Chicoric Acid from Health Foods Improves Motor Dysfunction in Zebrafish Parkinson’s Disease Model

Chicoric Acid from Health Foods Improves Motor Dysfunction in Zebrafish Parkinson’s Disease Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chicoric Acid from Health Foods Improves Motor Dysfunction in Zebrafish Parkinson’s Disease Model Chunyan Li, Mingyue Li, Ce Zhang, Huilin Zhang, Zhenhua Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2848281/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 Chicoric acid (CA) has been used as a nutritional supplement, health food, and medicine because of its antioxidant property. This study aimed to investigate whether CA can improve motor dysfunction in a zebrafish model of Parkinson’s disease (PD). AB-strain zebrafish larvae (24 hours post-fertilization) were incubated with 25 μM 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with or without CA for 5 days. The levels of malondialdehyde, glutathione, and reactive oxygen species were measured, as were the activities of antioxidant enzymes in brain tissue. Additionally, the expression of molecules from the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway were assayed by western blot. Compared with the control group, zebrafish in the MPTP group had impaired motor function. Treatment with CA improved motor dysfunction and attenuated dopaminergic neuronal injury in the PD zebrafish model. CA treatment also reduced the levels of malondialdehyde and reactive oxygen species and increased the antioxidant enzymes and glutathione level. Furthermore, CA augmented the expression of Nrf2, heme oxygenase, quinone oxidoreductase 1, and glutamate-cysteine ligase in the brain of the PD zebrafish model. CA from health foods also improved motor dysfunction and attenuated dopaminergic neuronal injury in the MPTP-induced PD zebrafish model. Our findings suggest that the mechanism of action of CA is related to its inhibition of oxidative stress via regulation of the Nrf2 pathway. Chicoric acid Motor function Zebrafish Oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Parkinson’s disease (PD) is a common degenerative disorder of the nervous system. The prevalence of PD increases with age; at 70 years of age or older, the prevalence may be as high as 550 per 100,000 people [1]. Most patients with PD present with signs of motor dysfunction such as bradykinesia, rigidity, resting tremor, and postural instability [2]. PD results from the loss of specific types of neurons that produce dopamine; the characteristic motor symptoms arise from the progressive loss of dopaminergic neurons in the brain. Generally, people lose at least 50% of dopaminergic neurons in the substantia nigra before noticing problems with their motor function [1]. The reason for the specific vulnerability of the dopaminergic neurons in the pathogenesis of PD is still unknown. Previous findings showed that MPTP could cause injury of dopaminergic cells, which made researches focusing on the environmental factors of PD. And recent results identified oxidative stress as an underlying mechanism [2]. Oxidative stress plays a key role in the pathophysiology of PD [3]. Enzymatic oxidation of dopamine produces free radical. External toxins also lead to oxidative stress. These factors are closely related to the causation and progression of PD [4]. Excessive oxygen free radicals react with lipids, DNA, and proteins, disrupting cellular metabolic homeostasis and causing oxidative stress. Cells have antioxidative stress systems, including non-enzymatic antioxidants such as glutathione (GSH) and vitamins, enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) [5]. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the antioxidant systematic reduction–oxidation balance by binding to antioxidant response elements and activating the expression of downstream antioxidant enzyme genes such as glutathione S-transferase (GST) and glutamate-cysteine ligase (GCL) and detoxification enzymes such as heme oxygenase (HO-1) and quinone oxidoreductase 1 (NQO1). Functional food are two major classes of food-related products that have health or medical benefits, such as the prevention or treatment of PD [6]. Previous studies have reported that dietary nutrients including carotenoids, vitamin E, vitamin C, anthocyanins, carvacrol, and curcumin have antioxidant properties; the daily consumption of foods with these nutrients might slow the progression of PD [7, 8]. Chicoric acid (CA) is a natural phenolic acid that is found in a variety of edible plants such as chicory ( Cichorium intybus ), purple coneflower ( Echinacea purpurea ), dandelion, and basil. A number of these plants have historically been consumed as food supplements or alternative medicines [9]. For example, Egyptians cultivated chicory as a vegetable crop, and in Europe, the roots of chicory and purple coneflower are baked, ground, and used as a coffee substitute. In addition, chicory has often been used to relieve the symptoms of digestive disorders, such as abdominal fullness, indigestion, and loss of appetite. Thus, CA has often been used as a functional food in Europe and Africa [10]. More recently, CA has a range of pharmacological effects including antioxidant, anti-inflammatory, and antiviral activities. For example, studies have reported that CA reduces liver oxidative degeneration in high-fat mice [11] and attenuates hyperglycemia-induced endothelial dysfunction through the inhibition of oxidative stress [12]. Moreover, CA reportedly protects zebrafish larvae from oxidative damage caused by Pb 2+ /H 2 O 2 [13, 14]. This experiment aimed to investigate whether CA can improve motor dysfunction in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced zebrafish PD model. Materials And Methods Regents and Chemicals Chicoric acid (purity ≥ 98%), MPTP, and 2’,7’-dichlorofluorescin diacetate (DCFH-DA) were from Sigma-Aldrich (St. Louis, MO, USA). Kits of glutathione peroxidase (GSH-Px, A005-1-2), glutathione (GSH, A006-2-1), superoxide dismutase (SOD, A001-3-2), catalase (CAT, A007-1-1), and malondialdehyde (MDA, A003-4-1) were purchased from Jiancheng Bioengineering Institute (Jiangsu, China). Primary antibodies of Nrf2, HO-1, NQO1, GAPDH, and secondary antibodies were obtained from Cell Signalling Technology (Boston, USA). Primary antibodies of glutamate cysteine ligase modifier subunit (GCLM) and glutamate cysteine ligase catalytic subunit (GCLC) were from Abcam (MA, USA). Primary antibody of tyrosine hydroxylase (TH) was from Merck (NJ, USA). BCA kit (P0010) was from Beyotime Institute of Biotechnology (Shanghai, China). ECL detection reagent was from Thermo Fisher Scientific company (Shanghai, China). Fish Maintenance Zebrafish is a freshwater fish. The ventral diencephalon of zebrafish is homologous to the substantia nigra of mammals. The components of the dopaminergic pathways in mammals are well represented in zebrafish. Recently, zebrafish models for PD have been widely used to study the pathogenesis and therapeutic agents of PD [15]. AB strain zebrafish (Danio rerio) were from the Northern Centre of the National Zebrafish Model Animals. Zebrafish was raised in a circulating system (Beijing Aisheng Biotechnology Co., Ltd., Beijing, China) which filtered and aerated the water to maintain an appropriate aquatic environment. Zebrafish was maintained under conditions as previous report [16]. In brief, the temperature of water was 26–28.5℃, pH = 7.5, 14 h light/10 h night. Fertilized embryos were collected after a natural mating. Larvae at 24 hours post-fertilization (hpf) were used in the following experiments. All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (No. 8023, revised in 1996). And this study was approved by the Animal Care and Use Committee of Yantai University (YTDX20210425). Drug Treatment Normal 24 hpf zebrafish embryos were assigned to plates containing E3 medium (3 mL). E3 medium is composed of KCl 0.127 g, NaCl 2.867 g, MgSO 4 ·7H 2 O 0.817 g, and CaCl 2 0.365 g. E3 medium was used as solvent for all the agents in