Moxibustion Pretreatment Attenuate epileptic seizures by mediation P2X7 receptor

Moxibustion Pretreatment Attenuate epileptic seizures by mediation P2X7 receptor | 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 Moxibustion Pretreatment Attenuate epileptic seizures by mediation P2X7 receptor Hong-Wei Zhang, Jia-Jia Li, YuLiHan Tang, Mao-Lin Tian, Sheng Huang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4672161/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Sep, 2024 Read the published version in Purinergic Signalling → Version 1 posted 8 You are reading this latest preprint version Abstract The P2X7 receptor (P2X7R) is involved in long-term brain hyperexcitability driven by epilepsy and inflammation. It is currently unclear whether moxibustion pretreatment by the purine signaling pathway interference in P2X7R is involved in the mechanism of epilepsy treatment. Male C57BL/6 mice received moxibustion pretreatments at the acupoints of Zusanli (ST36) and Dazhui (GV14) once a day for 7, 14, or 21 days and immediately after an intraperitoneal injection of KA (30 mg/kg) and recording the behavior changes follow the Racine scales. Moxibustion pretreatment stimulation at ST36 and GV14 of 14 or 21 days significantly reduced the KA-induced seizure. In addition, there was no difference between treatment 14 or 21 days in epileptic mice. Then, we aimed to investigate the role of P2X7R in epileptogenesis in vivo using agonist (BzATP) or inhibitor (A438079) to influence the expression of this receptor. P2X7R agonist BzATP can aggravate seizure. We found moxibustion pretreatment can attenuate epileptic seizures via confronting BzATP. The result implied that Moxibustion pretreatment at ST36 and GV14 acupoints for 14 days has anti-epileptic effects, which may be related to inflammation mediated by P2X7 receptors. Moxibustion P2X7 purinergic receptors kainic acid mice epileptic seizures Figures Figure 1 Figure 2 Figure 3 Introduction Epilepsy is a common chronic brain disease characterized by a long-lasting predisposition to recurrent epileptic seizures [ 1 ]. Research estimates the point prevalence of active epilepsy in the world (as of Feb 2023)to be about 51 million people, and about 4.9 million people develop new-onset epilepsy every year [ 2 ]. Seizures are due to an imbalance of excitatory and inhibitory neuronal firing, which is closely related to abnormal ion channel function, imbalance of neurotransmitter secretion, irregular glial system, neuroinflammation and oxidative stress, and abnormal neural circuits [ 3 ]. However, the specific pathogenesis of epilepsy is not entirely clear. More and more studies have focused on the crucial role of purine signaling ATP-gated purinergic P2X7 receptor (P2X7R) mediated neuroinflammatory response in epilepsy [ 4 , 5 ]. At present, there are various ways of treating epilepsy in traditional Chinese medicine (TCM), and it will have more significant potential in the clinical application of epilepsy [ 6 ]. The latest research shows that acupuncture and moxibustion combined with Western medicine are effective in treating epilepsy [ 7 ]. Moxibustion is a common and simple TCM treatment. The treatment process of moxibustion is to directly or indirectly apply ignited Artemisia (Artemisia vulgaris from TCM) to acupoints or other specific body parts to treat or prevent various diseases. Previous studies have shown that moxibustion therapy can reduce the frequency and grade of seizures [ 8 , 9 ]. However, the specific mechanism by which moxibustion therapy controls epilepsy is still unclear. Therefore, we are concerned about whether moxibustion pretreatment has an anti-epileptic effect on P2X7R-mediated neuroinflammation. Methods Animals For all experiments reported in this paper, we used adult 3-month-old C57BL/6J male mice weighing 20 ± 2 g (from Chengdu Dossy Experimental Animal Co., Chengdu, China). The animals were kept under standard husbandry conditions with free access to water and food. The light/dark cycle was 12 hours (light from 7 a.m. to 7 p.m.), and the ambient temperature was maintained at 22 ± 2°C.The animal study was reviewed and approved by the Institutional Review Board of Center for Brain Disorders, TongRen Municipal People's Hospital, Tongren, China Appl.NO.: ZMU21-2403-068). All methods were implemented in accordance with relevant guidelines and regulations. Groups of study C57B/J mice (8-week-old) were randomized into model group (KA group, n = 7) and moxibustion 7 days group (Moxi − 7 days group, n = 7), moxibustion 14 days group (Moxi − 14 days group, n = 7), and moxibustion 21 days group (Moxi − 21 days group, n = 7) (Fig. 3 ). The use of P2X7R agonists or antagonists was divided into four groups: control group (PBS group, n = 7), P2X7R activation group (BzATP group, n = 7), P2X7R antagonist group (A438079 group, n = 7), and P2X7R activation group after moxibustion pretreatment 14 days (Moxi + BzATP group ,n = 7) (Fig. 3 ). Bilateral cannulas implantation Mice were anesthetized using an inhalation anesthesia machine (isoflurane concentration: 1-1.5%, oxygen concentration: 0.4 L/min) and fixed on a stereotactic device (RWD Life Science Co., Ltd., China). Stereotactic implantation of cannulas (1.6 mm in length and 3.7 mm in diameter) into hippocampal CA1 area (from bregma: anteroposterior, − 1.85 mm; mediolateral, ± 2.15 mm; dorsoventral, − 1.6 mm) and fixed to the skull with dental cement. After surgery, wait for the mice to recover for two weeks before conducting experimental pretreatment. Experimental pretreatment Moxi − 7 days group: After binding and fixing the mice, moxibustion treatment was performed at a distance of 1cm from the bilateral Zusanli (ST36) and Dazhui (GV14) acupoints, with a duration of Once daily for 20 minutes, for 7 consecutive days. The pretreatment methods for Moxi − 14 days group and Moxi − 21 days group were the same as those for Moxi − 7 days group and were treated for 14 and 21 days, respectively. The fixation method and duration of KA group are the same as those of Moxi group. Moxi + BzATP group moxibustion pretreatment method is the same as for the Moxi − 14 days group, while