Effusol, a unique Juncus effusus phenanthrene, ameliorates amyloid β1-42-mediated neurotoxicity in mice

Effusol, a unique Juncus effusus phenanthrene, ameliorates amyloid β1-42-mediated neurotoxicity in mice | 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 Effusol, a unique Juncus effusus phenanthrene, ameliorates amyloid β1-42-mediated neurotoxicity in mice Haruna Tamano, Daichi Murakami, Toshiyuki Fukuda, Yasuhito Akagi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6492242/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Dehydroeffusol, a unique Juncus effusus phenanthrene, ameliorates amyloid β 1-42 (Aβ 1-42 )-mediated neurotoxicity. We tested the ameliorating effect of effusol, another unique Juncus effusus phenanthrene. Effusol (15 mg/kg) was orally delivered to mice once a day for 2 days and mice were subjected to intracerebroventricular (ICV) administration of Aβ 1-42 1 day after the last deliver of effusol in the same procedure as dehydroeffusol. Fourteen days later, effusol ameliorated neurotoxicity in the dentate granule cell layer evaluated by propidium iodide. Effusol did not increase the synthesis of metallothioneins (MTs) for capturing toxic Zn 2+ ferried by Aβ 1-42 , unlike the effect of dehydroeffusol via MT synthesis. However, effusol reduced both intracellular increases in Zn 2+ and reactive oxygen species (ROS) by Aβ 1-42 . To test the effect of post-intake of effusol, mice were subjected to ICV administration of Aβ 1-42 and effusol was delivered to mice in the same manner. Effusol ameliorated Aβ 1-42 -mediated neurotoxicity. These results first suggest that effusol ameliorates Aβ 1-42 -mediated neurotoxicity in the dentate gyrus by reducing intracellular ROS generation, which is induced by Zn 2+ dysregulation. A possible effect of effusol on Aβ 1-42 neurotoxicity is that effusol may reduce Aβ-induced synaptic hyperexcitation induced by glutamate exocytosis via activating GABA A receptors followed by ameliorating Zn 2+ dysregulation. Effusol Juncus effusus Zn2+ reactive oxygen species amyloid β1-42 Alzheimer’s disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Amyloid-β (Aβ) peptides, which derived from amyloid precursor protein, a membrane protein, contribute to the Alzheimer disease (AD) pathogenesis [ 1 , 2 ]. Aβ 1-40 and Aβ 1-42 are two major peptides. In the extracellular fluid, the concentration of Aβ 1-40 is much higher than that of Aβ 1-42 , while aggregation of Aβ 1-42 is more facilitated than that of Aβ 1-40 followed by higher neurotoxicity of Aβ 1-42 [ 3 , 4 ]. Furthermore, the aggregation of Aβ 1-42 is strongly facilitated in the presence of extracellular Zn 2+ in vivo. When Aβ 1-42 is accumulated to approximately 500 pM in the extracellular fluid of rat brain, Aβ 1-42 readily captures Zn 2+ and Zn-Aβ 1-42 complexes are preferentially passes through dentate gyrus neurons in the hippocampus. Aβ 1-42 uptake by dentate gyrus neurons is closely linked with more vulnerability to Aβ 1-42 neurotoxicity in the hippocampus followed by memory loss and cell death [ 5 – 8 ]. Aβ 1-42 neurotoxicity is avoided by either co-administrating CaEDTA for capturing extracellular Zn 2+ or ZnAF-2DA for capturing intracellular Zn 2+ [ 8 ]: Neuronal Aβ 1-42 accumulation is avoided by the conversion of CaEDTA into ZnEDTA, while intracellular ZnAF-2 captures Zn 2+ released from intracellular Zn-Aβ 1-42 complexes followed by reducing Aβ 1-42 neurotoxicity. Zn 2+ dysregulation induced by Aβ 1-42 serves as a trigger in the Aβ 1-42 pathogenesis [ 9 ]. Therefore, we paid attention to metallothioneins (MTs), intracellular Zn 2+ -proteins, which capture excess (toxic) Zn 2+ under physiological condition [ 9 ]. Memory loss and cell death by Aβ 1-42 are avoided by the pre-induction of MTs with dexamethasone, a blood-brain barrier-permeable steroid hormone, which increases the capacity for capturing excess Zn 2+ [ 8 , 10 ]. Capturing excess Zn 2+ ferried by Aβ 1-42 may contribute to the rescuing effect of dexamethasone-induced MTs on Aβ 1-42 neurotoxicity in vivo. Traditional Chinese medicines contain active natural ingredients and have been recognized for their medication effects such as anti-cancer and anti-oxidative activities. Many researchers have paid attention to phenanthrene derivatives, one of the active ingredients in Chinese herbal medicines, e.g., Juncaceae [ 11 ]. Effusol and dehydroeffusol are unique and major phenanthrene derivatives isolated from Juncus effusus , a member of Juncaceae family. We reported that oral intake of dehydroeffusol blocks cell death in the dentate gyrus of mouse hippocampus after administering Aβ 1-42 into the lateral ventricle [ 12 ]. MT synthesis by dehydroeffusol is increased via activation of adrenergic β receptors and reduces excess Zn 2+ ferried by Aβ 1-42 followed by the preventing effect against Aβ 1-42 neurotoxicity [ 13 ]. In contrast, Effusol concentration-dependently enhances GABA A receptor-mediated current in an in vitro system [ 14 ]. Effusol reduces intracellular Zn 2+ dysregulation, which is induced by corticosterone-induced accumulation of extracellular glutamate, followed by ameliorating adverse events of corticosterone [ 15 ]. Therefore, it is possible that effusol increases MT synthesis and/or regulates glutamate excitotoxicity followed the rescuing effect against Aβ 1-42 neurotoxicity. Biological half-life of MTs is 18–20 h [ 16 ]. In the case of dexamethasone-induced MTs, MTs reaches the maximum level 24 h after subcutaneous injection of dexamethasone once a day for two days followed by increase in capacity for capturing Zn 2+ [ 10 ]. On the basis of evidence on the beneficial period of newly synthesized MTs, it is estimated that oral intake of dehydroeffusol prevents Aβ 1-42 neurotoxicity [ 13 ]. In the present study, effusol was orally delivered to mice once a day for 2 days and then mice were subjected to intracerebroventricular (ICV) administration of Aβ 1-42 1 day after the last deliver of effusol according to the same procedure as dehydroeffusol [ 13 ]. 