Limonin attenuates neuroinflammation and enhances neurogenesis in a tMCAO mouse model of ischemic stroke

Limonin attenuates neuroinflammation and enhances neurogenesis in a tMCAO mouse model of ischemic stroke | 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 Limonin attenuates neuroinflammation and enhances neurogenesis in a tMCAO mouse model of ischemic stroke YANG YANG, Nan Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6879079/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neurogenesis in the subventricular zone (SVZ) is an effective way for brain repair after ischemic stroke. But neuroinflammation caused by cerebral ischemia would inhibit the effect of brain self-repair. As a Broadly active anti-inflammatory drugs, Limonin (LM) has a beneficial effect on ischemia-reperfusion(I/R) injury.However, the effect of LM on neurogenesis in the later stages of cerebral infarction is unknown. We speculate LM could generate anti-inflammation effect at the early stage of ischemic stroke and promote the subsequent neurogenesis. In our study, we used a transient middle cerebral artery occlusion (tMCAO) mouse model. We found LM treatment reduced the expression of iNOS and IL-1β proteins on day 3 after tMCAO. On day 7 after tMCAO, the number of BrdU/Nestin-positive cells around SVZ and BrdU/doublecortin (DCX)-positive cells in SVZ and the expression of Nestin, DCX proteins were increased through LM treatment. Moreover, on day 14 after tMCAO, the number of BrdU/DCX-positive cells in SVZ and peri-infarct area and the expression of DCX protein were increased in LM treated tMCAO mice. And LM treated tMCAO mice had fewer Cleaved-Caspase 3/DCX-positive cells in the peri-infarction zone compared to saline treated tMCAO mice 14 days after tMCAO. Finally, LM treatment increased the number of BrdU/NeuN-positive cells in the peri-infarct region and the expression of BDNF, GDNF, NGF proteins on day 14 after tMCAO. Our findings demonstrate that LM inhibits neuroinflammation and promotes neurogenesis after ischemic stroke. Biological sciences/Neuroscience/Regeneration and repair in the nervous system Health sciences/Medical research Health sciences/Medical research/Drug development limonin Ischemic stroke Neuroinflammation Neurogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Ischemic stroke is a leading cause of disability worldwide with high mortality and morbidity, and the effective treatment is limited [ 1 ]. A disruption in cerebral blood supply results in massive neural cell death along with loss of neurological function associated with the affected area [ 2 ]. Previous studies have revealed that neurogenesis in the subventricular zone (SVZ) is a promising endogenous and beneficial process for ischemic stroke [ 3 , 4 ]. Neural stem cells (NSCs) are located in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in hippocampus [ 5 ]. These NSCs retain the capacity to self-renew and possibly differentiate into neurons which are able to integrate in functional circuits [ 6 ]. Normally, the NSCs from SVZ could proliferate and differentiate into neural progenitor cells that migrate to other brain areas where they generate functional neurons for brain repair [ 7 ]. After ischemic stroke, NSCs from the SVZ can proliferate and generate doublecortin (DCX)-positive neuroblasts. Neuroblasts migrate to the ischemic zone and differentiate into mature neurons, which replace dead or injured neurons, integrate into the neuronal circuits, or secrete neuroprotective cytokines [ 2 ]. Thus, enhancing the efficiency of endogenous neurogenesis may provide a potential therapeutic method for ischemic stroke treatment. Limonin (LM) is an abundant natural tetracyclic triterpenoid compound with rich biological activities. LM has a wide range of anti-inflammatory, anti-microbial and anti-viral effects in mammalian systems [ 8 ]. LM has neuroprotective effects by inhibiting neuronal apoptosis and damage in cells and rat models[ 9 , 10 ].As we all known, excessive production of ROS may lead to oxidative stress injury and disrupts homeostasis in the brain. Then we assumed LM could inhibit neuroinflammation through reducing generation of ROS and then generate a stable microenvironment for ischemic brain. Previous study has shown that stroke could stimulate adult neurogenesis to against ischemic damage [ 11 – 13 ]. But ischemic injury could also activate immune cells such as microglia and macrophages, these cells can produce a large number of proinflammatory cytokines that can disrupt neural cells and inhibit neurogenesis [ 14 – 16 ]. Evidences have shown inflammation reduced the survival rate of new-born striatal neurons and new hippocampal neurons [ 17 , 18 ]. Therefore, a suitable inflammatory environment may promote neurogenesis after ischemic stroke and the effect of LM on the subsequent process of brain repair remain uncleared. In this study, we investigated whether LM could inhibit neuroinflammation and enhance SVZ neurogenesis after ischemic stroke in a mouse model of transient middle cerebral artery occlusion (tMCAO). 2. Methods 2.1. Animals We bought adult, male C57BL/6 mice (11–14 weeks old, 25–30 g) from the Animal Experimental Center of Zhengzhou University. All experimental protocols were approved by the Ethics Committee of the Second Affiliated Hospital of Zhengzhou University. All methods were carried out in accordance with relevant guidelines and regulations. This study is reported in accordance with ARRIVE guidelines..All mice were maintained at a constant temperature in the animal room with free access to food and water. All efforts were made to minimize the number of animals used and their suffering.After the anesthesia was completed, the mice were euthanized by dislocating their cervical vertebrae and crushing them. 2.2. Transient middle cerebral artery occlusion Model The intraluminal filament technique was used to made the transient middle cerebral artery occlusion (tMCAO) mice model as we previously described[ 16 ]. Firstly,surgical anesthesia was achieved with isoflurane (4% induction, 1.5% maintenance in 70% N₂O/30% O₂),then we made a midline neck incision to expose the carotid arteries. We inserted a 6.0 nylon suture monofilament with silicone-coated tip into the right internal carotid artery to block the origin of the middle cerebral artery. Lastly, we pulled out approximately 10 mm of the monofilament for blood reperfusion 60 minutes later. We defined a successful tMCAO as a decrease in cerebral blood flow of more than 80% as measured by laser Doppler flowmetry (Moor Instruments, Devon, UK). The mice in sham groups repeated the steps described above except that the filament was advanced to the origin of the middle cerebral artery and immediately withdrawn. 