the other three groups(PBS, A438079 and BzATP) only used the same fixed method and time as Moxi − 14 days group but did not receive moxibustion intervention. Kainic acid model protocol and behavioral tests Before building the kainic acid (KA) model, place the animals in a transparent acrylic box in a quiet environment for 30 minutes, one per box, at room temperature 22 ± 2°C. The PBS group injected 2 µL phosphate-buffered saline (PBS; Hyclone Labs., UT, Logan, USA) into the bilateral hippocampal CA1 region of mice, respectively. The BzATP group and the Moxi + BzATP group were both injected with 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (Bz-ATP; 10.5nmol BzATP = 0.00215g + 1ml PBS), or the A438079 group was injected with 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride (A-438079; 1.75nmol A438079 = 0.34261g + 1ml PBS) using the same method and dosage as the PBS group. After the above groups of drugs were injected, all study group mice were subjected to KA model construction. Based on previous studies[ 10 , 11 ], all epileptic seizure models of mice were immediately videotaped after intraperitoneal injection of KA (30 mg/kg) to observe the behavioral changes within 60 min. The Racine scale was used for the evaluation of epilepsy behavior grading [ 12 ]. Behavior was scored as follows: 0, behavioral arrest (motionless), piloerection, excitement and rapid breathing; 1, movement of the mouth, lips tongue and vibrissae, salivation; 2, head and eye clonus; 3, forelimb clonus, “wet dog shakes”; 4, clonic rearing; 5, clonic rearing with loss of postural control and uncontrolled jumping. The complete experimental process is summarized in Fig. 1 . Data analysis All data were expressed as means ± Standard error of the mean (SEM) of 7 observations. There was no prior sample size calculation. We set the number of animals in all experiments to 7. GraphPad Prism 9.0 was used for statistical analysis of the data. Use the Shapiro-Wilk normality test for normality testing. When all sampled distributions satisfied the normality and equal variance criteria, multiple comparisons between data were performed by the parametric one-way analysis of variance (ANOVA) followed by the Holm-Sidak test. The data in Fig. 3 had two variables (e.g., PBS versus moxibustion pretreatment after intracerebral injection of BzATP); therefore, 2-way ANOVA followed by the Tukey test was used for statistical evaluation. The post hoc tests were run only if F achieved P < 0.05. We considered the P -value less than 0.05 as statistically significant. Results Moxibustion pretreatment for 14 or 21 days can significantly alleviate the epilepsy behavior in mice The seizure stage following KA administration was significantly decreased in the Moxi − 7 days, Moxi − 14 days or Moxi − 21 days groups when compared to KA group (For Moxi − 7 days: p = 0.0107, for Moxi − 14 days: p = 0.0027 and for Moxi − 21 days: p = 0.0044) (Fig. 2 a,d, and g). The highest seizure stage was significantly lower in Moxi − 14 days or Moxi − 21 days groups than that inKAgroup (For Moxi − 14 days: p = 0.0013 and for Moxi − 21 days: p = 0.0013) (Fig. 2 e and h). Seizure severity was classified according to Racine’s scale 4–5. The duration of SE was decreased in the Moxi − 14 days or Moxi − 21 days groups when compared toKAgroup (For Moxi − 14 days: p = 0.0165 and for Moxi − 21 days: p = 0.0024) (Fig. 2 f and i). However, no difference was observed in the highest seizure stage or duration of SE betweenKAgroup and Moxi − 7 days group (For the highest seizure stage: p = 0.6261 and for the duration of SE: p = 0.4501) (Fig. 2 b and c). In addition, there was no difference in seizure stage, highest seizure stage, and duration of SE between the Moxi − 14 days and Moxi − 21 days groups (For the highest seizure stage: p > 0.9999 and for the duration of SE: p = 0.9329) (Fig. 2 j, k, and l). Moxibustion pretreatment for 14 days mediate the epilepsy behavior of mice after injection of P2X7R agonist or inhibitor The seizure stage was significantly decreased in A438079 or Moxi + BzATP groups than that in the PBS or BzATP groups (For A438079: p = 0.0072 and Moxi + BzATP: p = 0.0001, respectively) (Fig. 3 a). The seizure stage was significantly lower in Moxi + BzATP group compared to A438079 group ( p = 0.0005) (Fig. 3 a). Compared with PBS group, the duration of SE in A438079 or Moxi + BzATP groups decreased (For A438079: p = 0.0347 and for Moxi + BzATP: p = 0.0001), while the BzATP group increased ( p = 0.0087) (Fig. 3 b). The duration of SE was decreased in the A438079 group than that BzATP group ( p = 0.0001) (Fig. 3 b). In addition, there was a significant decrease in the duration of SE in the Moxi + BzATP compared with the A438079 or BzATP groups ( p = 0.0001, p = 0.0001, respectively) (Fig. 3 b). Discussion Moxibustion pretreatment refers to moxibustion treatment in advance during the period of no disease or disease omen, which belongs to a type of acupuncture and moxibustion pretreatment and has the function of disease prevention. Currently, research on acupuncture and moxibustion pretreatment has involved various diseases. A study has found that electroacupuncture pretreatment can effectively induce cerebral ischemic tolerance [ 13 ]. Li et al. revealed that acupuncture and moxibustion pretreatment can inhibit injection of amyloid-beta protein Induced neuronal damage and degeneration in hippocampus of rats [ 14 ]. Kim et al. showed that acupuncture pretreatment can reduce the content of inflammatory factor interleukin-1β (IL-1β), thereby alleviating epileptic seizures behavior in mice [ 15 ]. Another study has shown that acupuncture pretreatment can increase the expression of glutamate decarboxylase (GAD)-67 in hippocampus and inhibit KA induced seizures and hippocampal cell death [ 16 ]. However, there is limited research on the treatment of epilepsy with moxibustion pretreatment both domestically and internationally, and further research is needed. A study has shown that electroacupuncture stimulation of Dazhui (GV 14) and Baihui (Du 20) acupoints in epileptic rats can inhibit the inflammatory response mediated by microglia and have a