this experiment. CA with the final concentration at 1, 5, or 25 nM was added into the dishes containing 24 hpf zebrafish embryos. Two hours later, MPTP at concentration of 25 µM was added to induce a PD model. The culture plates were maintained in an incubator with 28℃ and the culture medium was changed every day for 5 days. Locomotor Activity The 60 zebrafish in each group were used for locomotor activity assessment. The experiment was repeated for 6 times, and the mean value of zebrafish in each group was analysed. At 7 dpf, the larvae were transferred to a room (28 ± 2℃). After 10-min acclimation, locomotor activity of zebrafish was recorded using Noldus Ethovision XT system (Beijing Noldus Biotechnology Co., Ltd., Beijing, China) for 10 min. Then immobility time, mean speed, total distance, angular velocity, maximum acceleration, and absolute turn angle were analysed. Immunohistochemistry Zebrafish larvae were fixed with 4% (v/v) paraformaldehyde for 5 h. Next, they were rinsed and stored at -20℃. Immunohistochemistry was performed. Briefly, fixed-samples were cut into section with 5 µm and then incubated with primary anti-TH antibody (1:100) at 4℃ overnight. Washed with phosphate-buffered saline, the samples were incubated with secondary antibody (1:200). Then the sections of zebrafish brain were photographed. ROS Measurement After locomotor activity evaluation, the zebrafish larvae were incubated with 10 µM DCFH-DA for 30 min. Then, the zebrafish were washed with 0.01 M phosphate-buffered saline. The fluorescence of the larva was detected with a confocal microscope (Leica, Germany) and quantified using Image J software (NIH, USA). The ROS levels are calculated with the percentage of fluorescence intensity of the control group. Measurement of CAT, SOD, GSH, GSH-Px, and MDA After locomotor activity evaluation, the zebrafish were collected and 400 µL 0.01 M phosphate-buffered saline was added. After homogenization, the homogenate of zebrafish was centrifuged (4500 rpm, 10 min, 4°C). The CAT, SOD, GSH, GSH-Px, and MDA in the supernatant were measured. The content of protein in the supernatant was assayed using BCA kit. Western Blotting Total proteins of tissues of zebrafish were extracted. To evaluate the expression of protein with different molecular weights, the samples were run with individual gel. The samples were separated with SDS-PAGE and polyvinylidene difluoride membranes. Then, the membranes were cut horizontally according to markers. Blocked with 5% BSA, the membranes were incubated with primary antibody: rabbit anti-TH (1:1000), rabbit anti-Nrf2 (1:1000), rabbit anti-NQO1 (1:1000), rabbit anti-HO-1(1:1000), rabbit anti-GCLC (1:1000), rabbit anti-GCLM (1:1000), or rabbit anti-GAPDH (1:1000). After incubation with horseradish peroxidase-labelled secondary antibody (1:1500), the bands were detected using ECL detection reagents. Finally, the images were captured and the relative density of the proteins was analysed using Image J software (NIH, Bethesda, USA). Glycer-aldehyde 3-phosphate dehydrogenase served as the control. Statistical Analysis Data were expressed as means ± SD. One-way analysis of variance followed by LSD post hoc test were carried out using SPSS 20.0 software (IBM, USA) and GraphPad Prism 8.4.3 (GraphPad Software, USA). P < 0.05 was considered statistically significant. Results Effects of CA on the MPTP-Induced Motor Dysfunction in Zebrafish Motor dysfunction is closely related to dopaminergic nerve injury in PD. In order to evaluate the effects of CA on motor dysfunction in zebrafish, we investigated the locomotor activity of zebrafish. The representative swimming patterns of zebrafish in each group were shown in Fig. 1 a. Compared with the control group, the total distance, the mean speed, and the maximum acceleration in the MPTP group were decreased ( P < 0.01). Compared with the MPTP group, the total distance, the mean speed, and the maximum acceleration in CA groups were increased ( P < 0.05 or P < 0.01), (Fig. 1 b-d). Compared with the control group, the immobility time, the absolute turn angle, and the angle velocity of zebrafish in the MPTP group was increased ( P < 0.01). Compared with the MPTP group, the immobility time, the absolute turn angle, and the angle velocity in CA groups were decreased ( P < 0.05 or P < 0.01), (Fig. 1 e-g). Effect of CA on Dopaminergic Nerve Injury in the MPTP-Induced Zebrafish PD Model TH-positive dopaminergic nerve and TH protein level were detected. Compared with the control group, TH-positive dopaminergic nerve and TH protein level were decreased in the MPTP group ( P < 0.01). Compared with the MPTP group, CA treatment not only inhibited the loss of TH-positive dopaminergic nerve but also increased TH protein level in zebrafish PD model ( P < 0.05 or P < 0.01), (Fig. 2 a-c). Effects of CA on Levels of ROS and MDA in the MPTP-Induced Zebrafish PD Model Figure 3 a and b shows the effect of CA on ROS level in the MPTP-induced zebrafish PD model. The level of ROS of MPTP group was higher than that of the control group ( P < 0.01). However, CA treatment markedly decreased the level of ROS ( P < 0.05 or P < 0.01). MDA is a lipid peroxidation product. Compared with the control group, the level of MDA was increased after MPTP exposure ( P < 0.01). Compared with the MPTP group, CA treatment reduced the content of MDA ( P < 0.01), (Fig. 3 c). Effects of CA on CAT, SOD, GSH-Px, and GSH in the MPTP-Induced Zebrafish PD Model Compared with the control group, the activities of CAT, SOD, GSH-Px and the level of GSH in the MPTP group were reduced ( P < 0.01). Compared with the MPTP group, administration of CA augmented the activities of CAT, SOD, GSH-Px and the level of GSH in the MPTP-induced zebrafish PD model ( P < 0.05 or P < 0.01), (Fig. 4 a-d). Effects of CA on the Expression of Nrf2, NQO1, HO-1, GCLC, and GCLM in the MPTP-Induced Zebrafish PD Model Compared with the control group, the expression of Nrf2, NQO1, HO-1, GCLC, and GCLM were decreased markedly after MPTP exposure ( P < 0.05 or P < 0.01). Compared with the MPTP group, CA treatment significantly augmented the expression of Nrf2, NQO1, HO-1, GCLC, and GCLM ( P < 0.05 or P < 0.01), (Fig. 5 a-e). Discussion Recent studies have indicated that many nutraceuticals, such as quercetin, flavonoids, ascorbic acid, β-carotene, and phenolic acid, have neuroprotective effects and may alleviate the symptoms of PD [17, 18]. This study investigated the effects of CA on the MPTP-induced zebrafish PD model. The administration of CA improved MPTP-induced motor dysfunction and attenuated dopaminergic neuronal injury in zebrafish. The mechanism of action of CA was related to oxidative stress inhibition via regulation of the Nrf2 pathway. Zebrafish are increasingly important for biological research. They have a similar genetic structure and the same major organs and tissues as humans. These characteristics make zebrafish a valuable model for studying human disease [19]. The zebrafish brain is comparable to the mammalian brain and displays physiological similarities to the human brain. Zebrafish are therefore often used to study neurodegenerative diseases such as PD [20]. The MPTP-induced zebrafish PD model exhibits motor dysfunction, such as reduced swimming speed and abnormal swimming behavior, that is similar to the motor symptoms of PD patients [21]. In the current study, mean speed, maximum acceleration, the total distance, and swimming time were reduced in the zebrafish PD model. Furthermore, MPTP exposure caused changes in swimming patterns. Swimming pattern abnormalities induced by MPTP are characterized by frequent direction changes, such as increased absolute turn angles and angle velocities [22]. This study demonstrated that treatment with CA ameliorated motor deficits including the increased swimming time, distance, speed, and acceleration. CA also attenuated swimming pattern abnormalities by reducing the absolute turn angle and angular velocity. Furthermore, the zebrafish PD model had decreased immunopositivity of dopaminergic