Neuroprotective Effects of Gallic Acid in an MPP⁺-Induced SH-SY5Y Cell Model of Parkinson’s Disease

Neuroprotective Effects of Gallic Acid in an MPP⁺-Induced SH-SY5Y Cell Model of Parkinson’s Disease | 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 Neuroprotective Effects of Gallic Acid in an MPP⁺-Induced SH-SY5Y Cell Model of Parkinson’s Disease Hasan Ulusal, Hatice Kubra Yigit Dumrul, Sibel Dagli, Mehmet Tarakcioglu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8653441/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version Abstract Background Parkinson’s Disease (PD) represents the second most prevalent neurodegenerative condition which leads to the progressive destruction of dopaminergic neurons in the substantia nigra through oxidative stress mechanisms. The research evaluated Gallic Acid (GA) as a natural polyphenol with proven antioxidant properties for its ability to protect cells from 1-methyl-4-phenylpyridinium (MPP⁺)-induced neurotoxicity in SH-SY5Y dopaminergic cell models. Methods and Results The research used SH-SY5Y cells which received 1 mM MPP⁺ treatment alongside different GA concentrations (25,50 and 100 µM) for 24 and 48 hours. The CCK-8 assay measured cell viability while flow cytometry evaluated apoptosis and SOD and MDA levels determined oxidative status through SOD and Catalase and NO measurements. The addition of MPP⁺ resulted in a 32.74% decrease in cell viability at 48 hours while simultaneously decreasing SOD and Catalase and NO levels and increasing MDA levels. The addition of 25 µM GA protected cells from damage by increasing their viability to 86.53% at 48 hours and decreasing apoptotic cell numbers. The protective effects of 25–50 µM GA were observed through its ability to stop Catalase and NO decreases and its successful reduction of MDA levels (GA 25 µM decreased MDA from 21.18 to 9.64 nM/mg prot at 48 hours). The research indicates that GA functions as an antioxidant by rebuilding the body's natural defense system against oxidative stress. Conclusions The study demonstrates that GA provides strong protection against peroxidation by maintaining essential antioxidant enzyme activities in MPP⁺-exposed cells. The research demonstrates that GA shows potential as an effective Gallic acid MPP+ Neuroprotection Oxidative Stress Parkinson Disease Figures Figure 1 Figure 2 Figure 3 1. Introduction Parkinson’s disease (PD) ranks as the second most prevalent neurodegenerative condition after Alzheimer’s disease while its incidence rate increases with the increasing number of elderly people in the world [ 1 – 3 ]. The worldwide number of PD patients reached 6.1 million in 2016. The main neurological feature of Parkinson's disease involves the continuous deterioration of dopaminergic neurons which reside in the substantia nigra pars compacta [ 2 ]. Research shows that multiple cellular mechanisms including mitochondrial damage and α-synuclein accumulation and lysosomal breakdown and immune system dysregulation and programmed cell death and oxidative stress damage dopaminergic neurons [ 4 , 5 ]. The death of dopaminergic neurons that originate from the substantia nigra pars compacta and project to the striatum results in the development of Parkinson's disease motor symptoms including tremor and bradykinesia and rigidity and postural instability. The development of non-motor symptoms in Parkinson's disease patients occurs before their motor symptoms become apparent [ 6 , 7 ]. Scientists now understand that Parkinson's disease develops through the combination of genetic factors and environmental elements instead of being caused by environmental factors alone [ 7 ]. The process of neurodegeneration in Parkinson's disease primarily results from excessive oxidative stress in the brain. The body produces too many reactive oxygen species which exceed its natural antioxidant defenses. The enzymatic antioxidants superoxide dismutase (SOD) and catalase and glutathione function as free radical neutralizers but their breakdown results in oxidative damage to lipids and proteins and DNA which leads to cell death that scientists link to Parkinson's disease development [ 8 – 10 ]. Research indicates that PD shows elevated oxidative stress levels during its initial stages before noticeable neuronal deterioration which supports the theory that uncontrolled reactive oxygen species (ROS) production starts dopaminergic neuron degeneration instead of being a progression marker [ 11 ]. The human neuroblastoma cell line SH-SY5Y serves as a common laboratory model for studying dopaminergic neuronal function because it displays catecholaminergic characteristics. The Parkinsonian toxin MPTP produces its active compound 1-methyl-4-phenylpyridinium (MPP⁺) which scientists use to create neurotoxicity in laboratory experiments that mimic PD-related cellular damage [ 12 ]. Natural polyphenolic compounds have attracted increasing interest as potential therapeutic agents for neurodegenerative diseases because of their strong antioxidant and anti-inflammatory properties. Gallic acid (GA; 3,4,5-trihydroxybenzoic acid) is a widely distributed plant-derived phenolic compound found in grapes, tea, walnuts, and various fruits [ 13 ]. GA has been shown to scavenge free radicals, modulate intracellular signaling pathways, inhibit oxidative stress–induced apoptosis, and attenuate neuroinflammation [ 13 , 14 ]. Previous in vitro and in vivo studies have reported that GA and its derivatives can reduce oxidative damage in SH-SY5Y cells and ameliorate oxidative stress–induced neurotoxicity in animal models [ 13 , 15 – 17 ]. Based on this evidence, the present study aims to investigate the potential neuroprotective effects of GA through its antioxidant activity in an MPP⁺-induced SH-SY5Y cell model of Parkinson’s disease. Specifically, this study seeks to determine whether GA can counteract MPP⁺-induced neurotoxicity by modulating oxidative stress–related pathways. We hypothesize that GA exerts neuroprotective effects in the MPP⁺-induced SH-SY5Y cell model by mitigating oxidative stress, lowering intracellular ROS accumulation, restoring antioxidant defense capacity, and consequently preventing mitochondrial dysfunction and cell death. 2. Materials and Methods 2.1. Reagent and Chemicals All materials were used without further purification. Gaziantep University Medical Biochemistry Department provided SH-SY5Ycell line. Fetal bovine serum (FBS) (US Origin, Product No: SH30071.03), phosphate buffer saline (PBS) (Without Calcium, magnesium, Product No: SH30256.01), and Dulbecco’s Modified Eagle Medium (DMEM, high glucose with L-glutamine, Product No: SH30022.FS) medium were purchased from HyClone. CCK-8 (Catalog No: KTA1020) was purchased from Abbkine. GA (Prodcut No: 8.42649), Butylated Hydroxytoluene (BHT) (Prodcut No: W218405), Trichloroacetic Acid (TCA) (Prodcut No: 8.22342), Ethylenediaminetetraacetic Acid (EDTA) (Prodcut No: 798681) and Thiobarbituric Acid (TBA) (Prodcut No: T5500) were purchased from Merck. 2.2. Cell Culture Human neuroblastoma SH-SY5Y cells (ATCC® CRL-2266™) were cultured DMEM supplemented with 10% fetal bovine serum FBS and 1% penicillin-streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ and subcultured upon reaching approximately 80% confluence. Frozen cells were rapidly thawed at 37°C, centrifuged, resuspended in fresh medium, and seeded in culture flasks. Cell viability and density were determined using trypan blue exclusion and a Neubauer chamber. For experiments, cells were seeded at a density of 5 × 10³ cells per well in 96-well plates and incubated for 24 h prior to treatments. 