protective effect on hippocampal neurons [ 17 ]. Acupuncture at point GV 14 can significantly reduce the mortality rate of rats during seizures [ 18 ]. Acupuncture at the ST36 and GV14 acupoints may have a protective effect on brain cells during epileptic seizures by affecting the expression of interleukin-8 (IL-8) and interleukin-10 (IL-10) in epileptic rats [ 19 ]. The effective mechanism of moxibustion at ST36 acupoint in treating epileptic rats may be related to reducing interleukin-2 (IL-2) in serum [ 20 ]. Based on the above analysis, this study selected at ST36 and GV14 acupoints for moxibustion pretreatment. P2X7R belongs to the ATP-sensitive ion channel type purinergic receptor. P2X7Rs are expressed throughout the central nervous system, including brain areas implicated in epilepsy development, such as the frontal cortex, hippocampus and amygdala, where they have been reported to be functional on all cell types [ 21 ]. Over the past few decades, a substantial body of evidence has been accumulated demonstrating a role for P2X7R during seizure generation and epilepsy [ 22 ]. Research has shown that intracerebral injection of ATP or BZ-ATP, an agonist at the P2X7R, into the mouse brain transiently enhanced electrographic seizures during status epilepticus. In contrast, Central injection of two chemically distinct P2X7R antagonists, brilliant blue G(BBG) and A-438079, reduced seizures during status epilepticus [ 23 ]. These results are consistent with the results of this study. The physiological role of P2X7R in epilepsy includes driving inflammatory processes, involvement in neuronal excitability, and astrocyte function [ 24 , 25 ], however the specific mechanism remains to be further elucidated. In recent years, research has shown that inflammatory response is closely related to epilepsy. Impaired regulation of the activation and resolution of inflammatory cells and molecules in the injured neuronal tissue is a critical factor to the development of epilepsy [ 26 ]. Transforming growth factor-β pathway inhibition prevents the activation of astrocytes during epileptogenesis, leading to a reduction in spontaneous seizure activity and brain inflammation [ 27 ]. Inflammation in the brain can increase the permeability of the blood-brain barrier and promote the transmission of glutamate neurotransmitters [ 28 ]. Glutamic acid and γ- Imbalance of aminobutyric acid can induce epilepsy [ 29 ], while glutamate receptors are mainly expressed in the hippocampal CA1 region [ 30 ]. In previous studies, it has been proved that acupuncture and moxibustion intervention can reduce the death of neurons in KA-induced epilepsy mice. This possible mechanism is that the neuroprotective effect of hippocampal neurons is that the γ-aminobutyric acid-mediated signal pathway increases Glutamic acid decarboxylase (GAD) [ 16 ]. A study has found that electroacupuncture at ST36 possess some curative effect on epileptic rats, related with change of GAD-67 mRNA level in dentate gyrus region [ 31 ]. In addition, the study indicates that moxibustion therapy mainly targets the effects of ST36 on epilepsy, and the possible mechanism of occurrence is to improve clinical symptoms by decreasing serum Tumor Necrosis Factor-α (TNF-α) and interleukin-6 (IL-6) levels [ 8 ]. These studies show that acupuncture and moxibustion can reduce the brain damage caused by KA-induced seizures. According to our experimental results, moxibustion pretreatment at ST36 and GV14 acupoints in mice for 14 and 21 days can significantly improve the seizure stage, reduce seizure stage and decrease duration of SE in KA model mice. However, moxibustion pretreatment for 7 days did not yield the same results. So, it is probably means that moxibustion pretratment can help to protect the damage from epileptic seizures but need to accumulative effect. In the experiment of using a P2X7R agonist (BzATP) or antagonist (A438079) to intervene in epileptic seizures in mice, it was found that BzATP aggravated seizure stage, increased highest seizure stage and prolonged duration of SE while using A438079 showed opposite results. Preconditioning the same acupoints with moxibustion for 14 days can reduce the level of seizures in mice using BzATP, decreased seizure stage, alleviated highest seizure stage, and reduced the duration of SE. From this result we can make a hypothesis, moxibustion pretreatment attenuate epileptic seizures may via P2X7R to mediate inflammatory response. Conclusion Moxibustion pretreatment at ST36 and GV14 acupoints for 14 or 21 days has anti-epileptic seizures effects, which may be related to inflammation mediated by P2X7 receptors.This work provides more insights for an in-depth study of the molecular mechanism of moxibustion pretreatment in treatment of epileptic seizures. Further study is need to investigate moxibustion pretreatment how to influence the signalling pathway of P2X7R for kainic acids- induced epileptic seizures. Declarations Funding This work was supported by the grants from NSFC (82305422) Author contribution All authors contributed to the study conception and design. Data collection: HWZ, JJL,YLHT, MLT. Data analysis: HWZ, MJS, Manuscript written: HWZ, MJS, and SH. All authors read and approved the final manuscript. Data availability All data included in this study are available from the corresponding authors on reasonable request. Conflict of interest The authors declare no competing interests. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. References Fisher RS, van Emde Boas W, Blume W, Elger C, Genton P, Lee P, Engel J Jr (2005) Epileptic seizures and epilepsy: definitions proposed by the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE). Epilepsia 46(4):470–472. https://doi.org/10.1111/j.0013-9580.2005.66104.x Asadi-Pooya AA, Brigo F, Lattanzi S, Blumcke I (2023) Adult epilepsy. 