2.3. Treatment and Groups We randomly divided all mice into four groups: sham-operated mice treated with saline (Sham + saline), sham-operated mice treated with LM (Sham + LM), tMCAO mice treated with saline (tMCAO + saline), tMCAO mice treated with LM (tMCAO + LM). LM was diluted to 10% (w/v) with saline. We chose 6 mg/kg/min as the optimal dose and mice were continuously infused with saline or LM by intravenous (i.v.) infusion via tail vein 10 minutes before the operation until the reperfusion begins. Furthermore, the mice used for immunofluorescence to detect cell proliferation were received of 5-bromo2′-deoxyuridine (BrdU; 50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) by intraperitoneal injections once daily for 7 days or 14 days beginning 24 h after surgery 2.4. Immunofluorescence We performed immunofluorescence analysis on the brain on day 7 and 14 after tMCAO as previously described [ 19 ]. First, we anesthetized mice, then transcardially perfused them with PBS followed by 4% paraformaldehyde. Then we carefully removed the whole brains and fixed them in 4% paraformaldehyde overnight at 4° C. After that we immersed the brains in 30% sucrose/PBS to dehydrate until they sank. We sliced each brain into 20-µm-thick sections by cryoultramicrotomy (CM1100, Leica Biosystems, Germany). Moreover, the brain sections used for BrdU detection were incubated in 2 mol/L HCl for 45 minutes and 0.1 mol/L sodium borate buffer (pH8.5) for 10 minutes at room temperature. Then we used PBS to wash these brain sections three times for 5 minutes and incubated them in PBST (0.3% Triton X-100 in PBS) for 30 minutes. After that, these brain sections were blocked in 1% bovine serum albumin (BSA) / PBST for 30minutes and we incubated them overnight at 4°C with antibodies against BrdU (1:500, Abcam, USA), DCX (1:300, Novus Biologicals, USA), NeuN (1:500, Novus Biologicals, USA), Cleaved-Caspase 3 (1:500, Affinity, China), Nestin (1:500, Affinity, China). All sections were washed in PBS three times for 5 minutes in the second day and we used secondary antibodies to incubate them for 2h at room temperature. Then these sections were washed in PBS three times for 5 minutes again. Finally, we put these brain sections on microslides, covered them with 4′,6-diamidino2-phenylindole (DAPI; Santa Cruz Biotech, Dallas, TX, USA) after drying and an investigator observed them with a fluorescence microscope (ZEISS Scope A1, ZEISS, Germany). For each section that contained brain infarction, an investigator randomly chose three non-overlapping 10 × fields around SVZ to quantify BrdU/Nestin-positive cells, three non-overlapping 20 × fields were chosen to quantify BrdU/DCX-positive cells in SVZ and BrdU/DCX-positive cells, BrdU/NeuN-positive cells, Cleaved-Caspase 3/DCX-positive cells in peri-infarction zones. And brain sections from sham-operation groups repeated the steps described above. All investigators were blinded to the treatments and groups. 2.5. Western blot analysis We sacrificed mice for Western blot analysis on days 3, 7 and 14 after tMCAO as previously described [ 4 ]. Protein was extracted from the whole right hemisphere and separated on 10% glycine gel and transferred onto polyvinylidene difluoride membranes. Firstly, we carefully removed the brains from deeply anesthetized mice, and protein samples were extracted from the whole right hemisphere in every groups and separated on 10% glycine gel. Then we transferred protein to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). And we used 5% nonfat milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) to block the membranes for 45 minutes at room temperature. After that, the membranes were incubated with primary antibodies against iNOS (1:1500, Affinity, China), IL-1β(1:1000, Affinity, China), DCX (1:1000, Novus Biologicals, USA), Nestin (1:1000, Affinity, China), BDNF (1:2000, Novus Biologicals, USA), NGF (1:2000, Abcam, USA), GDNF (1:1500, Abcam, USA), β-actin (1:2000, Affinity, USA) over night at 4°C. Then we washed these membranes with TBST three times for 5 minutes in the second day and incubated them with secondary antibodies for 2 h at room temperature. Lastly, these membranes were washed with TBST three times for 5 minutes again and protein bands were visualized by enhanced chemiluminescence detection kit (CWBIO, Beijing, China). An investigator blinded to the animal group quantified the optical density of the protein bands through Gel Analysis V 2.02 software (Clinx Science Instruments, Shanghai, China). β-actin served as a loading control. 2.6. Statistical analysis Statistical analysis was carried out with SPSS version 13.0. All results are expressed as mean ± SD. We used Student’s t test or one-way ANOVA to analyze the differences in date from Western blot analysis and immunofluorescence. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. LM treatment can inhibit neuroinflammation in the early stage of ischemic stroke, reduce the area of cerebral infarction, and prolong survival time.The survival time of the tMCAO + saline group was shorter than that of the tMCAO + LM group(Fig. 1 . A, B, C).Western blot analysis illustrated that the expression of iNOS and IL-1β in the tMCAO + saline group were higher that the tMCAO + LM group on day 3 after operation, and LM treatment did not obviously change iNOS and IL-1β protein levels in the sham mice (Fig. 1 . D, E, F).The area of cerebral infarction in the tMCAO + saline group was larger than that in the tMCAO + LM group (Fig. 1 . G, H). 3.2. LM treatment could promote neural stem cells proliferation on day 7 after ischemic stroke.In comparison with the tMCAO + LM group, the tMCAO + saline group had significantly fewer Brdu/Nestin-positive cells around SVZ on day 7. No difference was apparent between the saline-treated and LM-treated sham mice (Fig. 2 .A, B). Western blot analysis showed that the protein levels of Nestin in the tMCAO + saline group were lower that the tMCAO + LM group, and LM treatment did not obviously change Nestin protein levels in the sham mice (Fig. 2 .C, D). 3.3. LM treatment could promote neuroblasts proliferation on day 7 after ischemic stroke.Immunofluorescence staining indicated that the tMCAO + saline group had significantly fewer BrdU/DCX-positive cells compared to the tMCAO + LM group in SVZ on day 7. No difference was apparent between the saline-treated and LM-treated sham mice (Fig. 3 .A, B). Western blot analysis showed that the protein levels of DCX in the tMCAO + saline group were lower that the tMCAO + LM group, and LM treatment did not obviously change DCX protein levels in the sham mice (Fig. 3 .C, D). 3.4. LM can promote neuroblasts proliferation in SVZ and the migration of neuroblasts in peri-infract region on day 14 after ischemic stroke.Through the results of immunofluorescence staining, we find the number of BrdU/DCX-positive cells in the tMCAO + LM group were significant increased compared to the tMCAO + saline group in SVZ and peri-infarction zone on day 14. No difference was apparent between the saline-treated and LM-treated sham mice (Fig. 4 .A, B, D, E). Western blot analysis showed that the protein levels of DCX in the tMCAO + saline group were lower that the tMCAO + LM group, and LM treatment did not obviously change DCX protein levels in the sham mice (Fig. 4 .C, F). 3.5. LM could inhibit the apoptosis of neuroblasts during migration after ischemic stroke.Through the results of immunofluorescence staining, we find the number of Cleaved-Caspase 3/DCX-positive cells in the tMCAO + LM group were significant increased compared to the tMCAO + saline group in the peri-infarction zone on day 14(Fig. 5 .A,B). 