2.3. Cell Viability Assay Cells were seeded into 96-well culture plates at a density of 5x10^3 cells/well (in 100 µL of complete culture medium) and allowed to adhere overnight. After 24 hours, the cells reached approximately 70–80% confluence, and the morphology was confirmed under an inverted microscope. Cells were first treated with 1 and 2 mM MPP + and then, 2 hours later, with different concentrations of GA (25, 50, and 100 µM). Additionally, MPP + (1 and 2 mM) and GA (25, 50, and 100 µM) were applied alone, without being combined. Untreated cells served as the control. Cells were incubated for 24 and 48 hours. Cell viability was determined using the CCK-8 (Cell Counting Kit-8) assay. At the end of the incubation periods, 10 µL of the CCK-8 reagent was added to each well and incubated at 37 o C for 120 minutes (or until optimal color change). The optical density (OD) was measured at a wavelength of 450 nm using a microplate reader (Thermo Scientific, Multiskan SkyHigh, Singapore) following a brief 10-second orbital shaking step. All experimental conditions were performed with three technical replicates. 2.4. Assessment of Oxidative Stress and Antioxidant Enzymes For oxidative stress analyses, cells were seeded at 1 × 10⁶ cells per well in 6-well plates. Following MPP⁺ and/or GA treatment, cells were collected and analyzed for catalase, nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. Catalase (Elabscience, Catalog No: E-BC-K031-S, USA), NO (Elabscience, Catalog No: E-BC-K035-M, USA) and SOD (Elabscience, Catalog No: E-BC-K020-M, USA) levels were quantified using commercial colorometric assay kits. Measurement of Lipid Peroxidation (TBARS Assay) Lipid peroxidation was quantified by measuring the level of MDA using the Thiobarbituric Acid Reactive Substances (TBARS) assay [ 18 ]. Briefly, 0.2 mL of cell lysate was sequentially mixed with 0.8 mL of phosphate buffer (pH 7.4), 0.025 mL of BHT (at 0.88% concentration, to prevent further oxidation), and 0.5 mL of TCA (at 30.0% concentration). Following vortex mixing, the tubes were incubated on ice for 2 hours to ensure complete protein precipitation. The resulting mixture was then centrifuged at 2,000 rpm for 15 minutes. One mL of the supernatant was carefully transferred to a new tube, followed by the addition of 0.075 mL of EDTA (at 0.1 M concentration) and 0.25 mL of TBA (at 1.0% mM concentration). The tubes were briefly vortexed and then incubated in a boiling water bath for 15 minutes to form the characteristic pink MDA-TBA adduct. After cooling to room temperature, the (Thermo Scientific, Multiskan SkyHigh, Singapore). MDA concentration was calculated using the molar extinction coefficient (ε) of the MDA-TBA complex, which is 1.56 x 10 5 cm − 1 M − 1 at 532 nm. The final MDA level was expressed as nmol/mg protein. 2.5. Apoptosis Analysis by Flow Cytometry Apoptotic cell death was evaluated using Annexin V-FITC and Propidium Iodide (PI) staining with a commercial kit (Biolegend 640932, USA). Early apoptotic cells were identified by phosphatidylserine externalization detected by fluorochrome-labeled Annexin V, whereas PI staining indicated late apoptosis or necrosis. SH-SY5Y cells were treated with MPP⁺, GA, or their combination prior to analysis. 2.6. Statistical Analysis Statistical analyses were performed using SPSS Statistics version 27.0 (IBM Corp., Armonk, NY, USA). The distributional characteristics of variables were assessed using the Shapiro–Wilk test. Normality was further evaluated through inspection of skewness and kurtosis values, histogram shapes, Q–Q plots, and detrended Q–Q plots. For variables that met the normality assumption, group differences in mean values were examined using one-way analysis of variance (One-Way ANOVA). Homogeneity of variances was verified with Levene’s test, and when the assumption was satisfied, Tukey test was applied for post-hoc pairwise comparisons. All statistical tests were two-tailed, and a p-value < 0.05 was considered statistically significant. 3. Results 3.1. CCK-8 Cell Viability Assay The CCK-8 assay demonstrated that GA protected SH-SY5Y cells from MPP⁺-induced cytotoxicity in a concentration-dependent manner. In cells treated with 1 mM MPP⁺, 25 µM GA increased cell viability by 34.7% and 55% at 24 h and 48 h, respectively, compared to MPP⁺ alone (Table 1 and Table 2 , Figs. 1 and 2 ). Treatment with 2 mM MPP⁺ resulted in extensive cell death (> 80%), indicating that this concentration was too cytotoxic for further apoptosis assays. Table 1 Effect of GA on MPP⁺-Induced SH-SY5Y Cell Viability GA (µM) MPP (mM) 24h 48h Control 100 100 100 - 32,93** 18,28 ** 50 - 97,80 70,44 ** 25 - 94,43 78,12 * - 1 49,87** 32,74 ** 100 1 21,30 **# 13,44 **# 50 1 69,21 * 47,01 ** 25 1 82,94 ## 86,53 ## - 2 24,84 ** 13,52 ** 100 2 18,87 ** 12,34 ** 50 2 32,51 ** 18,50 ** 25 2 39,79 **# 39,73 **## CCK-8 cell viability results following treatment with MPP⁺ and GA *p ≤ 0,05; ** p ≤ 0,001: Denote statistically significant differences compared with the control group. #p ≤ 0,05; ## p ≤ 0,001: Denote statistically significant differences between the GA + MPP⁺ groups and the MPP⁺ group. *p ≤ 0,05; ** p ≤ 0,001: Denote statistically significant differences compared with the control group. #p ≤ 0,05; ## p ≤ 0,001: Denote statistically significant differences between the GA + MPP⁺ groups and the MPP⁺ group. 3.2. Apoptosis Analysis Based on the CCK-8 results, 1 mM MPP⁺ was selected for apoptosis assays. Flow cytometry revealed that 25 µM GA provided the most protective effect against MPP⁺-induced apoptosis. The percentage of viable cells increased from 11.1% and 8.4% in MPP⁺-treated cells at 24 h and 48 h, respectively, to 14.8% and 13.4% upon GA co-treatment (Fig. 2 and Fig. 3 ). 3.3. Oxidative Stress and Antioxidant Enzyme Levels Gallic acid treatment significantly enhanced the antioxidant defense system of SH-SY5Y cells exposed to MPP⁺. Both catalase and nitric oxide (NO) levels were elevated in GA-treated groups compared to MPP⁺-only cells, indicating that GA mitigates MPP⁺-induced oxidative stress (Table 2 ). Table 2 Effect of GA on oxidative stress parameters in MPP⁺-Induced SH-SY5Y cells GA (µM) MPP (mM) MDA (nM/mg prot) %NO SOD (U/mg prot) Catalase (U/mg prot) 24h 48h 24h 48h 24h 48h 24h 48h Control 7,18 9,65 100 100,00 17,15 23,68 30,01 34,24 100 - 16,28 ** 19,09 ** 15,06 ** 12,54 ** 10,50 ** 13,41 17,74 ** 16,37 ** 50 - 11,34 * 14,04 * 53,98 ** 42,46 ** 13,14 * 16,29 22,27 ** 25,72 ** 25 - 7,32 11,75 77,02 ** 64,66 ** 17,30 27,01 34,00 39,31 - 1 18,67 ** 21,18 ** 17,96 ** 12,98 ** 11,24 ** 9,69* 19,04 ** 14,84 ** 100 1 19,90 ** 31,14 **## 9,92 **# 7,69 **# 11,62 ** 8,69* 19,14 ** 16,11 ** 50 1 10,91 *## 13,29 ## 51,36 **## 63,01 **## 13,27 * 15,31 26,73 ## 24,11 **## 25 1 8,30 ## 9,64 ## 79,65 **## 86,07 **## 16,10 ## 14,25 29,28 ## 29,98 ## *p ≤ 0.05; ** p ≤ 0.001: Denote statistically significant differences compared with the control group. #p ≤ 0.05; ## p ≤ 0.001: Denote statistically significant differences between the GA + MPP⁺ groups and the MPP⁺ group. 