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Cite Share Download PDF Status: Published Journal Publication published 13 Sep, 2024 Read the published version in Purinergic Signalling → Version 1 posted Editorial decision: Revision requested 09 Jul, 2024 Reviews received at journal 08 Jul, 2024 Reviewers agreed at journal 04 Jul, 2024 Reviewers agreed at journal 04 Jul, 2024 Reviewers invited by journal 04 Jul, 2024 Editor assigned by journal 02 Jul, 2024 Submission checks completed at journal 02 Jul, 2024 First submitted to journal 02 Jul, 2024 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-4672161","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":324765516,"identity":"fa79fff8-8a41-49f9-8ce4-c7156ce429cc","order_by":0,"name":"Hong-Wei Zhang","email":"","orcid":"","institution":"TongRen Municipal People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hong-Wei","middleName":"","lastName":"Zhang","suffix":""},{"id":324765517,"identity":"a2a4ba45-5297-458d-9649-0318cda9d916","order_by":1,"name":"Jia-Jia Li","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jia-Jia","middleName":"","lastName":"Li","suffix":""},{"id":324765518,"identity":"c7a79232-20db-4074-9707-e358d3a99264","order_by":2,"name":"YuLiHan Tang","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"YuLiHan","middleName":"","lastName":"Tang","suffix":""},{"id":324765519,"identity":"f79211dc-6caa-4f96-ad81-cd172261d335","order_by":3,"name":"Mao-Lin Tian","email":"","orcid":"","institution":"TongRen Municipal People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mao-Lin","middleName":"","lastName":"Tian","suffix":""},{"id":324765520,"identity":"9f134837-386a-4fbf-8cfe-a5ff97e49478","order_by":4,"name":"Sheng Huang","email":"","orcid":"","institution":"TongRen Municipal People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Huang","suffix":""},{"id":324765521,"identity":"86e43539-8248-40cd-8911-308e206630a9","order_by":5,"name":"Meng-Juan Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYJACZgYDBgZ+9gYGBokKCTl+orVI9hwAajljYSzZQJQWIDC4kcDAwNhWkbiBkBb5GbmHPxcU3JEzOHP86QbLeRKMGxiYHz66gUeLwY28NOkZBs+MJY/3mN2Q3CbBbM7AZmycg0+LRI4ZM4/B4cS+M2fYQFrYLBt42KTxaZGfkWP8GailvuFG+rMbknMkeAwOENDCcCPHQBqoJUHgRgLQYQ0SEgS1GJx5YwbSYjiz54zZDYljEgaSzQT8It8Octifw/L87O3PbkvU1NX3szc/fIzXYciAWQJMEqscBBg/kKJ6FIyCUTAKRgwAALrwS+ZlJabKAAAAAElFTkSuQmCC","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Meng-Juan","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-07-02 07:16:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4672161/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4672161/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11302-024-10050-x","type":"published","date":"2024-09-13T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61131308,"identity":"1eb25ed4-3238-43e7-b00f-19e11753654b","added_by":"auto","created_at":"2024-07-26 04:13:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":732561,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental process diagram\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4672161/v1/d0e657573684417829e8233d.png"},{"id":61131307,"identity":"918fa10c-d8cd-4d9e-b166-57281f88c41a","added_by":"auto","created_at":"2024-07-26 04:13:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1110257,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral effects of moxibustion pretreatment with different days on epileptic seizures mice \u0026nbsp;\u003cstrong\u003ea\u003c/strong\u003eThe seizure stage within 60 minutes in KA and Moxi - 7 days groups. \u003cstrong\u003eb\u003c/strong\u003e The highest seizure stage within 60 minutes in KA and Moxi - 7 days groups. \u003cstrong\u003ec\u003c/strong\u003e The duration of status epilepticus (SE) in KA and Moxi - 7 days groups. \u003cstrong\u003ed\u003c/strong\u003e The seizure stage within 60 minutes in KA and Moxi - 14 days groups. \u003cstrong\u003ee\u003c/strong\u003e The highest seizure stage within 60 minutes in KA and Moxi - 14 daysgroups. \u003cstrong\u003ef \u003c/strong\u003eThe duration of SE in KA and Moxi - 7 days groups.\u003cstrong\u003e g\u003c/strong\u003e The seizure stage within 60 minutes in KA and Moxi - 21 days groups. \u003cstrong\u003eh\u003c/strong\u003e The highest seizure stage within 60 minutes in KA and Moxi - 21 days groups. \u003cstrong\u003ei\u003c/strong\u003eThe duration of SE in KA and Moxi - 21 days groups.\u003cstrong\u003e j\u003c/strong\u003e The seizure stage within 60 minutes in Moxi - 14 daysand Moxi - 21 daysgroups. \u003cstrong\u003ek\u003c/strong\u003e The highest seizure stage within 60 minutes in Moxi - 14 days and Moxi - 21 days groups. \u003cstrong\u003el\u003c/strong\u003eThe duration of SE in Moxi - 14 days and Moxi - 21 daysgroups. Bars represent the means \u003cem\u003e± \u003c/em\u003eStandard error of the mean deviation for each group (each group \u003cem\u003en=7\u003c/em\u003e). ns: no significance, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4672161/v1/e04e0ad7dab7fa7a67e6f414.png"},{"id":61131570,"identity":"ce50b227-5917-4bb9-b57b-4179db95019c","added_by":"auto","created_at":"2024-07-26 04:21:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":353321,"visible":true,"origin":"","legend":"\u003cp\u003eMoxibustion pretreatment affects epileptic seizures by mediation P2X7 receptor \u003cstrong\u003ea\u003c/strong\u003e The seizure stage within 60 minutes in the PBS, A438079, BzATP and Moxi + BzATP groups. \u003cstrong\u003eb\u003c/strong\u003e The duration of status epilepticus (SE) in the PBS, A438079, BzATP and Moxi + BzATP groups. Bars represent the means \u003cem\u003e± \u003c/em\u003eStandard error of the mean deviation for each group (each group \u003cem\u003en=7\u003c/em\u003e). *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4672161/v1/4027ab94831c20918f35f5a0.png"},{"id":64619946,"identity":"97bcbdaa-4b04-4bc5-9a58-b7bbb1678cc6","added_by":"auto","created_at":"2024-09-16 16:17:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2643379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4672161/v1/21665449-52f7-4126-b2c5-843f3650b342.