{"paper_id":"31cf9dbf-e5af-4b90-9c03-fd794bff7115","body_text":"Chinese Prescription Kangen-karyu Attenuates Neuronal Damage and Improves Cognitive Function in Ischemic Stroke by Regulating ROS-Mediated MAPK Activation | 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 Article Chinese Prescription Kangen-karyu Attenuates Neuronal Damage and Improves Cognitive Function in Ischemic Stroke by Regulating ROS-Mediated MAPK Activation Dong Hyuk Youn, Sung Woo Han, Ji Hyeon Lee, Jong-Tae Kim, Seongwon Pak, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8423120/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 16 You are reading this latest preprint version Abstract OBJECTIVE Ischemic stroke is a leading cause of morbidity and mortality worldwide, largely due to the limited and inadequate therapeutic options currently available. As a result, developing new therapeutic approaches is crucial. This study aims to investigate the potential therapeutic effects of Kangen-karyu (KK), a compound that has garnered significant attention for its diverse biological functions, in mitigating brain damage induced by cerebral ischemia/reperfusion. METHODS Seven to eight-week-old male C57BL/6J mice underwent either sham surgery or bilateral common carotid artery occlusion (BCCAO) for 10 minutes, followed by reperfusion to induce cerebral ischemia/reperfusion injury. The mice were treated orally with either a vehicle, KK (200 mg/kg), or nimodipine (NP, 30 mg/kg), administered 2 hours before (Pre-KK) or after (Post-KK, Post-NP) BCCAO. The neurobehavioral performance of the mice was assessed. Additionally, histopathological changes, oxidative stress, inflammation, and apoptotic parameters were evaluated in brain tissues. RESULTS Infarct volume, brain water content, DHE, and Fluoro-Jade B staining confirmed that post-KK significantly ameliorated BCCAO-induced histopathological damage and neurobehavioral deficits compared to the pre-KK and post-NP groups. Additionally, administration of post-KK reduced the protein expression levels of phospho-c-Jun N-terminal kinase (p-JNK), phospho-p38, inducible nitric oxide synthase (iNOS), Bax, and caspase 3. In the cognition tests, BCCAO showed a decreased preference index and an increased alteration rate, which together indicate improved cognitive performance after post-KK. CONCLUSION Our study suggests that Kangen-karyu (KK) exerts a neuroprotective effect by reducing oxidative stress, inflammation, and apoptosis via the JNK/p38 pathway, indicating its potential as a therapeutic candidate for mitigating ischemia/reperfusion-induced brain damage. Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Health sciences/Neurology Biological sciences/Neuroscience Kangen-karyu Cerebral ischemia/reperfusion JNK/p38 neuroprotective cognition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Stroke is a serious, life-threatening illness that occurs when the blood supply to part of the brain is blocked, making it is a medical emergency that requires treatment necessitates. Like all organs, the brain needs oxygen and nutrients from the blood to function properly. There are two main causes of stroke: strokes caused by blockage of an artery (ischemic) and an artery in the brain leaks or bursts (hemorrhagic stroke). Most strokes are ischemic stroke. This occurs when blood vessels in the brain become narrowed or blocked, severely reducing blood flow. Blockage or narrowing of blood vessels is caused by fatty deposits or blood clots that build up in the blood vessels 1 – 3 . Many factors can increase the risk of stroke: unhealthy lifestyle, being overweight or obese, lack of exercise, binge or heavy drinking, and illicit drug use. In addition, medical risk points (hypertension, cigarette or passive smoking, high blood cholesterol, diabetes, obstructive sleep apnea, cardiovascular disease, transient ischemic attack, heart attack, and family or personal medical history of stroke) are mentioned. Other risk factors that contribute to causing a stroke include: race or ethnicity, sex, hormones, and age 4 – 6 . Many novel treatment options have emerged from experimental research based on the pathogenic factors of stroke, including clot-busting medicines, antiplatelets, anticoagulants, and blood pressure medicines, but despite the widespread application of numerous therapeutic approaches treatments focusing on the management of the above-mentioned factors, the incidence of stroke remains high. Herbal medicines, including traditional medicines using natural products, have attracted much attention due to their significant and varied pharmacological actions and bioactive phytoconstituents without side effects or toxicity 7 , 8 . Medicinal plants, also called medicinal herbs, have been used against various diseases for thousands of years around the world. Kangen-karyu [(Guan-Yuan-Ke-Li in Chinese and developed in Japan by the alteration of medicinal plants of Kan-shin no. 2 (Guan-xin no. 2 in Chinese)], one of our major interests among traditional Chinese medicine agents, is a crude drug consisting of six herbs and has been used clinically for treating cerebrovascular diseases 9 . Previous in vivo and in vitro research results have demonstrated that Kangen-karyu exhibit lower high blood pressure, platelet aggregation inhibition, learning and memory improvement, and antidementia and neuroprotective activities 10 – 12 . These results suggest that Kangen-karyu promotes blood flow and resolves blood stasis in the brain. However, neuroprotective effect against ischemic stroke and the corresponding mechanisms of Kangen-karyu have not been. The aim of the current study was to systematically evaluate whether Kangen-karyu protects against acute brain injury following ischemia/reperfusion in mice and elucidate the mechanisms underlying Kangen-karyu activity. Materials and Methods Materials 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St. Louis, MO, USA). Dichloro-dihydro-fluorescein diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO, USA). Fluoro-Jade B was purchased from Histo-Chem Inc. (Jefferson, AR, USA). phosphor-JNK (p-JNK), p-p38, inducible NO synthase (iNOS), Bax, caspase 3, and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Preparation of Kangen-karyu extract Powdered granules of an aqueous extract of Kangen-karyu were prepared and supplied by Iskra Industry Co., Ltd. (Tokyo, Japan). Kangenkaryu was made up as follows (values refer to the ratio of each herbal medicine expressed as part-to-whole). Carthami Flos 2, Paeoniae Radix 2, Cnidii Rhizoma 2, Cyperi Rhizoma 1, Aucklandiae Radix 1, and Salviae Miltiorrhizae Radix 4, and was extracted in water with 25-fold the total amount of these six crude drugs at 100 ℃ for 1 h. The final extract obtained by filtration was evaporated in vacuo giving 44%, by weight, of the raw materials. In our previous study, the major chemical compounds of these granules were verified as: phenolic acids (rosmarinic acid, lithospermic acid, and lithospermic acid B), phenolic glycoside (pentagalloyl glucose), and monoterpene glycoside (paeoniflorin) (Fig. 6 A and B) 13 . A voucher specimen has been deposited in Hallym University. Experimental animals and surgery of ischemic stroke All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Hallym University (approval no. HallymR1 2021-95) and were performed in accordance with the relevant guidelines and regulations, including the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, the Institute for Laboratory Animal Research (ILAR) guidelines, and the Principles of Laboratory Animal Care. Eight- to nine-week-old male C57BL/6 mice of specific pathogen-free (SPF) grade were used. The mice were group-housed (four per cage) with access to commercial pellets and tap water, maintained on a 12-hour light/dark cycle, and kept at 24°C with 55 ± 5% humidity. Bilateral common carotid artery occlusion (BCCAO) was performed as previously described. Briefly, mice were anesthetized with 2.5% isoflurane, and the carotid arteries were exposed and