neurons and TH expression; CA treatment mitigated this dopaminergic neuronal injury in the zebrafish PD model. Together, these results indicate that CA improves motor function in the zebrafish PD model by protecting the dopaminergic nervous system. ROS are produced by exogenous chemicals or endogenous metabolic processes. The overproduction of ROS may oxidize DNA, lipids, and proteins in brain tissue, thus leading to dopaminergic neuronal injury [23]. Under physiological conditions, ROS are scavenged by the antioxidant system in the body. Antioxidant proteins such as SOD and CAT can eliminate superoxide radicals and hydrogen peroxide [24]. Moreover, GSH-Px catalyzes the reduction of hydroperoxides to water via the oxidation of reduced GSH into its bisulfide form. GSH-Px is also involved in the termination reaction of the ROS pathway. Increasing endogenous GSH levels is a promising strategy for attenuating oxidative stress-induced pathologies [25]. In the present study, MPTP exposure resulted in excessive ROS production, which led to redox imbalance and oxidative stress. CA increased not only the activities of SOD, CAT, and GSH-Px, but also the levels of GSH. Furthermore, CA inhibited ROS production and reduced MDA content. These findings suggest that the antioxidant properties of CA play a key role in its neuroprotective effects in the zebrafish PD model. The Nrf2-mediated activation of antioxidant signaling pathway is a cardinal mechanism for maintaining cellular redox balance [26]. The expression of antioxidant genes is regulated by Nrf2. In the cytoplasm, Nrf2 is bound to the inhibitory protein Keap1. Oxidative stress causes Nrf2 to move into the nucleus and regulates the expression of antioxidant molecules [27]. Furthermore, the activation of Nrf2 upregulates antioxidant genes such as HO-1, NQO1, GCL, SOD, and CAT [5]. HO-1 is an antioxidant enzyme. HO-1 has a property of clearing ROS and repairing DNA [28]. NQO1 is a ubiquitous enzyme. It is involved in phase II detoxification and inhibits the redox reaction of quinone and ROS formation [29]. GCL is comprised of a catalytic subunit and a modulatory subunit, and plays a key role in GSH biosynthesis in the cytoplasm [30]. The findings of this study demonstrate that CA increases Nrf2 levels and thus upregulates the expression of NQO1, HO-1, and subunits of GCL. It is therefore reasonable to speculate that the antioxidant effects of CA are associated with Nrf2 activation. Conclusions In the present study, CA from health foods improved MPTP-induced motor dysfunction and attenuated dopaminergic neuronal injury in a zebrafish PD model. Our findings indicate that the mechanism of action of CA is related to its inhibition of oxidative stress via regulation of the Nrf2 pathway. Declarations Acknowledgments We thank Bronwen Gardner, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn/), for editing the English text of a draft of this manuscript. Author Contributions CL, ML. Methodology, Visualization, Investigation, Writing – original draft. HZ. Data curation, Formal analysis. TW. Supervision, Investigation. CZ. Visualization, Investigation. ZW. Formal analysis. GZ. Conceptualization, Validation, Writing – review & editing. BH. Conceptualization, Data curation, Supervision, Validation, Writing – review & editing. All authors read and approved the final manuscript. Funding This work was supported by the Natural Science Foundation of Shandong Province (Grant numbers ZR2020MH377). Data Availability Data will be made available on request. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Ethical Approval All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (No. 8023, revised in 1996). And this study was approved by the Animal Care and Use Committee of Yantai University (YTDX20210425). References Rabiei Z, Solati K, Amini-Khoei H (2019) Phytotherapy in treatment of Parkinson’s disease: a review. Pharm Biol 57:355-362. https://doi.org/10.1080/13880209.2019.1618344. Doyle JM, Croll RP (2022) A Critical Review of Zebrafish Models of Parkinson's Disease. Front Pharmacol 13:835827. https://doi.org/10.3389/fphar.2022.835827. Aborode AT, Pustake M, Awuah WA, Alwerdani M, Shah P, Yarlagadda R, Ahmad S, Silva Correia IF, Chandra A, Nansubuga EP, Abdul-Rahman T, Mehta A, Ali O, Amaka SO, Zuniga YMH, Shkodina AD, Inya OC, Shen B, Alexiou A (2022) Targeting Oxidative Stress Mechanisms to Treat Alzheimer's and Parkinson's Disease: A Critical Review. Oxid Med Cell Longev 2022:7934442. https://doi.org/10.1155/2022/7934442. Cacabelos R (2017) Parkinson's Disease: From Pathogenesis to Pharmacogenomics. Int J Mol Sci 18(3):551. https://doi.org/ARTN55110.3390/ijms18030551. Wang Y, Gao L, Chen J, Li Q, Huo L, Wang Y, Wang H, Du J (2021) Pharmacological Modulation of Nrf2/HO-1 Signaling Pathway as a Therapeutic Target of Parkinson’s Disease. Front Pharmacol 12:757161. https://doi.org/10.3389/fphar.2021.757161. Ciulla M, Marinelli L, Cacciatore I, Stefano AD (2019) Role of Dietary Supplements in the Management of Parkinson's Disease. Biomolecules 9(7):271. https://doi.org/10.3390/biom9070271. Talebi S, Ghoreishy SM, Jayedi A, Travica N, Mohammadi H (2022) Dietary Antioxidants and Risk of Parkinson's Disease: A Systematic Review and Dose-Response Meta-analysis of Observational Studies. Adv Nutr 13:1493-1504. https://doi.org/10.1093/advances/nmac001. Agarwal P, Wang Y, Buchman AS, Holland TM, Bennett DA, Morris MC (2022) Dietary antioxidants associated with slower progression of parkinsonian signs in older adults. Nutr Neurosci 25:550-557. https://doi.org/10.1080/1028415X.2020.1769411. Street RA, Sidana J, Prinsloo G (2013) Cichorium intybus: Traditional Uses, Phytochemistry, Pharmacology, and Toxicology. Evid Based Complement Alternat Med 2013:579319. https://doi.org/10.1155/2013/579319. Peng Y, Sun Q, Park Y (2019) The Bioactive Effects of Chicoric Acid As a Functional Food Ingredient. J Med Food 22:645-652. https://doi.org/10.1089/jmf.2018.0211. Ding X, Jian T, Li J, Lv H, Tong B, Li J, Meng X, Ren B, Chen J (2020) Chicoric Acid Ameliorates Nonalcoholic Fatty Liver Disease via the AMPK/Nrf2/NFκB Signaling Pathway and Restores Gut Microbiota in High-Fat-Diet-Fed Mice. Oxid Med Cell Longev 2020:9734560. https://doi.org/10.1155/2020/9734560. Ma X, Zhang J, Wu Z, Wang X (2021) Chicoric acid attenuates hyperglycemia-induced endothelial dysfunction through AMPK-dependent inhibition of oxidative/nitrative stresses. J Recept Signal Transduct Res 41(4):378-392. https://doi.org/10.1080/10799893.2020.1817076. Mu Y, Yu J, Ji W, Chen L, Wang X, Yan B (2019) Alleviation of Pb2+ pollution-induced oxidative stress and toxicity in microglial cells and zebrafish larvae by chicoric acid. Ecotoxicol Environ Saf 180:396-402. https://doi.org/10.1016/j.ecoenv.2019.05.040. Ma J, Li M, Kalavagunta PK, Li J, He Q, Zhang Y, Ahmad O, Yin H, Wang T, Shang J (2018) Protective effects of cichoric acid on H2O2-induced oxidative injury in hepatocytes and larval zebrafish models. Biomed Pharmacother 104:679-685.. https://doi.org/10.1016/j.biopha.2018.05.081. Najib NHM, Nies YH, Abd Halim SAS, Yahaya MF, Das S, Lim WL, Teoh SL (2020) Modeling Parkinson's Disease in Zebrafish. CNS Neurol Disord Drug Targets 19(5):386-399. https://doi.org/10.2174/1871527319666200708124117. Avdesh A, Chen M, Martin-Iverson MT, Mondal A, Ong D, Rainey-Smith S, Taddei K, Lardelli M, Groth DM, Verdile G, Martins RN (2012) Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction. J Vis Exp (69):e4196. https://doi.org/10.3791/4196. Mundkar M, Bijalwan A, Soni D, Kumar P (2022) Neuroprotective potential of Moringa oleifera mediated by NF-kB/Nrf2/HO-1 signaling pathway: A review. J Food Biochem e14451. https://doi.org/10.1111/jfbc.14451. Vishwas S, Kumar R, Khursheed R, Ramanunny AK, Kumar R, Awasthi A, Corrie L, Porwal O, Arshad MF, Alshammari MK, Alghitran AA, Qumayri AN, Alkhaldi SM, Alshammari AK, Chellappan DK, Gupta G, Collet T, Adams J, Dua K, Gulati M, Singh SK (2022) Expanding Arsenal against Neurodegenerative Diseases using Quercetin based Nanoformulations: Breakthroughs and Bottlenecks. Curr Neuropharmacol Online ahead of print. https://doi.org/10.2174/1570159x20666220810105421. de Abreu MS, Genario R, Giacomini ACVV, Demin KA, Lakstygal AM, Amstislavskaya TG, Fontana BD, Parker MO, Kalueff AV (2020) Zebrafish as a Model of Neurodevelopmental Disorders. Neuroscience 445:3-11. https://doi.org/https://doi.org/10.1016/j.neuroscience.2019.08.034. Bretaud S, Lee S, Guo S (2004) Sensitivity of zebrafish to environmental toxins implicated in Parkinson's disease. Neurotoxicol Teratol 26(6):857-64. https://doi.org/10.1016/j.ntt.2004.06.014. Razali K, Othman N, Mohd Nasir MH, Doolaanea AA, Kumar J, Ibrahim WN, Mohamed Ibrahim N, Mohamed WMY (2021) The Promise of the Zebrafish Model for Parkinson’s Disease: Today’s Science and Tomorrow’s Treatment. Front Genet 12:655550. 