MPTP/p had significantly lower levels of p-PI3K and p-Akt (Ser473) than the controls (p < 0.05). By inducing the anti-apoptotic action of Akt, GSK3β, its downstream substrate, was phosphorylated. The unphosphorylated version of GSK3β was compared to its phosphorylated variants at serine position 9 in these groups. Control group Vs MPTP/p treatment reduced in the levels of phosphorylated-GSK3β (p < 0.05). The decrease was stopped by pre-treating with ML 100 mg/kg (Fig. 5B). In mTOR indicated a significant downregulate in the phospho-mTOR, phospho-p70S6K, and phospho-4E-BP1 expression in the chronic MPTP/p mice model. The three proteins' phosphorylation had increased dramatically after pretreatment with ML. (Fig. 6 ). DISCUSSION Previous research has shown a clear correlation between the degree of dopamine cell death in the SN and the impairment of motor behaviour [ 20 ]. Loss of motor capabilities is the outcome of DA neurotransmitter release being reduced concomitant with DA neuron degeneration [ 21 ]. Mice administered with MPTP/p demonstrated reduced activity and movement in the open field test, indicating a hypo-activity status. During the narrow beam walking test, rats injected with MPTP/p exhibited impaired muscle coordination, balance, and vestibular integrity, as evidenced by longer crossing times and a larger frequency of foot slip errors [ 22 , 23 ]. The main causes of the loss of motor function observed in PD patients are decreased DAT and VMAT-2 expression and decreased levels of catecholamines [ 2 ]. Prior research has demonstrated the neuroprotective impact of BDNF in both patients and PD models [ 24 ]. For DA neurons, GDNF is thought to be a potent neuroprotector [ 25 ]. Similar to GDNF, VEGF has been shown to have neuroprotective effects on Parkinson's disease [ 26 ]. Tsai et al., 2019 demonstrated that pharmaco kinetic and phramacothermodyanamic studies showed that ML administered via orally of ML at larger dosages (20, 50, and 100 mg kg − 1) markedly reduced the animal's locomotor activity, but not at lower doses (5 or 10 mg kg − 1) [ 29 ]. In our investigation, ML pre-treatment improved DA levels and the expressions of NTFs and dopaminergic markers to mitigate behavioral abnormalities caused by MPTP/p. Growing data points to the critical role mitochondrial kinases, such as MAPK, play in dopaminergic neurodegeneration [ 25 ]. Three signaling pathways JNK, P38, and ERK that are important for a range of physiological responses make up the MAPK subfamily. JNK and P38 are linked to cell differentiation and survival as well as apoptosis [ 26 ]. Rapid and transitory pERK activation has been shown to improve survival [ 27 ], whereas prolonged and delayed pERK activation has been linked to cell death [ 28 ]. Prior research has indicated that oxidative stress can trigger ERK activation, which also plays a role in cell death [ 29 ]. Released from the mitochondria, free radicals enter the cytoplasm and phosphorylate JNK that has been activated c-Jun, which results in excitotoxicity and the death of dopaminergic neurons [ 30 ]. ML reduced ROS, which in turn downregulated the expression of p-JNK, p38, and pERK [ 31 ]. According to earlier research, treating ML prevented the production and build-up of ROS in cardiomyocytes caused by lactate [ 32 ], oxygen-glucose deprivation, cognitive impairments [ 33 ], and experimental models of spinal cord injury [ 34 ]. One of the other essential signalling pathways linked to neuronal survival, development, and function is the PI3K/Akt pathway [ 36 ]. In a physiological sense, PI3K activates the Akt pathway in reaction to cell stress, cytokines, growth factors, insulin, and NTFs. GSK3β being recognized to be important in the pathophysiology of neurodegenerative illnesses as well as the induction of neuronal death produced by oxidative stress [ 38 ]. By raising GSK3β phosphorylation on serines 9 and 216 [ 39 ], Akt suppresses GSK3β activities and reduces apoptosis. The present investigation revealed that the administration of ML therapy mitigated the decrease in active P13K and Akt signaling markers generated by MPTP/p, thereby augmenting the activation of GSK3β, a neuroprotective factor. For the regulation of motor function, mTOR activity is crucial. Ribosomal p70SK and translation repressor 4E-BP1 [ 40 ]. Neuronal survival may be enhanced by downstream protein activation and mTOR activation. The phosphorylation of 4E-BP1 and p70S6K was found to be significantly elevated with ML treatment, indicating that the mTOR pathway was activated in this