2. Materials and method 2.1. Chemicals Human Aβ 1−42 (ChinaPeptides, Shanghai, China) dissolved in saline was immediately used in every experiment. ZnAF-2DA (Sekisui Medical Co., LTD, Hachimantai, Japan) is plasma membrane-permeable and captures intracellular Zn 2+ . ZnAF-2DA was dissolved in dimethyl sulfoxide and diluted with Ringer solution, which consisted of 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO 4 , 1.0 mM NaH 2 PO 4 , 2.5 mM CaCl 2 , 26.2 mM NaHCO 3 , and 11 mM D-glucose (pH 7.3). Aminophenyl Fluorescein (APF, Goryochemical, Sapporo, Japan) is plasma membrane-permeable and captures intracellular reactive oxygen species (ROS), i.e., ・ OH and ONOO¯. APF dissolved in N, N-dimethylformamide was also used by diluting with Ringer solution. 2.2. Experimental animals Mice (ddY male, 10 weeks of age, Japan SLC, Hamamatsu, Japan) was maintained under the standard conditions (a diurnal 12-h light cycle; room temperature, 23 ± 1°C; relative humidity, 55 ± 5%). A laboratory diet and water were freely given to mice. Mice were subcutaneously injected with chloral hydrate (30 mg/kg) and anesthetized prior to surgery for administering Aβ 1−42 and also decapitation for all experiments. The present animal experiments were done based on the Guidelines, which were made for the Care and Use of Laboratory Animals in the University of Shizuoka after permission by the Ethics Committee of Experimental Animals in the University of Shizuoka (Ethical approval no. 196418, 2019). 2.3. Isolation of effusol and administration Effusol was purified from Juncus effusus (Rush, Igusa) obtained from HAGIHARA & CO., LTD. (Kurashiki, Japan). The structure of effusol was identified by spectroscopic methods including 1D and 2D NMR ( 1 H, 13 C, COSY, HMQC, and HMBC) and mass spectrometry (ESI-MS) as reported previously (Fig. 1 ) [ 15 ]. On the basis of the data on the minimum effective dose (15 mg/kg body weight in mice) of dehydroeffusol [ 17 ], effusol (15 mg/kg) was dissolved in saline containing 0.5% carboxymethyl cellulose sodium and delivered to mice orally once a day for 2 days in the same procedure as dehydroeffusol [ 13 ]. 2.4. ICV administration of Aβ 1-42 To induce preferential neurodegeneration in the dentate granule cell layer by Aβ 1−42 , vehicle (saline) and Aβ 1−42 (ChinaPeptides, Shanghai, China) in saline were administered into the mouse lateral ventricle for 40 min at the rate of 0.5 µL/min through a cannula (25 µM, 20 µL, 500 pmol/mouse) under anesthetization as reported previously [ 8 , 12 , 13 ]. 2.5. Propidium iodide (PI) imaging Two weeks after ICV administrating Aβ 1−42 , PI fluorescence (Ex/Em: 535 nm/617 nm) of brain slices, which was used to assess neuronal damage, was observed in the dentate gyrus by using a Nikon A1 confocal microscopic system (Nikon Corp., Japan) [ 8 , 12 , 13 ]. 2.6. MT immunostaining One day after the last delivering effusol, MT immunostaining of brain slices was done by using anti-MT antibody [UC1MT] ab12228 (Abcam) and Alexa Fluor 488 goat anti-mouse secondary antibody (Thermo Fisher Scientific). MT images (Ex/Em: 495 nm/519 nm) were observed in the dentate gyrus [ 13 ]. 2.7. Zn 2+ imaging One hour after ICV administering saline and Aβ 1−42 in saline, the fluorescence of brain slices with ZnAF-2DA, which was used for assess intracellular Zn 2+ level, was observed in the dentate gyrus (Ex/Em: 488 nm/505–530 nm) [ 13 ]. 2.8. Reactive oxygen species (ROS) imaging One hour after ICV administration of saline containing APF (50 µM) and Aβ 1−42 in saline containing Aminophenyl Fluorescein (APF, 50 µM), the fluorescence of APF of brain slices, which was used for assess the level of intracellular ROS, i.e., ・ OH and ONOO - , was observed in the dentate gyrus (Ex/Em: 490 nm/500–550 nm). 2.9. Data analysis Student’s paired t -test was used for comparing the means of paired data (means ± standard error). One-way ANOVA followed by post hoc testing using the Tukey’s test (the statistical software, GraphPad Prism 5) was used for multiple comparisons between treatments. 3. Results and discussion Because phenanthrene derivatives, which are water-insoluble and lipophilic low-molecular weights, are blood-brain barrier-permeable as reported in morphine [ 18 , 19 ], it is estimated that water-insoluble effusol passes through the blood-brain barrier and is transported into the brain extracellular fluid. On the basis of the data on the minimum effective dose of dehydroeffusol (15 mg/kg body weight in mice) [ 17 ], effusol (15 mg/kg) was delivered to mice orally once a day for 2 days in the same procedure as dehydroeffusol [ 13 ]. In the resent study, Aβ 1−42 was administered 1 day after the second delivering effusol. Effusol blocked Aβ 1−42 toxicity in the dentate granule cell layer by PI fluorescence, an index of dead cells (Fig. 1 ). However, effusol did not elevate the level of MT synthesis by MT immunostaining, unlike the case of dehydroeffusol [ 13 ] (Fig. 2 ). When intracellular Zn 2+ in the dentate granule cell layer was assessed by ZnAF-2 fluorescence, effusol reduced increase in intracellular Zn 2+ induced by Aβ 1−42 , which was observed 1 h after administering Aβ 1−42 (Fig. 3 ). Intracellular Zn 2+ dysregulation induced by Aβ 1−42 may lead to the increase in ROS generation, as well as intracellular Zn 2+ dysregulation induced by Zn 2+ influx through GluR2-lacking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors [ 9 , 20 ]. It is likely that Zn 2+ dysregulation leads to mitochondrial Zn 2+ accumulation