β-caryophyllene to relieve inflammation by inhibiting HMGB1 signaling in ischemic stroke mice

β-caryophyllene to relieve inflammation by inhibiting HMGB1 signaling in ischemic stroke mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article β-caryophyllene to relieve inflammation by inhibiting HMGB1 signaling in ischemic stroke mice Yuchun Wang, Yang Yang, Tuo Meng, Shengwei Liu, Jingdong Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4898492/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2025 Read the published version in Molecular Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Characterized by high mortality and high disability rate, ischemic stroke accounts for the vast majority of current stroke patients. Reperfusion after surgical treatment will cause serious secondary damage to the ischemic stroke patient, but there is still no specific drug for the clinical treatment of ischemic stroke. Anti-inflammatory disease is an important part of ischemia and reperfusion injury, and it is urgent to find new anti-inflammatory targets and drugs. High-mobility group box-1(HMGB1) is abundant in both neuronal cell bodies and axons, and has been found to have late pro-inflammatory effects, becoming one of the hot research topics in critical care medicine recently. The increase of HMGB1 expression leads to the aggravation of inflammatory reaction after ischemia stroke. B-caryophyllene (BCP) is a natural drug with anti-inflammatory effects. Whether the anti-inflammatory mechanism of BCP is related to HMGB1 is still unknown. We aimed to investigate the relationship and potential signaling mechanisms between HMGB1 and BCP in ischemia stroke model in vivo and in vitro. Methods: Establishment of middle cerebral artery embolism model in mice by thread thrombus and primary neurons were exposed to oxygen-glucose deprivation and re-oxygenation (OGD/R) in vitro. In vitro, transfection of HMGB1 DNA overexpression virus（GV-HMGB1）the same time, transfectionHMGB1 DNA silencing virus（RNAi-HMGB1）the same, in vivo , injection of GV-HMGB1 into the lateral ventricle of mice , injection of RNAi-HMGB1 into another group of mice. Results: It was found that HMGB1 increased after ischemic stroke, and further affected the expression of TLR4, RAGE and other related inflammatory factors, thus reducing the inflammatory response and finally protecting the injury. The results confirmed the effect of HMGB1 in effecting TLR4/RAGE signaling and subsequently regulating inflammation, oxidative stress and apoptosis. Furthermore, BCP alleviates ischemic brain damage potentially by suppressing HMGB1/ TLR4/RAGE signaling, reducing expression of IL-1β/IL-6/TNF-α，inhibiting neuronal death and inflammatory response. Conclusion: These data indicated that BCP exerted a protective effect against ischemia stroke inflammatory injury by adjusting the HMGB1/TLR4/RAGE signaling pathway, which provided new insights into the mechanisms of this therapeutic candidate for the treatment of ischemia stroke. ischemic stroke inflammatory HMGB1 BCP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Stroke is a local or global brain injury caused by complex causes, mainly including ischemic stroke and hemorrhagic stroke, among which ischemic stroke accounts for the vast majority(Ajoolabady, et al. 2021). Since the 1990s, the incidence of stroke has increased year by year, and the onset age has decreased year by year(Rosengren, et al. 2013). In some countries, about 10% of all deaths die from a stroke(Goyal, et al. 2015). With high morbidity and mortality, ischemic stroke can affect people of all ages, accounts for 88% of stroke(Fu, et al. 2019). All of these seriously affect the normal life of stroke patients and their families. At present, the clinical treatment mode for stroke patients is mainly surgical treatment and drug treatment(Jovin, et al. 2022). There is no specific drug for drug treatment in ischemic stroke. While thrombolysis by surgery is usually used in the treatment of acute ischemic stroke, which can lead to serious ischemia reperfusion injury again. In ischemia stroke and reperfusion process, the injury leads to a severe inflammatory reaction and neuronal death. Therefore, reducing inflammatory response and protecting neuron cells has become an important purpose before and especially after thrombolysis in patients with ischemic stroke. New and more effective drugs need to be found. High-mobility group box-1 (HMGB1) is a nuclear protein with late proinflammatory effects (Zhang, et al. 2020)that is widely distributed in mammalian cells. Recent studies have found that necrotic cells can release HMGB1(Scaffidi, et al. 2002; Zhao, et al. 2023) from outside the nucleus to induce an inflammatory response. When damaged cells from HMGB1 mice were cultured with macrophages(Xue, et al. 2020), the intensity of inflammatory response was significantly reduced. It can be known that the passive release of HMGB1 has an important role in the tissue necrotizing inflammatory response. To reduce HMGB1 