12https://doi.org/10.3389/fgene.2021.655550. Zhang W, Fan R, Luo S, Ying l, Jin Y, Li Y, Xiong M, Yuan X, Jia L, Chen Y (2022) Antagonistic effects and mechanisms of carbendazim and chlorpyrifos on the neurobehavior of larval zebrafish. Chemosphere 293:133522. https://doi.org/10.1016/j.chemosphere.2022.133522. Manzanilla B, Robles J (2022) Antiradical properties of curcumin, caffeic acid phenethyl ester, and chicoric acid: a DFT study. J Mol Model 28(3):68. https://doi.org/10.1007/s00894-022-05056-4. Wang M, Zhao J, Wu L, Ma T (2021) Effects of 4-epianhydrotetracycline on oxidative stress in zebrafish (Danio rerio) embryos. Sci Total Environ 796:149047. https://doi.org/10.1016/j.scitotenv.2021.149047. Kagan DB, Liu H, Hutnik CM (2012) Efficacy of various antioxidants in the protection of the retinal pigment epithelium from oxidative stress. Clin Ophthalmol 6:1471-1476. https://doi.org/10.2147/opth.S35139. You L, Peng H, Liu J, Cai M, Wu H, Zhang Z, Bai J, Yao Y, Dong X, Yin X, Ni J (2021) Catalpol Protects ARPE-19 Cells against Oxidative Stress via Activation of the Keap1/Nrf2/ARE Pathway. Cells 10(10):2635. https://doi.org/10.3390/cells10102635. Magesh S, Chen Y, Hu L (2012) Small Molecule Modulators of Keap1-Nrf2-ARE Pathway as Potential Preventive and Therapeutic Agents. Med Res Rev 32(4):687-726. https://doi.org/10.1002/med.21257. Maines MD, Panahian N (2001) The heme oxygenase system and cellular defense mechanisms. Do HO-1 and HO-2 have different functions? Exp Med Biol 502:249-272. https://doi.org/10.1007/978-1-4757-3401-0_17. Liu Z, Zhang F, Zhao L, Zhang X, Li Y, Liu L (2020) Protective Effect of Pravastatin on Myocardial Ischemia Reperfusion Injury by Regulation of the miR-93/Nrf2/ARE Signal Pathway. Drug Des Devel Ther 14:3853-3864. https://doi.org/10.2147/dddt.S251726. Jung KA, Kwak MK (2010) The Nrf2 system as a potential target for the development of indirect antioxidants. Molecules 15(10):7266-7291. https://doi.org/10.3390/molecules15107266. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2848281","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":194740285,"identity":"affe59bf-4134-44a9-b0eb-309851efe64e","order_by":0,"name":"Chunyan Li","email":"","orcid":"","institution":"Center for Mitochondria and Healthy Aging, College of Life Science, Yantai University, Yantai 264005, PR China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Li","suffix":""},{"id":194740286,"identity":"5e82f1f9-f766-45de-8b60-8f1daef79184","order_by":1,"name":"Mingyue Li","email":"","orcid":"","institution":"R \u0026 D Department, Yinfeng Biological Group Co., Ltd., Jinan, Shandong 250102, RP China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingyue","middleName":"","lastName":"Li","suffix":""},{"id":194740287,"identity":"076c53cf-f1be-4e62-a8a2-65978f5f3366","order_by":2,"name":"Ce Zhang","email":"","orcid":"","institution":"School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universit","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ce","middleName":"","lastName":"Zhang","suffix":""},{"id":194740288,"identity":"931e29cd-124e-4af2-b87b-4ebfe1092b83","order_by":3,"name":"Huilin Zhang","email":"","orcid":"","institution":"Center for Mitochondria and Healthy Aging, College of Life Science, Yantai University, Yantai 264005, PR China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huilin","middleName":"","lastName":"Zhang","suffix":""},{"id":194740289,"identity":"a83daeb3-ab7c-49bf-868c-5005a2ed26d9","order_by":4,"name":"Zhenhua Wang","email":"","orcid":"","institution":"Center for Mitochondria and Healthy Aging, College of Life Science, Yantai University, Yantai 264005, PR China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenhua","middleName":"","lastName":"Wang","suffix":""},{"id":194740290,"identity":"1bb0f362-7e75-4cd2-a698-ebe2004160bf","order_by":5,"name":"Tian Wang","email":"","orcid":"","institution":"School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universit","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Wang","suffix":""},{"id":194740291,"identity":"8c55a2e6-0bac-4d3b-884b-f7a25e800a66","order_by":6,"name":"Guirong Zhang","email":"","orcid":"","institution":"R \u0026 D Department, Yinfeng Biological Group Co., Ltd., Jinan, Shandong 250102, RP China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guirong","middleName":"","lastName":"Zhang","suffix":""},{"id":194740292,"identity":"17c1320d-c279-4514-bb0f-f62487e1b6cf","order_by":7,"name":"Bing Han","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYDACCSjNB8TMP37UyLGxtx8gTgsbSAtjzzFjPp4zCSRoYWBjTpwn4WCAV4e5dPOzTzdq7EDuuSZdwMOW3ibBkMDwo2IbTi2Wc44Zz845lmzMxnOmTHqGhUxum3TjAcaeM7dxajG4kWDMnMN2ILFNIidNgoeHLbdN5kACM2MbPi3pn5lz/sG0sDGns0kkGBDQkmPMnNsG0pJ+TBqoJYGgFssZOcXMuX1gvzBbzuw5ZtgGDOSD+PxiLpG+mTnnm50cP3v7wxsfftTIy7e3H3zwowKPwxBMHhNYHDEcwKkeVQv74w/4VI6CUTAKRsHIBQAM61LcP1kWaAAAAABJRU5ErkJggg==","orcid":"","institution":"Center for Mitochondria and Healthy Aging, College of Life Science, Yantai University, Yantai 264005, PR China","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Han","suffix":""}],"badges":[],"createdAt":"2023-04-22 11:44:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2848281/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2848281/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36346522,"identity":"d5a7605e-abac-4916-b044-fca465960b04","added_by":"auto","created_at":"2023-04-26 20:19:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82037,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CA on the MPTP-induced motor dysfunction in zebrafish. (a) The representative swimming patterns of zebrafish in each group. (b) The total distance. (c) The mean speed. (d) The maximum acceleration. (e) The immobility time. (f) The absolute turn angle. (g) The angular velocity. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the MPTP group. The 60 zebrafish were used in each group for locomotor activity assessment. The experiment was repeated for 6 times (n=6).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2848281/v1/05b73dc2a717b309caf13f3b.jpg"},{"id":36346523,"identity":"0d2b4660-17b5-4957-8712-a42196538d93","added_by":"auto","created_at":"2023-04-26 20:19:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":119388,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of CA on dopaminergic nerve injury in the MPTP-induced zebrafish PD model. (a) Representative photographs of TH-positive dopaminergic nerve in zebrafish brain. (b) Representative photographs of TH protein in Western blot. (c) Bar graphs of quantitative analysis of TH expression in zebrafish. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the MPTP group. The 60 zebrafish were used in each group for testing. The mean value of zebrafish in each group was statistically analysed. The experiment was repeated for 3 times (n=3).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2848281/v1/c09b65a8910473c71dba49f6.jpg"},{"id":36347011,"identity":"0a795233-c90f-4ce1-b716-d590e518a684","added_by":"auto","created_at":"2023-04-26 20:27:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":45660,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CA on levels of ROS and MDA in the MPTP-induced zebrafish PD model. (a) Representative photograph of ROS level in zebrafish. (b) Bar graphs of quantitative analysis of ROS level in zebrafish. (c) MDA level. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the MPTP group. The 60 zebrafish were used in each group for testing. The experiment was repeated for 6 times (n=6).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2848281/v1/936360f73004ea0be0b22bac.jpg"},{"id":36346525,"identity":"0a73ae69-a7c0-41a6-8183-e525cc655346","added_by":"auto","created_at":"2023-04-26 20:19:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51528,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CA on CAT, SOD, GSH-Px and GSH in the MPTP-induced zebrafish PD model. (a) CAT. (b) SOD. (c) GSH-Px. (d) GSH. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the MPTP group. The 60 zebrafish were used in each group for testing. The experiment was repeated for 6 times (n=6).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2848281/v1/06bee238e808405a58b58aa7.jpg"},{"id":36347012,"identity":"24840ce8-4b96-49b2-bf4e-a2a37d0605a7","added_by":"auto","created_at":"2023-04-26 20:27:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39759,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CA on the expression of Nrf2, NQO1, HO-1, GCLC, and GCLM in the MPTP-induced zebrafish PD model. (a), (b), (c), (d), (e) Bar graphs of quantitative analysis of Nrf2, NQO1, HO-1, GCLC, and GCLM expression in zebrafish. Typical representative pictures of Nrf2, NQO1, HO-1, GCLC, and GCLM in Western blot were shown on the above of each histogram. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus the MPTP group. The 60 zebrafish were used in each group for testing. The experiment was repeated for 3 times (n=3).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2848281/v1/7a2e2543ebb3ac92b0ac0c7a.jpg"},{"id":47266384,"identity":"c4842cfe-46f6-4b2b-9f85-4af3b9d38bba","added_by":"auto","created_at":"2023-11-29 09:16:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":606661,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2848281/v1/ba3c4794-a6f5-4875-8dec-49f3cd0d8ebd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chicoric Acid from Health Foods Improves Motor Dysfunction in Zebrafish Parkinson’s Disease Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a common degenerative disorder of the nervous system. The prevalence of PD increases with age; at 70 years of age or older, the prevalence may be as high as 550 per 100,000 people [1]. Most patients with PD present with signs of motor dysfunction such as bradykinesia, rigidity, resting tremor, and postural instability [2]. PD results from the loss of specific types of neurons that produce dopamine; the characteristic motor symptoms arise from the progressive loss of dopaminergic neurons in the brain. Generally, people lose at least 50% of dopaminergic neurons in the substantia nigra before noticing problems with their motor function [1].\u003c/p\u003e \u003cp\u003eThe reason for the specific vulnerability of the dopaminergic neurons in the pathogenesis of PD is still unknown. Previous findings showed that MPTP could cause injury of dopaminergic cells, which made researches focusing on the environmental factors of PD. And recent results identified oxidative stress as an underlying mechanism [2]. Oxidative stress plays a key role in the pathophysiology of PD [3]. Enzymatic oxidation of dopamine produces free radical. External toxins also lead to oxidative stress. These factors are closely related to the causation and progression of PD [4]. Excessive oxygen free radicals react with lipids, DNA, and proteins, disrupting cellular metabolic homeostasis and causing oxidative stress. Cells have antioxidative stress systems, including non-enzymatic antioxidants such as glutathione (GSH) and vitamins, enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) [5]. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the antioxidant systematic reduction\u0026ndash;oxidation balance by binding to antioxidant response elements and activating the expression of downstream antioxidant enzyme genes such as glutathione S-transferase (GST) and glutamate-cysteine ligase (GCL) and detoxification enzymes such as heme oxygenase (HO-1) and quinone oxidoreductase 1 (NQO1).\u003c/p\u003e \u003cp\u003eFunctional food are two major classes of food-related products that have health or medical benefits, such as the prevention or treatment of PD [6]. Previous studies have reported that dietary nutrients including carotenoids, vitamin E, vitamin C, anthocyanins, carvacrol, and curcumin have antioxidant properties; the daily consumption of foods with these nutrients might slow the progression of PD [7, 8]. Chicoric acid (CA) is a natural phenolic acid that is found in a variety of edible plants such as chicory (\u003cem\u003eCichorium intybus\u003c/em\u003e), purple coneflower (\u003cem\u003eEchinacea purpurea\u003c/em\u003e), dandelion, and basil. A number of these plants have historically been consumed as food supplements or alternative medicines [9]. For example, Egyptians cultivated chicory as a vegetable crop, and in Europe, the roots of chicory and purple coneflower are baked, ground, and used as a coffee substitute. In addition, chicory has often been used to relieve the symptoms of digestive disorders, such as abdominal fullness, indigestion, and loss of appetite. Thus, CA has often been used as a functional food in Europe and Africa [10]. More recently, CA has a range of pharmacological effects including antioxidant, anti-inflammatory, and antiviral activities. For example, studies have reported that CA reduces liver oxidative degeneration in high-fat mice [11] and attenuates hyperglycemia-induced endothelial dysfunction through the inhibition of oxidative stress [12]. Moreover, CA reportedly protects zebrafish larvae from oxidative damage caused by Pb\u003csup\u003e2+\u003c/sup\u003e/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [13, 14]. This experiment aimed to investigate whether CA can improve motor dysfunction in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced zebrafish PD model.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRegents and Chemicals\u003c/h2\u003e \u003cp\u003eChicoric acid (purity\u0026thinsp;\u0026ge;\u0026thinsp;98%), MPTP, and 2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescin diacetate (DCFH-DA) were from Sigma-Aldrich (St. Louis, MO, USA). Kits of glutathione peroxidase (GSH-Px, A005-1-2), glutathione (GSH, A006-2-1), superoxide dismutase (SOD, A001-3-2), catalase (CAT, A007-1-1), and malondialdehyde (MDA, A003-4-1) were purchased from Jiancheng Bioengineering Institute (Jiangsu, China). Primary antibodies of Nrf2, HO-1, NQO1, GAPDH, and secondary antibodies were obtained from Cell Signalling Technology (Boston, USA). Primary antibodies of glutamate cysteine ligase modifier subunit (GCLM) and glutamate cysteine ligase catalytic subunit (GCLC) were from Abcam (MA, USA). Primary antibody of tyrosine hydroxylase (TH) was from Merck (NJ, USA). BCA kit (P0010) was from Beyotime Institute of Biotechnology (Shanghai, China). ECL detection reagent was from Thermo Fisher Scientific company (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFish Maintenance\u003c/h2\u003e \u003cp\u003eZebrafish is a freshwater fish. The ventral diencephalon of zebrafish is homologous to the substantia nigra of mammals. The components of the dopaminergic pathways in mammals are well represented in zebrafish. Recently, zebrafish models for PD have been widely used to study the pathogenesis and therapeutic agents of PD [15]. AB strain zebrafish (Danio rerio) were from the Northern Centre of the National Zebrafish Model Animals. Zebrafish was raised in a circulating system (Beijing Aisheng Biotechnology Co., Ltd., Beijing, China) which filtered and aerated the water to maintain an appropriate aquatic environment. Zebrafish was maintained under conditions as previous report [16]. In brief, the temperature of water was 26\u0026ndash;28.5℃, pH\u0026thinsp;=\u0026thinsp;7.5, 14 h light/10 h night. Fertilized embryos were collected after a natural mating. Larvae at 24 hours post-fertilization (hpf) were used in the following experiments. All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (No. 8023, revised in 1996). And this study was approved by the Animal Care and Use Committee of Yantai University (YTDX20210425).