followed by mitochondrial ROS deneration. Intracellular ROS assessed by APF was increased 1 h after administering Aβ 1−42 , while the increase was also reduced by pre-oral intake of effusol (Fig. 4 ), suggesting that ROS generation induced by Zn 2+ dysregulation is linked with Aβ 1−42 neurotoxicity and that decrease in ROS generation by effusol contributes to neuroprotection against Aβ 1−42 [ 9 , 20 ]. Intracellular Zn 2+ dysregulation by Aβ 1−42 induces Zn 2+ accumulation and ROS generation in the mitochondria, followed by neurodegeneration [ 21 ]. Oxidative stress via ROS is associated with neurodegenerative diseases [ 22 ] and is also associated with the etiology of AD [ 23 , 24 ]. Furthermore, the effect of post-oral effusol intake was tested 1 day after administering Aβ 1−42 . Effusol also decreased Aβ 1−42 toxicity in the dentate granule cell layer by PI fluorescence (Fig. 5 ). It has been reported that affected vesicular exocytosis for releasing glutamate, which is induced by soluble Aβ oligomers, contributes to pathological events in neurological diseases, which are also linked with AD pathophysiology: Aβ 1−42 oligomers preferentially leads to accumulating extracellular glutamate followed by synaptic dyshomeostasis of glutamate, and initially induces synaptic hyperactivity by inhibiting glutamate reuptake [ 25 – 27 ] or enhancing glutamate release probability [ 28 ]. Soluble Aβ 1−42 oligomers markedly increase vesicular storage of glutamate and then increase vesicular exocytosis for releasing glutamate by the action potential in addition to the spontaneous exocytosis [ 29 ]. These events may decrease vesicular concentration of glutamate and lead to reduced release probability. Astrocytes serve a key role for the clearance and metabolism of glutamate in the synaptic clefts, while Aβ also affects vesicular release of neurotransmitters in the astrocyte model system [ 30 ]. It is likely that Aβ 1−42 -induced synaptic hyperexcitation accumulates glutamate in the brain extracellular fluid and that glutamate excitotoxicity leads to postsynaptic Zn 2+ dysregulation via excess influx of extracellular Zn 2+ through GluR2-lacking AMPA receptors, in addition to Zn 2+ dysregulation via ferry of extracellular Aβ 1−42 . In contrast, γ-aminobutyric acid (GABA) is a major inhibitory neurotransmitter and GABAergic system plays a key role in regulating glutamate excitotoxicity in neurological diseases including AD [ 31 , 32 ]. Effusol concentration-dependently enhance GABA A receptor-mediated current in an in vitro system [ 14 ], suggesting that GABA A receptor activation by effusol is involved in the effects on the traditional use of Juncus effuses in anxiety and insomnia. Effusol reduces glutamate-induced excitotoxicity in the hippocampus after exposure to corticosterone, perhaps via GABA A receptor activation followed by amelioration of Zn 2+ dysregulation [ 15 ]. A possible effect of effusol on Aβ 1−42 neurotoxicity is that effusol may reduce Aβ-induced synaptic hyperexcitation induced by glutamate exocytosis via activating GABA A receptors followed by ameliorating Zn 2+ dysregulation. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Jun, 2025 Reviews received at journal 01 Jun, 2025 Reviews received at journal 27 May, 2025 Reviews received at journal 26 May, 2025 Reviewers agreed at journal 20 May, 2025 Reviewers agreed at journal 19 May, 2025 Reviewers agreed at journal 19 May, 2025 Reviewers invited by journal 27 Apr, 2025 Editor assigned by journal 27 Apr, 2025 Submission checks completed at journal 24 Apr, 2025 First submitted to journal 20 Apr, 2025 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-6492242","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448722937,"identity":"59a2c6e5-0b19-484b-bd5a-1228d96a25f1","order_by":0,"name":"Haruna Tamano","email":"","orcid":"","institution":"University of Shizuoka","correspondingAuthor":false,"prefix":"","firstName":"Haruna","middleName":"","lastName":"Tamano","suffix":""},{"id":448722938,"identity":"48d5467d-86cd-484a-b9d7-86c349ca2958","order_by":1,"name":"Daichi Murakami","email":"","orcid":"","institution":"University of Shizuoka","correspondingAuthor":false,"prefix":"","firstName":"Daichi","middleName":"","lastName":"Murakami","suffix":""},{"id":448722939,"identity":"2dd5d33b-3f77-4ed7-a0f5-2ef148c62d32","order_by":2,"name":"Toshiyuki Fukuda","email":"","orcid":"","institution":"Satoen CO., LTD","correspondingAuthor":false,"prefix":"","firstName":"Toshiyuki","middleName":"","lastName":"Fukuda","suffix":""},{"id":448722940,"identity":"8ddfc60b-4099-4127-9772-c17bbb88feeb","order_by":3,"name":"Yasuhito Akagi","email":"","orcid":"","institution":"Hagihara \u0026 CO., LTD","correspondingAuthor":false,"prefix":"","firstName":"Yasuhito","middleName":"","lastName":"Akagi","suffix":""},{"id":448722942,"identity":"c1d09900-be02-4dde-a31c-1f92bb34f18e","order_by":4,"name":"Shinji Ikeura","email":"","orcid":"","institution":"Hagihara \u0026 CO., LTD","correspondingAuthor":false,"prefix":"","firstName":"Shinji","middleName":"","lastName":"Ikeura","suffix":""},{"id":448722943,"identity":"dd440ecf-9927-45da-9e73-89429cd6068f","order_by":5,"name":"Atsushi Takeda","email":"data:image/png;base64,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","orcid":"","institution":"University of Shizuoka","correspondingAuthor":true,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Takeda","suffix":""}],"badges":[],"createdAt":"2025-04-21 04:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6492242/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6492242/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81653565,"identity":"9a2c6ec3-9ec6-4667-b3d3-7705dc853c7a","added_by":"auto","created_at":"2025-04-29 