investigation. According to Fuller et al., the concentration of MPTP in the tissues from various organs varied after its administration [ 44 ]. It has been shown that systemic MPTP raises MPP + levels in the heart, brain, and lung [ 45 ]. Norepinephrine depletion in the cardiac region [ 47 ], and lung creation [ 46 ]. Probenecid is one drug that reduces urine volume, which increases the excretion of MPTP N-oxide. This means that it may be possible to increase the cerebral injury of MPTP and develop a dependable mouse model of parkinsonism. A growing body of research indicates that multiple pathways, such as free radical production, inflammation, a lower potential of the mitochondrial respiratory chain enzyme Complex I, changes to the Ca 2+ homeostasis and Glutamate dysfunction, the ubiquitin system, and altered signaling pathways that ultimately result in apoptosis, may connect by MPTP to parkinson's disease [ 48 ]. CONCLUSION This study demonstrated that pharmacological stimulation of the PI3K/Akt/mTOR/GSK-3β signalling treated ML might greatly provide a safer cerebral region against the loss of brain cells produced by MPTP/p. The findings of this investigation may point to a universal mechanism underpinning the neurotrophic therapy of Parkinson's disease with ML and related small-molecule medications. Declarations ETHICS APPROVAL AND CONSENT TO PARTICIPATE The Institutional Animal Ethics Committee of Shaanxi Provincial People's Hospital, China, approved all experimental animal procedures (Ethical No: 16036SX). HUMAN AND ANIMAL RIGHTS All the animal experimentation was performed according to the Guide for the Care and Use of Laboratory Animals. CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. FUNDING None. 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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-5906100","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":409020603,"identity":"2489b55f-9bbd-437e-b573-f829d92676bf","order_by":0,"name":"Ruili Chen","email":"","orcid":"","institution":"Shaanxi Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruili","middleName":"","lastName":"Chen","suffix":""},{"id":409020604,"identity":"28380638-dfbc-4cee-946e-f02991839309","order_by":1,"name":"Dongdong Zhang","email":"","orcid":"","institution":"Hospital of Norinco Group","correspondingAuthor":false,"prefix":"","firstName":"Dongdong","middleName":"","lastName":"Zhang","suffix":""},{"id":409020605,"identity":"5fa421c1-6dc4-4166-9576-2652172ac62b","order_by":2,"name":"Annamalai Vijayalakshmi","email":"","orcid":"","institution":"Galileovasan Offshore and Research and Development Pvt. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Annamalai","middleName":"","lastName":"Vijayalakshmi","suffix":""},{"id":409020606,"identity":"3111fd96-7924-4ef5-9acc-5dd069e39b19","order_by":3,"name":"Periyannan Velu","email":"","orcid":"","institution":"Galileovasan Offshore and Research and Development Pvt. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Periyannan","middleName":"","lastName":"Velu","suffix":""},{"id":409020607,"identity":"bd5ad6c2-aa3a-4f62-93cb-3bc07777457a","order_by":4,"name":"Nini Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBACPiA+8PGfBA8/e//zHx8qbBjYCGkBKmA8OIPNRk6y5wyD5IwzaURpYT7Mw5ZmbHDDh0Gat+UwYYexSfceODiD53DizBm8Bwx4G87b80k3P2D4UbENtxaZcwkHPkgcTuyX7ktIkNxxO7FN5pgBY8+Z27i1SOQYHJxhALRlzgGDA4ZnbiewSSQYMDO24ddymCfhcOKGGwmGDYlt5+zZJNI/EKHlAMj7OcYMB9sOMLYBRQhqOTizARTIx9IYG84kJwK1FBzE5xd+iRzjDx8bQFHZfIz5T4WdvfyM9I0PflTg1oIdHCBR/SgYBaNgFIwCNAAAMPJdtAa3zWwAAAAASUVORK5CYII=","orcid":"","institution":"Shaanxi Provincial People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Nini","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-01-26 11:38:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5906100/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5906100/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75366762,"identity":"b44349a8-efa6-4539-ad11-b07b46dbc88e","added_by":"auto","created_at":"2025-02-03 19:49:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284643,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images. A Experimental protocol for dose dependent study; B Experimental protocol for dose fixation study.