double-ligated 14 . Sham mice underwent the same procedure without ligation. Post-surgery, mice were monitored for physical condition during recovery. Mice were randomly assigned to five groups: (1) Sham (n = 20), (2) Vehicle (n = 20), (3) Pre-Kangen-karyu (n = 20), (4) Post-Kangen-karyu (n = 20), and (5) Post-nimodipine (n = 20). Kangen-karyu was administered at a dose of 200 mg/kg body weight via a stomach tube, while the vehicle group received water. Nimodipine, administered at 30 mg/kg, served as a positive control. The dose of Kangen-karyu was based on the results of a preliminary study. TTC staining and Brain water content To evaluate and visualize the extent of tissue damage, 2 mm thick brain slices were stained with a 2% solution of 2,3,5-triphenyl tetrazolium chloride (TTC). The damaged lesions were quantified using ImageJ™ software, with the injury volume calculated using the formula: corrected injury volume = [(contralateral hemispheric volume) - (injured hemispheric volume)] / [(contralateral hemispheric volume) × 2] × 100%. Brain water content was measured through dry/wet weight determination, using the following formula: % water content = 100 × (wet weight - dry weight) / wet weight. Brain tissue was sectioned at 30 µm thickness. DCFH-DA assay ROS was measured using the method reported by Ali et al. 15 . Brain tissues were homogenized on ice with 1 mM EDTA-50 mM sodium phosphate buffer (pH 7.4), and 25 mM DCFH-DA was then added to the homogenates. After incubation for 30 min, the changes in fluorescence were determined at an excitation and emission wavelength of 486 and 530 nm, respectively. Fluoro-Jade B staining The sections were incubated in a 0.06% potassium permanganate solution for 15 minutes, then washed with distilled water for 2 minutes. Next, the slides were stained with 0.001% Fluoro-Jade B (FJB; Histo-Chem Inc., Jefferson, AR, USA) solution for 45 minutes. After two 2-minute washes in distilled water, the slides were dried overnight in the dark at room temperature and cover-slipped with D.P.X. (Sigma-Aldrich Co.). The stained tissue was examined using a fluorescence microscope with a wavelength range of 450–490 nm (Carl Zeiss, Germany). ImageJ software was used to count the number of Fluoro-Jade B-positive cells. qRT-PCR and Western blotting RNA from brain tissue was extracted using the easy-BLUE kit (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was carried out using the Maxime Oligo RT PreMix kit (iNtRON Biotechnology, Korea), following the manufacturer's protocol. PCR amplifications were performed with a Rotor-Gene Q instrument (Qiagen) using the 2X Rotor-Gene SYBR Green PCR Master Mix (Qiagen, CA, USA). The PCR cycling conditions included 36 cycles of denaturation at 94°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 70°C for 30 seconds. Brain tissue was lysed in RIPA buffer with a proteinase inhibitor. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA), and equal amounts of protein were loaded for Western blot analysis 16 . Quantification from Western blotting was performed by measuring the optical density relative to β-actin. The lists the primer sequences and antibodies used in this study (Supplemental Tables S2A and B). Cognition function test Cognitive functions were assessed through two tests: the novel object recognition (NOR) task and the Y-maze. Our experiments were conducted following the methods outlined by Youn et al. 17 . All data were collected using a video tracking system and analyzed with heat-map images (Noldus Ethovision, Leesburg, VA, USA). In these maps, red indicates areas that were visited more frequently, while blue represents areas with less frequent visits. Each experiment was conducted at least three times, and the results were reviewed by blinded analysts. Statistical analysis Data was presented as means ± standard errors of the mean (SEM). A one-way ANOVA with a post-hoc Bonferroni correction was conducted to assess all possible pairwise comparisons. Statistical significance of < 0.05, 0.01, and 0.005 are denoted by *, **, and ***, respectively. GraphPad Prism software (v.8.02; GraphPad Software Inc., San Diego, CA, USA) was used for statistics. Result Comparison of physiological indicators across groups After the induction of the BCCAO mouse model, both body weight and food intake were significantly reduced compared to the sham group. Treatment with pre-Kangen-karyu, post-Kangen-karyu, and post-nimodipine did not result in any notable changes in body weight, but other parameters, such as water and food intake, were significantly reduced (Figs. 1 B-E, Supplemental Table S1A). Reduction of ischemic brain injury and neuronal degeneration TTC staining was used to assess the extent of ischemic brain injury. TTC-stained brain slices showed a decrease in brain volume and cerebral edema throughout the brain in BCCAO-injured mice treated with post-KK compared to sham or vehicle-treated mice administered pre-KK and post-NP (Figs. 2 A-C, Supplemental Table S1B). Fluoro-Jade B staining was used to detect neuronal degeneration and death. Post-KK treatment reduced the number of FJB-positive cells more than pre-KK and post-NP treatments (Figs. 4 A and B, Supplemental Table S1D) Alleviation of inflammation and apoptosis by ROS reduction Ischemic cerebral injury is known to involve a critical ROS-based inflammatory response that contributes to neuronal damage. Therefore, the current study aimed to clarify the effect of Kangen-karyu on the brain in this context. Post-KK treatment significantly reduced the mRNA expression of SOD1 and SOD2 compared to sham or vehicle-treated mice that received pre-KK and post-NP (Figs. 3 A and B, Supplemental Table S1C). This reduction suggests that Kangen-karyu may affect the regulation of antioxidant enzymes after ischemic stroke. Additionally, DCFH-DA levels were significantly decreased in the post-KK group, indicating reduced ROS production (Fig. 3 C, Supplemental Table S1C). This suggests that Kangen-karyu treatment directly lowers elevated ROS levels following ischemic stroke. Furthermore, post-KK treatment also significantly reduced the protein expression of COX-2, iNOS, BAX, and Caspase-3 compared to sham or vehicle-treated mice that received pre-KK and post-NP (Figs. 3 D-G, Figs. 4 C-F, Supplemental Tables S2C and D). These results highlight that Kangen-karyu treatment mitigates the ROS-based inflammatory response and apoptosis associated with ischemic cerebral injury. Regulation of MAPK signaling via the JNK/p38 Pathway A potential mechanism underlying the neuroprotective effects of Kangen-karyu may involve the JNK/p38 mitogen-activated protein kinase (MAPK) pathway. To investigate this, we assessed the levels of phosphorylated JNK and p38 through Western blot analysis. The BCCAO group exhibited elevated levels of phosphorylated JNK and p38 MAPK. In contrast, post-administration of Kangen-karyu significantly reduced the levels of phosphorylated JNK and p38 MAPK compared to the BCCAO group. However, when compared to post-KK administration, the reduction in these MAPK levels was minimal in the pre-administration and post-NP groups, with the post-KK group showing a more significant effect (Figs. 2 D-G, Supplemental Table S1B). Improvement of cognitive function BCCAO mice exhibited significantly reduced preference index scores in the NOR task and reduced alteration rates in the Y-maze, indicating cognitive impairment following BCCAO. However, BCCAO mice that received post-KK treatment performed better in cognitive tests compared to those that did not receive treatment, as well as those that received pre-KK or post-NP treatment. They showed improved alteration rates in the Y-maze test and better preference index scores in the NOR test (Figs. 5 A-D, Supplemental Table S1E). Discussion Ischemic stroke is the main cause of morbidity and death worldwide, especially since ischemic stroke management therapeutic choices are inadequate and limited; thus, the development of new therapeutic options is indispensable 18 . By