3.6. LM could promote neuroblasts differentiate into mature neurons and increases the expression of neurotrophic factors after ischemic stroke.The results of immunofluorescence analysis showed that the number of BrdU/NeuN-positive cells in the tMCAO + LM group were increased compared to the tMCAO + saline group on day 14 (Fig. 6 .A, C). Western blot analysis showed that the expression levels of BDNF, GDNF and NGF in the tMCAO + LM group were significantly higher than the tMCAO + saline group. There is no significant difference between those two sham groups(Fig. 6 .B,D,E,F). 4. Discussion In this study, we confirmed LM treatment could inhibit neuroinflammation and effectively promote neurogenesis in a mouse model of tMCAO. Our findings showed that LM treatment inhibited the expression of pro-inflammatory cytokines and enhanced the proliferation of neural stem cells around SVZ after ischemic stroke injury. Furthermore, LM treatment promoted the proliferation and migration of neuroblasts after cerebral infarction. And LM treatment can also attenuate the apoptosis of neuroblasts during migration to ischemic aera, promote neuroblasts differentiate into mature neurons and increase the expression of neurotrophic factors. After ischemic stroke, a disruption in cerebral blood supply results in cellular acidosis, mitochondrial overcharge and the production of high levels of ROS (Bang, Goyal, and Liebeskind 2015), leading to oxidative stress and vast neural cell death, and brain ischemia can also stimulate adult neurogenesis (Marques et al. 2019). Limonin exerts anti-inflammatory, anti-tumor and analgesic effects by regulating NF-κB, MAPK and JAK-STAT signaling pathways[ 20 ]. This function may generate a beneficial effect at the early stage of ischemic stroke. Our findings showed LM treatment decreased iNOS and IL-1β protein levels on day 3 after tMCAO. This result means LM has an anti-inflammatory effect on tMCAO mice at the early stage of ischemic stroke. Further, releasing of proinflammatory cytokines has a negative effect on brain repair and neurogenesis (Xiong, Liu, and Yang 2016). IL-1β overexpression results in chronic neuroinflammation and severely impair adult hippocampal neurogenesis [ 21 , 22 ]. iNOS is an intracellular marker of the M1 microglia, and the expression of iNOS was increased and result in neural apoptosis or necrosis, this effect leads to poor neurogenesis after stroke [ 23 – 25 ]. And a suitable inflammatory microenvironment is beneficial for the subsequent neurogenesis [ 16 ] and we suspected LM could promote neurogenesis after ischemic stroke. Neurogenesis is a process that the neural progenitor cells (NPCs) in neurogenic regions go through proliferation, migration, differentiation and integrating in the local circuitry, then the newborn neurons could exert their functional role in the brain [ 26 , 27 ]. Adult neurogenesis is important to brain normal function and this process provides a promising target for therapeutic strategies in various brain pathologies [ 28 ]. The SVZ and SGZ in the dentate gyrus are two main established neurogenic regions [ 29 , 30 ]. Evidences have shown that ischemic stroke could stimulate adult neurogenesis in order to relieve ischemic injury in the brain [ 31 – 33 ], and inhibition of stroke-induced neurogenesis results in a worsened functional outcome in rodent stroke models [ 34 , 35 ]. Thus, artificial enhance stroke-induced neurogenesis could be a therapeutic target for cerebral function recovery after stroke. In this study, we mainly focused our research on neurogenesis in SVZ after ischemic stroke. NSCs and NPCs have the ability of asymmetric cell division, so these cells could produce mature offspring neurons and maintain the stem cell and progenitor cell pools. This ability may allow for continued brain repair [ 36 ]. Ischemic stroke strongly stimulates NSCs proliferation in the SVZ and the proportion of mitotically active NSCs reaching a peak at 7 days after ischemic stroke [ 37 , 38 ]. In order to verify whether LM treatment could promote neurogenesis, we used BrdU/Nestin to label proliferation of NSCs. Our findings showed LM treatment increased the number of BrdU/Nestin-postive cells around SVZ on day 7 after tMCAO, and the expression of Nestin protein was also increased on day 7 after injury. These finding means LM treatment could promote the proliferation of NSCs around SVZ. NSCs in SVZ could differentiate towards neuroblasts which express doublecortin (DCX) [ 39 ]. DCX-positive neuroblasts could continue proliferate in SVZ and migrate to the damage area to form mature neurons after ischemic stroke [ 40 , 41 ]. Then we used DCX as a maker to stain the proliferation and migration of neuroblasts in our study. And we found the number of BrdU/DCX-positive cells in the SVZ and the expression of DCX protein were increased through LM treatment on day 7 after operation, this result showed us that LM treatment promoted the proliferation of neuroblasts. This conclusion was also confirmed in our subsequent study. We found the number of BrdU/DCX-positive cells in the SVZ were also increased through LM treatment on day 14 after operation. And the number of BrdU/DCX-positive cells in LM treated tMCAO mice in the peri-infarcted zone were increased compared to saline treated tMCAO mice, the expression of DCX protein was also increased. This result means LM treatment promoted the migration of neuroblasts from SVZ to injury aera after ischemic stroke. But the proliferative neuroblasts through a process of apoptosis during migration to the infracted area and neuroinflammation would aggravate neurons death [ 42 , 43 ]. Our result confirmed LM treatment could inhibit neuroinflammation and this effect may alleviate the death of neuroblasts during migration. Then we used Cleaved-Caspase 3 to labeled neuroblasts undergoing apoptosis. Our findings showed that the number of Cleaved-Caspase 3/DCX-positive cells were increased through LM treatment, which means LM treatment alleviated the process of apoptosis and promoted neuroblasts survival. At last, we found the number of BrdU/NeuN-positive cells in the peri-infarcted zone were increased through LM treatment, this showed us that LM treatment was beneficial for the process of differentiation. In addition, the expression level of BDNF, GDNF and NGF proteins were increased in LM treatmed tMCAO mice compared to saline treated tMCAO mice. BDNF, GDNF and NGF are endogenous nutrient factors that could improve neurorepair by enhancing survival of neural cells and preventing glial scar formation, allowing improved functional plasticity after stroke [ 44 , 45 ], and elevated levels of BDNF, GDNF and NGF could enhance endogenous neurogenesis after ischemic stroke[ 46 , 47 ]. The major limitation in our study was that we just focused on the effect of LM treatment, we did not explore the specific mechanism that LM inhibited neuroinflammation and promoted neurogenesis. We may continue to study the mechanism in the future. 