4. Discussion The exact origins of PD remain unknown but both inherited and spontaneous cases present identical neurological damage patterns which include mitochondrial breakdown and brain inflammation and toxic substance exposure. The production of excessive reactive oxygen species (ROS) serves as a common factor which leads to dopamine metabolism problems and substantia nigra pars compacta iron deposition of iron and results in dopaminergic neuron death. The combination of dopamine oxidation and mitochondrial damage and protein aggregate accumulation makes oxidative stress the primary factor responsible for Parkinson's disease development [ 12 , 19 , 20 ]. Research shows that dopaminergic neurons produce high levels of Reactive Oxygen Species (ROS) because dopamine has a high oxidation potential and brain tissue lacks sufficient antioxidant defenses [ 21 , 22 ]. The substantia nigra dopaminergic neurons in Parkinson's disease (PD) show exceptional sensitivity to oxidative stress which makes antioxidants essential for both disease prevention and treatment. The neurotoxin MPP⁺ shows specific toxicity toward dopaminergic neurons found in the substantia nigra. The compound enters mitochondrial structures where it blocks Complex I function which results in oxidative stress that damages cells. Scientists use this compound to create a cellular model of Parkinson's disease because it accurately replicates the disease process [ 23 – 25 ]. Research indicates that antioxidants show promise as therapeutic options for treating Parkinson's disease because they address multiple factors involved in the disease progression [ 19 , 26 ]. Scientists have focused on polyphenols because these compounds show promise for treating oxidative stress-related diseases. The phenolic structure of GA contains hydrogen atoms which make it an effective antioxidant because these atoms can easily transfer to free radicals. Research shows that GA protects neurons through its ability to reduce oxidative stress and inflammation [ 15 , 17 , 19 , 27 ]. GA demonstrates anti-inflammatory effects through its ability to neutralize superoxide anions and block myeloperoxidase release and activity and possibly disrupt NADPH oxidase formation. The combination of antioxidant and anti-inflammatory properties in GA makes it a promising substance for preventing oxidative stress in Parkinson's disease [ 14 , 27 – 30 ]. The current research evaluates GA's ability to protect cells from MPP⁺ toxicity in a Parkinson's disease cellular model. The study shows that GA protects MPP⁺-treated SH-SY5Y cells through its ability to boost antioxidant systems and reduce programmed cell death. The research results support established GA mechanisms of action while strengthening its potential as a therapeutic agent for treating PD [ 17 , 29 ]. The research findings show that MPP⁺ exposure causes neuroblastoma cell death while producing high levels of apoptotic cells which represents the severe oxidative damage and neurotoxic effects of MPP⁺ exposure [ 23 ]. The protective effects of GA become evident at 25 µM concentrations because they help cells survive and reduce programmed cell death according to previous research which demonstrated its direct free radical scavenging abilities and mitochondrial protective effects. The protective effects of GA against 6-OHDA-induced damage in SH-SY5Y cells have been demonstrated through its ability to prevent mitochondrial membrane potential loss and ROS production and apoptosis by maintaining Bax/Bcl-2 ratio equilibrium [ 29 ]. Secondly, and consistent with the literature, MPP⁺ exposure in SH-SY5Y cells led to a marked depletion of antioxidant enzymes (SOD and Catalase) and NO levels, concomitant with a significant elevation in MDA levels, a byproduct of lipid peroxidation [ 23 , 24 ]. The decrease in SOD enzyme activity results in superoxide radical (O₂⁻) accumulation inside cells which leads to accelerated oxidative tissue damage [ 31 ]. The insufficient activity of Catalase prevents the proper removal of H₂O₂ produced by SOD which results in increased damage to mitochondria and membrane lipids The insufficient activity of Catalase prevents the proper removal of H₂O₂ produced by SOD which results in increased damage to mitochondria and membrane lipids [ 32 ]. Similarly, the decline in NO—a molecule that serves as a neuroprotective signal modulator under physiological conditions—may predispose cells to unchecked damage from reactive nitrogen species such as peroxynitrite [ 33 ]. The rise in MDA concentrations together with decreased antioxidant enzyme activities shows that membrane lipid destruction accelerates while MPP⁺ causes damage to cellular membranes. The measurement of MDA serves as a well-established method to detect oxidative stress in cells [ 34 ]. OThe results show that GA restores the antioxidant defense systems of SH-SY5Y cells which were damaged by MPP⁺ exposure. The antioxidant enzyme systems of MPP⁺-exposed SH-SY5Y cells become active through GA treatment which demonstrates its dual role as a radical scavenger and endogenous enzyme activator. Research reviews about polyphenol neuroprotection at the mitochondrial level support the idea that these compounds activate endogenous antioxidant enzyme systems. Research shows that mitochondria-directed polyphenols including GA analogs protect neurons from degeneration through Nrf2/ARE pathway activation of antioxidant defenses [ 35 , 36 ]. The results from our cellular model support the idea that GA will perform better when it targets specific intracellular processes. The application of 25 µM GA to MPP⁺-treated SH-SY5Y cells during 24 and 48 hours resulted in SOD enzyme activity that matched control group levels. The application of 25 µM and 50 µM GA doses successfully blocked MPP⁺ from decreasing catalase and NO levels. Similarly, the increase in the lipid peroxidation marker, MDA, caused by MPP⁺ was significantly reduced by the 25 µM and 50 µM GA treatments, returning levels to near those of the control group. These results substantiate that GA effectively attenuates oxidative stress by potentiating antioxidant defense systems in the PD cell model. Thirdly, data from in vivo studies support our findings. A study conducted on a Rotenone-induced PD model in mice showed that when a high dose of GA (e.g., 100 mg/kg) was administered, the loss of dopaminergic neurons in the substantia nigra was reduced, motor functions were preserved, and lipid peroxidation levels were lowered [ 17 ]. The neuroprotective effects of GA have been proven through in vivo research which extends beyond cell culture experiments. The research contains specific restrictions which need evaluation. The study failed to identify the exact signaling pathways through which GA protects cells from MPP⁺ toxicity including Nrf2 and mitophagy and autophagy and inflammation pathways. Future research needs to investigate how GA effects on Nrf2/Keap1 and SIRT-1 and PGC-1α and mitochondrial biogenesis and autophagy markers in detail. The bioavailability and intracellular distribution of GA along with its metabolites need evaluation because transferring in vitro concentrations to in vivo settings becomes difficult. Conclusion In conclusion, our study demonstrates that GA supports the restoration of antioxidant enzyme activities, exerts anti-peroxidative effects by reducing lipid peroxidation, and exhibits protective activity on the viability of MPP+-induced PD cells. This protective action suggests GA may function by activating crucial endogenous antioxidant signaling pathways, such as the Nrf2/ARE axis. GA can be considered a promising potential therapeutic strategy for the prevention and treatment of Parkinson’s Disease. Abbreviations BHT Butylated Hydroxytoluene DMEM Dulbecco’s Modified Eagle Medium EDTA Ethylenediaminetetraacetic Acid FBS Fetal bovine serum GA Gallic acid MDA Malondialdehyde MPP+ 1-methyl-4-phenylpyridinium NO Nitric oxide OD Optical density PBS Phosphate buffer saline PD Parkinson’s disease ROS Reactive oxygen species SOD Superoxide dismutase TBA Thiobarbituric Acid TCA Trichloroacetic Acid Declarations Competing Interests The authors declare that there is no conflict of interest. Data Avaibility Statements Data will be made available on request. Ethics Statements There is no problem in terms of research and publication ethics. Funding The study was supported by the Gaziantep University (Project No: TF.HZP.23.44). Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hasan Ulusal, Hatice Kubra Yigit Dumrul and Mehmet Tarakcioglu. The first draft of the manuscript was written by Hasan Ulusal, Hatice Kubra Yigit Dumrul and Sibel Dagli, and all authors commented on previous versions of the manuscript. Project administration was led by Mehmet Tarakcioglu. All authors read and approved the final manuscript. Data Availability Data will be made available on request. References Balestrino R, Schapira AHV (2020) Parkinson disease. Eur J Neurol 27:27–42. 10.1111/ENE.14108 Hayes MT (2019) Parkinson's Disease and Parkinsonism, American Journal of Medicine. Elsevier Inc., pp 802–807 Marogianni C, Sokratous M, Dardiotis E, Hadjigeorgiou GM et al (2020) Neurodegeneration and inflammation—an interesting interplay in parkinson’s disease. Int J Mol Sci MDPI AG, pp. 1–15 Amro MS, Teoh SL, Norzana AG, Srijit D (2018) The potential role of herbal products in the treatment of Parkinson's disease. 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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-8653441","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581627030,"identity":"80912cc0-2bfa-486b-87cf-a401430f8968","order_by":0,"name":"Hasan Ulusal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBACAygtA8SMBz4wSCSAuTxEaAGrOTgDqIUHypUgSsthIElYizn78YefC4CKDc4ffnDY5pdFnr1EAuODt20MdeYN2LVY9uQYS88AabmRZnA4t0+imEcigdlwbhuDhMwBHA47kMMgzQPWwgDU0iOR2CORwCbNC9SCy2UG558//g3Wcv74h8OWEC3sv/FquZFgBrHlQI7BYYYfEFuY8Wt5Y2bNY5DOI3kjp+BgbwPQL2ceNkvOOSchOQOnw9If3+apsJbjO39844Mff+ry2NuTD354U2bDjzOUIRqhNGMbmGxgwBMt6OAPsQpHwSgYBaNgJAEAVbtWBI4hV1oAAAAASUVORK5CYII=","orcid":"","institution":"Gaziantep Islam Science and Technology University","correspondingAuthor":true,"prefix":"","firstName":"Hasan","middleName":"","lastName":"Ulusal","suffix":""},{"id":581627033,"identity":"cabadf4e-9665-46fa-86a7-537c22f15079","order_by":1,"name":"Hatice Kubra Yigit Dumrul","email":"","orcid":"","institution":"Ağrı İbrahim Çeçen University","correspondingAuthor":false,"prefix":"","firstName":"Hatice","middleName":"Kubra Yigit","lastName":"Dumrul","suffix":""},{"id":581627034,"identity":"4c2629ef-fbd2-4a8a-a44d-484d8a5355c1","order_by":2,"name":"Sibel Dagli","email":"","orcid":"","institution":"Sanko University","correspondingAuthor":false,"prefix":"","firstName":"Sibel","middleName":"","lastName":"Dagli","suffix":""},{"id":581627036,"identity":"edbf6500-2a82-4c7d-96ac-9834e04bd8bf","order_by":3,"name":"Mehmet Tarakcioglu","email":"","orcid":"","institution":"Gaziantep University","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"Tarakcioglu","suffix":""}],"badges":[],"createdAt":"2026-01-20 22:43:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8653441/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8653441/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101451259,"identity":"27f85aee-5778-4d63-a14e-6f7e8f3d3a29","added_by":"auto","created_at":"2026-01-29 20:24:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82558,"visible":true,"origin":"","legend":"\u003cp\u003eMPP\u003csup\u003e+\u003c/sup\u003e and Gallic acid CCK 8 results\u003c/p\u003e\n\u003cp\u003e*p ≤ 0,05; ** p ≤ 0,001: Denote statistically significant differences compared with the control group.\u003c/p\u003e\n\u003cp\u003e#p ≤ 0,05; ## p ≤ 0,001: Denote statistically significant differences between the GA + MPP⁺ groups and the MPP⁺ group.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8653441/v1/aa2223e5b7125042ca2384b4.jpg"},{"id":101451260,"identity":"ef42ecb2-ffab-4ec3-ae87-4588b111c077","added_by":"auto","created_at":"2026-01-29 20:24:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154775,"visible":true,"origin":"","legend":"\u003cp\u003e24-hour flow cytometry data\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8653441/v1/53431b36732003e7b7f2bc22.jpg"},{"id":101451261,"identity":"3bed3db3-941f-41fc-a094-80ffa3457b48","added_by":"auto","created_at":"2026-01-29 20:24:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":147388,"visible":true,"origin":"","legend":"\u003cp\u003e48-hour flow cytometry data\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8653441/v1/f32d15f246245bd3bdf1ae68.jpg"},{"id":102397413,"identity":"9ccee3ec-65f4-4498-af32-6d78c3bf78e9","added_by":"auto","created_at":"2026-02-11 10:16:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1106256,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8653441/v1/6bfa37d5-11e5-4287-88b4-a86675985846.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neuroprotective Effects of Gallic Acid in an MPP⁺-Induced SH-SY5Y Cell Model of Parkinson’s Disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) ranks as the second most prevalent neurodegenerative condition after Alzheimer\u0026rsquo;s disease while its incidence rate increases with the increasing number of elderly people in the world [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The worldwide number of PD patients reached 6.1\u0026nbsp;million in 2016. The main neurological feature of Parkinson's disease involves the continuous deterioration of dopaminergic neurons which reside in the substantia nigra pars compacta [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Research shows that multiple cellular mechanisms including mitochondrial damage and α-synuclein accumulation and lysosomal breakdown and immune system dysregulation and programmed cell death and oxidative stress damage dopaminergic neurons [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe death of dopaminergic neurons that originate from the substantia nigra pars compacta and project to the striatum results in the development of Parkinson's disease motor symptoms including tremor and bradykinesia and rigidity and postural instability. The development of non-motor symptoms in Parkinson's disease patients occurs before their motor symptoms become apparent [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Scientists now understand that Parkinson's disease develops through the combination of genetic factors and environmental elements instead of being caused by environmental factors alone [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe process of neurodegeneration in Parkinson's disease primarily results from excessive oxidative stress in the brain. The body produces too many reactive oxygen species which exceed its natural antioxidant defenses. The enzymatic antioxidants superoxide dismutase (SOD) and catalase and glutathione function as free radical neutralizers but their breakdown results in oxidative damage to lipids and proteins and DNA which leads to cell death that scientists link to Parkinson's disease development [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Research indicates that PD shows elevated oxidative stress levels during its initial stages before noticeable neuronal deterioration which supports the theory that uncontrolled reactive oxygen species (ROS) production starts dopaminergic neuron degeneration instead of being a progression marker [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe human neuroblastoma cell line SH-SY5Y serves as a common laboratory model for studying dopaminergic neuronal function because it displays catecholaminergic characteristics. The Parkinsonian toxin MPTP produces its active compound 1-methyl-4-phenylpyridinium (MPP⁺) which scientists use to create neurotoxicity in laboratory experiments that mimic PD-related cellular damage [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNatural polyphenolic compounds have attracted increasing interest as potential therapeutic agents for neurodegenerative diseases because of their strong antioxidant and anti-inflammatory properties. Gallic acid (GA; 3,4,5-trihydroxybenzoic acid) is a widely distributed plant-derived phenolic compound found in grapes, tea, walnuts, and various fruits [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. GA has been shown to scavenge free radicals, modulate intracellular signaling pathways, inhibit oxidative stress\u0026ndash;induced apoptosis, and attenuate neuroinflammation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Previous in vitro and in vivo studies have reported that GA and its derivatives can reduce oxidative damage in SH-SY5Y cells and ameliorate oxidative stress\u0026ndash;induced neurotoxicity in animal models [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on this evidence, the present study aims to investigate the potential neuroprotective effects of GA through its antioxidant activity in an MPP⁺-induced SH-SY5Y cell model of Parkinson\u0026rsquo;s disease. Specifically, this study seeks to determine whether GA can counteract MPP⁺-induced neurotoxicity by modulating oxidative stress\u0026ndash;related pathways.