ischemia/reperfusion-induced damage, neurons are damaged and/or die due to ischemic stroke, followed by re-establishing blood flow to ischemic zones, aggravating brain damage and further deteriorating the condition 19 . Since the late 1970s, various animal stroke models have been developed for the purpose of identifying the mechanisms of developing new agents for ischemia therapy 20 . In 1972, the BCCAO model was first proposed by Eklöf and Siesjö in lightly anesthetized rats and has been modified on many occasions since 21 . Therefore, this study was planned to explore the neuroprotective effect of Kangen-karyu in the BCCAO model. This animal model was validated through biochemical and histological data, along with significant neurobehavioral changes. BCCAO also leads to serious neuronal cell damage. BCCAO mice increased the number of Fluoro-Jade B–stained positive cells, degenerating neurons, while Kangen-karyu-treated mice reduced the number of Fluoro-Jade B–stained positive cells. Therefore, we demonstrated that Kangen-karyu exerts neuroprotective effects against BCCAO-induced injury in an animal model. Neuronal cells are particularly vulnerable to cerebral ischemia. Neuronal injury is predominantly attributed to factors including inflammation, oxidative stress, energy failure, and excitotoxicity 22 , 23 . Neuronal degeneration and necrosis have been identified as associated with behavioral deficits 24 , 25 . The present study demonstrated that not only neuronal loss and degeneration but also infarct volume and edema were improved following treatment with Kangen-karyu. Ischemic stroke caused by various mechanisms involved in the ischemic process, and ultimately leads to neuronal cell death and damage through multiple signaling pathways 26 . In addition, brain is more vulnerable to oxidative stress than other tissues due to low levels of natural antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase, and catalase, which protect cells from damage caused by free radicles by forming a cellular defense mechanism against ROS 27 . In the present study, Kangen-karyu suppressed the brain ROS level, suggesting that Kangen-karyu offers protection against oxidative stress damage in the BCCAO-treated brain. The MAPK signaling pathway is involved in inflammatory processes as it is one of the main signaling mechanisms that regulate neuroinflammation. The activated MAPK causes the overproduction of pro-inflammatory factors 16 , 28 In this study, the phosphorylation of JNK and p38 was clearly amplified in BCCAO mice, indicating that the p-JNK and p-p38 signaling pathways were triggered. However, Kangen-karyu inhibited the phosphorylation of p-JNK and p-p38, which contributed to lowering the production as well as iNOS of pro-inflammatory factors. The critical pro-inflammatory enzyme is iNOS. During cerebral ischemia, iNOS expression elevates, leading to excessive NO production, which results in irreversible cell injury by deterring the mitochondrial respiratory chain and making peroxynitrite with superoxide anions 29 . In addition, several studies showed that ischemia-induced neurotoxicity might be alleviated by iNOS inhibitors 30 . Production of ROS affect significant effects on cellular processes, such as cell growth, apoptosis, migration, and extracellular matrix modeling. Apoptosis induces cell suicide process and, eventually, loss of function in tissues due to mitochondrial dysfunction including loss of membrane potential and the upregulation of Bax and cytochrome c 31 . Bax induces permeability and the release of cytochrome c from the intermembrane space into the intracellurar fluid. Release of cytochrome c from mitochondria is an important stage in the apoptotic cascade, and it leads to the release of downstream caspases, such as caspase 3, which have been related in the pathogenesis of tissue injury and may be blocked by antioxidants 32 . The results presented here suggest that Kangen-karyu could prevent apoptosis-induced brain injury, at least in part, through the amelioration of oxidative stress-induced dysfunction. Improving cognitive function following cerebral ischemia is crucial because cognitive impairments are one of the most debilitating consequences of brain injury, significantly affecting patients' quality of life. Early restoration of cognitive function can play a key role in overall brain recovery and long-term outcomes. In this experiment, our findings confirmed that locomotor and sensorimotor dysfunctions induced by cerebral ischemia/reperfusion were significantly improved with Kangen-karyu administration. The deteriorated performance scores of BCCAO mice in the NOR task and Y-maze showed marked improvement, suggesting that Kangen-karyu not only helps improve cognitive function following cerebral ischemia, which is a crucial aspect of brain injury recovery, but also has neuroprotective effects against BCCAO-induced behavioral and motor impairments 33 . In this study, treatment with post-Kangen-karyu showed the greatest efficacy in improving behavioral and histological findings compared to pre-Kangen-karyu and post-nimodipine treatments. Additionally, nimodipine, which is used for cerebral infarction after subarachnoid hemorrhage, showed some effectiveness, although it was similar to the pre-Kangen-karyu treatment and less effective than post-Kangen-karyu treatment. In summary, both pre-Kangen-karyu and nimodipine showed effects, but post-Kangen-karyu demonstrated the greatest efficacy. Nevertheless, several limitations of our study should be considered when interpreting the results. First, we used a global cerebral ischemia model, which differs from focal ischemia models like the MCAO model. While focal ischemia affects specific brain regions, our model induces ischemia throughout the entire brain, which may limit the generalizability of our findings. Second, the therapeutic effects of the drug were evaluated at an early stage of treatment. While early intervention is crucial, the long-term effects of Kangen-karyu on ischemic stroke remain unclear and require further investigation. Additionally, our study only used male mice, which excludes the potential influence of estrogen on the results. Future research should account for gender differences, as these may affect treatment efficacy. Third, Kangen-karyu is a complex formulation made from multiple herbs that interact with one another to produce therapeutic effects. Drug studies typically assess the effects of individual compounds, but the interactions among the ingredients in a complex formulation make it challenging to attribute the observed effects to a single component. Therefore, further research is needed to determine how each ingredient contributes to the overall therapeutic outcome. Finally, ischemic stroke accounts for about 87% of all stroke cases and is associated with high morbidity and mortality rates. In our study, we observed significant changes in physiological indicators following ischemic stroke, which could pose a life-threatening risk (Figs. 1 B-E, Supplementary Table S1A). These changes are closely associated with mortality. Therefore, further studies are needed to determine whether Kangen-karyu can reduce the mortality rate associated with ischemic stroke, and additional clinical research, particularly on ischemic stroke patients, is essential to validate these findings. Conclusions Our study revealed the neuroprotective effects of Kangen-karyu in the BCCAO model of ischemic stroke. Kangen-karyu alleviated behavioral impairments, reduced neuronal damage, and modulated oxidative stress, inflammation, and apoptosis through the regulation of ROS-mediated MAPK signaling (Fig. 7 ). These findings suggest that Kangen-karyu may serve as a potential therapeutic agent for the prevention and mitigation of ischemic stroke-induced brain injury, contributing to the scientific body of research on Kangen-karyu, a key compound in traditional Chinese medicine. Declarations Author Contributions Conceptualization, D.H.Y., C.H.P. and J.P.J.; methodology, S.W.H., S.W.P., J.H.L., and D.H.Y.; animal ischemic stroke surgery, S.W.H., J.H.L., and D.H.Y.; investigation, J.T.K., J.H.K., S.W.P., H.Y.C., J.S.C., J.D.L., E.H.L., K.S., and D.H.Y.; writing—original draft preparation, D.H.Y. and C.H.P.; writing—review and editing, D.H.Y., J.P.J., and C.H.P.; funding acquisition, G.S.H., Y.A., M.S., M.O., J.P.J., and C.H.P. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by the Japan Association for Study of Chinese Traditional Medicines, Hallym University Research Fund, and this research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2022-NR071171) Institutional Review Board Statement All experiments were approved by the Institutional Animal Care and Use Committee of the participating university (approval no. HallymR1 2021-95) Informed Consent Statement Not applicable Data Availability Statement The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding author. Acknowledgments The authors are grateful to the Prof. Takako Yokozawa at Graduate School of Science and Engineering for Research, University of Toyama, Toyama, Japan, for their assistance with Experimental Design. Conflicts of interest All authors declare no competing interests. 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09:08:23\",\"extension\":\"png\",\"order_by\":22,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":97934,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Onlinefloatimage9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/4a6d3a0496cdc21eeaf3d8d2.png\"},{\"id\":100566133,\"identity\":\"fd12a419-1b7c-4ae9-8b04-a3a3ffdd08b5\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 09:08:24\",\"extension\":\"xml\",\"order_by\":23,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":100724,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"4cb3bfbcf474419885972bd54cd86c311structuring.xml\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/bf41f17b8e1220f9f24dfdc5.xml\"},{\"id\":100595311,\"identity\":\"7d716f48-338d-431c-9ce1-0bf21743daf2\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 13:48:11\",\"extension\":\"html\",\"order_by\":24,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":118466,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"earlyproof.html\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/4c0027407642e98df25bf0c3.html\"},{\"id\":100566092,\"identity\":\"1dbdf5c2-97a1-4f55-b07e-ba0ac7478da1\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 09:08:23\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":187544,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExperimental design \\u003cstrong\\u003e(A)\\u003c/strong\\u003e and examination of survival rate \\u003cstrong\\u003e(B)\\u003c/strong\\u003e, body weight \\u003cstrong\\u003e(C)\\u003c/strong\\u003e, water intake \\u003cstrong\\u003e(D)\\u003c/strong\\u003e, and food intake \\u003cstrong\\u003e(E)\\u003c/strong\\u003e. All animal experiments were conducted with n=9 per group. Error bars, mean ± SEM, *P\\u0026lt;0.05, ** P\\u0026lt;0.01, and *** P\\u0026lt;0.005. KK indicates kangen-karyu.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/489cb0831aaeb34e2524bd01.jpg\"},{\"id\":100566150,\"identity\":\"f65bb791-dc84-44c5-b082-a680240fa834\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 09:08:24\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":676790,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eOptical visible image of TTC staining \\u003cstrong\\u003e(A and B, n=4)\\u003c/strong\\u003e, brain water content \\u003cstrong\\u003e(C, n=4)\\u003c/strong\\u003e. Western blotting of BAX and Caspase 3 according to KK treatment \\u003cstrong\\u003e(D-G, n=6)\\u003c/strong\\u003e. The result was confirmed in mouse brains. Error bars, mean ± SEM, *P\\u0026lt;0.05, ** P\\u0026lt;0.01, and *** P\\u0026lt;0.005. KK indicates kangen-karyu.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/ed49e6c2eb784f107e714cf6.png\"},{\"id\":100595847,\"identity\":\"b5a8b60d-2a50-4f06-80f1-888db5770760\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 13:49:33\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":427774,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDifferences in mRNA experssion \\u003cstrong\\u003e(A and B)\\u003c/strong\\u003e, DCFH-DA fluorescence intensity \\u003cstrong\\u003e(C)\\u003c/strong\\u003e, and Western blotting of COX-2, and iNOS according to KK treatment \\u003cstrong\\u003e(D-G)\\u003c/strong\\u003e. All animal experiments were conducted with n=6 per group. The result was confirmed in mouse brains. Error bars, mean ± SEM, *P\\u0026lt;0.05, ** P\\u0026lt;0.01, and *** P\\u0026lt;0.005.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/6cf8a77e796f25aeb8f36dc4.png\"},{\"id\":100595171,\"identity\":\"ee1b054e-1668-446d-841f-cab72bf02015\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 13:47:45\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":487790,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRepresentative images showing FJB-positive cells in CA1, CA3, and DG regions of the brain \\u003cstrong\\u003e(A-B)\\u003c/strong\\u003e. Western blotting of p-JNK and p-p38 according to KK treatment \\u003cstrong\\u003e(C-F)\\u003c/strong\\u003e. All animal experiments were conducted with n=6 per group. The result was confirmed in mouse brains. Scale bar is 200 μm. Error bars, mean ± SEM, *P\\u0026lt;0.05, ** P\\u0026lt;0.01, and *** P\\u0026lt;0.005. CA1 indicates corna ammonis 1; CA3, corna ammonis 3; DG, dentate gyrus.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/9caf1704f501ca24fb7c6947.png\"},{\"id\":100566085,\"identity\":\"880ee8f3-85b8-462e-a778-09dea3826047\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 09:08:23\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":394512,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCognitive function test results (NOR and Y-maze) following pre- and post-KK administration in a mouse model of BCCAO \\u003cstrong\\u003e(A and D)\\u003c/strong\\u003e. All animal experiments were conducted with n=6 per group. Error bars, mean ± SEM, *P\\u0026lt;0.05, ** P\\u0026lt;0.01, and *** P\\u0026lt;0.005. NOR indicates novel objection recognition.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/c87bd58d09cf934f9aa30f0d.png\"},{\"id\":100566079,\"identity\":\"8cfca30d-74e2-4443-bafe-ad88ca3854f3\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 09:08:22\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":93918,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eHigh-performance liquid chromatography (HPLC) of Kangen-karyu extract showing its major compounds \\u003cstrong\\u003e(A)\\u003c/strong\\u003e. Chemical structures of 3-(3,4-dihydroxyphenyl)-lactic acid, paeoniflorin, rosmarinic acid, lithospermic acid, and lithospermic acid B \\u003cstrong\\u003e(B)\\u003c/strong\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/68be958d492b6f7476a77fb6.jpg\"},{\"id\":100595492,\"identity\":\"cab9daa8-f5ca-4663-831e-8a40410aaf66\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 13:48:36\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":398313,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMAPK signaling in brain tissue following pre-KK, post-KK, and post-NP treatments in response to BCCAO-induced ischemic stroke.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/f95ba24c07fd30da5b59d3f9.png\"},{\"id\":100803993,\"identity\":\"1c3c9a52-3e68-437f-93ef-b46f981fc1e3\",\"added_by\":\"auto\",\"created_at\":\"2026-01-21 14:33:41\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3336289,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/5db32d3b-932a-42c6-987b-890a3317b237.pdf\"},{\"id\":100566129,\"identity\":\"6fdbe1b3-7666-4b92-bfcb-47a4bd493a46\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 09:08:24\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":952874,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SUPPLEMENTALDATA.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8423120/v1/647bc6f958da1f7d7724498d.