5. Conclusion In conclusion, we found LM treatment could inhibit neuroinflammation at the early stage of ischemic stroke. And LM treatment promoted the proliferation of NSCs and neuroblasts, promoted neuroblasts migration from SVZ to damage area. Meanwhile, the apoptosis of neuroblasts during migration was weaken through LM treatment, and LM promoted neuroblasts differentiate into mature neurons and increased the expression of neurotrophic factors. These findings indicate that LM treatment may be an effective therapy for neuronal function recovery after ischemic stroke. Declarations Funding: This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 81701162).We are grateful for thoughtful comments on the manuscript from Zhen Li, and have benefited from additional fruitful discussions involving Xin Li,Bailu Wu,Qiankun Zhang.We sincerely thank the proof-readers and editors for their meticulous review and valuable suggestions, which significantly improved the quality of this work. Author contributions: Conceptualization: Yang Yang,Nan Li Methodology: Yang Yang, Supervision: Nan Li Writing—original draft: Yang Yang,Nan Li Writing—review & editing: Nan Li Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Competing interests The authors declare no competing interests. References Benjamin, E. et al. Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation 135 (10), e146–e603 (2017). Marques, B. et al. The role of neurogenesis in neurorepair after ischemic stroke. Semin. Cell Dev. Biol. 95 , 98–110 (2019). Wang, J. et al. Effects of crenolanib, a nonselective inhibitor of PDGFR, in a mouse model of transient middle cerebral artery occlusion. Neuroscience 364 , 202–211 (2017). Lan, X. et al. Modulators of microglial activation and polarization after intracerebral haemorrhage. Nat. Rev. Neurol. 13 (7), 420–433 (2017). 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Stem Cells . 26 (1), 254–265 (2008). Liao, W. et al. Therapeutic effect of human umbilical cord multipotent mesenchymal stromal cells in a rat model of stroke. Transplantation 87 (3), 350–359 (2009). Jeong, C. H. et al. Mesenchymal stem cells expressing brain-derived neurotrophic factor enhance endogenous neurogenesis in an ischemic stroke model. Biomed Res Int, 2014: p. 129145. (2014). Additional Declarations No competing interests reported. Supplementary Files supplementarymaterials.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6879079","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":474736577,"identity":"db25c52b-a118-41fd-a27a-23b5eb77bd40","order_by":0,"name":"YANG YANG","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"YANG","middleName":"","lastName":"YANG","suffix":""},{"id":474736578,"identity":"61105566-077d-48d6-9337-e9d689de5866","order_by":1,"name":"Nan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACefnzHx98/GcjZ9/eQKQWwxkMxoYz2NKMDXgOEGvNDQYzYR62w4kGEglE6mCc3ZDGzMOTlmAu+XjjDYYam2iCWthlDhx7OEfCJs9ydlqxBcOxtNwGgrY0JLYbvDFIK2a4nWMmwdhwmLAWhgPJbBI8CYcTG26eIVbLjTQ2SZ4DhxM33OAhUothzxlmw5kNacaSPUC/JBDjF3n2HsYHHxts5PjZD2+88aHGhgiHIQHiowZJC6k6RsEoGAWjYGQAAARRQfF9gP62AAAAAElFTkSuQmCC","orcid":"","institution":"The Second Affiliated Hospital of Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Nan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-06-12 09:53:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6879079/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6879079/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85385927,"identity":"b1096506-a490-4ba0-a84c-6f470948f1d5","added_by":"auto","created_at":"2025-06-25 09:52:18","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":125601,"visible":true,"origin":"","legend":"\u003cp\u003e(A,B,C) LM treatment can prolong survival time. (D) Western blot analysis iNOS and IL-1β. (E, F) Quantification of iNOS and IL-1β protein levels in each group (*p \u0026lt; 0.05 vs. the Sham + saline group; #p \u0026lt; 0.05 vs. tMCAO + LM group; n = 10/group). Data are shown as mean ± SD).(G, H)LM treatment can reduce the size of the infarcted area((*p \u0026lt; 0.05 vs. the Sham + saline group).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6879079/v1/d9959dbba4df7738bcc047ce.jpeg"},{"id":85385932,"identity":"f6d46e32-e494-4b0b-bbf2-685459abc1bb","added_by":"auto","created_at":"2025-06-25 09:52:19","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":253114,"visible":true,"origin":"","legend":"\u003cp\u003eLM promoted neural stem cells proliferation around SVZ on day 7 after ischemic stroke. (A) Representative images of double immunofluorescence staining illustrated the BrdU/Nestin-positive cells. Scale bar = 50μm. (B) Quantification of immunofluorescence staining (*p \u0026lt; 0.05, n = 10/group). (C) Western blot analysis Nestin. (D) Quantification of Nestin protein levels in each group (*p \u0026lt; 0.05 vs. the Sham + saline group; #p \u0026lt; 0.05 vs. tMCAO + LM group; n = 10/group). Data are shown as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6879079/v1/e45bc68f1b4f58321c38662f.jpeg"},{"id":85385928,"identity":"8aab6bdd-2da4-4533-99fd-639e04190868","added_by":"auto","created_at":"2025-06-25 09:52:18","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":262251,"visible":true,"origin":"","legend":"\u003cp\u003eLM promoted neuroblasts proliferation in SVZ on day 7 after ischemic stroke. (A) Representative images of double immunofluorescence staining illustrated the BrdU/DCX-positive cells. Scale bar = 50μm. (B) Quantification of immunofluorescence staining (*p \u0026lt; 0.05, n = 10/group). (C) Western blot analysis DCX. (D) Quantification of DCX protein levels in each group (*p \u0026lt; 0.05 vs. the Sham+saline group; #p \u0026lt; 0.05 vs. tMCAO + LM group; n = 10/group). Data are shown as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6879079/v1/13147d594ab12961af04f217.jpeg"},{"id":85385938,"identity":"098166bc-8b13-4c81-a290-324076f1ca3a","added_by":"auto","created_at":"2025-06-25 09:52:19","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":236416,"visible":true,"origin":"","legend":"\u003cp\u003eLM promoted neuroblasts proliferation in SVZ and the migration of neuroblasts in peri-infract region on day 14 after ischemic stroke. (A, B) Representative images of double immunofluorescence staining illustrated the BrdU/DCX-positive cells in SVZ and peri-infract region. White arrowheads point out BrdU/DCX-positive cells in peri-infraction zone. Scale bar = 50μm. (C) Western blot analysis DCX. (D) Quantification of immunofluorescence staining in SVZ (*p \u0026lt; 0.05 vs. the Sham + saline group; #p \u0026lt; 0.05 vs. tMCAO + LM group; n = 10/group). (E) Quantification of immunofluorescence staining in peri-infract region (*p \u0026lt; 0.05, n = 10/group). (F) Quantification of DCX protein levels in each group (*p \u0026lt; 0.05 vs. the Sham + saline group; #p \u0026lt; 0.05 vs. tMCAO + LM group; n = 10/group). Data are shown as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6879079/v1/7b62bc99de4ab819906ff7ac.jpeg"},{"id":85385931,"identity":"381acb99-d418-4aba-8c85-53cd54bbe6b0","added_by":"auto","created_at":"2025-06-25 09:52:19","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":88214,"visible":true,"origin":"","legend":"\u003cp\u003eLM inhibited the apoptosis of neuroblasts during migration. (A) Representative images of double immunofluorescence staining illustrated