17:12:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1007693,"visible":true,"origin":"","legend":"\u003cp\u003eEffusol ameliorates Aβ\u003csub\u003e1-42\u003c/sub\u003e-mediated neurotoxicity\u003c/p\u003e\n\u003cp\u003eAβ\u003csub\u003e1-42\u003c/sub\u003e and vehicle (saline) were administered to mice 1 day after oral delivery of effusol (EF). Fourteen days later, Aβ\u003csub\u003e1-42\u003c/sub\u003e-induced neurotoxicity by PI fluorescence was determined in the dentate granule cell layer (GCL) indicated by the dotted line (upper). Bar; 50 µm. Structure of effusol (lower light). The data showed the rate (%) of PI fluorescence of vehicle/Aβ\u003csub\u003e1-42\u003c/sub\u003e (n=12 mice) and effusol/Aβ\u003csub\u003e1-42\u003c/sub\u003e (n=11 mice) groups to that of vehicle/saline (n=17 mice) group showed as 100% (lower right). *, p\u0026lt;0.05, vs. vehicle/saline, \u003csup\u003e##\u003c/sup\u003e, p\u0026lt;0.01, vs. vehicle/Aβ (Tukey’s test).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6492242/v1/7cf9d0d5d98d1f7d92aa47c5.png"},{"id":81653631,"identity":"4cf59ad8-f08f-4b09-bc5e-a954aae60d67","added_by":"auto","created_at":"2025-04-29 17:20:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":640306,"visible":true,"origin":"","legend":"\u003cp\u003eEffusol does not increase MT synthesis\u003c/p\u003e\n\u003cp\u003eMT immunostaining was determined in the GCL 1 day after oral delivery of effusol (EF) (left). Bar; 50 µm. The data showed the rate (%) of MT staining of effusol group (n=8 mice) to that of vehicle group showed as 100% (right) (n=12 mice).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6492242/v1/2f5b35de6537959d05291b7e.png"},{"id":81653567,"identity":"65d7a984-5dc4-425e-ad9c-4b3105b7f908","added_by":"auto","created_at":"2025-04-29 17:12:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1598815,"visible":true,"origin":"","legend":"\u003cp\u003eEffusol suppresses increase in intracellular Zn\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAβ\u003csub\u003e1-42\u003c/sub\u003e and vehicle (saline) were administered to mice 1 day after oral delivery of effusol (EF). Intracellular Zn\u003csup\u003e2+\u003c/sup\u003e level with ZnAF-2 fluorescence was determined in the GCL 1 h after adminstering Aβ\u003csub\u003e1-42\u003c/sub\u003e (Upper). Bar; 50 µm. The data showed the rate (%) of intracellular ZnAF-2 intensity of vehicle/Aβ\u003csub\u003e1-42\u003c/sub\u003e (n=12) and effusol/Aβ\u003csub\u003e1-42\u003c/sub\u003e (n=12 mice) groups to that of vehicle/saline (n=12 mice) group showed as 100% (lower). **, p\u0026lt;0.01, vs. vehicle/saline, \u003csup\u003e#\u003c/sup\u003e, P\u0026lt;0.05, vs. vehicle/Aβ (Tukey’s test).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6492242/v1/70ede323ea49956e2991c66d.png"},{"id":81653573,"identity":"1d11a3fe-163f-4e2e-af16-ba0db678ffbf","added_by":"auto","created_at":"2025-04-29 17:12:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1379241,"visible":true,"origin":"","legend":"\u003cp\u003eEffusol reduces increase in intracellular ROS\u003c/p\u003e\n\u003cp\u003eAβ\u003csub\u003e1-42\u003c/sub\u003e and saline with APF were administered to mice 1 day after oral delivery of effusol (EF). Intracellular ROS with APF fluorescence was determined in the GCL 1 h after administering Aβ\u003csub\u003e1-42\u003c/sub\u003e. (Upper). Bar; 50 µm. The data showed the rate (%) of intracellular APF intensity of vehicle/Aβ\u003csub\u003e1-42\u003c/sub\u003e (n=12 mice) and effusol/Aβ\u003csub\u003e1-42\u003c/sub\u003e (n=9 mice) groups to that of vehicle/saline (n=7 mice) group showed as 100% (lower). ***, p\u0026lt;0.01, vs. vehicle/saline; \u003csup\u003e#\u003c/sup\u003e, P\u0026lt;0.05, vs. vehicle/Aβ (Tukey’s test).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6492242/v1/dd55a46a830a8897c669c788.png"},{"id":81653571,"identity":"df70234c-79cb-4439-af88-3534775c4485","added_by":"auto","created_at":"2025-04-29 17:12:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1238223,"visible":true,"origin":"","legend":"\u003cp\u003ePost-oral intake of effusol decreases Aβ\u003csub\u003e1-42\u003c/sub\u003e-induced neurotoxicity\u003c/p\u003e\n\u003cp\u003eOne day after administering Aβ\u003csub\u003e1-42\u003c/sub\u003e and saline (vehicle), effusol (EF) and vehicle were orally delivered to mice. Aβ\u003csub\u003e1-42\u003c/sub\u003e-induced neurotoxicity by PI fluorescence was determined in the GCL 14 day after administering Aβ\u003csub\u003e1-42\u003c/sub\u003e (left). Bar; 50 µm. The data showed the rate (%) of PI fluorescence of Aβ/vehicle (n=24 mice) and Aβ/effusol (n=12 mice) groups to that of saline/vehicle (n=28 mice) group showed as 100% (right). *, p\u0026lt;0.05, vs. saline/vehicle (Tukey’s test).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6492242/v1/0ff65a63a9a846f80449b59c.png"},{"id":81653998,"identity":"70051136-2593-4083-a537-93337e68875b","added_by":"auto","created_at":"2025-04-29 17:28:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5823625,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6492242/v1/8e4aa669-4340-4b13-982d-9199c7d9602d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effusol, a unique Juncus effusus phenanthrene, ameliorates amyloid β1-42-mediated neurotoxicity in mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAmyloid-β (Aβ) peptides, which derived from amyloid precursor protein, a membrane protein, contribute to the Alzheimer disease (AD) pathogenesis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Aβ\u003csub\u003e1-40\u003c/sub\u003e and Aβ\u003csub\u003e1-42\u003c/sub\u003e are two major peptides. In the extracellular fluid, the concentration of Aβ\u003csub\u003e1-40\u003c/sub\u003e is much higher than that of Aβ\u003csub\u003e1-42\u003c/sub\u003e, while aggregation of