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Chinese Prescription Kangen-karyu Attenuates Neuronal Damage and Improves Cognitive Function in Ischemic Stroke by Regulating ROS-Mediated MAPK Activation\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eStroke is a serious, life-threatening illness that occurs when the blood supply to part of the brain is blocked, making it is a medical emergency that requires treatment necessitates. Like all organs, the brain needs oxygen and nutrients from the blood to function properly. There are two main causes of stroke: strokes caused by blockage of an artery (ischemic) and an artery in the brain leaks or bursts (hemorrhagic stroke). Most strokes are ischemic stroke. This occurs when blood vessels in the brain become narrowed or blocked, severely reducing blood flow. Blockage or narrowing of blood vessels is caused by fatty deposits or blood clots that build up in the blood vessels \\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR2\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eMany factors can increase the risk of stroke: unhealthy lifestyle, being overweight or obese, lack of exercise, binge or heavy drinking, and illicit drug use. In addition, medical risk points (hypertension, cigarette or passive smoking, high blood cholesterol, diabetes, obstructive sleep apnea, cardiovascular disease, transient ischemic attack, heart attack, and family or personal medical history of stroke) are mentioned. Other risk factors that contribute to causing a stroke include: race or ethnicity, sex, hormones, and age \\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR5\\\" citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. Many novel treatment options have emerged from experimental research based on the pathogenic factors of stroke, including clot-busting medicines, antiplatelets, anticoagulants, and blood pressure medicines, but despite the widespread application of numerous therapeutic approaches treatments focusing on the management of the above-mentioned factors, the incidence of stroke remains high.\\u003c/p\\u003e \\u003cp\\u003eHerbal medicines, including traditional medicines using natural products, have attracted much attention due to their significant and varied pharmacological actions and bioactive phytoconstituents without side effects or toxicity \\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. Medicinal plants, also called medicinal herbs, have been used against various diseases for thousands of years around the world. Kangen-karyu [(Guan-Yuan-Ke-Li in Chinese and developed in Japan by the alteration of medicinal plants of Kan-shin no. 2 (Guan-xin no. 2 in Chinese)], one of our major interests among traditional Chinese medicine agents, is a crude drug consisting of six herbs and has been used clinically for treating cerebrovascular diseases \\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e. Previous \\u003cem\\u003ein vivo\\u003c/em\\u003e and \\u003cem\\u003ein vitro\\u003c/em\\u003e research results have demonstrated that Kangen-karyu exhibit lower high blood pressure, platelet aggregation inhibition, learning and memory improvement, and antidementia and neuroprotective activities \\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR11\\\" citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. These results suggest that Kangen-karyu promotes blood flow and resolves blood stasis in the brain. However, neuroprotective effect against ischemic stroke and the corresponding mechanisms of Kangen-karyu have not been.\\u003c/p\\u003e \\u003cp\\u003eThe aim of the current study was to systematically evaluate whether Kangen-karyu protects against acute brain injury following ischemia/reperfusion in mice and elucidate the mechanisms underlying Kangen-karyu activity.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMaterials\\u003c/h2\\u003e \\u003cp\\u003e2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St. Louis, MO, USA). Dichloro-dihydro-fluorescein diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO, USA). Fluoro-Jade B was purchased from Histo-Chem Inc. (Jefferson, AR, USA). phosphor-JNK (p-JNK), p-p38, inducible NO synthase (iNOS), Bax, caspase 3, and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003ePreparation of Kangen-karyu extract\\u003c/h3\\u003e\\n \\u003cp\\u003ePowdered granules of an aqueous extract of Kangen-karyu were prepared and supplied by Iskra Industry Co., Ltd. (Tokyo, Japan). Kangenkaryu was made up as follows (values refer to the ratio of each herbal medicine expressed as part-to-whole). Carthami Flos 2, Paeoniae Radix 2, Cnidii Rhizoma 2, Cyperi Rhizoma 1, Aucklandiae Radix 1, and Salviae Miltiorrhizae Radix 4, and was extracted in water with 25-fold the total amount of these six crude drugs at 100 ℃ for 1 h. The final extract obtained by filtration was evaporated \\u003cem\\u003ein vacuo\\u003c/em\\u003e giving 44%, by weight, of the raw materials. In our previous study, the major chemical compounds of these granules were verified as: phenolic acids (rosmarinic acid, lithospermic acid, and lithospermic acid B), phenolic glycoside (pentagalloyl glucose), and monoterpene glycoside (paeoniflorin) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA and B) \\u003csup\\u003e13\\u003c/sup\\u003e. A voucher specimen has been deposited in Hallym University.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eExperimental animals and surgery of ischemic stroke\\u003c/h3\\u003e\\n\\u003cp\\u003e All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Hallym University (approval no. HallymR1 2021-95) and were performed in accordance with the relevant guidelines and regulations, including the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, the Institute for Laboratory Animal Research (ILAR) guidelines, and the Principles of Laboratory Animal Care. Eight- to nine-week-old male C57BL/6 mice of specific pathogen-free (SPF) grade were used. The mice were group-housed (four per cage) with access to commercial pellets and tap water, maintained on a 12-hour light/dark cycle, and kept at 24°C with 55 ± 5% humidity. Bilateral common carotid artery occlusion (BCCAO) was performed as previously described. Briefly, mice were anesthetized with 2.5% isoflurane, and the carotid arteries were exposed and double-ligated \\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. Sham mice underwent the same procedure without ligation. Post-surgery, mice were monitored for physical condition during recovery. Mice were randomly assigned to five groups: (1) Sham (n = 20), (2) Vehicle (n = 20), (3) Pre-Kangen-karyu (n = 20), (4) Post-Kangen-karyu (n = 20), and (5) Post-nimodipine (n = 20). Kangen-karyu was administered at a dose of 200 mg/kg body weight via a stomach tube, while the vehicle group received water. Nimodipine, administered at 30 mg/kg, served as a positive control. The dose of Kangen-karyu was based on the results of a preliminary study.\\u003c/p\\u003e\\n\\u003ch3\\u003eTTC staining and Brain water content\\u003c/h3\\u003e\\n\\u003cp\\u003eTo evaluate and visualize the extent of tissue damage, 2 mm thick brain slices were stained with a 2% solution of 2,3,5-triphenyl tetrazolium chloride (TTC). The damaged lesions were quantified using ImageJ™ software, with the injury volume calculated using the formula: corrected injury volume = [(contralateral hemispheric volume) - (injured hemispheric volume)] / [(contralateral hemispheric volume) × 2] × 100%. Brain water content was measured through dry/wet weight determination, using the following formula: % water content = 100 × (wet weight - dry weight) / wet weight. Brain tissue was sectioned at 30 µm thickness.\\u003c/p\\u003e\\n\\u003ch3\\u003eDCFH-DA assay\\u003c/h3\\u003e\\n\\u003cp\\u003eROS was measured using the method reported by Ali et al. \\u003csup\\u003e15\\u003c/sup\\u003e. Brain tissues were homogenized on ice with 1 mM EDTA-50 mM sodium phosphate buffer (pH 7.4), and 25 mM DCFH-DA was then added to the homogenates. After incubation for 30 min, the changes in fluorescence were determined at an excitation and emission wavelength of 486 and 530 nm, respectively.