the Cleaved-Caspase 3/DCX-positive cells in peri-infract region. White arrowheads point out the Cleaved-Caspase 3/DCX-positive cells. Scale bar = 50μm. (B) Quantification of immunofluorescence staining in peri-infract region (*p \u0026lt; 0.05, n = 10/group)\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6879079/v1/4ad385a09cb71e6283ae5414.jpeg"},{"id":85385935,"identity":"1add36cf-c75f-490c-bf38-f04b0d08d5cd","added_by":"auto","created_at":"2025-06-25 09:52:19","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":179746,"visible":true,"origin":"","legend":"\u003cp\u003eLM promoted the differentiation of neuroblasts to mature neurons and increased the expression of neurotrophic factors. (A) Representative images of double immunofluorescence staining illustrated the BrdU/NeuN-positive cells in peri-infract region. White arrowheads point out BrdU/NeuN-positive cells. Scale bar = 50μm. (B) Western blot analysis BDNF, NGF and NeuN. (C) Quantification of immunofluorescence staining in peri-infract region (*p \u0026lt; 0.05, n = 10/group). (D, E, F) Quantification of BDNF, NGF and NeuN protein levels in each group (*p \u0026lt; 0.05 vs. the Sham+saline group; #p \u0026lt; 0.05 vs. tMCAO + LM group; n = 10/group). Data are shown as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6879079/v1/d52e522312ab97cef09ef166.jpeg"},{"id":98609322,"identity":"d4ec4ffd-00a4-4848-a666-00e0a39c5126","added_by":"auto","created_at":"2025-12-19 14:09:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1700008,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6879079/v1/be089457-879f-4fbb-8309-07039b977b86.pdf"},{"id":85385986,"identity":"ff6dae7a-7cf4-4e48-afb7-919342d633ec","added_by":"auto","created_at":"2025-06-25 09:52:26","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":169730292,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterials.zip","url":"https://assets-eu.researchsquare.com/files/rs-6879079/v1/192d3b82ba21c625f54c03ec.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eLimonin attenuates neuroinflammation and enhances neurogenesis in a tMCAO mouse model of ischemic stroke\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIschemic stroke is a leading cause of disability worldwide with high mortality and morbidity, and the effective treatment is limited [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A disruption in cerebral blood supply results in massive neural cell death along with loss of neurological function associated with the affected area [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Previous studies have revealed that neurogenesis in the subventricular zone (SVZ) is a promising endogenous and beneficial process for ischemic stroke [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Neural stem cells (NSCs) are located in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in hippocampus [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These NSCs retain the capacity to self-renew and possibly differentiate into neurons which are able to integrate in functional circuits [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Normally, the NSCs from SVZ could proliferate and differentiate into neural progenitor cells that migrate to other brain areas where they generate functional neurons for brain repair [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. After ischemic stroke, NSCs from the SVZ can proliferate and generate doublecortin (DCX)-positive neuroblasts. Neuroblasts migrate to the ischemic zone and differentiate into mature neurons, which replace dead or injured neurons, integrate into the neuronal circuits, or secrete neuroprotective cytokines [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Thus, enhancing the efficiency of endogenous neurogenesis may provide a potential therapeutic method for ischemic stroke treatment.\u003c/p\u003e \u003cp\u003eLimonin (LM) is an abundant natural tetracyclic triterpenoid compound with rich biological activities. LM has a wide range of anti-inflammatory, anti-microbial and anti-viral effects in mammalian systems [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. LM has neuroprotective effects by inhibiting neuronal apoptosis and damage in cells and rat models[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].As we all known, excessive production of ROS may lead to oxidative stress injury and disrupts homeostasis in the brain. Then we assumed LM could inhibit neuroinflammation through reducing generation of ROS and then generate a stable microenvironment for ischemic brain. Previous study has shown that stroke could stimulate adult neurogenesis to against ischemic damage [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. But ischemic injury could also activate immune cells such as microglia and macrophages, these cells can produce a large number of proinflammatory cytokines that can disrupt neural cells and inhibit neurogenesis [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Evidences have shown inflammation reduced the survival rate of new-born striatal neurons and new hippocampal neurons [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, a suitable inflammatory environment may promote neurogenesis after ischemic stroke and the effect of LM on the subsequent process of brain repair remain uncleared. In this study, we investigated whether LM could inhibit neuroinflammation and enhance SVZ neurogenesis after ischemic stroke in a mouse model of transient middle cerebral artery occlusion (tMCAO).\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eWe bought adult, male C57BL/6 mice (11\u0026ndash;14 weeks old, 25\u0026ndash;30 g) from the Animal Experimental Center of Zhengzhou University. All experimental protocols were approved by the Ethics Committee of the Second Affiliated Hospital of Zhengzhou University. All methods were carried out in accordance with relevant guidelines and regulations. This study is reported in accordance with ARRIVE guidelines..All mice were maintained at a constant temperature in the animal room with free access to food and water. All efforts were made to minimize the number of animals used and their suffering.After the anesthesia was completed, the mice were euthanized by dislocating their cervical vertebrae and crushing them.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Transient middle cerebral artery occlusion Model\u003c/h2\u003e \u003cp\u003eThe intraluminal filament technique was used to made the transient middle cerebral artery occlusion (tMCAO) mice model as we previously described[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Firstly,surgical anesthesia was achieved with isoflurane (4% induction, 1.5% maintenance in 70% N₂O/30% O₂),then we made a midline neck incision to expose the carotid arteries. We inserted a 6.0 nylon suture monofilament with silicone-coated tip into the right internal carotid artery to block the origin of the middle cerebral artery. Lastly, we pulled out approximately 10 mm of the monofilament for blood reperfusion 60 minutes later. We defined a successful tMCAO as a decrease in cerebral blood flow of more than 80% as measured by laser Doppler flowmetry (Moor Instruments, Devon, UK). The mice in sham groups repeated the steps described above except that the filament was advanced to the origin of the middle cerebral artery and immediately withdrawn.