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5906100/v1/15d01622143c42f631b7e937.png"},{"id":75366404,"identity":"5f96d81c-dd08-48c3-a60b-791dab2319e5","added_by":"auto","created_at":"2025-02-03 19:33:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":259947,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ML on impairment of spontaneous movement caused by MPTP/p and activity during a five-minute open field test. A) When compared to control, MPTP/p injection significantly reduced in central and peripheral motions and activities related to grooming and raising. ML pretreatment, however, lessened these motor deficits. B) Both the b foot slip errors and the time it took to traverse the beam higher in the MPTP/p treated mice Vs the control group. ML pretreatment to MPTP/p greatly lowered foot slip mistakes and the amount of time needed to cross the beam. C) ML impact on neurochem DoPAC, HVA, and depletion of striatal dopamine was induced by Res 25 MPTP/p. We present the data as Mean ± SD. Values not sharing common superscript letter differ statistically significant at \u003csup\u003e#,$,*,**\u003c/sup\u003ep\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5906100/v1/5764b70303f13ca634ad8798.png"},{"id":75366409,"identity":"8335fdad-d5e4-41fa-8c51-c98a9e6e46ca","added_by":"auto","created_at":"2025-02-03 19:33:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":161231,"visible":true,"origin":"","legend":"\u003cp\u003eA) Western blot shows DAT and VMAT-2 in the spinal cords of experimental and control mice. B) Western blot study of VMAT2 and DAT in the ST of experimental and control mice. Scanning densitometry was used to measure the band density. A bar graph illustrates the comparison between the groups. β-actin internal control for measuring the expression of proteins. Values not sharing common superscript letter differ statistically significant at \u003csup\u003e#,$,*,**\u003c/sup\u003ep\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5906100/v1/758d4658a5fd89cce03bb0bd.png"},{"id":75366412,"identity":"99bdb4bd-b72c-42db-8ef2-3579f72fa051","added_by":"auto","created_at":"2025-02-03 19:33:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105889,"visible":true,"origin":"","legend":"\u003cp\u003eShows a western blot examination of TrkB, GDND, VEGF, and BDNF in the SN of experimental and control mice. Scanning densitometry was used to measure the band density. Comparisons between groups are displayed in a bar graph. β-actin internal control for measuring the expression of proteins. Values not sharing common superscript letter differ statistically significant at \u003csup\u003e*, #\u003c/sup\u003ep\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5906100/v1/3541b2ef7f206603509752cd.png"},{"id":75366414,"identity":"e3e6b7d6-cc1e-4d7e-8600-ce9a0c4f0605","added_by":"auto","created_at":"2025-02-03 19:33:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":230398,"visible":true,"origin":"","legend":"\u003cp\u003eA) Shows a western blot examination of p-JNK/JNK, p-ERK/ERK, and p-P38 MAPK/P38 MAPK in the supernatant of experimental animals. B) PI3K, p-PI3K, Akt, p-Akt, GSK-3β, and p-GSK-3β in the supernatants of experimental animals were analyzed using Western blots. Scanning densitometry was used to measure the band density. Comparisons between groups are displayed in a bar graph. β-actin served as an internal control for measuring the expression of proteins. Values not sharing common superscript letter differ statistically significant at \u003csup\u003e*, #\u003c/sup\u003ep\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5906100/v1/9155670591bfff2b96ec7bbe.png"},{"id":75366411,"identity":"4f7802b5-1159-4dc4-b433-075071958771","added_by":"auto","created_at":"2025-02-03 19:33:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":122038,"visible":true,"origin":"","legend":"\u003cp\u003eThe western blot test detected phospho-mTOR/mTOR, phospho-p70S6K/p70S6K, and phospho-4E-BP1/4E-BP1, and the SN showed that an ML treatment activated mTOR. The band density was measured using scanning densitometry. A bar graph illustrates the comparison between the groups. β-actin served as an internal control for measuring the expression of proteins. Values not sharing common superscript letter differ statistically significant at \u003csup\u003e*, #\u003c/sup\u003ep\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5906100/v1/4e7d2c9a217a14da59f7d015.png"},{"id":75367073,"identity":"365b1c4b-9e8b-4bbe-906c-83e4acda4ab9","added_by":"auto","created_at":"2025-02-03 20:05:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1831632,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5906100/v1/1f525cb8-871e-49a1-be73-9782ea6c637e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neurotrophic effect magnolol on MPTP/p induced parkinsons disease by regulation of PI3K-AKT-GSK3β via MAPK/mTOR