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFluoro-Jade B staining\\u003c/h2\\u003e \\u003cp\\u003eThe sections were incubated in a 0.06% potassium permanganate solution for 15 minutes, then washed with distilled water for 2 minutes. Next, the slides were stained with 0.001% Fluoro-Jade B (FJB; Histo-Chem Inc., Jefferson, AR, USA) solution for 45 minutes. After two 2-minute washes in distilled water, the slides were dried overnight in the dark at room temperature and cover-slipped with D.P.X. (Sigma-Aldrich Co.). The stained tissue was examined using a fluorescence microscope with a wavelength range of 450–490 nm (Carl Zeiss, Germany). ImageJ software was used to count the number of Fluoro-Jade B-positive cells.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eqRT-PCR and Western blotting\\u003c/h3\\u003e\\n \\u003cp\\u003eRNA from brain tissue was extracted using the easy-BLUE kit (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was carried out using the Maxime Oligo RT PreMix kit (iNtRON Biotechnology, Korea), following the manufacturer's protocol. PCR amplifications were performed with a Rotor-Gene Q instrument (Qiagen) using the 2X Rotor-Gene SYBR Green PCR Master Mix (Qiagen, CA, USA). The PCR cycling conditions included 36 cycles of denaturation at 94°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 70°C for 30 seconds. Brain tissue was lysed in RIPA buffer with a proteinase inhibitor. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA), and equal amounts of protein were loaded for Western blot analysis \\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e. Quantification from Western blotting was performed by measuring the optical density relative to β-actin. The lists the primer sequences and antibodies used in this study (Supplemental Tables S2A and B).\\u003c/p\\u003e\\n\\u003ch3\\u003eCognition function test\\u003c/h3\\u003e\\n\\u003cp\\u003eCognitive functions were assessed through two tests: the novel object recognition (NOR) task and the Y-maze. Our experiments were conducted following the methods outlined by Youn et al. \\u003csup\\u003e17\\u003c/sup\\u003e. All data were collected using a video tracking system and analyzed with heat-map images (Noldus Ethovision, Leesburg, VA, USA). In these maps, red indicates areas that were visited more frequently, while blue represents areas with less frequent visits. Each experiment was conducted at least three times, and the results were reviewed by blinded analysts.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eData was presented as means ± standard errors of the mean (SEM). A one-way ANOVA with a post-hoc Bonferroni correction was conducted to assess all possible pairwise comparisons. Statistical significance of \\u0026lt; 0.05, 0.01, and 0.005 are denoted by *, **, and ***, respectively. GraphPad Prism software (v.8.02; GraphPad Software Inc., San Diego, CA, USA) was used for statistics.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section3\\\"\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Result\",\"content\":\"\\u003ch2\\u003eComparison of physiological indicators across groups\\u003c/h2\\u003e\\u003cp\\u003eAfter the induction of the BCCAO mouse model, both body weight and food intake were significantly reduced compared to the sham group. Treatment with pre-Kangen-karyu, post-Kangen-karyu, and post-nimodipine did not result in any notable changes in body weight, but other parameters, such as water and food intake, were significantly reduced (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB-E, Supplemental Table S1A).\\u003c/p\\u003e\\u003cp\\u003e \\u003c/p\\u003e\\u003ch2\\u003eReduction of ischemic brain injury and neuronal degeneration\\u003c/h2\\u003e\\u003cp\\u003eTTC staining was used to assess the extent of ischemic brain injury. TTC-stained brain slices showed a decrease in brain volume and cerebral edema throughout the brain in BCCAO-injured mice treated with post-KK compared to sham or vehicle-treated mice administered pre-KK and post-NP (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA-C, Supplemental Table S1B). Fluoro-Jade B staining was used to detect neuronal degeneration and death. Post-KK treatment reduced the number of FJB-positive cells more than pre-KK and post-NP treatments (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA and B, Supplemental Table S1D)\\u003c/p\\u003e\\u003cp\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003c/p\\u003e\\u003ch2\\u003eAlleviation of inflammation and apoptosis by ROS reduction\\u003c/h2\\u003e\\u003cp\\u003eIschemic cerebral injury is known to involve a critical ROS-based inflammatory response that contributes to neuronal damage. Therefore, the current study aimed to clarify the effect of Kangen-karyu on the brain in this context. Post-KK treatment significantly reduced the mRNA expression of SOD1 and SOD2 compared to sham or vehicle-treated mice that received pre-KK and post-NP (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA and B, Supplemental Table S1C). This reduction suggests that Kangen-karyu may affect the regulation of antioxidant enzymes after ischemic stroke. Additionally, DCFH-DA levels were significantly decreased in the post-KK group, indicating reduced ROS production (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC, Supplemental Table S1C). This suggests that Kangen-karyu treatment directly lowers elevated ROS levels following ischemic stroke. Furthermore, post-KK treatment also significantly reduced the protein expression of COX-2, iNOS, BAX, and Caspase-3 compared to sham or vehicle-treated mice that received pre-KK and post-NP (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD-G, Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC-F, Supplemental Tables S2C and D). These results highlight that Kangen-karyu treatment mitigates the ROS-based inflammatory response and apoptosis associated with ischemic cerebral injury.\\u003c/p\\u003e\\u003cp\\u003e \\u003c/p\\u003e\\u003ch2\\u003eRegulation of MAPK signaling via the JNK/p38 Pathway\\u003c/h2\\u003e\\u003cp\\u003eA potential mechanism underlying the neuroprotective effects of Kangen-karyu may involve the JNK/p38 mitogen-activated protein kinase (MAPK) pathway. To investigate this, we assessed the levels of phosphorylated JNK and p38 through Western blot analysis. The BCCAO group exhibited elevated levels of phosphorylated JNK and p38 MAPK. In contrast, post-administration of Kangen-karyu significantly reduced the levels of phosphorylated JNK and p38 MAPK compared to the BCCAO group. However, when compared to post-KK administration, the reduction in these MAPK levels was minimal in the pre-administration and post-NP groups, with the post-KK group showing a more significant effect (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD-G, Supplemental Table S1B).\\u003c/p\\u003e\\u003ch2\\u003eImprovement of cognitive function\\u003c/h2\\u003e\\u003cp\\u003eBCCAO mice exhibited significantly reduced preference index scores in the NOR task and reduced alteration rates in the Y-maze, indicating cognitive impairment following BCCAO. However, BCCAO mice that received post-KK treatment performed better in cognitive tests compared to those that did not receive treatment, as well as those that received pre-KK or post-NP treatment. They showed improved alteration rates in the Y-maze test and better preference index scores in the NOR test (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA-D, Supplemental Table S1E).