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Treatment and Groups\u003c/h2\u003e \u003cp\u003eWe randomly divided all mice into four groups: sham-operated mice treated with saline (Sham\u0026thinsp;+\u0026thinsp;saline), sham-operated mice treated with LM (Sham\u0026thinsp;+\u0026thinsp;LM), tMCAO mice treated with saline (tMCAO\u0026thinsp;+\u0026thinsp;saline), tMCAO mice treated with LM (tMCAO\u0026thinsp;+\u0026thinsp;LM). LM was diluted to 10% (w/v) with saline. We chose 6 mg/kg/min as the optimal dose and mice were continuously infused with saline or LM by intravenous (i.v.) infusion via tail vein 10 minutes before the operation until the reperfusion begins. Furthermore, the mice used for immunofluorescence to detect cell proliferation were received of 5-bromo2\u0026prime;-deoxyuridine (BrdU; 50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) by intraperitoneal injections once daily for 7 days or 14 days beginning 24 h after surgery\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Immunofluorescence\u003c/h2\u003e \u003cp\u003eWe performed immunofluorescence analysis on the brain on day 7 and 14 after tMCAO as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. First, we anesthetized mice, then transcardially perfused them with PBS followed by 4% paraformaldehyde. Then we carefully removed the whole brains and fixed them in 4% paraformaldehyde overnight at 4\u0026deg; C. After that we immersed the brains in 30% sucrose/PBS to dehydrate until they sank. We sliced each brain into 20-\u0026micro;m-thick sections by cryoultramicrotomy (CM1100, Leica Biosystems, Germany). Moreover, the brain sections used for BrdU detection were incubated in 2 mol/L HCl for 45 minutes and 0.1 mol/L sodium borate buffer (pH8.5) for 10 minutes at room temperature. Then we used PBS to wash these brain sections three times for 5 minutes and incubated them in PBST (0.3% Triton X-100 in PBS) for 30 minutes. After that, these brain sections were blocked in 1% bovine serum albumin (BSA) / PBST for 30minutes and we incubated them overnight at 4\u0026deg;C with antibodies against BrdU (1:500, Abcam, USA), DCX (1:300, Novus Biologicals, USA), NeuN (1:500, Novus Biologicals, USA), Cleaved-Caspase 3 (1:500, Affinity, China), Nestin (1:500, Affinity, China). All sections were washed in PBS three times for 5 minutes in the second day and we used secondary antibodies to incubate them for 2h at room temperature. Then these sections were washed in PBS three times for 5 minutes again. Finally, we put these brain sections on microslides, covered them with 4\u0026prime;,6-diamidino2-phenylindole (DAPI; Santa Cruz Biotech, Dallas, TX, USA) after drying and an investigator\u003c/p\u003e \u003cp\u003eobserved them with a fluorescence microscope (ZEISS Scope A1, ZEISS, Germany). For each section that contained brain infarction, an investigator randomly chose three non-overlapping 10 \u0026times; fields around SVZ to quantify BrdU/Nestin-positive cells, three non-overlapping 20 \u0026times; fields were chosen to quantify BrdU/DCX-positive cells in SVZ and BrdU/DCX-positive cells, BrdU/NeuN-positive cells, Cleaved-Caspase 3/DCX-positive cells in peri-infarction zones. And brain sections from sham-operation groups repeated the steps described above. All investigators were blinded to the treatments and groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Western blot analysis\u003c/h2\u003e \u003cp\u003eWe sacrificed mice for Western blot analysis on days 3, 7 and 14 after tMCAO as previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Protein was extracted from the whole right hemisphere and separated on 10% glycine gel and transferred onto polyvinylidene difluoride membranes. Firstly, we carefully removed the brains from deeply anesthetized mice, and protein samples were extracted from the whole right hemisphere in every groups and separated on 10% glycine gel. Then we transferred protein to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). And we used 5% nonfat milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) to block the membranes for 45 minutes at room temperature. After that, the membranes were incubated with primary antibodies against iNOS (1:1500, Affinity, China), IL-1β(1:1000, Affinity, China), DCX (1:1000, Novus Biologicals, USA), Nestin (1:1000, Affinity, China), BDNF (1:2000, Novus Biologicals, USA), NGF (1:2000, Abcam, USA), GDNF (1:1500, Abcam, USA), β-actin (1:2000, Affinity, USA) over night at 4\u0026deg;C. Then we washed these membranes with TBST three times for 5 minutes in the second day and incubated them with secondary antibodies for 2 h at room temperature. Lastly, these membranes were washed with TBST three times for 5 minutes again and protein bands were visualized by enhanced chemiluminescence detection kit (CWBIO, Beijing, China). An investigator blinded to the animal group quantified the optical density of the protein bands through Gel Analysis V 2.02 software (Clinx Science Instruments, Shanghai, China). β-actin served as a loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was carried out with SPSS version 13.0. All results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. We used Student\u0026rsquo;s t test or one-way ANOVA to analyze the differences in date from Western blot analysis and immunofluorescence. Differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1. LM treatment can inhibit neuroinflammation in the early stage of ischemic stroke, reduce the area of cerebral infarction, and prolong survival time.The survival time of the tMCAO\u0026thinsp;+\u0026thinsp;saline group was shorter than that of the tMCAO\u0026thinsp;+\u0026thinsp;LM group(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A, B, C).Western blot analysis illustrated that the expression of iNOS and IL-1β in the tMCAO\u0026thinsp;+\u0026thinsp;saline group were higher that the tMCAO\u0026thinsp;+\u0026thinsp;LM group on day 3 after operation, and LM treatment did not obviously change iNOS and IL-1β protein levels in the sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. D, E, F).The area of cerebral infarction in the tMCAO\u0026thinsp;+\u0026thinsp;saline group was larger than that in the tMCAO\u0026thinsp;+\u0026thinsp;LM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. G, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.2. LM treatment could promote neural stem cells proliferation on day 7 after ischemic stroke.In comparison with the tMCAO\u0026thinsp;+\u0026thinsp;LM group, the tMCAO\u0026thinsp;+\u0026thinsp;saline group had significantly fewer Brdu/Nestin-positive cells around SVZ on day 7. No difference was apparent between the saline-treated and LM-treated sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A, B). Western blot analysis showed that the protein levels of Nestin in the tMCAO\u0026thinsp;+\u0026thinsp;saline group were lower that the tMCAO\u0026thinsp;+\u0026thinsp;LM group, and LM treatment did not obviously change Nestin protein levels in the sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.C, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.3. LM treatment could promote neuroblasts proliferation on day 7 after ischemic stroke.Immunofluorescence staining indicated that the tMCAO\u0026thinsp;+\u0026thinsp;saline group had significantly fewer BrdU/DCX-positive cells compared to the tMCAO\u0026thinsp;+\u0026thinsp;LM group in SVZ on day 7. No difference was apparent between the saline-treated and LM-treated sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A, B). Western blot analysis showed that the protein levels of DCX in the tMCAO\u0026thinsp;+\u0026thinsp;saline group were lower that the tMCAO\u0026thinsp;+\u0026thinsp;LM group, and LM treatment did not obviously change DCX protein levels in the sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.C, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.4. LM can promote neuroblasts proliferation in SVZ and the migration of neuroblasts in peri-infract region on day 14 after ischemic stroke.Through the results of immunofluorescence staining, we find the number of BrdU/DCX-positive cells in the tMCAO\u0026thinsp;+\u0026thinsp;LM group were significant increased compared to the tMCAO\u0026thinsp;+\u0026thinsp;saline group in SVZ and peri-infarction zone on day 14. No difference was apparent between the saline-treated and LM-treated sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.A, B, D, E). Western blot analysis showed that the protein levels of DCX in the tMCAO\u0026thinsp;+\u0026thinsp;saline group were lower that the tMCAO\u0026thinsp;+\u0026thinsp;LM group, and LM treatment did not obviously change DCX protein levels in the sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.C, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.5. LM could inhibit the apoptosis of neuroblasts during migration after ischemic stroke.Through the results of immunofluorescence staining, we find the number of Cleaved-Caspase 3/DCX-positive cells in the tMCAO\u0026thinsp;+\u0026thinsp;LM group were significant increased compared to the tMCAO\u0026thinsp;+\u0026thinsp;saline group in the peri-infarction zone on day 14(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.A,B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.6. LM could promote neuroblasts differentiate into mature neurons and increases the expression of neurotrophic factors after ischemic stroke.The results of immunofluorescence analysis showed that the number of BrdU/NeuN-positive cells in the tMCAO\u0026thinsp;+\u0026thinsp;LM group were increased compared to the tMCAO\u0026thinsp;+\u0026thinsp;saline group on day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.A, C). Western blot analysis showed that the expression levels of BDNF, GDNF and NGF in the tMCAO\u0026thinsp;+\u0026thinsp;LM group were significantly higher than the tMCAO\u0026thinsp;+\u0026thinsp;saline group. There is no significant difference between those two sham groups(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.B,D,E,F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we confirmed LM treatment could inhibit neuroinflammation and effectively promote neurogenesis in a mouse model of tMCAO. Our findings showed that LM treatment inhibited the expression of pro-inflammatory cytokines and enhanced the proliferation of neural stem cells around SVZ after ischemic stroke injury. Furthermore, LM treatment promoted the proliferation and migration of neuroblasts after cerebral infarction. And LM treatment can also attenuate the apoptosis of neuroblasts during migration to ischemic aera, promote neuroblasts differentiate into mature neurons and increase the expression of neurotrophic factors.\u003c/p\u003e \u003cp\u003eAfter ischemic stroke, a disruption in cerebral blood supply results in cellular acidosis, mitochondrial overcharge and the production of high levels of ROS (Bang, Goyal, and Liebeskind 2015), leading to oxidative stress and vast neural cell death, and brain ischemia can also stimulate adult neurogenesis (Marques et al. 2019). Limonin exerts anti-inflammatory, anti-tumor and analgesic effects by regulating NF-κB, MAPK and JAK-STAT signaling pathways[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This function may generate a beneficial effect at the early stage of ischemic stroke. Our findings showed LM treatment decreased iNOS and IL-1β protein levels on day 3 after tMCAO. This result means LM has an anti-inflammatory effect on tMCAO mice at the early stage of ischemic stroke. Further, releasing of proinflammatory cytokines has a negative effect on brain repair and neurogenesis (Xiong, Liu, and Yang 2016). IL-1β overexpression results in chronic neuroinflammation and severely impair adult hippocampal neurogenesis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. iNOS is an intracellular marker of the M1 microglia, and the expression of iNOS was increased and result in neural apoptosis or necrosis, this effect leads to poor neurogenesis after stroke [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. And a suitable inflammatory microenvironment is beneficial for the subsequent neurogenesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and we suspected LM could promote neurogenesis after ischemic stroke.\u003c/p\u003e \u003cp\u003eNeurogenesis is a process that the neural progenitor cells (NPCs) in neurogenic regions go through proliferation, migration, differentiation and integrating in the local circuitry, then the newborn neurons could exert their functional role in the brain [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Adult neurogenesis is important to brain normal function and this process provides a promising target for therapeutic strategies in various brain pathologies [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The SVZ and SGZ in the dentate gyrus are two main established neurogenic regions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Evidences have shown that ischemic stroke could stimulate adult neurogenesis in order to relieve ischemic injury in the brain [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and inhibition of stroke-induced neurogenesis results in a worsened functional outcome in rodent stroke models [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Thus, artificial enhance stroke-induced neurogenesis could be a therapeutic target for cerebral function recovery after stroke.