signalling pathway","fulltext":[{"header":"Background","content":"\u003cp\u003eParkinson's disease (PD), deteriorating degenerative motor disease over time [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The current approaches to treating Parkinson's disease (PD) focus on raising dopamine levels by giving levodopa (L-Dopa), a dopamine counterpart, and/or dopamine agonists to contribute to get the motor circuitry functioning normally again [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This method does not protect the remaining dopaminergic neurons from injury and is unable to restore the injured neurons [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Rather of treating the clinical symptoms, researchers are increasingly spending more time on treatment techniques that slow reduction or terminate the neurotoxic process [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn PD animal models, VEGF, produced through the circulatory path, has been shown to support the proliferation of DA nerve cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Brain-derived neurotrophic factor (BDNF) stimulates brain generation process by regulating transcriptional proteins [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Many therapeutic experiments using the direct supply of trophic elements to the PD brain have inconsistent outcomes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This could be because different injection or infusion procedures were used, as well as the site and manner of delivery. Approaches to raise endogenous trophic factor expression or augment their transmission function are receiving greater attention recently as potential PD therapy choices.\u003c/p\u003e \u003cp\u003ePlant extract is mostly utilized in Chinese medicine, magnolil is a binaphthalene polyphenolic substance that was extracted from \u003cem\u003eMagnolia officinalis\u003c/em\u003e. The structural isomer of magnolol is honokiol.Which was first isolated from the magnolia root in 1930 by Sugii, a Japanese scientist [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. According to Ho et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], by promoting apoptosis, a smooth muscle relaxant, ML inhibits the control, accommodation, and production of thrombus in vascular smooth muscle cells. This allows ML to reach the therapeutic level for cardiovascular diseases, protecting high-risk patients from cardiovascular diseases. ML significantly decreased the cytotoxicity and ROS production caused by MPP+ [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. ML's neurotrophic qualities have not yet been established, nevertheless.\u003c/p\u003e \u003cp\u003eTherefore, the goal of this work is to assess ML potential for neuroprotection against MPTP/p-caused neurodegeneration in a Parkinson's by examining motor impairments, neurochemical modifications, the expression of different NTFs, and its downstream pathway.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies\u003c/h2\u003e \u003cp\u003eWe bought MPTP, ML, and Probenecid from Sigma Aldrich in the United States. The antibodies and secondary antibodies listed below were acquired from Cell Signaling in the United States. Analytical-grade chemicals were employed for all other purposes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eIn this work, adult male C57BL/6 mice (25\u0026ndash;30 g of weight) were purchased. They were kept in an atmosphere that was 22\u0026deg;C, had a 12-hour light and dark cycle, and had limitless access to water. The Institution's local Institutional Animal Ethical Committee approved each experiment.\u003c/p\u003e\n\u003ch3\u003eExperimental design for dose fixation\u003c/h3\u003e\n\u003cp\u003eTwelve mice per experimental group were used for the concentration-dependent investigation (phase I) and 6 rats/group were used for the brain-protective study (phase II) to analyze the effective dose.\u003c/p\u003e\n\u003ch3\u003ePhase I\u003c/h3\u003e\n\u003cp\u003eThe animals had prior training in both narrow beam and open field walking tests after a seven-day acclimatization period, and this process continued and then achieved. As a control, the Group-1 received saline treatment. Experimental design 1 (Phase I) is diagrammatically explained in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Two days following the experimental phase, animals were put to death and their brains were removed for additional research on neurochemical and immunoblot analysis investigations. Behavior assessments were carried out, including the dose-dependent study's narrow beam walking test and the open field test.