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDrug Treatment\u003c/h2\u003e \u003cp\u003eNormal 24 hpf zebrafish embryos were assigned to plates containing E3 medium (3 mL). E3 medium is composed of KCl 0.127 g, NaCl 2.867 g, MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO 0.817 g, and CaCl\u003csub\u003e2\u003c/sub\u003e 0.365 g. E3 medium was used as solvent for all the agents in this experiment. CA with the final concentration at 1, 5, or 25 nM was added into the dishes containing 24 hpf zebrafish embryos. Two hours later, MPTP at concentration of 25 \u0026micro;M was added to induce a PD model. The culture plates were maintained in an incubator with 28℃ and the culture medium was changed every day for 5 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLocomotor Activity\u003c/h2\u003e \u003cp\u003eThe 60 zebrafish in each group were used for locomotor activity assessment. The experiment was repeated for 6 times, and the mean value of zebrafish in each group was analysed. At 7 dpf, the larvae were transferred to a room (28\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃). After 10-min acclimation, locomotor activity of zebrafish was recorded using Noldus Ethovision XT system (Beijing Noldus Biotechnology Co., Ltd., Beijing, China) for 10 min. Then immobility time, mean speed, total distance, angular velocity, maximum acceleration, and absolute turn angle were analysed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eZebrafish larvae were fixed with 4% (v/v) paraformaldehyde for 5 h. Next, they were rinsed and stored at -20℃. Immunohistochemistry was performed. Briefly, fixed-samples were cut into section with 5 \u0026micro;m and then incubated with primary anti-TH antibody (1:100) at 4℃ overnight. Washed with phosphate-buffered saline, the samples were incubated with secondary antibody (1:200). Then the sections of zebrafish brain were photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eROS Measurement\u003c/h2\u003e \u003cp\u003eAfter locomotor activity evaluation, the zebrafish larvae were incubated with 10 \u0026micro;M DCFH-DA for 30 min. Then, the zebrafish were washed with 0.01 M phosphate-buffered saline. The fluorescence of the larva was detected with a confocal microscope (Leica, Germany) and quantified using Image J software (NIH, USA). The ROS levels are calculated with the percentage of fluorescence intensity of the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of CAT, SOD, GSH, GSH-Px, and MDA\u003c/h2\u003e \u003cp\u003eAfter locomotor activity evaluation, the zebrafish were collected and 400 \u0026micro;L 0.01 M phosphate-buffered saline was added. After homogenization, the homogenate of zebrafish was centrifuged (4500 rpm, 10 min, 4\u0026deg;C). The CAT, SOD, GSH, GSH-Px, and MDA in the supernatant were measured. The content of protein in the supernatant was assayed using BCA kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blotting\u003c/h2\u003e \u003cp\u003eTotal proteins of tissues of zebrafish were extracted. To evaluate the expression of protein with different molecular weights, the samples were run with individual gel. The samples were separated with SDS-PAGE and polyvinylidene difluoride membranes. Then, the membranes were cut horizontally according to markers. Blocked with 5% BSA, the membranes were incubated with primary antibody: rabbit anti-TH (1:1000), rabbit anti-Nrf2 (1:1000), rabbit anti-NQO1 (1:1000), rabbit anti-HO-1(1:1000), rabbit anti-GCLC (1:1000), rabbit anti-GCLM (1:1000), or rabbit anti-GAPDH (1:1000). After incubation with horseradish peroxidase-labelled secondary antibody (1:1500), the bands were detected using ECL detection reagents. Finally, the images were captured and the relative density of the proteins was analysed using Image J software (NIH, Bethesda, USA). Glycer-aldehyde 3-phosphate dehydrogenase served as the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. One-way analysis of variance followed by LSD post hoc test were carried out using SPSS 20.0 software (IBM, USA) and GraphPad Prism 8.4.3 (GraphPad Software, USA). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of CA on the MPTP-Induced Motor Dysfunction in Zebrafish\u003c/h2\u003e \u003cp\u003eMotor dysfunction is closely related to dopaminergic nerve injury in PD. In order to evaluate the effects of CA on motor dysfunction in zebrafish, we investigated the locomotor activity of zebrafish. The representative swimming patterns of zebrafish in each group were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003eCompared with the control group, the total distance, the mean speed, and the maximum acceleration in the MPTP group were decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the MPTP group, the total distance, the mean speed, and the maximum acceleration in CA groups were increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003eCompared with the control group, the immobility time, the absolute turn angle, and the angle velocity of zebrafish in the MPTP group was increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the MPTP group, the immobility time, the absolute turn angle, and the angle velocity in CA groups were decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of CA on Dopaminergic Nerve Injury in the MPTP-Induced Zebrafish PD Model\u003c/h2\u003e \u003cp\u003eTH-positive dopaminergic nerve and TH protein level were detected. Compared with the control group, TH-positive dopaminergic nerve and TH protein level were decreased in the MPTP group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the MPTP group, CA treatment not only inhibited the loss of TH-positive dopaminergic nerve but also increased TH protein level in zebrafish PD model (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of CA on Levels of ROS and MDA in the MPTP-Induced Zebrafish PD Model\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and b shows the effect of CA on ROS level in the MPTP-induced zebrafish PD model. The level of ROS of MPTP group was higher than that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, CA treatment markedly decreased the level of ROS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). MDA is a lipid peroxidation product. Compared with the control group, the level of MDA was increased after MPTP exposure (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the MPTP group, CA treatment reduced the content of MDA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffects of CA on CAT, SOD, GSH-Px, and GSH in the MPTP-Induced Zebrafish PD Model\u003c/h2\u003e \u003cp\u003eCompared with the control group, the activities of CAT, SOD, GSH-Px and the level of GSH in the MPTP group were reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the MPTP group, administration of CA augmented the activities of CAT, SOD, GSH-Px and the level of GSH in the MPTP-induced zebrafish PD model (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of CA on the Expression of Nrf2, NQO1, HO-1, GCLC, and GCLM in the MPTP-Induced Zebrafish PD Model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared with the control group, the expression of Nrf2, NQO1, HO-1, GCLC, and GCLM were decreased markedly after MPTP exposure (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the MPTP group, CA treatment significantly augmented the expression of Nrf2, NQO1, HO-1, GCLC, and GCLM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecent studies have indicated that many nutraceuticals, such as quercetin, flavonoids, ascorbic acid, β-carotene, and phenolic acid, have neuroprotective effects and may alleviate the symptoms of PD [17, 18]. This study investigated the effects of CA on the MPTP-induced zebrafish PD model. The administration of CA improved MPTP-induced motor dysfunction and attenuated dopaminergic neuronal injury in zebrafish. The mechanism of action of CA was related to oxidative stress inhibition via regulation of the Nrf2 pathway.