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Moxibustion Pretreatment Attenuate epileptic seizures by mediation P2X7 receptor","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpilepsy is a common chronic brain disease characterized by a long-lasting predisposition to recurrent epileptic seizures [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Research estimates the point prevalence of active epilepsy in the world (as of Feb 2023)to be about 51\u0026nbsp;million people, and about 4.9\u0026nbsp;million people develop new-onset epilepsy every year [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Seizures are due to an imbalance of excitatory and inhibitory neuronal firing, which is closely related to abnormal ion channel function, imbalance of neurotransmitter secretion, irregular glial system, neuroinflammation and oxidative stress, and abnormal neural circuits [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the specific pathogenesis of epilepsy is not entirely clear. More and more studies have focused on the crucial role of purine signaling ATP-gated purinergic P2X7 receptor (P2X7R) mediated neuroinflammatory response in epilepsy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. At present, there are various ways of treating epilepsy in traditional Chinese medicine (TCM), and it will have more significant potential in the clinical application of epilepsy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The latest research shows that acupuncture and moxibustion combined with Western medicine are effective in treating epilepsy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moxibustion is a common and simple TCM treatment. The treatment process of moxibustion is to directly or indirectly apply ignited Artemisia (Artemisia vulgaris from TCM) to acupoints or other specific body parts to treat or prevent various diseases. Previous studies have shown that moxibustion therapy can reduce the frequency and grade of seizures [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the specific mechanism by which moxibustion therapy controls epilepsy is still unclear. Therefore, we are concerned about whether moxibustion pretreatment has an anti-epileptic effect on P2X7R-mediated neuroinflammation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eFor all experiments reported in this paper, we used adult 3-month-old C57BL/6J male mice weighing 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g (from Chengdu Dossy Experimental Animal Co., Chengdu, China). The animals were kept under standard husbandry conditions with free access to water and food. The light/dark cycle was 12 hours (light from 7 a.m. to 7 p.m.), and the ambient temperature was maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C.The animal study was reviewed and approved by the Institutional Review Board of Center for Brain Disorders, TongRen Municipal People's Hospital, Tongren, China Appl.NO.: ZMU21-2403-068). All methods were implemented in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGroups of study\u003c/h2\u003e \u003cp\u003eC57B/J mice (8-week-old) were randomized into model group (KA group, n\u0026thinsp;=\u0026thinsp;7) and moxibustion 7 days group (Moxi \u0026minus;\u0026thinsp;7 days group, n\u0026thinsp;=\u0026thinsp;7), moxibustion 14 days group (Moxi \u0026minus;\u0026thinsp;14 days group, n\u0026thinsp;=\u0026thinsp;7), and moxibustion 21 days group (Moxi \u0026minus;\u0026thinsp;21 days group, n\u0026thinsp;=\u0026thinsp;7) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The use of P2X7R agonists or antagonists was divided into four groups: control group (PBS group, n\u0026thinsp;=\u0026thinsp;7), P2X7R activation group (BzATP group, n\u0026thinsp;=\u0026thinsp;7), P2X7R antagonist group (A438079 group, n\u0026thinsp;=\u0026thinsp;7), and P2X7R activation group after moxibustion pretreatment 14 days (Moxi\u0026thinsp;+\u0026thinsp;BzATP group ,n\u0026thinsp;=\u0026thinsp;7) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBilateral cannulas implantation\u003c/h2\u003e \u003cp\u003eMice were anesthetized using an inhalation anesthesia machine (isoflurane concentration: 1-1.5%, oxygen concentration: 0.4 L/min) and fixed on a stereotactic device (RWD Life Science Co., Ltd., China). Stereotactic implantation of cannulas (1.6 mm in length and 3.7 mm in diameter) into hippocampal CA1 area (from bregma: anteroposterior, \u0026minus;\u0026thinsp;1.85 mm; mediolateral, \u0026plusmn;\u0026thinsp;2.15 mm; dorsoventral, \u0026minus;\u0026thinsp;1.6 mm) and fixed to the skull with dental cement. After surgery, wait for the mice to recover for two weeks before conducting experimental pretreatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExperimental pretreatment\u003c/h2\u003e \u003cp\u003eMoxi \u0026minus;\u0026thinsp;7 days group: After binding and fixing the mice, moxibustion treatment was performed at a distance of 1cm from the bilateral Zusanli (ST36) and Dazhui (GV14) acupoints, with a duration of Once daily for 20 minutes, for 7 consecutive days. The pretreatment methods for Moxi \u0026minus;\u0026thinsp;14 days group and Moxi \u0026minus;\u0026thinsp;21 days group were the same as those for Moxi \u0026minus;\u0026thinsp;7 days group and were treated for 14 and 21 days, respectively. The fixation method and duration of KA group are the same as those of Moxi group. Moxi\u0026thinsp;+\u0026thinsp;BzATP group moxibustion pretreatment method is the same as for the Moxi \u0026minus;\u0026thinsp;14 days group, while the other three groups(PBS, A438079 and BzATP) only used the same fixed method and time as Moxi \u0026minus;\u0026thinsp;14 days group but did not receive moxibustion intervention.