\u003c/p\u003e\n\u003ch3\u003ePhase II\u003c/h3\u003e\n\u003cp\u003eBased on the DA level we determined the effective dose of ML to be 100 mg/kg bw., and we selected this dosage for additional trials. To put it briefly, twenty-four male C57BL/6 mice were sacrificed, split up into 4 groups (n\u0026thinsp;=\u0026thinsp;6). Experimental design 2 (Phase II) explained diagrammatically in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOpen field test\u003c/h2\u003e \u003cp\u003eA wooden box with dimensions of W100 \u0026times; D100 \u0026times; H4 cm was utilized in an open field test. A rexin cloth covered the floor, which was then divided into 16 smaller squares. After being carefully placed in the open field's right corner, the animals were given five minutes to investigate. When the animal used both of its forelimbs to enter a square, a count was made. The animal's assessed motions have been separated into two main groups: periphery (outer central 16 squares) and core (inner central 9 squares). The quantity of raising (i.e., resting on the wall with the forelegs while standing on hind limbs, sniffing, and looking around) and preparation for activities was also counted [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNarrow beam walking\u003c/h3\u003e\n\u003cp\u003eThe beam walk test was carried out using Rajasankar et al. methodology [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Before the experiment, mice were taught by being placed in an L100 cm \u0026times; W1 cm narrow beam and having to crawl to a pitch-black goal box. The narrow beam apparatus was illuminated with a strong light. 48 hours after the final MPTP/p injection, every mouse was put in its spot and given the freedom to move. The mice's trip time from the start region to the dark box as well as the quantity of foot slip faults were noted. The treatment groups were hidden from the observer who graded the mice's behavior. The striatum (ST) and SN Franklin and Paxinos [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] were tested using a mouse brain atlas after behavioural changes.\u003c/p\u003e\n\u003ch3\u003eAnalysis of striatal DA and its metabolites\u003c/h3\u003e\n\u003cp\u003eTo measure the amount of DA and its metabolites by following the procedure [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The Beckman Gold 118 integrator was used to record and quantify the signals. By comparing peak levels with external norms, DA levels were determined. The moist weight of the brain tissue was reported as ng/mg for the results.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eFollowing the completion of experiments protein expression levels in the kidney tissues were examined by western blotting [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The nuclear extraction kit was used to extract the protein according to the company\u0026rsquo;s guidelines. Nuclear Fyn and Nrf2 were determined using Western blot according to guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIBM SPSS Statistics for Windows, version 25 (IBM Corp., Armonk, N.Y., USA) and GraphPad Prism v. 8.0.2 (GraphPad Software, San Diego, USA) were used to evaluate the data from each group statistically. The data were deemed statistically significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eML on long-term behavioural deficits\u003c/h2\u003e \u003cp\u003eProlonged MPTP/p therapy impeded movement in the open field test and diminished balance in the narrow beam walking test. Open field test observed that MPTP/p-injected mice drastically lowered their peripheral, central, rearing and grooming, and other activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, compared to control mice, they took longer to reach the goal box due to better foot slip errors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The behavioral issues caused by MPTP/p were substantially alleviated by ML co-administration at doses of 50 and 100 mg/kg; ML (25 mg/kg) had no impact. Nevertheless, no appreciable variations were discovered between the ML and control animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of ML reduces the persistent MPTP/p-induced depletion of DA\u003c/h2\u003e \u003cp\u003eIn ST-tissue, chronic MPTP/p-injected animals significantly decrease in DA, DOPAC, and HVA. When compared to PD mice treated with high doses of ML, the amounts of striatal DA and its by products in mice receiving MPTP/p were not significantly increased by ML used orally at