\u003c/p\u003e \u003cp\u003eZebrafish are increasingly important for biological research. They have a similar genetic structure and the same major organs and tissues as humans. These characteristics make zebrafish a valuable model for studying human disease [19]. The zebrafish brain is comparable to the mammalian brain and displays physiological similarities to the human brain. Zebrafish are therefore often used to study neurodegenerative diseases such as PD [20]. The MPTP-induced zebrafish PD model exhibits motor dysfunction, such as reduced swimming speed and abnormal swimming behavior, that is similar to the motor symptoms of PD patients [21]. In the current study, mean speed, maximum acceleration, the total distance, and swimming time were reduced in the zebrafish PD model. Furthermore, MPTP exposure caused changes in swimming patterns. Swimming pattern abnormalities induced by MPTP are characterized by frequent direction changes, such as increased absolute turn angles and angle velocities [22]. This study demonstrated that treatment with CA ameliorated motor deficits including the increased swimming time, distance, speed, and acceleration. CA also attenuated swimming pattern abnormalities by reducing the absolute turn angle and angular velocity. Furthermore, the zebrafish PD model had decreased immunopositivity of dopaminergic neurons and TH expression; CA treatment mitigated this dopaminergic neuronal injury in the zebrafish PD model. Together, these results indicate that CA improves motor function in the zebrafish PD model by protecting the dopaminergic nervous system.\u003c/p\u003e \u003cp\u003eROS are produced by exogenous chemicals or endogenous metabolic processes. The overproduction of ROS may oxidize DNA, lipids, and proteins in brain tissue, thus leading to dopaminergic neuronal injury [23]. Under physiological conditions, ROS are scavenged by the antioxidant system in the body. Antioxidant proteins such as SOD and CAT can eliminate superoxide radicals and hydrogen peroxide [24]. Moreover, GSH-Px catalyzes the reduction of hydroperoxides to water via the oxidation of reduced GSH into its bisulfide form. GSH-Px is also involved in the termination reaction of the ROS pathway. Increasing endogenous GSH levels is a promising strategy for attenuating oxidative stress-induced pathologies [25]. In the present study, MPTP exposure resulted in excessive ROS production, which led to redox imbalance and oxidative stress. CA increased not only the activities of SOD, CAT, and GSH-Px, but also the levels of GSH. Furthermore, CA inhibited ROS production and reduced MDA content. These findings suggest that the antioxidant properties of CA play a key role in its neuroprotective effects in the zebrafish PD model.\u003c/p\u003e \u003cp\u003eThe Nrf2-mediated activation of antioxidant signaling pathway is a cardinal mechanism for maintaining cellular redox balance [26]. The expression of antioxidant genes is regulated by Nrf2. In the cytoplasm, Nrf2 is bound to the inhibitory protein Keap1. Oxidative stress causes Nrf2 to move into the nucleus and regulates the expression of antioxidant molecules [27]. Furthermore, the activation of Nrf2 upregulates antioxidant genes such as HO-1, NQO1, GCL, SOD, and CAT [5]. HO-1 is an antioxidant enzyme. HO-1 has a property of clearing ROS and repairing DNA [28]. NQO1 is a ubiquitous enzyme. It is involved in phase II detoxification and inhibits the redox reaction of quinone and ROS formation [29]. GCL is comprised of a catalytic subunit and a modulatory subunit, and plays a key role in GSH biosynthesis in the cytoplasm [30]. The findings of this study demonstrate that CA increases Nrf2 levels and thus upregulates the expression of NQO1, HO-1, and subunits of GCL. It is therefore reasonable to speculate that the antioxidant effects of CA are associated with Nrf2 activation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, CA from health foods improved MPTP-induced motor dysfunction and attenuated dopaminergic neuronal injury in a zebrafish PD model. Our findings indicate that the mechanism of action of CA is related to its inhibition of oxidative stress via regulation of the Nrf2 pathway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e We thank Bronwen Gardner, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn/), for editing the English text of a draft of this manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions \u003c/strong\u003eCL, ML. Methodology, Visualization, Investigation, Writing \u0026ndash; original draft. HZ. Data curation, Formal analysis. TW. Supervision, Investigation. CZ. Visualization, Investigation. ZW. Formal analysis. GZ. Conceptualization, Validation, Writing \u0026ndash; review \u0026amp; editing. BH. Conceptualization, Data curation, Supervision, Validation, Writing \u0026ndash; review \u0026amp; editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003eThis work was supported by the Natural Science Foundation of Shandong Province (Grant numbers ZR2020MH377). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e Data will be made available on request.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interests \u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (No. 8023, revised in 1996). And this study was approved by the Animal Care and Use Committee of Yantai University (YTDX20210425).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRabiei Z, Solati K, Amini-Khoei H (2019) Phytotherapy in treatment of Parkinson\u0026rsquo;s disease: a review. Pharm Biol 57:355-362. https://doi.org/10.1080/13880209.2019.1618344.\u003c/li\u003e\n\u003cli\u003eDoyle JM, Croll RP (2022) A Critical Review of Zebrafish Models of Parkinson\u0026apos;s Disease. Front Pharmacol 13:835827. https://doi.org/10.3389/fphar.2022.835827.\u003c/li\u003e\n\u003cli\u003eAborode AT, Pustake M, Awuah WA, Alwerdani M, Shah P, Yarlagadda R, Ahmad S, Silva Correia IF, Chandra A, Nansubuga EP, Abdul-Rahman T, Mehta A, Ali O, Amaka SO, Zuniga YMH, Shkodina AD, Inya OC, Shen B, Alexiou A (2022) Targeting Oxidative Stress Mechanisms to Treat Alzheimer\u0026apos;s and Parkinson\u0026apos;s Disease: A Critical Review. Oxid Med Cell Longev 2022:7934442. https://doi.org/10.1155/2022/7934442.\u003c/li\u003e\n\u003cli\u003eCacabelos R (2017) Parkinson\u0026apos;s Disease: From Pathogenesis to Pharmacogenomics. Int J Mol Sci 18(3):551. https://doi.org/ARTN55110.3390/ijms18030551.\u003c/li\u003e\n\u003cli\u003eWang Y, Gao L, Chen J, Li Q, Huo L, Wang Y, Wang H, Du J (2021) Pharmacological Modulation of Nrf2/HO-1 Signaling Pathway as a Therapeutic Target of Parkinson\u0026rsquo;s Disease. Front Pharmacol 12:757161. https://doi.org/10.3389/fphar.2021.757161.\u003c/li\u003e\n\u003cli\u003eCiulla M, Marinelli L, Cacciatore I, Stefano AD (2019) Role of Dietary Supplements in the Management of Parkinson\u0026apos;s Disease. Biomolecules 9(7):271. https://doi.org/10.3390/biom9070271.\u003c/li\u003e\n\u003cli\u003eTalebi S, Ghoreishy SM, Jayedi A, Travica N, Mohammadi H (2022) Dietary Antioxidants and Risk of Parkinson\u0026apos;s Disease: A Systematic Review and Dose-Response Meta-analysis of Observational Studies. Adv Nutr 13:1493-1504. https://doi.org/10.1093/advances/nmac001.\u003c/li\u003e\n\u003cli\u003eAgarwal P, Wang Y, Buchman AS, Holland TM, Bennett DA, Morris MC (2022) Dietary antioxidants associated with slower progression of parkinsonian signs in older adults. Nutr Neurosci 25:550-557. https://doi.org/10.1080/1028415X.2020.1769411.\u003c/li\u003e\n\u003cli\u003eStreet RA, Sidana J, Prinsloo G (2013) Cichorium intybus: Traditional Uses, Phytochemistry, Pharmacology, and Toxicology. Evid Based Complement Alternat Med 2013:579319. https://doi.org/10.1155/2013/579319.\u003c/li\u003e\n\u003cli\u003ePeng Y, Sun Q, Park Y (2019) The Bioactive Effects of Chicoric Acid As a Functional Food Ingredient. J Med Food 22:645-652. https://doi.org/10.1089/jmf.2018.0211.