Aβ\u003csub\u003e1-42\u003c/sub\u003e is more facilitated than that of Aβ\u003csub\u003e1-40\u003c/sub\u003e followed by higher neurotoxicity of Aβ\u003csub\u003e1-42\u003c/sub\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, the aggregation of Aβ\u003csub\u003e1-42\u003c/sub\u003e is strongly facilitated in the presence of extracellular Zn\u003csup\u003e2+\u003c/sup\u003e in vivo. When Aβ\u003csub\u003e1-42\u003c/sub\u003e is accumulated to approximately 500 pM in the extracellular fluid of rat brain, Aβ\u003csub\u003e1-42\u003c/sub\u003e readily captures Zn\u003csup\u003e2+\u003c/sup\u003e and Zn-Aβ\u003csub\u003e1-42\u003c/sub\u003e complexes are preferentially passes through dentate gyrus neurons in the hippocampus. Aβ\u003csub\u003e1-42\u003c/sub\u003e uptake by dentate gyrus neurons is closely linked with more vulnerability to Aβ\u003csub\u003e1-42\u003c/sub\u003e neurotoxicity in the hippocampus followed by memory loss and cell death [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Aβ\u003csub\u003e1-42\u003c/sub\u003e neurotoxicity is avoided by either co-administrating CaEDTA for capturing extracellular Zn\u003csup\u003e2+\u003c/sup\u003e or ZnAF-2DA for capturing intracellular Zn\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]: Neuronal Aβ\u003csub\u003e1-42\u003c/sub\u003e accumulation is avoided by the conversion of CaEDTA into ZnEDTA, while intracellular ZnAF-2 captures Zn\u003csup\u003e2+\u003c/sup\u003e released from intracellular Zn-Aβ\u003csub\u003e1-42\u003c/sub\u003e complexes followed by reducing Aβ\u003csub\u003e1-42\u003c/sub\u003e neurotoxicity. Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation induced by Aβ\u003csub\u003e1-42\u003c/sub\u003e serves as a trigger in the Aβ\u003csub\u003e1-42\u003c/sub\u003e pathogenesis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, we paid attention to metallothioneins (MTs), intracellular Zn\u003csup\u003e2+\u003c/sup\u003e-proteins, which capture excess (toxic) Zn\u003csup\u003e2+\u003c/sup\u003e under physiological condition [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Memory loss and cell death by Aβ\u003csub\u003e1-42\u003c/sub\u003e are avoided by the pre-induction of MTs with dexamethasone, a blood-brain barrier-permeable steroid hormone, which increases the capacity for capturing excess Zn\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Capturing excess Zn\u003csup\u003e2+\u003c/sup\u003e ferried by Aβ\u003csub\u003e1-42\u003c/sub\u003e may contribute to the rescuing effect of dexamethasone-induced MTs on Aβ\u003csub\u003e1-42\u003c/sub\u003e neurotoxicity in vivo.\u003c/p\u003e \u003cp\u003eTraditional Chinese medicines contain active natural ingredients and have been recognized for their medication effects such as anti-cancer and anti-oxidative activities. Many researchers have paid attention to phenanthrene derivatives, one of the active ingredients in Chinese herbal medicines, e.g., \u003cem\u003eJuncaceae\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Effusol and dehydroeffusol are unique and major phenanthrene derivatives isolated from \u003cem\u003eJuncus effusus\u003c/em\u003e, a member of \u003cem\u003eJuncaceae\u003c/em\u003e family. We reported that oral intake of dehydroeffusol blocks cell death in the dentate gyrus of mouse hippocampus after administering Aβ\u003csub\u003e1-42\u003c/sub\u003e into the lateral ventricle [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. MT synthesis by dehydroeffusol is increased via activation of adrenergic β receptors and reduces excess Zn\u003csup\u003e2+\u003c/sup\u003e ferried by Aβ\u003csub\u003e1-42\u003c/sub\u003e followed by the preventing effect against Aβ\u003csub\u003e1-42\u003c/sub\u003e neurotoxicity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In contrast, Effusol concentration-dependently enhances GABA\u003csub\u003eA\u003c/sub\u003e receptor-mediated current in an in vitro system [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Effusol reduces intracellular Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation, which is induced by corticosterone-induced accumulation of extracellular glutamate, followed by ameliorating adverse events of corticosterone [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, it is possible that effusol increases MT synthesis and/or regulates glutamate excitotoxicity followed the rescuing effect against Aβ\u003csub\u003e1-42\u003c/sub\u003e neurotoxicity.\u003c/p\u003e \u003cp\u003eBiological half-life of MTs is 18\u0026ndash;20 h [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the case of dexamethasone-induced MTs, MTs reaches the maximum level 24 h after subcutaneous injection of dexamethasone once a day for two days followed by increase in capacity for capturing Zn\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. On the basis of evidence on the beneficial period of newly synthesized MTs, it is estimated that oral intake of dehydroeffusol prevents Aβ\u003csub\u003e1-42\u003c/sub\u003e neurotoxicity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the present study, effusol was orally delivered to mice once a day for 2 days and then mice were subjected to intracerebroventricular (ICV) administration of Aβ\u003csub\u003e1-42\u003c/sub\u003e 1 day after the last deliver of effusol according to the same procedure as dehydroeffusol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eHuman Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (ChinaPeptides, Shanghai, China) dissolved in saline was immediately used in every experiment. ZnAF-2DA (Sekisui Medical Co., LTD, Hachimantai, Japan) is plasma membrane-permeable and captures intracellular Zn\u003csup\u003e2+\u003c/sup\u003e. ZnAF-2DA was dissolved in dimethyl sulfoxide and diluted with Ringer