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eKainic acid model protocol and behavioral tests\u003c/h2\u003e \u003cp\u003eBefore building the kainic acid (KA) model, place the animals in a transparent acrylic box in a quiet environment for 30 minutes, one per box, at room temperature 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe PBS group injected 2 \u0026micro;L phosphate-buffered saline (PBS; Hyclone Labs., UT, Logan, USA) into the bilateral hippocampal CA1 region of mice, respectively. The BzATP group and the Moxi\u0026thinsp;+\u0026thinsp;BzATP group were both injected with 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (Bz-ATP; 10.5nmol BzATP\u0026thinsp;=\u0026thinsp;0.00215g\u0026thinsp;+\u0026thinsp;1ml PBS), or the A438079 group was injected with 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride (A-438079; 1.75nmol A438079\u0026thinsp;=\u0026thinsp;0.34261g\u0026thinsp;+\u0026thinsp;1ml PBS) using the same method and dosage as the PBS group. After the above groups of drugs were injected, all study group mice were subjected to KA model construction. Based on previous studies[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], all epileptic seizure models of mice were immediately videotaped after intraperitoneal injection of KA (30 mg/kg) to observe the behavioral changes within 60 min. The Racine scale was used for the evaluation of epilepsy behavior grading [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Behavior was scored as follows: 0, behavioral arrest (motionless), piloerection, excitement and rapid breathing; 1, movement of the mouth, lips tongue and vibrissae, salivation; 2, head and eye clonus; 3, forelimb clonus, \u0026ldquo;wet dog shakes\u0026rdquo;; 4, clonic rearing; 5, clonic rearing with loss of postural control and uncontrolled jumping. The complete experimental process is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eAll data were expressed as \u003cem\u003emeans\u0026thinsp;\u0026plusmn;\u003c/em\u003e\u0026thinsp;Standard error of the mean (SEM) of 7 observations. There was no prior sample size calculation. We set the number of animals in all experiments to 7. GraphPad Prism 9.0 was used for statistical analysis of the data. Use the Shapiro-Wilk normality test for normality testing. When all sampled distributions satisfied the normality and equal variance criteria, multiple comparisons between data were performed by the parametric one-way analysis of variance (ANOVA) followed by the Holm-Sidak test. The data in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e had two variables (e.g., PBS versus moxibustion pretreatment after intracerebral injection of BzATP); therefore, 2-way ANOVA followed by the Tukey test was used for statistical evaluation. The post hoc tests were run only if \u003cem\u003eF\u003c/em\u003e achieved \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. We considered the \u003cem\u003eP\u003c/em\u003e-value less than 0.05 as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMoxibustion pretreatment for 14 or 21 days can significantly alleviate the epilepsy behavior in mice\u003c/h2\u003e \u003cp\u003eThe seizure stage following KA administration was significantly decreased in the Moxi \u0026minus;\u0026thinsp;7 days, Moxi \u0026minus;\u0026thinsp;14 days or Moxi \u0026minus;\u0026thinsp;21 days groups when compared to KA group (For Moxi \u0026minus;\u0026thinsp;7 days: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0107, for Moxi \u0026minus;\u0026thinsp;14 days: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0027 and for Moxi \u0026minus;\u0026thinsp;21 days: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0044) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,d, and g). The highest seizure stage was significantly lower in Moxi \u0026minus;\u0026thinsp;14 days or Moxi \u0026minus;\u0026thinsp;21 days groups than that inKAgroup (For Moxi \u0026minus;\u0026thinsp;14 days: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0013 and for Moxi \u0026minus;\u0026thinsp;21 days: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0013) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and h). Seizure severity was classified according to Racine\u0026rsquo;s scale 4\u0026ndash;5. The duration of SE was decreased in the Moxi \u0026minus;\u0026thinsp;14 days or Moxi \u0026minus;\u0026thinsp;21 days groups when compared toKAgroup (For Moxi \u0026minus;\u0026thinsp;14 days: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0165 and for Moxi \u0026minus;\u0026thinsp;21 days: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0024) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and i). However, no difference was observed in the highest seizure stage or duration of SE betweenKAgroup and Moxi \u0026minus;\u0026thinsp;7 days group (For the highest seizure stage: p\u0026thinsp;=\u0026thinsp;0.6261 and for the duration of SE: p\u0026thinsp;=\u0026thinsp;0.4501) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and c). In addition, there was no difference in seizure stage, highest seizure stage, and duration of SE between the Moxi \u0026minus;\u0026thinsp;14 days and Moxi \u0026minus;\u0026thinsp;21 days groups (For the highest seizure stage: p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999 and for the duration of SE: p\u0026thinsp;=\u0026thinsp;0.9329) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ej, k, and l).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMoxibustion pretreatment for 14 days mediate the epilepsy behavior of mice after injection of P2X7R agonist or inhibitor\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe seizure stage was significantly decreased in A438079 or Moxi\u0026thinsp;+\u0026thinsp;BzATP groups than that in the PBS or BzATP groups (For A438079: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0072 and Moxi\u0026thinsp;+\u0026thinsp;BzATP: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The seizure stage was significantly lower in Moxi\u0026thinsp;+\u0026thinsp;BzATP group compared to A438079 group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Compared with PBS group, the duration of SE in A438079 or Moxi\u0026thinsp;+\u0026thinsp;BzATP groups decreased (For A438079: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0347 and for Moxi\u0026thinsp;+\u0026thinsp;BzATP: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001), while the BzATP group increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0087) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The duration of SE was decreased in the A438079 group than that BzATP group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In addition, there was a significant decrease in the duration of SE in the Moxi\u0026thinsp;+\u0026thinsp;BzATP compared with the A438079 or BzATP groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMoxibustion pretreatment refers to moxibustion treatment in advance during the period of no disease or disease omen, which belongs to