\u003c/p\u003e \u003cp\u003eIn this study, we mainly focused our research on neurogenesis in SVZ after ischemic stroke. NSCs and NPCs have the ability of asymmetric cell division, so these cells could produce mature offspring neurons and maintain the stem cell and progenitor cell pools. This ability may allow for continued brain repair [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Ischemic stroke strongly stimulates NSCs proliferation in the SVZ and the proportion of mitotically active NSCs reaching a peak at 7 days after ischemic stroke [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In order to verify whether LM treatment could promote neurogenesis, we used BrdU/Nestin to label proliferation of NSCs. Our findings showed LM treatment increased the number of BrdU/Nestin-postive cells around SVZ on day 7 after tMCAO, and the expression of Nestin protein was also increased on day 7 after injury. These finding means LM treatment could promote the proliferation of NSCs around SVZ. NSCs in SVZ could differentiate towards neuroblasts which express doublecortin (DCX) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. DCX-positive neuroblasts could continue proliferate in SVZ and migrate to the damage area to form mature neurons after ischemic stroke [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Then we used DCX as a maker to stain the proliferation and migration of neuroblasts in our study. And we found the number of BrdU/DCX-positive cells in the SVZ and the expression of DCX protein were increased through LM treatment on day 7 after operation, this result showed us that LM treatment promoted the proliferation of neuroblasts. This conclusion was also confirmed in our subsequent study. We found the number of BrdU/DCX-positive cells in the SVZ were also increased through LM treatment on day 14 after operation. And the number of BrdU/DCX-positive cells in LM treated tMCAO mice in the peri-infarcted zone were increased compared to saline treated tMCAO mice, the expression of DCX protein was also increased. This result means LM treatment promoted the migration of neuroblasts from SVZ to injury aera after ischemic stroke. But the proliferative neuroblasts through a process of apoptosis during migration to the infracted area and neuroinflammation would aggravate neurons death [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our result confirmed LM treatment could inhibit neuroinflammation and this effect may alleviate the death of neuroblasts during migration. Then we used Cleaved-Caspase 3 to labeled neuroblasts undergoing apoptosis. Our findings showed that the number of Cleaved-Caspase 3/DCX-positive cells were increased through LM treatment, which means LM treatment alleviated the process of apoptosis and promoted neuroblasts survival. At last, we found the number of BrdU/NeuN-positive cells in the peri-infarcted zone were increased through LM treatment, this showed us that LM treatment was beneficial for the process of differentiation. In addition, the expression level of BDNF, GDNF and NGF proteins were increased in LM treatmed tMCAO mice compared to saline treated tMCAO mice. BDNF, GDNF and NGF are endogenous nutrient factors that could improve neurorepair by enhancing survival of neural cells and preventing glial scar formation, allowing improved functional plasticity after stroke [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], and elevated levels of BDNF, GDNF and NGF could enhance endogenous neurogenesis after ischemic stroke[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe major limitation in our study was that we just focused on the effect of LM treatment, we did not explore the specific mechanism that LM inhibited neuroinflammation and promoted neurogenesis. We may continue to study the mechanism in the future.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, we found LM treatment could inhibit neuroinflammation at the early stage of ischemic stroke. And LM treatment promoted the proliferation of NSCs and neuroblasts, promoted neuroblasts migration from SVZ to damage area. Meanwhile, the apoptosis of neuroblasts during migration was weaken through LM treatment, and LM promoted neuroblasts differentiate into mature neurons and increased the expression of neurotrophic factors. These findings indicate that LM treatment may be an effective therapy for neuronal function recovery after ischemic stroke.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 81701162).We are grateful for thoughtful comments on the manuscript from Zhen Li, and have benefited from additional fruitful discussions involving Xin Li,Bailu Wu,Qiankun Zhang.We sincerely thank the proof-readers and editors for their meticulous review and valuable suggestions, which significantly improved the quality of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization: Yang Yang,Nan Li\u003c/p\u003e\n\u003cp\u003eMethodology: Yang Yang,\u003c/p\u003e\n\u003cp\u003eSupervision: Nan Li\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;original draft: Yang Yang,Nan Li\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;review \u0026amp; editing: Nan Li\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBenjamin, E. et al. 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(2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"limonin, Ischemic stroke, Neuroinflammation, Neurogenesis","lastPublishedDoi":"10.21203/rs.3.rs-6879079/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6879079/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeurogenesis in the subventricular zone (SVZ) is an effective way for brain repair after ischemic stroke. But neuroinflammation caused by cerebral ischemia would inhibit the effect of brain self-repair. As a Broadly active anti-inflammatory drugs, Limonin (LM) has a beneficial effect on ischemia-reperfusion(I/R) injury.However, the effect of LM on neurogenesis in the later stages of cerebral infarction is unknown. We speculate LM could generate anti-inflammation effect at the early stage of ischemic stroke and promote the subsequent neurogenesis. In our study, we used a transient middle cerebral artery occlusion (tMCAO) mouse model. We found LM treatment reduced the expression of iNOS and IL-1β proteins on day 3 after tMCAO. On day 7 after tMCAO, the number of BrdU/Nestin-positive cells around SVZ and BrdU/doublecortin (DCX)-positive cells in SVZ and the expression of Nestin, DCX proteins were increased through LM treatment. Moreover, on day 14 after tMCAO, the number of BrdU/DCX-positive cells in SVZ and peri-infarct area and the expression of DCX protein were increased in LM treated tMCAO mice. And LM treated tMCAO mice had fewer Cleaved-Caspase 3/DCX-positive cells in the peri-infarction zone compared to saline treated tMCAO mice 14 days after tMCAO. Finally, LM treatment increased the number of BrdU/NeuN-positive cells in the peri-infarct region and the expression of BDNF, GDNF, NGF proteins on day 14 after tMCAO. Our findings demonstrate that LM inhibits neuroinflammation and promotes neurogenesis after ischemic stroke.\u003c/p\u003e","manuscriptTitle":"Limonin attenuates neuroinflammation and enhances neurogenesis in a tMCAO mouse model of ischemic stroke","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 09:52:14","doi":"10.21203/rs.3.rs-6879079/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53487deb-fa74-42e0-89b8-eabba5b4342f","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50569120,"name":"Biological sciences/Neuroscience/Regeneration and repair in the nervous system"},{"id":50569121,"name":"Health sciences/Medical research"},{"id":50569122,"name":"Health sciences/Medical research/Drug development"}],"tags":[],"updatedAt":"2025-12-19T14:08:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-25 09:52:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6879079","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6879079","identity":"rs-6879079","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}