\u003c/p\u003e \u003cp\u003eWe hypothesize that GA exerts neuroprotective effects in the MPP⁺-induced SH-SY5Y cell model by mitigating oxidative stress, lowering intracellular ROS accumulation, restoring antioxidant defense capacity, and consequently preventing mitochondrial dysfunction and cell death.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagent and Chemicals\u003c/h2\u003e \u003cp\u003eAll materials were used without further purification. Gaziantep University Medical Biochemistry Department provided SH-SY5Ycell line. Fetal bovine serum (FBS) (US Origin, Product No: SH30071.03), phosphate buffer saline (PBS) (Without Calcium, magnesium, Product No: SH30256.01), and Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, high glucose with L-glutamine, Product No: SH30022.FS) medium were purchased from HyClone. CCK-8 (Catalog No: KTA1020) was purchased from Abbkine. GA (Prodcut No: 8.42649), Butylated Hydroxytoluene (BHT) (Prodcut No: W218405), Trichloroacetic Acid (TCA) (Prodcut No: 8.22342), Ethylenediaminetetraacetic Acid (EDTA) (Prodcut No: 798681) and Thiobarbituric Acid (TBA) (Prodcut No: T5500) were purchased from Merck.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell Culture\u003c/h2\u003e \u003cp\u003eHuman neuroblastoma SH-SY5Y cells (ATCC\u0026reg; CRL-2266\u0026trade;) were cultured DMEM supplemented with 10% fetal bovine serum FBS and 1% penicillin-streptomycin. Cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂ and subcultured upon reaching approximately 80% confluence. Frozen cells were rapidly thawed at 37\u0026deg;C, centrifuged, resuspended in fresh medium, and seeded in culture flasks. Cell viability and density were determined using trypan blue exclusion and a Neubauer chamber. For experiments, cells were seeded at a density of 5 \u0026times; 10\u0026sup3; cells per well in 96-well plates and incubated for 24 h prior to treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell Viability Assay\u003c/h2\u003e \u003cp\u003eCells were seeded into 96-well culture plates at a density of 5x10^3 cells/well (in 100 \u0026micro;L of complete culture medium) and allowed to adhere overnight. After 24 hours, the cells reached approximately 70\u0026ndash;80% confluence, and the morphology was confirmed under an inverted microscope. Cells were first treated with 1 and 2 mM MPP\u003csup\u003e+\u003c/sup\u003e and then, 2 hours later, with different concentrations of GA (25, 50, and 100 \u0026micro;M). Additionally, MPP\u003csup\u003e+\u003c/sup\u003e (1 and 2 mM) and GA (25, 50, and 100 \u0026micro;M) were applied alone, without being combined. Untreated cells served as the control. Cells were incubated for 24 and 48 hours. Cell viability was determined using the CCK-8 (Cell Counting Kit-8) assay. At the end of the incubation periods, 10 \u0026micro;L of the CCK-8 reagent was added to each well and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 120 minutes (or until optimal color change). The optical density (OD) was measured at a wavelength of 450 nm using a microplate reader (Thermo Scientific, Multiskan SkyHigh, Singapore) following a brief 10-second orbital shaking step. All experimental conditions were performed with three technical replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Assessment of Oxidative Stress and Antioxidant Enzymes\u003c/h2\u003e \u003cp\u003eFor oxidative stress analyses, cells were seeded at 1 \u0026times; 10⁶ cells per well in 6-well plates. Following MPP⁺ and/or GA treatment, cells were collected and analyzed for catalase, nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. Catalase (Elabscience, Catalog No: E-BC-K031-S, USA), NO (Elabscience, Catalog No: E-BC-K035-M, USA) and SOD (Elabscience, Catalog No: E-BC-K020-M, USA) levels were quantified using commercial colorometric assay kits.\u003c/p\u003e \u003cp\u003eMeasurement of Lipid Peroxidation (TBARS Assay) Lipid peroxidation was quantified by measuring the level of MDA using the Thiobarbituric Acid Reactive Substances (TBARS) assay [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Briefly, 0.2 mL of cell lysate was sequentially mixed with 0.8 mL of phosphate buffer (pH 7.4), 0.025 mL of BHT (at 0.88% concentration, to prevent further oxidation), and 0.5 mL of TCA (at 30.0% concentration). Following vortex mixing, the tubes were incubated on ice for 2 hours to ensure complete protein precipitation. The resulting mixture was then centrifuged at 2,000 rpm for 15 minutes. One mL of the supernatant was carefully transferred to a new tube, followed by the addition of 0.075 mL of EDTA (at 0.1 M concentration) and 0.25 mL of TBA (at 1.0% mM concentration). The tubes were briefly vortexed and then incubated in a boiling water bath for 15 minutes to form the characteristic pink MDA-TBA adduct. After cooling to room temperature, the (Thermo Scientific, Multiskan SkyHigh, Singapore). MDA concentration was calculated using the molar extinction coefficient (ε) of the MDA-TBA complex, which is 1.56 x 10\u003csup\u003e5\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 532 nm. The final MDA level was expressed as nmol/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Apoptosis Analysis by Flow Cytometry\u003c/h2\u003e \u003cp\u003eApoptotic cell death was evaluated using Annexin V-FITC and Propidium Iodide (PI) staining with a commercial kit (Biolegend 640932, USA). Early apoptotic cells were identified by phosphatidylserine externalization detected by fluorochrome-labeled Annexin V, whereas PI staining indicated late apoptosis or necrosis. SH-SY5Y cells were treated with MPP⁺, GA, or their combination prior to analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS Statistics version 27.0 (IBM Corp., Armonk, NY, USA). The distributional characteristics of variables were assessed using the Shapiro\u0026ndash;Wilk test. Normality was further evaluated through inspection of skewness and kurtosis values, histogram shapes, Q\u0026ndash;Q plots, and detrended Q\u0026ndash;Q plots. For variables that met the normality assumption, group differences in mean values were examined using one-way analysis of variance (One-Way ANOVA). Homogeneity of variances was verified with Levene\u0026rsquo;s test, and when the assumption was satisfied, Tukey test was applied for post-hoc pairwise comparisons. All statistical tests were two-tailed, and a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. CCK-8 Cell Viability Assay\u003c/h2\u003e \u003cp\u003eThe CCK-8 assay demonstrated that GA protected SH-SY5Y cells from MPP⁺-induced cytotoxicity in a concentration-dependent manner. In cells treated with 1 mM MPP⁺, 25 \u0026micro;M GA increased cell viability by 34.7% and 55% at 24 h and 48 h, respectively, compared to MPP⁺ alone (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Treatment with 2 mM MPP⁺ resulted in extensive cell death (\u0026gt;\u0026thinsp;80%), indicating that this concentration was too cytotoxic for further apoptosis assays.