a type of acupuncture and moxibustion pretreatment and has the function of disease prevention. Currently, research on acupuncture and moxibustion pretreatment has involved various diseases. A study has found that electroacupuncture pretreatment can effectively induce cerebral ischemic tolerance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Li et al. revealed that acupuncture and moxibustion pretreatment can inhibit injection of amyloid-beta protein Induced neuronal damage and degeneration in hippocampus of rats [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Kim et al. showed that acupuncture pretreatment can reduce the content of inflammatory factor interleukin-1β (IL-1β), thereby alleviating epileptic seizures behavior in mice [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Another study has shown that acupuncture pretreatment can increase the expression of glutamate decarboxylase (GAD)-67 in hippocampus and inhibit KA induced seizures and hippocampal cell death [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, there is limited research on the treatment of epilepsy with moxibustion pretreatment both domestically and internationally, and further research is needed.\u003c/p\u003e \u003cp\u003eA study has shown that electroacupuncture stimulation of Dazhui (GV 14) and Baihui (Du 20) acupoints in epileptic rats can inhibit the inflammatory response mediated by microglia and have a protective effect on hippocampal neurons [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Acupuncture at point GV 14 can significantly reduce the mortality rate of rats during seizures [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Acupuncture at the ST36 and GV14 acupoints may have a protective effect on brain cells during epileptic seizures by affecting the expression of interleukin-8 (IL-8) and interleukin-10 (IL-10) in epileptic rats [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The effective mechanism of moxibustion at ST36 acupoint in treating epileptic rats may be related to reducing interleukin-2 (IL-2) in serum [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Based on the above analysis, this study selected at ST36 and GV14 acupoints for moxibustion pretreatment.\u003c/p\u003e \u003cp\u003eP2X7R belongs to the ATP-sensitive ion channel type purinergic receptor. P2X7Rs are expressed throughout the central nervous system, including brain areas implicated in epilepsy development, such as the frontal cortex, hippocampus and amygdala, where they have been reported to be functional on all cell types [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Over the past few decades, a substantial body of evidence has been accumulated demonstrating a role for P2X7R during seizure generation and epilepsy [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Research has shown that intracerebral injection of ATP or BZ-ATP, an agonist at the P2X7R, into the mouse brain transiently enhanced electrographic seizures during status epilepticus. In contrast, Central injection of two chemically distinct P2X7R antagonists, brilliant blue G(BBG) and A-438079, reduced seizures during status epilepticus [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These results are consistent with the results of this study. The physiological role of P2X7R in epilepsy includes driving inflammatory processes, involvement in neuronal excitability, and astrocyte function [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], however the specific mechanism remains to be further elucidated.\u003c/p\u003e \u003cp\u003eIn recent years, research has shown that inflammatory response is closely related to epilepsy. Impaired regulation of the activation and resolution of inflammatory cells and molecules in the injured neuronal tissue is a critical factor to the development of epilepsy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Transforming growth factor-β pathway inhibition prevents the activation of astrocytes during epileptogenesis, leading to a reduction in spontaneous seizure activity and brain inflammation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Inflammation in the brain can increase the permeability of the blood-brain barrier and promote the transmission of glutamate neurotransmitters [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Glutamic acid and γ- Imbalance of aminobutyric acid can induce epilepsy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], while glutamate receptors are mainly expressed in the hippocampal CA1 region [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In previous studies, it has been proved that acupuncture and moxibustion intervention can reduce the death of neurons in KA-induced epilepsy mice. This possible mechanism is that the neuroprotective effect of hippocampal neurons is that the γ-aminobutyric acid-mediated signal pathway increases Glutamic acid decarboxylase (GAD) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A study has found that electroacupuncture at ST36 possess some curative effect on epileptic rats, related with change of GAD-67 mRNA level in dentate gyrus region [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In addition, the study indicates that moxibustion therapy mainly targets the effects of ST36 on epilepsy, and the possible mechanism of occurrence is to improve clinical symptoms by decreasing serum Tumor Necrosis Factor-α (TNF-α) and interleukin-6 (IL-6) levels [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These studies show that acupuncture and moxibustion can reduce the brain damage caused by KA-induced seizures.\u003c/p\u003e \u003cp\u003eAccording to our experimental results, moxibustion pretreatment at ST36 and GV14 acupoints in mice for 14 and 21 days can significantly improve the seizure stage, reduce seizure stage and decrease duration of SE in KA model mice. However, moxibustion pretreatment for 7 days did not yield the same results. So, it is probably means that moxibustion pretratment can help to protect the damage from epileptic seizures but need to accumulative effect.