\u003c/li\u003e\n\u003cli\u003eDing X, Jian T, Li J, Lv H, Tong B, Li J, Meng X, Ren B, Chen J (2020) Chicoric Acid Ameliorates Nonalcoholic Fatty Liver Disease via the AMPK/Nrf2/NF\u0026kappa;B Signaling Pathway and Restores Gut Microbiota in High-Fat-Diet-Fed Mice. Oxid Med Cell Longev 2020:9734560. https://doi.org/10.1155/2020/9734560.\u003c/li\u003e\n\u003cli\u003eMa X, Zhang J, Wu Z, Wang X (2021) Chicoric acid attenuates hyperglycemia-induced endothelial dysfunction through AMPK-dependent inhibition of oxidative/nitrative stresses. J Recept Signal Transduct Res 41(4):378-392. https://doi.org/10.1080/10799893.2020.1817076.\u003c/li\u003e\n\u003cli\u003eMu Y, Yu J, Ji W, Chen L, Wang X, Yan B (2019) Alleviation of Pb2+ pollution-induced oxidative stress and toxicity in microglial cells and zebrafish larvae by chicoric acid. Ecotoxicol Environ Saf 180:396-402. https://doi.org/10.1016/j.ecoenv.2019.05.040.\u003c/li\u003e\n\u003cli\u003eMa J, Li M, Kalavagunta PK, Li J, He Q, Zhang Y, Ahmad O, Yin H, Wang T, Shang J (2018) Protective effects of cichoric acid on H2O2-induced oxidative injury in hepatocytes and larval zebrafish models. Biomed Pharmacother 104:679-685.. https://doi.org/10.1016/j.biopha.2018.05.081.\u003c/li\u003e\n\u003cli\u003eNajib NHM, Nies YH, Abd Halim SAS, Yahaya MF, Das S, Lim WL, Teoh SL (2020) Modeling Parkinson\u0026apos;s Disease in Zebrafish. CNS Neurol Disord Drug Targets 19(5):386-399. https://doi.org/10.2174/1871527319666200708124117.\u003c/li\u003e\n\u003cli\u003eAvdesh A, Chen M, Martin-Iverson MT, Mondal A, Ong D, Rainey-Smith S, Taddei K, Lardelli M, Groth DM, Verdile G, Martins RN (2012) Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction. J Vis Exp (69):e4196. https://doi.org/10.3791/4196.\u003c/li\u003e\n\u003cli\u003eMundkar M, Bijalwan A, Soni D, Kumar P (2022) Neuroprotective potential of Moringa oleifera mediated by NF-kB/Nrf2/HO-1 signaling pathway: A review. J Food Biochem e14451. https://doi.org/10.1111/jfbc.14451.\u003c/li\u003e\n\u003cli\u003eVishwas S, Kumar R, Khursheed R, Ramanunny AK, Kumar R, Awasthi A, Corrie L, Porwal O, Arshad MF, Alshammari MK, Alghitran AA, Qumayri AN, Alkhaldi SM, Alshammari AK, Chellappan DK, Gupta G, Collet T, Adams J, Dua K, Gulati M, Singh SK (2022) Expanding Arsenal against Neurodegenerative Diseases using Quercetin based Nanoformulations: Breakthroughs and Bottlenecks. Curr Neuropharmacol Online ahead of print. https://doi.org/10.2174/1570159x20666220810105421.\u003c/li\u003e\n\u003cli\u003ede Abreu MS, Genario R, Giacomini ACVV, Demin KA, Lakstygal AM, Amstislavskaya TG, Fontana BD, Parker MO, Kalueff AV (2020) Zebrafish as a Model of Neurodevelopmental Disorders. Neuroscience 445:3-11. https://doi.org/https://doi.org/10.1016/j.neuroscience.2019.08.034.\u003c/li\u003e\n\u003cli\u003eBretaud S, Lee S, Guo S (2004) Sensitivity of zebrafish to environmental toxins implicated in Parkinson\u0026apos;s disease. Neurotoxicol Teratol 26(6):857-64. https://doi.org/10.1016/j.ntt.2004.06.014.\u003c/li\u003e\n\u003cli\u003eRazali K, Othman N, Mohd Nasir MH, Doolaanea AA, Kumar J, Ibrahim WN, Mohamed Ibrahim N, Mohamed WMY (2021) The Promise of the Zebrafish Model for Parkinson\u0026rsquo;s Disease: Today\u0026rsquo;s Science and Tomorrow\u0026rsquo;s Treatment. Front Genet 12:655550. 12https://doi.org/10.3389/fgene.2021.655550.\u003c/li\u003e\n\u003cli\u003eZhang W, Fan R, Luo S, Ying l, Jin Y, Li Y, Xiong M, Yuan X, Jia L, Chen Y (2022) Antagonistic effects and mechanisms of carbendazim and chlorpyrifos on the neurobehavior of larval zebrafish. Chemosphere 293:133522. https://doi.org/10.1016/j.chemosphere.2022.133522.\u003c/li\u003e\n\u003cli\u003eManzanilla B, Robles J (2022) Antiradical properties of curcumin, caffeic acid phenethyl ester, and chicoric acid: a DFT study. J Mol Model 28(3):68. https://doi.org/10.1007/s00894-022-05056-4.\u003c/li\u003e\n\u003cli\u003eWang M, Zhao J, Wu L, Ma T (2021) Effects of 4-epianhydrotetracycline on oxidative stress in zebrafish (Danio rerio) embryos. Sci Total Environ 796:149047. https://doi.org/10.1016/j.scitotenv.2021.149047.\u003c/li\u003e\n\u003cli\u003eKagan DB, Liu H, Hutnik CM (2012) Efficacy of various antioxidants in the protection of the retinal pigment epithelium from oxidative stress. Clin Ophthalmol 6:1471-1476. https://doi.org/10.2147/opth.S35139.\u003c/li\u003e\n\u003cli\u003eYou L, Peng H, Liu J, Cai M, Wu H, Zhang Z, Bai J, Yao Y, Dong X, Yin X, Ni J (2021) Catalpol Protects ARPE-19 Cells against Oxidative Stress via Activation of the Keap1/Nrf2/ARE Pathway. Cells 10(10):2635. https://doi.org/10.3390/cells10102635.\u003c/li\u003e\n\u003cli\u003eMagesh S, Chen Y, Hu L (2012) Small Molecule Modulators of Keap1-Nrf2-ARE Pathway as Potential Preventive and Therapeutic Agents. Med Res Rev 32(4):687-726. https://doi.org/10.1002/med.21257.\u003c/li\u003e\n\u003cli\u003eMaines MD, Panahian N (2001) The heme oxygenase system and cellular defense mechanisms. Do HO-1 and HO-2 have different functions? Exp Med Biol 502:249-272. https://doi.org/10.1007/978-1-4757-3401-0_17.\u003c/li\u003e\n\u003cli\u003eLiu Z, Zhang F, Zhao L, Zhang X, Li Y, Liu L (2020) Protective Effect of Pravastatin on Myocardial Ischemia Reperfusion Injury by Regulation of the miR-93/Nrf2/ARE Signal Pathway. Drug Des Devel Ther 14:3853-3864. https://doi.org/10.2147/dddt.S251726.\u003c/li\u003e\n\u003cli\u003eJung KA, Kwak MK (2010) The Nrf2 system as a potential target for the development of indirect antioxidants. Molecules 15(10):7266-7291. https://doi.org/10.3390/molecules15107266.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chicoric acid, Motor function, Zebrafish, Oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-2848281/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2848281/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChicoric acid (CA) has been used as a nutritional supplement, health food, and medicine because of its antioxidant property. This study aimed to investigate whether CA can improve motor dysfunction in a zebrafish model of Parkinson’s disease (PD). AB-strain zebrafish larvae (24 hours post-fertilization) were incubated with 25 μM 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with or without CA for 5 days. The levels of malondialdehyde, glutathione, and reactive oxygen species were measured, as were the activities of antioxidant enzymes in brain tissue. Additionally, the expression of molecules from the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway were assayed by western blot. Compared with the control group, zebrafish in the MPTP group had impaired motor function. Treatment with CA improved motor dysfunction and attenuated dopaminergic neuronal injury in the PD zebrafish model. CA treatment also reduced the levels of malondialdehyde and reactive oxygen species and increased the antioxidant enzymes and glutathione level. Furthermore, CA augmented the expression of Nrf2, heme oxygenase, quinone oxidoreductase 1, and glutamate-cysteine ligase in the brain of the PD zebrafish model. CA from health foods also improved motor dysfunction and attenuated dopaminergic neuronal injury in the MPTP-induced PD zebrafish model. Our findings suggest that the mechanism of action of CA is related to its inhibition of oxidative stress via regulation of the Nrf2 pathway.\u003c/p\u003e","manuscriptTitle":"Chicoric Acid from Health Foods Improves Motor Dysfunction in Zebrafish Parkinson’s Disease Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-26 20:19:08","doi":"10.21203/rs.3.rs-2848281/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":"0f0f52b6-376c-4a15-b577-47a67ba40687","owner":[],"postedDate":"April 26th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-29T09:16:41+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-26 20:19:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2848281","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2848281","identity":"rs-2848281","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}