\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\u003eEffect of GA on MPP⁺-Induced SH-SY5Y Cell Viability\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGA (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMPP\u003c/p\u003e \u003cp\u003e(mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48h\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32,93**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18,28\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97,80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70,44\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94,43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78,12\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49,87**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32,74\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21,30\u003csup\u003e**#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13,44\u003csup\u003e**#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69,21\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47,01\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82,94\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86,53\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24,84\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13,52\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18,87\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12,34\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32,51\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18,50\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39,79\u003csup\u003e**#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39,73\u003csup\u003e**##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCCK-8 cell viability results following treatment with MPP⁺ and GA\u003c/p\u003e \u003cp\u003e*p\u0026thinsp;\u0026le;\u0026thinsp;0,05; ** p\u0026thinsp;\u0026le;\u0026thinsp;0,001: Denote statistically significant differences compared with the control group.\u003c/p\u003e \u003cp\u003e#p\u0026thinsp;\u0026le;\u0026thinsp;0,05; ## p\u0026thinsp;\u0026le;\u0026thinsp;0,001: Denote statistically significant differences between the GA\u0026thinsp;+\u0026thinsp;MPP⁺ groups and the MPP⁺ group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e*p\u0026thinsp;\u0026le;\u0026thinsp;0,05; ** p\u0026thinsp;\u0026le;\u0026thinsp;0,001: Denote statistically significant differences compared with the control group.\u003c/p\u003e \u003cp\u003e#p\u0026thinsp;\u0026le;\u0026thinsp;0,05; ## p\u0026thinsp;\u0026le;\u0026thinsp;0,001: Denote statistically significant differences between the GA\u0026thinsp;+\u0026thinsp;MPP⁺ groups and the MPP⁺ group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Apoptosis Analysis\u003c/h2\u003e \u003cp\u003eBased on the CCK-8 results, 1 mM MPP⁺ was selected for apoptosis assays. Flow cytometry revealed that 25 \u0026micro;M GA provided the most protective effect against MPP⁺-induced apoptosis. The percentage of viable cells increased from 11.1% and 8.4% in MPP⁺-treated cells at 24 h and 48 h, respectively, to 14.8% and 13.4% upon GA co-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Oxidative Stress and Antioxidant Enzyme Levels\u003c/h2\u003e \u003cp\u003eGallic acid treatment significantly enhanced the antioxidant defense system of SH-SY5Y cells exposed to MPP⁺. Both catalase and nitric oxide (NO) levels were elevated in GA-treated groups compared to MPP⁺-only cells, indicating that GA mitigates MPP⁺-induced oxidative stress (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\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\u003eEffect of GA on oxidative stress parameters in MPP⁺-Induced SH-SY5Y cells\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGA (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMPP\u003c/p\u003e \u003cp\u003e(mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eMDA (nM/mg prot)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e%NO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eSOD (U/mg prot)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eCatalase (U/mg prot)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e48h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e24h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e48h\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7,18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9,65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17,15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e23,68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e30,01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e34,24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16,28\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19,09\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15,06\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12,54\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10,50\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13,41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e17,74\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e16,37\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11,34\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14,04\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53,98\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42,46\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13,14\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16,29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e22,27\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e25,72\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7,32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11,75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77,02\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64,66\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17,30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e27,01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e34,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e39,31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18,67\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21,18\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17,96\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12,98\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11,24\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9,69*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e19,04\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e14,84\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19,90\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31,14\u003csup\u003e**##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9,92\u003csup\u003e**#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7,69\u003csup\u003e**#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11,62\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8,69*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e19,14\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e16,11\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10,91\u003csup\u003e*##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13,29\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51,36\u003csup\u003e**##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63,01\u003csup\u003e**##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13,27\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15,31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e26,73\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24,11\u003csup\u003e**##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8,30\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9,64\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79,65\u003csup\u003e**##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e86,07\u003csup\u003e**##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16,10\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14,25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e29,28\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e29,98\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*p\u0026thinsp;\u0026le;\u0026thinsp;0.05; ** p\u0026thinsp;\u0026le;\u0026thinsp;0.001: Denote statistically significant differences compared with the control group.