solution, which consisted of 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 1.0 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 26.2 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, and 11 mM D-glucose (pH 7.3). Aminophenyl Fluorescein (APF, Goryochemical, Sapporo, Japan) is plasma membrane-permeable and captures intracellular reactive oxygen species (ROS), i.e., \u003csup\u003e・\u003c/sup\u003eOH and ONOO\u0026macr;. APF dissolved in N, N-dimethylformamide was also used by diluting with Ringer solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental animals\u003c/h2\u003e \u003cp\u003eMice (ddY male, 10 weeks of age, Japan SLC, Hamamatsu, Japan) was maintained under the standard conditions (a diurnal 12-h light cycle; room temperature, 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C; relative humidity, 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5%). A laboratory diet and water were freely given to mice. Mice were subcutaneously injected with chloral hydrate (30 mg/kg) and anesthetized prior to surgery for administering Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e and also decapitation for all experiments. The present animal experiments were done based on the Guidelines, which were made for the Care and Use of Laboratory Animals in the University of Shizuoka after permission by the Ethics Committee of Experimental Animals in the University of Shizuoka (Ethical approval no. 196418, 2019).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Isolation of effusol and administration\u003c/h2\u003e \u003cp\u003eEffusol was purified from \u003cem\u003eJuncus effusus\u003c/em\u003e (Rush, Igusa) obtained from HAGIHARA \u0026amp; CO., LTD. (Kurashiki, Japan). The structure of effusol was identified by spectroscopic methods including 1D and 2D NMR (\u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC, COSY, HMQC, and HMBC) and mass spectrometry (ESI-MS) as reported previously (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the basis of the data on the minimum effective dose (15 mg/kg body weight in mice) of dehydroeffusol [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], effusol (15 mg/kg) was dissolved in saline containing 0.5% carboxymethyl cellulose sodium and delivered to mice orally once a day for 2 days in the same procedure as dehydroeffusol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. ICV administration of Aβ\u003csub\u003e1-42\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eTo induce preferential neurodegeneration in the dentate granule cell layer by Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e, vehicle (saline) and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (ChinaPeptides, Shanghai, China) in saline were administered into the mouse lateral ventricle for 40 min at the rate of 0.5 \u0026micro;L/min through a cannula (25 \u0026micro;M, 20 \u0026micro;L, 500 pmol/mouse) under anesthetization as reported previously [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Propidium iodide (PI) imaging\u003c/h2\u003e \u003cp\u003eTwo weeks after ICV administrating Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e, PI fluorescence (Ex/Em: 535 nm/617 nm) of brain slices, which was used to assess neuronal damage, was observed in the dentate gyrus by using a Nikon A1 confocal microscopic system (Nikon Corp., Japan) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. MT immunostaining\u003c/h2\u003e \u003cp\u003eOne day after the last delivering effusol, MT immunostaining of brain slices was done by using anti-MT antibody [UC1MT] ab12228 (Abcam) and Alexa Fluor 488 goat anti-mouse secondary antibody (Thermo Fisher Scientific). MT images (Ex/Em: 495 nm/519 nm) were observed in the dentate gyrus [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Zn\u003csup\u003e2+\u003c/sup\u003e imaging\u003c/h2\u003e \u003cp\u003eOne hour after ICV administering saline and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e in saline, the fluorescence of brain slices with ZnAF-2DA, which was used for assess intracellular Zn\u003csup\u003e2+\u003c/sup\u003e level, was observed in the dentate gyrus (Ex/Em: 488 nm/505\u0026ndash;530 nm) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Reactive oxygen species (ROS) imaging\u003c/h2\u003e \u003cp\u003eOne hour after ICV administration of saline containing APF (50 \u0026micro;M) and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e in saline containing Aminophenyl Fluorescein (APF, 50 \u0026micro;M), the fluorescence of APF of brain slices, which was used for assess the level of intracellular ROS, i.e., \u003csup\u003e・\u003c/sup\u003eOH and ONOO\u003csup\u003e-\u003c/sup\u003e, was observed in the dentate gyrus (Ex/Em: 490 nm/500\u0026ndash;550 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Data analysis\u003c/h2\u003e \u003cp\u003eStudent\u0026rsquo;s paired \u003cem\u003et\u003c/em\u003e-test was used for comparing the means of paired data (means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error). One-way ANOVA followed by post hoc testing using the Tukey\u0026rsquo;s test (the statistical software, GraphPad Prism 5) was used for multiple comparisons between treatments.