low or medium doses. Thus, the effective dose of ML was determined to be 100 mg/kg, and more research was done (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eML effect on DAT and VMAT-2 expressions\u003c/h2\u003e \u003cp\u003eIn comparison to the control group, MPTP/p dramatically reduced the expression of DAT and VMAT2 in both SN and ST. In the meantime, ML therapy clearly restored these protein expressions in contrast to the MPTP/p group. The control and ML-only treatment groups did not vary in any noticeable ways (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026amp;B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImpact of ML on NTF depletion induced by MPTP/p\u003c/h2\u003e \u003cp\u003eIn comparison to matched control mice, the SN region's expressions of BDNF, GDNF, VEGF, and TrkB were dramatically reduced following a prolonged course of MPTP/p treatment. Treatment of mice with MPTP/p alone. The SN region of the injected animals showed significantly higher levels of BDNF, GDNF, VEGF, and TrkB expressions when ML was co-administered. Research on BDNF, GDNF, VEGF, and TrkB protein expression in mice given with saline as a control group and with ML alone did not reveal any significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImpact of ML on expressions of the p38 MAPK signaling cascade\u003c/h2\u003e \u003cp\u003eComparing the MPTP/p treatment group to the control group, Western blot analysis showed that p-P38, MAPK, p-ERK, and p-JNK all had noticeably elevated expressions. MPTP/p group vs treatment with ML resulted in a significant decrease in p-P38 MAPK, p-ERK, and p-JNK. These findings imply that JNK, ERK, and P38 MAPK-mediated signalling pathways were inhibited in order for ML to have neuroprotective effects on MPTP/p-induced neurotoxicity (Fig.\u0026nbsp;5A).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5A\u003c/b\u003e) Shows a western blot examination of p-JNK/JNK, p-ERK/ERK, and p-P38 MAPK/P38 MAPK in the supernatant of experimental animals. B) PI3K, p-PI3K, Akt, p-Akt, GSK-3β, and p-GSK-3β in the supernatants of experimental animals were analyzed using Western blots. Scanning densitometry was used to measure the band density. Comparisons between groups are displayed in a bar graph. β-actin served as an internal control for measuring the expression of proteins. Values not sharing common superscript letter differ statistically significant at \u003csup\u003e*, #\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eActivation of ML treatment GSK3β/mTOR/PI3K/AKT signaling\u003c/h2\u003e \u003cp\u003eMice treated with MPTP/p had significantly lower levels of p-PI3K and p-Akt (Ser473) than the controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). By inducing the anti-apoptotic action of Akt, GSK3β, its downstream substrate, was phosphorylated. The unphosphorylated version of GSK3β was compared to its phosphorylated variants at serine position 9 in these groups. Control group Vs MPTP/p treatment reduced in the levels of phosphorylated-GSK3β (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The decrease was stopped by pre-treating with ML 100 mg/kg (Fig.\u0026nbsp;5B). In mTOR indicated a significant downregulate in the phospho-mTOR, phospho-p70S6K, and phospho-4E-BP1 expression in the chronic MPTP/p mice model. The three proteins' phosphorylation had increased dramatically after pretreatment with ML. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003ePrevious research has shown a clear correlation between the degree of dopamine cell death in the SN and the impairment of motor behaviour [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Loss of motor capabilities is the outcome of DA neurotransmitter release being reduced concomitant with DA neuron degeneration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Mice administered with MPTP/p demonstrated reduced activity and movement in the open field test, indicating a hypo-activity status. During the narrow beam walking test, rats injected with MPTP/p exhibited impaired muscle coordination, balance, and vestibular integrity, as evidenced by longer crossing times and a larger frequency of foot slip errors [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The main causes of the loss of motor function observed in PD patients are decreased DAT and VMAT-2 expression and decreased levels of catecholamines [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Prior research has demonstrated the neuroprotective impact of BDNF in both patients and PD models [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For DA neurons, GDNF is thought to be a potent neuroprotector [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similar to GDNF, VEGF has been shown to have neuroprotective effects on Parkinson's disease [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTsai et al., 2019 demonstrated that pharmaco kinetic and phramacothermodyanamic studies showed that ML administered via orally of ML at larger dosages (20, 50, and 100 mg kg\u0026thinsp;\u0026minus;\u0026thinsp;1) markedly reduced the animal's locomotor activity, but not at lower doses (5 or 10 mg kg\u0026thinsp;\u0026minus;\u0026thinsp;1) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In our investigation, ML pre-treatment improved DA levels and the expressions of NTFs and dopaminergic markers to mitigate behavioral abnormalities caused by MPTP/p.\u003c/p\u003e \u003cp\u003eGrowing data points to the critical role mitochondrial kinases, such as MAPK, play in dopaminergic neurodegeneration [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Three signaling pathways JNK, P38, and ERK that are important for a range of physiological responses make up the MAPK subfamily. JNK and P38 are linked to cell differentiation and survival as well as apoptosis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Rapid and transitory pERK activation has been shown to improve survival [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], whereas prolonged and delayed pERK activation has been linked to cell death [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Prior research has indicated that oxidative stress can trigger ERK activation, which also plays a role in cell death [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReleased from the mitochondria, free radicals enter the cytoplasm and phosphorylate JNK that has been activated c-Jun, which results in excitotoxicity and the death of dopaminergic neurons [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. ML reduced ROS, which in turn downregulated the expression of p-JNK, p38, and pERK [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. According to earlier research, treating ML prevented the production and build-up of ROS in cardiomyocytes caused by lactate [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], oxygen-glucose deprivation, cognitive impairments [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and experimental models of spinal cord injury [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the other essential signalling pathways linked to neuronal survival, development, and function is the PI3K/Akt pathway [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In a physiological sense, PI3K activates the Akt pathway in reaction to cell stress, cytokines, growth factors, insulin, and NTFs. GSK3β being recognized to be important in the pathophysiology of neurodegenerative illnesses as well as the induction of neuronal death produced by oxidative stress [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. By raising GSK3β phosphorylation on serines 9 and 216 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], Akt suppresses GSK3β activities and reduces apoptosis. The present investigation revealed that the administration of ML therapy mitigated the decrease in active P13K and Akt signaling markers generated by MPTP/p, thereby augmenting the activation of GSK3β, a neuroprotective factor.\u003c/p\u003e \u003cp\u003eFor the regulation of motor function, mTOR activity is crucial. Ribosomal p70SK and translation repressor 4E-BP1 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Neuronal survival may be enhanced by downstream protein activation and mTOR activation. The phosphorylation of 4E-BP1 and p70S6K was found to be significantly elevated with ML treatment, indicating that the mTOR pathway was activated in this investigation.\u003c/p\u003e \u003cp\u003eAccording to Fuller et al., the concentration of MPTP in the tissues from various organs varied after its administration [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It has been shown that systemic MPTP raises MPP\u0026thinsp;+\u0026thinsp;levels in the heart, brain, and lung [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Norepinephrine depletion in the cardiac region [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and lung creation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Probenecid is one drug that reduces urine volume, which increases the excretion of MPTP N-oxide. This means that it may be possible to increase the