\u003c/p\u003e \u003cp\u003e#p\u0026thinsp;\u0026le;\u0026thinsp;0.05; ## p\u0026thinsp;\u0026le;\u0026thinsp;0.001: Denote statistically significant differences between the GA\u0026thinsp;+\u0026thinsp;MPP⁺ groups and the MPP⁺ group.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe exact origins of PD remain unknown but both inherited and spontaneous cases present identical neurological damage patterns which include mitochondrial breakdown and brain inflammation and toxic substance exposure. The production of excessive reactive oxygen species (ROS) serves as a common factor which leads to dopamine metabolism problems and substantia nigra pars compacta iron deposition of iron and results in dopaminergic neuron death. The combination of dopamine oxidation and mitochondrial damage and protein aggregate accumulation makes oxidative stress the primary factor responsible for Parkinson's disease development [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearch shows that dopaminergic neurons produce high levels of Reactive Oxygen Species (ROS) because dopamine has a high oxidation potential and brain tissue lacks sufficient antioxidant defenses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The substantia nigra dopaminergic neurons in Parkinson's disease (PD) show exceptional sensitivity to oxidative stress which makes antioxidants essential for both disease prevention and treatment. The neurotoxin MPP⁺ shows specific toxicity toward dopaminergic neurons found in the substantia nigra. The compound enters mitochondrial structures where it blocks Complex I function which results in oxidative stress that damages cells. Scientists use this compound to create a cellular model of Parkinson's disease because it accurately replicates the disease process [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Research indicates that antioxidants show promise as therapeutic options for treating Parkinson's disease because they address multiple factors involved in the disease progression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eScientists have focused on polyphenols because these compounds show promise for treating oxidative stress-related diseases. The phenolic structure of GA contains hydrogen atoms which make it an effective antioxidant because these atoms can easily transfer to free radicals. Research shows that GA protects neurons through its ability to reduce oxidative stress and inflammation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. GA demonstrates anti-inflammatory effects through its ability to neutralize superoxide anions and block myeloperoxidase release and activity and possibly disrupt NADPH oxidase formation. The combination of antioxidant and anti-inflammatory properties in GA makes it a promising substance for preventing oxidative stress in Parkinson's disease [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The current research evaluates GA's ability to protect cells from MPP⁺ toxicity in a Parkinson's disease cellular model. The study shows that GA protects MPP⁺-treated SH-SY5Y cells through its ability to boost antioxidant systems and reduce programmed cell death. The research results support established GA mechanisms of action while strengthening its potential as a therapeutic agent for treating PD [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe research findings show that MPP⁺ exposure causes neuroblastoma cell death while producing high levels of apoptotic cells which represents the severe oxidative damage and neurotoxic effects of MPP⁺ exposure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The protective effects of GA become evident at 25 \u0026micro;M concentrations because they help cells survive and reduce programmed cell death according to previous research which demonstrated its direct free radical scavenging abilities and mitochondrial protective effects. The protective effects of GA against 6-OHDA-induced damage in SH-SY5Y cells have been demonstrated through its ability to prevent mitochondrial membrane potential loss and ROS production and apoptosis by maintaining Bax/Bcl-2 ratio equilibrium [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSecondly, and consistent with the literature, MPP⁺ exposure in SH-SY5Y cells led to a marked depletion of antioxidant enzymes (SOD and Catalase) and NO levels, concomitant with a significant elevation in MDA levels, a byproduct of lipid peroxidation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe decrease in SOD enzyme activity results in superoxide radical (O₂⁻) accumulation inside cells which leads to accelerated oxidative tissue damage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The insufficient activity of Catalase prevents the proper removal of H₂O₂ produced by SOD which results in increased damage to mitochondria and membrane lipids The insufficient activity of Catalase prevents the proper removal of H₂O₂ produced by SOD which results in increased damage to mitochondria and membrane lipids [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, the decline in NO\u0026mdash;a molecule that serves as a neuroprotective signal modulator under physiological conditions\u0026mdash;may predispose cells to unchecked damage from reactive nitrogen species such as peroxynitrite [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe rise in MDA concentrations together with decreased antioxidant enzyme activities shows that membrane lipid destruction accelerates while MPP⁺ causes damage to cellular membranes. The measurement of MDA serves as a well-established method to detect oxidative stress in cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOThe results show that GA restores the antioxidant defense systems of SH-SY5Y cells which were damaged by MPP⁺ exposure. The antioxidant enzyme systems of MPP⁺-exposed SH-SY5Y cells become active through GA treatment which demonstrates its dual role as a radical scavenger and endogenous enzyme activator. Research reviews about polyphenol neuroprotection at the mitochondrial level support the idea that these compounds activate endogenous antioxidant enzyme systems. Research shows that mitochondria-directed polyphenols including GA analogs protect neurons from degeneration through Nrf2/ARE pathway activation of antioxidant defenses [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The results from our cellular model support the idea that GA will perform better when it targets specific intracellular processes.\u003c/p\u003e \u003cp\u003eThe application of 25 \u0026micro;M GA to MPP⁺-treated SH-SY5Y cells during 24 and 48 hours resulted in SOD enzyme activity that matched control group levels. The application of 25 \u0026micro;M and 50 \u0026micro;M GA doses successfully blocked MPP⁺ from decreasing catalase and NO levels. Similarly, the increase in the lipid peroxidation marker, MDA, caused by MPP⁺ was significantly reduced by the 25 \u0026micro;M and 50 \u0026micro;M GA treatments, returning levels to near those of the control group. These results substantiate that GA effectively attenuates oxidative stress by potentiating antioxidant defense systems in the PD cell model.