\u003c/p\u003e \u003cp\u003eIn the experiment of using a P2X7R agonist (BzATP) or antagonist (A438079) to intervene in epileptic seizures in mice, it was found that BzATP aggravated seizure stage, increased highest seizure stage and prolonged duration of SE while using A438079 showed opposite results. Preconditioning the same acupoints with moxibustion for 14 days can reduce the level of seizures in mice using BzATP, decreased seizure stage, alleviated highest seizure stage, and reduced the duration of SE. From this result we can make a hypothesis, moxibustion pretreatment attenuate epileptic seizures may via P2X7R to mediate inflammatory response.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMoxibustion pretreatment at ST36 and GV14 acupoints for 14 or 21 days has anti-epileptic seizures effects, which may be related to inflammation mediated by P2X7 receptors.This work provides more insights for an in-depth study of the molecular mechanism of moxibustion pretreatment in treatment of epileptic seizures. Further study is need to investigate moxibustion pretreatment how to influence the signalling pathway of P2X7R for kainic acids- induced epileptic seizures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the grants from NSFC (82305422) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Data collection: HWZ, JJL,YLHT, MLT. Data analysis: HWZ, MJS, \u0026nbsp;Manuscript written: HWZ, MJS, and SH. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available from the corresponding authors on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFisher RS, van Emde Boas W, Blume W, Elger C, Genton P, Lee P, Engel J Jr (2005) Epileptic seizures and epilepsy: definitions proposed by the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE). Epilepsia 46(4):470\u0026ndash;472. https://doi.org/10.1111/j.0013-9580.2005.66104.x\u003c/li\u003e\n\u003cli\u003eAsadi-Pooya AA, Brigo F, Lattanzi S, Blumcke I (2023) Adult epilepsy. Lancet (London, England) 402(10399):412\u0026ndash;424. https://doi.org/10.1016/S0140-6736(23)01048-6\u003c/li\u003e\n\u003cli\u003eHu T, Zhang J, Wang J, Sha L, Xia Y, Ortyl TC, Tian X, Chen L (2023) Advances in Epilepsy: Mechanisms, Clinical Trials, and Drug Therapies. 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Brain Res 1188:165-172. https://doi.org/10.1016/j.brainres.2007.10.017\u003c/li\u003e\n\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":"purinergic-signalling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pusi","sideBox":"Learn more about [Purinergic Signalling](http://link.springer.com/journal/11302)","snPcode":"11302","submissionUrl":"https://submission.nature.com/new-submission/11302/3","title":"Purinergic Signalling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Moxibustion, P2X7 purinergic receptors, kainic acid, mice, epileptic seizures","lastPublishedDoi":"10.21203/rs.3.rs-4672161/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4672161/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe P2X7 receptor (P2X7R) is involved in long-term brain hyperexcitability driven by epilepsy and inflammation. It is currently unclear whether moxibustion pretreatment by the purine signaling pathway interference in P2X7R is involved in the mechanism of epilepsy treatment. Male C57BL/6 mice received moxibustion pretreatments at the acupoints of Zusanli (ST36) and Dazhui (GV14) once a day for 7, 14, or 21 days and immediately after an intraperitoneal injection of KA (30 mg/kg) and recording the behavior changes follow the Racine scales. Moxibustion pretreatment stimulation at ST36 and GV14 of 14 or 21 days significantly reduced the KA-induced seizure. In addition, there was no difference between treatment 14 or 21 days in epileptic mice. Then, we aimed to investigate the role of P2X7R in epileptogenesis in vivo using agonist (BzATP) or inhibitor (A438079) to influence the expression of this receptor. P2X7R agonist BzATP can aggravate seizure. We found moxibustion pretreatment can attenuate epileptic seizures via confronting BzATP. The result implied that Moxibustion pretreatment at ST36 and GV14 acupoints for 14 days has anti-epileptic effects, which may be related to inflammation mediated by P2X7 receptors.\u003c/p\u003e","manuscriptTitle":"Moxibustion Pretreatment Attenuate epileptic seizures by mediation P2X7 receptor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-26 04:13:29","doi":"10.21203/rs.3.rs-4672161/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-09T14:13:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-08T20:58:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84483980309574845294824669083569811408","date":"2024-07-05T02:43:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194651942503502703681643179380994856735","date":"2024-07-04T09:50:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-04T08:45:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-03T01:09:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-03T01:09:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Purinergic Signalling","date":"2024-07-02T07:15:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"purinergic-signalling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pusi","sideBox":"Learn more about [Purinergic Signalling](http://link.springer.com/journal/11302)","snPcode":"11302","submissionUrl":"https://submission.nature.com/new-submission/11302/3","title":"Purinergic Signalling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"aa86a122-a7d2-4648-bd50-8d3f05ec019e","owner":[],"postedDate":"July 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:11:47+00:00","versionOfRecord":{"articleIdentity":"rs-4672161","link":"https://doi.org/10.1007/s11302-024-10050-x","journal":{"identity":"purinergic-signalling","isVorOnly":false,"title":"Purinergic Signalling"},"publishedOn":"2024-09-13 15:57:39","publishedOnDateReadable":"September 13th, 2024"},"versionCreatedAt":"2024-07-26 04:13:29","video":"","vorDoi":"10.1007/s11302-024-10050-x","vorDoiUrl":"https://doi.org/10.1007/s11302-024-10050-x","workflowStages":[]},"version":"v1","identity":"rs-4672161","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4672161","identity":"rs-4672161","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}