cerebral injury of MPTP and develop a dependable mouse model of parkinsonism. A growing body of research indicates that multiple pathways, such as free radical production, inflammation, a lower potential of the mitochondrial respiratory chain enzyme Complex I, changes to the Ca\u003csup\u003e2+\u003c/sup\u003ehomeostasis and Glutamate dysfunction, the ubiquitin system, and altered signaling pathways that ultimately result in apoptosis, may connect by MPTP to parkinson's disease [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study demonstrated that pharmacological stimulation of the PI3K/Akt/mTOR/GSK-3β signalling treated ML might greatly provide a safer cerebral region against the loss of brain cells produced by MPTP/p. The findings of this investigation may point to a universal mechanism underpinning the neurotrophic therapy of Parkinson's disease with ML and related small-molecule medications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/h2\u003e \u003cp\u003e The Institutional Animal Ethics Committee of Shaanxi Provincial People's Hospital, China, approved all experimental animal procedures (Ethical No: 16036SX).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eHUMAN AND ANIMAL RIGHTS\u003c/h2\u003e \u003cp\u003e All the animal experimentation was performed according to the Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCONSENT FOR PUBLICATION\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest, financial or otherwise.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eNone.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRC \u0026amp; DZ - research concept and design; AV \u0026amp; PV- collection and/or assembly of data; PNL\u0026amp; RC \u0026ndash; data analysis and interpretation; DZ \u0026ndash; writing the article; AV \u0026ndash; critical revision of the article; PV \u0026ndash; final approval of the article\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eDeclared none.\u003c/p\u003e\u003ch2\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/h2\u003e \u003cp\u003eThe data and supportive information are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKouli A, Torsney KM, Kuan WL. 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Metabolites. 2023;13(1):84.\u003c/span\u003e\u003c/li\u003e\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":"ML, MPTP/p, NTFs, cell proliferation, Apoptosis, Parkinson disease","lastPublishedDoi":"10.21203/rs.3.rs-5906100/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5906100/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction\u003c/h2\u003e \u003cp\u003eParkinson's disease (PD) dopaminergic loss may be slowed down by neurotrophic factors (NTFs) activating at a higher level by regulating several signalling pathways, including PI3K, AKT, and Ras-MAPK. Therefore, the goal of the current investigation is to ascertain how neurotrophic magnolol (ML) is about the neurotoxicity of MPTP/p in Parkinson's disease.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFive weeks of ML treatment markedly reduced the motor impairments, decrease in tyrosine kinase receptor expression (TrKB) and dopamine deficiency and NTFs caused by MPTP/p.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAdditionally discovered that ML treatment markedly reduced the activation of MAPK/P38/JNK-related proteins. Additionally, ML therapy enhanced phosphorylation of PI3K, Akt, GSK-3β, and mTOR, indicating ML regulated the PI3K/Akt/mTOR signaling pathway, this reason ML has protected the Brain system. In a chronic parkinson's disease, the current study provides more comprehensive \u003cem\u003ein vivo\u003c/em\u003e evidence supporting the neuroprotective action of ML on dopaminergic neurons.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIt also raises the idea of employing ML as a novel chemotherapeutic medication.\u003c/p\u003e","manuscriptTitle":"Neurotrophic effect magnolol on MPTP/p induced parkinsons disease by regulation of PI3K-AKT-GSK3β via MAPK/mTOR signalling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-03 19:33:46","doi":"10.21203/rs.3.rs-5906100/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":"d080cca7-c85c-477c-ad43-7216acb9ddec","owner":[],"postedDate":"February 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T19:33:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-03 19:33:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5906100","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5906100","identity":"rs-5906100","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}