\u003c/p\u003e \u003cp\u003eThirdly, data from in vivo studies support our findings. A study conducted on a Rotenone-induced PD model in mice showed that when a high dose of GA (e.g., 100 mg/kg) was administered, the loss of dopaminergic neurons in the substantia nigra was reduced, motor functions were preserved, and lipid peroxidation levels were lowered [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The neuroprotective effects of GA have been proven through in vivo research which extends beyond cell culture experiments.\u003c/p\u003e \u003cp\u003eThe research contains specific restrictions which need evaluation. The study failed to identify the exact signaling pathways through which GA protects cells from MPP⁺ toxicity including Nrf2 and mitophagy and autophagy and inflammation pathways. Future research needs to investigate how GA effects on Nrf2/Keap1 and SIRT-1 and PGC-1α and mitochondrial biogenesis and autophagy markers in detail. The bioavailability and intracellular distribution of GA along with its metabolites need evaluation because transferring in vitro concentrations to in vivo settings becomes difficult.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study demonstrates that GA supports the restoration of antioxidant enzyme activities, exerts anti-peroxidative effects by reducing lipid peroxidation, and exhibits protective activity on the viability of MPP+-induced PD cells. This protective action suggests GA may function by activating crucial endogenous antioxidant signaling pathways, such as the Nrf2/ARE axis. GA can be considered a promising potential therapeutic strategy for the prevention and treatment of Parkinson\u0026rsquo;s Disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBHT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eButylated Hydroxytoluene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEthylenediaminetetraacetic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGallic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPP+\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1-methyl-4-phenylpyridinium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNitric oxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate buffer saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eParkinson\u0026rsquo;s disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSuperoxide dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTBA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThiobarbituric Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTrichloroacetic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eData Avaibility Statements\u003c/h2\u003e \u003cp\u003eData will be made available on request.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics Statements\u003c/strong\u003e \u003cp\u003eThere is no problem in terms of research and publication ethics.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was supported by the Gaziantep University (Project No: TF.HZP.23.44).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hasan Ulusal, Hatice Kubra Yigit Dumrul and Mehmet Tarakcioglu. The first draft of the manuscript was written by Hasan Ulusal, Hatice Kubra Yigit Dumrul and Sibel Dagli, and all authors commented on previous versions of the manuscript. Project administration was led by Mehmet Tarakcioglu. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBalestrino R, Schapira AHV (2020) Parkinson disease. 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Journal of Translational Medicine 2025 23:1, 23 572 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/S12967-025-06605-0\u003c/span\u003e\u003cspan address=\"10.1186/S12967-025-06605-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKung HC, Lin KJ, Kung CT, Lin TK (2021) Oxidative Stress, Mitochondrial Dysfunction, and Neuroprotection of Polyphenols with Respect to Resveratrol in Parkinson's Disease. Biomedicines 9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/BIOMEDICINES9080918\u003c/span\u003e\u003cspan address=\"10.3390/BIOMEDICINES9080918\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gallic acid, MPP+, Neuroprotection, Oxidative Stress, Parkinson Disease","lastPublishedDoi":"10.21203/rs.3.rs-8653441/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8653441/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eParkinson\u0026rsquo;s Disease (PD) represents the second most prevalent neurodegenerative condition which leads to the progressive destruction of dopaminergic neurons in the substantia nigra through oxidative stress mechanisms. The research evaluated Gallic Acid (GA) as a natural polyphenol with proven antioxidant properties for its ability to protect cells from 1-methyl-4-phenylpyridinium (MPP⁺)-induced neurotoxicity in SH-SY5Y dopaminergic cell models.\u003c/p\u003e\u003ch2\u003eMethods and Results\u003c/h2\u003e \u003cp\u003eThe research used SH-SY5Y cells which received 1 mM MPP⁺ treatment alongside different GA concentrations (25,50 and 100 \u0026micro;M) for 24 and 48 hours. The CCK-8 assay measured cell viability while flow cytometry evaluated apoptosis and SOD and MDA levels determined oxidative status through SOD and Catalase and NO measurements. The addition of MPP⁺ resulted in a 32.74% decrease in cell viability at 48 hours while simultaneously decreasing SOD and Catalase and NO levels and increasing MDA levels. The addition of 25 \u0026micro;M GA protected cells from damage by increasing their viability to 86.53% at 48 hours and decreasing apoptotic cell numbers. The protective effects of 25\u0026ndash;50 \u0026micro;M GA were observed through its ability to stop Catalase and NO decreases and its successful reduction of MDA levels (GA 25 \u0026micro;M decreased MDA from 21.18 to 9.64 nM/mg prot at 48 hours). The research indicates that GA functions as an antioxidant by rebuilding the body's natural defense system against oxidative stress.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe study demonstrates that GA provides strong protection against peroxidation by maintaining essential antioxidant enzyme activities in MPP⁺-exposed cells. The research demonstrates that GA shows potential as an effective\u003c/p\u003e","manuscriptTitle":"Neuroprotective Effects of Gallic Acid in an MPP⁺-Induced SH-SY5Y Cell Model of Parkinson’s Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 20:24:49","doi":"10.21203/rs.3.rs-8653441/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-06T11:13:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-02T16:25:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133541249189716176194819312133668347154","date":"2026-02-01T17:43:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-31T11:34:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T08:15:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16655132290166488643224203623058606892","date":"2026-01-27T09:53:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282797046962080085098088153480388477312","date":"2026-01-27T06:43:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78117230747291765197039483765416234723","date":"2026-01-27T03:34:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216986619365235052337055651291935346048","date":"2026-01-27T01:07:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313337390639865320685801860617225682484","date":"2026-01-27T01:03:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T00:41:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-22T12:48:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-22T12:46:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2026-01-20T22:28:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9c078f98-e533-4954-bd3a-14892564d6c1","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-02-06T11:24:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 20:24:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8653441","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8653441","identity":"rs-8653441","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}