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIschemic stroke is the main cause of morbidity and death worldwide, especially since ischemic stroke management therapeutic choices are inadequate and limited; thus, the development of new therapeutic options is indispensable \\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e. By ischemia/reperfusion-induced damage, neurons are damaged and/or die due to ischemic stroke, followed by re-establishing blood flow to ischemic zones, aggravating brain damage and further deteriorating the condition \\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. Since the late 1970s, various animal stroke models have been developed for the purpose of identifying the mechanisms of developing new agents for ischemia therapy \\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. In 1972, the BCCAO model was first proposed by Ekl\\u0026ouml;f and Siesj\\u0026ouml; in lightly anesthetized rats and has been modified on many occasions since \\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, this study was planned to explore the neuroprotective effect of Kangen-karyu in the BCCAO model.\\u003c/p\\u003e \\u003cp\\u003eThis animal model was validated through biochemical and histological data, along with significant neurobehavioral changes. BCCAO also leads to serious neuronal cell damage. BCCAO mice increased the number of Fluoro-Jade B\\u0026ndash;stained positive cells, degenerating neurons, while Kangen-karyu-treated mice reduced the number of Fluoro-Jade B\\u0026ndash;stained positive cells. Therefore, we demonstrated that Kangen-karyu exerts neuroprotective effects against BCCAO-induced injury in an animal model.\\u003c/p\\u003e \\u003cp\\u003eNeuronal cells are particularly vulnerable to cerebral ischemia. Neuronal injury is predominantly attributed to factors including inflammation, oxidative stress, energy failure, and excitotoxicity \\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. Neuronal degeneration and necrosis have been identified as associated with behavioral deficits \\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e. The present study demonstrated that not only neuronal loss and degeneration but also infarct volume and edema were improved following treatment with Kangen-karyu.\\u003c/p\\u003e \\u003cp\\u003eIschemic stroke caused by various mechanisms involved in the ischemic process, and ultimately leads to neuronal cell death and damage through multiple signaling pathways \\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. In addition, brain is more vulnerable to oxidative stress than other tissues due to low levels of natural antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase, and catalase, which protect cells from damage caused by free radicles by forming a cellular defense mechanism against ROS \\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. In the present study, Kangen-karyu suppressed the brain ROS level, suggesting that Kangen-karyu offers protection against oxidative stress damage in the BCCAO-treated brain.\\u003c/p\\u003e \\u003cp\\u003eThe MAPK signaling pathway is involved in inflammatory processes as it is one of the main signaling mechanisms that regulate neuroinflammation. The activated MAPK causes the overproduction of pro-inflammatory factors \\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e In this study, the phosphorylation of JNK and p38 was clearly amplified in BCCAO mice, indicating that the p-JNK and p-p38 signaling pathways were triggered. However, Kangen-karyu inhibited the phosphorylation of p-JNK and p-p38, which contributed to lowering the production as well as iNOS of pro-inflammatory factors. The critical pro-inflammatory enzyme is iNOS. During cerebral ischemia, iNOS expression elevates, leading to excessive NO production, which results in irreversible cell injury by deterring the mitochondrial respiratory chain and making peroxynitrite with superoxide anions \\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. In addition, several studies showed that ischemia-induced neurotoxicity might be alleviated by iNOS inhibitors \\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eProduction of ROS affect significant effects on cellular processes, such as cell growth, apoptosis, migration, and extracellular matrix modeling. Apoptosis induces cell suicide process and, eventually, loss of function in tissues due to mitochondrial dysfunction including loss of membrane potential and the upregulation of Bax and cytochrome \\u003cem\\u003ec\\u003c/em\\u003e \\u003csup\\u003e31\\u003c/sup\\u003e. Bax induces permeability and the release of cytochrome \\u003cem\\u003ec\\u003c/em\\u003e from the intermembrane space into the intracellurar fluid. Release of cytochrome \\u003cem\\u003ec\\u003c/em\\u003e from mitochondria is an important stage in the apoptotic cascade, and it leads to the release of downstream caspases, such as caspase 3, which have been related in the pathogenesis of tissue injury and may be blocked by antioxidants \\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. The results presented here suggest that Kangen-karyu could prevent apoptosis-induced brain injury, at least in part, through the amelioration of oxidative stress-induced dysfunction.\\u003c/p\\u003e \\u003cp\\u003eImproving cognitive function following cerebral ischemia is crucial because cognitive impairments are one of the most debilitating consequences of brain injury, significantly affecting patients' quality of life. Early restoration of cognitive function can play a key role in overall brain recovery and long-term outcomes. In this experiment, our findings confirmed that locomotor and sensorimotor dysfunctions induced by cerebral ischemia/reperfusion were significantly improved with Kangen-karyu administration. The deteriorated performance scores of BCCAO mice in the NOR task and Y-maze showed marked improvement, suggesting that Kangen-karyu not only helps improve cognitive function following cerebral ischemia, which is a crucial aspect of brain injury recovery, but also has neuroprotective effects against BCCAO-induced behavioral and motor impairments \\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eIn this study, treatment with post-Kangen-karyu showed the greatest efficacy in improving behavioral and histological findings compared to pre-Kangen-karyu and post-nimodipine treatments.\\u003c/p\\u003e \\u003cp\\u003eAdditionally, nimodipine, which is used for cerebral infarction after subarachnoid hemorrhage, showed some effectiveness, although it was similar to the pre-Kangen-karyu treatment and less effective than post-Kangen-karyu treatment. In summary, both pre-Kangen-karyu and nimodipine showed effects, but post-Kangen-karyu demonstrated the greatest efficacy.\\u003c/p\\u003e \\u003cp\\u003eNevertheless, several limitations of our study should be considered when interpreting the results. First, we used a global cerebral ischemia model, which differs from focal ischemia models like the MCAO model. While focal ischemia affects specific brain regions, our model induces ischemia throughout the entire brain, which may limit the generalizability of our findings. Second, the therapeutic effects of the drug were evaluated at an early stage of treatment. While early intervention is crucial, the long-term effects of Kangen-karyu on ischemic stroke remain unclear and require further investigation. Additionally, our study only used male mice, which excludes the potential influence of estrogen on the results. Future research should account for gender differences, as these may affect treatment efficacy. Third, Kangen-karyu is a complex formulation made from multiple herbs that interact with one another to produce therapeutic effects. Drug studies typically assess the effects of individual compounds, but the interactions among the ingredients in a complex formulation make it challenging to attribute the observed effects to a single component. Therefore, further research is needed to determine how each ingredient contributes to the overall therapeutic outcome. Finally, ischemic stroke accounts for about 87% of all stroke cases and is associated with high morbidity and mortality rates. In our study, we observed significant changes in physiological indicators following ischemic stroke, which could pose a life-threatening risk (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB-E, Supplementary Table S1A). These changes are closely associated with mortality. Therefore, further studies are needed to determine whether Kangen-karyu can reduce the mortality rate associated with ischemic stroke, and additional clinical research, particularly on ischemic stroke patients, is essential to validate these findings.