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eBecause phenanthrene derivatives, which are water-insoluble and lipophilic low-molecular weights, are blood-brain barrier-permeable as reported in morphine [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], it is estimated that water-insoluble effusol passes through the blood-brain barrier and is transported into the brain extracellular fluid. On the basis of the data on the minimum effective dose of dehydroeffusol (15 mg/kg body weight in mice) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], effusol (15 mg/kg) was delivered to mice orally once a day for 2 days in the same procedure as dehydroeffusol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the resent study, Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e was administered 1 day after the second delivering effusol. Effusol blocked Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e toxicity in the dentate granule cell layer by PI fluorescence, an index of dead cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, effusol did not elevate the level of MT synthesis by MT immunostaining, unlike the case of dehydroeffusol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen intracellular Zn\u003csup\u003e2+\u003c/sup\u003e in the dentate granule cell layer was assessed by ZnAF-2 fluorescence, effusol reduced increase in intracellular Zn\u003csup\u003e2+\u003c/sup\u003e induced by Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e, which was observed 1 h after administering Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Intracellular Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation induced by Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e may lead to the increase in ROS generation, as well as intracellular Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation induced by Zn\u003csup\u003e2+\u003c/sup\u003e influx through GluR2-lacking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It is likely that Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation leads to mitochondrial Zn\u003csup\u003e2+\u003c/sup\u003e accumulation followed by mitochondrial ROS deneration.\u003c/p\u003e \u003cp\u003eIntracellular ROS assessed by APF was increased 1 h after administering Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e, while the increase was also reduced by pre-oral intake of effusol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting that ROS generation induced by Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation is linked with Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e neurotoxicity and that decrease in ROS generation by effusol contributes to neuroprotection against Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Intracellular Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation by Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e induces Zn\u003csup\u003e2+\u003c/sup\u003e accumulation and ROS generation in the mitochondria, followed by neurodegeneration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Oxidative stress via ROS is associated with neurodegenerative diseases [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and is also associated with the etiology of AD [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, the effect of post-oral effusol intake was tested 1 day after administering Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e. Effusol also decreased Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e toxicity in the dentate granule cell layer by PI fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt has been reported that affected vesicular exocytosis for releasing glutamate, which is induced by soluble Aβ oligomers, contributes to pathological events in neurological diseases, which are also linked with AD pathophysiology: Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e oligomers preferentially leads to accumulating extracellular glutamate followed by synaptic dyshomeostasis of glutamate, and initially induces synaptic hyperactivity by inhibiting glutamate reuptake [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] or enhancing glutamate release probability [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Soluble Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e oligomers markedly increase vesicular storage of glutamate and then increase vesicular exocytosis for releasing glutamate by the action potential in addition to the spontaneous exocytosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These events may decrease vesicular concentration of glutamate and lead to reduced release probability. Astrocytes serve a key role for the clearance and metabolism of glutamate in the synaptic clefts, while Aβ also affects vesicular release of neurotransmitters in the astrocyte model system [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is likely that Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e-induced synaptic hyperexcitation accumulates glutamate in the brain extracellular fluid and that glutamate excitotoxicity leads to postsynaptic Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation via excess influx of extracellular Zn\u003csup\u003e2+\u003c/sup\u003e through GluR2-lacking AMPA receptors, in addition to Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation via ferry of extracellular Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, γ-aminobutyric acid (GABA) is a major inhibitory neurotransmitter and GABAergic system plays a key role in regulating glutamate excitotoxicity in neurological diseases including AD [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Effusol concentration-dependently enhance GABA\u003csub\u003eA\u003c/sub\u003e receptor-mediated current in an in vitro system [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], suggesting that GABA\u003csub\u003eA\u003c/sub\u003e receptor activation by effusol is involved in the effects on the traditional use of \u003cem\u003eJuncus effuses\u003c/em\u003e in anxiety and insomnia. Effusol reduces glutamate-induced excitotoxicity in the hippocampus after exposure to corticosterone, perhaps via GABA\u003csub\u003eA\u003c/sub\u003e receptor activation followed by amelioration of Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A possible effect of effusol on Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e neurotoxicity is that effusol may reduce Aβ-induced synaptic hyperexcitation induced by glutamate exocytosis via activating GABA\u003csub\u003eA\u003c/sub\u003e receptors followed by ameliorating Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation.\u003c/p\u003e \u003cp\u003eIn conclusion, the present paper first suggests that effusol protects dentate gyrus neuron death via reducing ROS generation, which is induced by intracellular increase in Zn\u003csup\u003e2+\u003c/sup\u003e after exposure to Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e. It is likely that oral intake of effusol may protectively act on the Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e pathogenesis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eAuthors declare no conflicts of interest.\u003c/p\u003e \u003ch2\u003eFUNDING DECLARATION\u003c/h2\u003e \u003cp\u003eNo Funding\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e\"A.T. wrote the main manuscript text and H.T. prepared figures 1-5. All authors reviewed the manuscript.\"\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePerrin RJ, Fagan AM, Holtzman DM. Multimodal techniques for diagnosis and prognosis of Alzheimer's disease. Nature. 2009;461(7266):916\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKepp KP. Alzheimer's disease due to loss of function: A new synthesis of the available data. 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Exp Neurol. 2025;384:115050.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nutrire","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Nutrire](https://www.springer.com/journal/41110)","snPcode":"41110","submissionUrl":"https://submission.nature.com/new-submission/41110/3","title":"Nutrire","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Effusol, Juncus effusus, Zn2+, reactive oxygen species, amyloid β1-42, Alzheimer’s disease","lastPublishedDoi":"10.21203/rs.3.rs-6492242/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6492242/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDehydroeffusol, a unique \u003cem\u003eJuncus effusus\u003c/em\u003e phenanthrene, ameliorates amyloid β\u003csub\u003e1-42\u003c/sub\u003e (Aβ\u003csub\u003e1-42\u003c/sub\u003e)-mediated neurotoxicity. We tested the ameliorating effect of effusol, another unique \u003cem\u003eJuncus effusus\u003c/em\u003e phenanthrene. Effusol (15 mg/kg) was orally delivered to mice once a day for 2 days and mice were subjected to intracerebroventricular (ICV) administration of Aβ\u003csub\u003e1-42\u003c/sub\u003e 1 day after the last deliver of effusol in the same procedure as dehydroeffusol. Fourteen days later, effusol ameliorated neurotoxicity in the dentate granule cell layer evaluated by propidium iodide. Effusol did not increase the synthesis of metallothioneins (MTs) for capturing toxic Zn\u003csup\u003e2+\u003c/sup\u003e ferried by Aβ\u003csub\u003e1-42\u003c/sub\u003e, unlike the effect of dehydroeffusol via MT synthesis. However, effusol reduced both intracellular increases in Zn\u003csup\u003e2+\u003c/sup\u003e and reactive oxygen species (ROS) by Aβ\u003csub\u003e1-42\u003c/sub\u003e. To test the effect of post-intake of effusol, mice were subjected to ICV administration of Aβ\u003csub\u003e1-42\u003c/sub\u003e and effusol was delivered to mice in the same manner. Effusol ameliorated Aβ\u003csub\u003e1-42\u003c/sub\u003e-mediated neurotoxicity. These results first suggest that effusol ameliorates Aβ\u003csub\u003e1-42\u003c/sub\u003e-mediated neurotoxicity in the dentate gyrus by reducing intracellular ROS generation, which is induced by Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation. A possible effect of effusol on Aβ\u003csub\u003e1-42\u003c/sub\u003e neurotoxicity is that effusol may reduce Aβ-induced synaptic hyperexcitation induced by glutamate exocytosis via activating GABA\u003csub\u003eA\u003c/sub\u003e receptors followed by ameliorating Zn\u003csup\u003e2+\u003c/sup\u003e dysregulation.\u003c/p\u003e","manuscriptTitle":"Effusol, a unique Juncus effusus phenanthrene, ameliorates amyloid β1-42-mediated neurotoxicity in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 17:12:00","doi":"10.21203/rs.3.rs-6492242/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-02T23:30:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-01T12:05:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-27T21:20:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-26T19:25:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46219820130292261002463144351652670777","date":"2025-05-20T13:06:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273374738974615016012762544701749163927","date":"2025-05-19T16:14:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74191606617242452184064896402088549958","date":"2025-05-19T05:50:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-27T22:37:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-27T22:35:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-24T10:54:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Nutrire","date":"2025-04-21T03:59:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nutrire","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Nutrire](https://www.springer.com/journal/41110)","snPcode":"41110","submissionUrl":"https://submission.nature.com/new-submission/41110/3","title":"Nutrire","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0bf8b460-ff14-416d-bb49-54df11db5703","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-08T18:53:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-29 17:12:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6492242","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6492242","identity":"rs-6492242","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}