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eOur study revealed the neuroprotective effects of Kangen-karyu in the BCCAO model of ischemic stroke. Kangen-karyu alleviated behavioral impairments, reduced neuronal damage, and modulated oxidative stress, inflammation, and apoptosis through the regulation of ROS-mediated MAPK signaling (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). These findings suggest that Kangen-karyu may serve as a potential therapeutic agent for the prevention and mitigation of ischemic stroke-induced brain injury, contributing to the scientific body of research on Kangen-karyu, a key compound in traditional Chinese medicine.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization, D.H.Y., C.H.P. and J.P.J.; methodology, S.W.H., S.W.P., J.H.L., and D.H.Y.; animal ischemic stroke surgery, S.W.H., J.H.L., and D.H.Y.; investigation, J.T.K., J.H.K., S.W.P., H.Y.C., J.S.C., J.D.L., E.H.L., K.S., and D.H.Y.; writing—original draft preparation, D.H.Y. and C.H.P.; writing—review and editing, D.H.Y., J.P.J., and C.H.P.; funding acquisition, G.S.H., Y.A., M.S., M.O., J.P.J., and C.H.P. All authors have read and agreed to the published version of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the Japan Association for Study of Chinese Traditional Medicines, Hallym University Research Fund, and this research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2022-NR071171)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInstitutional Review Board Statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll experiments were approved by the Institutional Animal Care and Use Committee of the participating university (approval no. HallymR1 2021-95)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInformed Consent Statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability Statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding author.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors are grateful to the Prof. Takako Yokozawa at Graduate School of Science and Engineering for Research, University of Toyama, Toyama, Japan, for their assistance with Experimental Design.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflicts of interest\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors declare no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eFeigin, V. 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Comput.\\u003c/em\\u003e \\u003cb\\u003e33\\u003c/b\\u003e, 398\\u0026ndash;414. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.3758/bf03195394\\u003c/span\\u003e\\u003cspan address=\\\"10.3758/bf03195394\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e (2001).\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Kangen-karyu, Cerebral ischemia/reperfusion, JNK/p38, neuroprotective, cognition\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8423120/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8423120/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eOBJECTIVE\\u003c/h2\\u003e \\u003cp\\u003eIschemic stroke is a leading cause of morbidity and mortality worldwide, largely due to the limited and inadequate therapeutic options currently available. As a result, developing new therapeutic approaches is crucial. This study aims to investigate the potential therapeutic effects of Kangen-karyu (KK), a compound that has garnered significant attention for its diverse biological functions, in mitigating brain damage induced by cerebral ischemia/reperfusion.\\u003c/p\\u003e\\u003ch2\\u003eMETHODS\\u003c/h2\\u003e \\u003cp\\u003eSeven to eight-week-old male C57BL/6J mice underwent either sham surgery or bilateral common carotid artery occlusion (BCCAO) for 10 minutes, followed by reperfusion to induce cerebral ischemia/reperfusion injury. The mice were treated orally with either a vehicle, KK (200 mg/kg), or nimodipine (NP, 30 mg/kg), administered 2 hours before (Pre-KK) or after (Post-KK, Post-NP) BCCAO. The neurobehavioral performance of the mice was assessed. Additionally, histopathological changes, oxidative stress, inflammation, and apoptotic parameters were evaluated in brain tissues.\\u003c/p\\u003e\\u003ch2\\u003eRESULTS\\u003c/h2\\u003e \\u003cp\\u003eInfarct volume, brain water content, DHE, and Fluoro-Jade B staining confirmed that post-KK significantly ameliorated BCCAO-induced histopathological damage and neurobehavioral deficits compared to the pre-KK and post-NP groups. Additionally, administration of post-KK reduced the protein expression levels of phospho-c-Jun N-terminal kinase (p-JNK), phospho-p38, inducible nitric oxide synthase (iNOS), Bax, and caspase 3. In the cognition tests, BCCAO showed a decreased preference index and an increased alteration rate, which together indicate improved cognitive performance after post-KK.\\u003c/p\\u003e\\u003ch2\\u003eCONCLUSION\\u003c/h2\\u003e \\u003cp\\u003eOur study suggests that Kangen-karyu (KK) exerts a neuroprotective effect by reducing oxidative stress, inflammation, and apoptosis via the JNK/p38 pathway, indicating its potential as a therapeutic candidate for mitigating ischemia/reperfusion-induced brain damage.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Chinese Prescription Kangen-karyu Attenuates Neuronal Damage and Improves Cognitive Function in Ischemic Stroke by Regulating ROS-Mediated MAPK Activation\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-01-19 09:08:07\",\"doi\":\"10.21203/rs.3.rs-8423120/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-01-20T01:00:45+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-01-19T09:25:43+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"311623288901716442816008507899921092607\",\"date\":\"2026-01-15T09:49:35+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"323773878736201485751697255417560601209\",\"date\":\"2026-01-15T08:31:08+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-01-15T06:39:47+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"104439182722155184903342306955690289513\",\"date\":\"2026-01-15T06:14:09+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-01-14T15:49:38+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"290094190344601518028968598377908701139\",\"date\":\"2026-01-14T14:59:43+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"224649383639045248726153406066754575420\",\"date\":\"2026-01-14T14:41:29+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"118504436872461299337073754294804873900\",\"date\":\"2026-01-14T14:36:32+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"201589660152727728538797524595038241024\",\"date\":\"2026-01-14T08:37:25+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-01-14T07:51:47+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-01-14T07:48:45+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2026-01-07T21:32:25+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-01-03T03:37:12+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2026-01-03T03:32:03+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"07231251-91cd-45f2-a518-bd028a814cb6\",\"owner\":[],\"postedDate\":\"January 19th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"in-revision\",\"subjectAreas\":[{\"id\":61278156,\"name\":\"Health sciences/Diseases\"},{\"id\":61278157,\"name\":\"Biological sciences/Drug discovery\"},{\"id\":61278158,\"name\":\"Health sciences/Medical research\"},{\"id\":61278159,\"name\":\"Health sciences/Neurology\"},{\"id\":61278160,\"name\":\"Biological sciences/Neuroscience\"}],\"tags\":[],\"updatedAt\":\"2026-01-20T01:11:18+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-01-19 09:08:07\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8423120\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8423120\",\"identity\":\"rs-8423120\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}