expression may be important for suppressing the inflammatory response in ischemic stroke. β-caryophyllene (BCP) is a kind of sesquiterpenoids, which has strong anti-inflammatory activity and inhibits inflammatory cascade reaction(Agnes, et al. 2023). Our previous studies have shown that BCP can reduce the volume of cerebral infarction after CIR injury in rats. While the effect of BCP on HMGB1 in cerebral ischemia has not been studied. It has been confirmed that the pro-inflammatory effect of HMGB1 is related to cell surface receptors, including Toll-like receptors (TLR4) and advanced glycosylation end product receptors (RAGE)(Yang, et al. 2017). Bioinformatics analysis also found that HMGB1, TLR4 and RAGE receptors were closely related, or even directly related. Therefore, this study also wants to explore whether there is a relationship between the core of HMGB1 and downstream TLR4/RAGE. And hope to find the potential drug on this basis. In this study, we explore the relationship between HMGB1/TLR4/RAGE and the effect of BCP in vivo and in vitro to provide a theoretical basis for the application of BCP in stroke. Methods Materials β-caryophyllene, 2,3,5-triphenyltetrazolium chloride (TTC), cytosine arabinoside (Ara-C), L-glutamine, and poly-L-lysine (0.1%) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). All cell culture medium and fetal bovine serum (FBS) were obtained from GIBCO (Life Technologies, Grand Island, NY, USA). ELISA kit against interleukin-1b (IL-1b), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) were obtained from USCN (Life Science Inc., Harrington Oakland,CA, USA). RNAi-HMGB1 and GV-HMGB1 were from Gene (Shanghai). Animals Newborn C57BL/6 mice in 24 hours and adult male mice C57BL/6 (20–25 g) in a specific pathogen-free (SPF) grade were obtained from the Experimental Animal Center, Chongqing Medical University (Chongqing, China). All animal procedures were approved by the Experimental Ethics Committee of Chongqing Medical University (Reference Number: 2015027) and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All surgeries were performed under anesthesia, and all efforts were made to minimize the animals’ suffering. Primary Neuron Cultures Primary cortical neurons were prepared from newborn mice (in 24 hours) as described previously. Cortex were minced and dissected with trypsin-EDTA (0.125mg/mL) in Hank’s balanced salt solution (HBSS). Neurons were cultured in plates, precoated with 0.01%poly-L-lysine, with Dulbecco’s Modified Eagle’s Medium medium containing 10% FBS. After 4 h of incubation, neurons were maintained in neurobasal A medium supplemented with 2% B27 and 1% glutamine (2mM). Every 3 days, 50% of the culture medium was changed. Microtubule-associated protein-2 (MAP 2), the specific marker of neuron, was used to identify the purity of primary neurons by immunofluorescence. The primary cells, which commonly consist of > 95% neurons, were used in the experiments on the 7th day in vitro. Experimental design The experimental design consists of three parts. In the first part, the animals were divided into the sham group and the I/ R group, and the cells were divided into normal and OGD groups, to explore and confirm the occurrence of injury and the change of the key factors in the ischemic model. In the second part, the animals were divided into four groups: sham group, I/R group, RNAi-HMGB1 group, GV-HMGB1 group. The cells were divided into four groups: normal group, OGD group, RNAi-HMGB1 group, GV-HMGB1 group. The third part is to explore the relationship between BCP and HMGB1 pathway. The animals were divided into sham group, I/R group, and I/R BCP (72mg/kg) groups, while the cells were divided into normal group, OGD group, and OGD BCP (10µM) groups. Oxygen-Glucose Deprivation and Re-oxygenation Treatments in vitro/ Transient Focal Cerebral Ischemia in vivo Oxygen-glucose deprivation and re-oxygenation (OGD/R) were used as an in vitro model for ischemia (Zhang, et al. 2007). Briefly, at the seventh in vitro, neurons were washed and incubated with glucose-free medium, subsequently transferred to an anaerobic incubator equilibrated with 94% N2, 5% CO2, and 1% O2 at 37◦C for 1 h. The cells were then returned to the normoxic incubator with 25mM glucose without serum for 24h. Control neurons were cultured in the same medium supplemented with 25mM glucose in a normoxic incubator. Male mice underwent procedures to cause transient focal cerebral ischemia via right middle cerebral artery occlusion (MCAO) (Sugo, et al. 2002) . Briefly, mice were anesthetized with isoflurane (induced with 3% and maintained by 1.0–1.5%) mixed with oxygen and nitrogen using a facemask. The right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were separated carefully under an operating microscope. A 6 − 0 nylon monofilament (Guangzhou Jialing Biotechnology Co., Ltd., Guangzhou, China) was inserted through the stump of ECA into the ICA and advanced into the middle cerebral artery until light resistance was felt (∼8–12mm). After 1 h of MCAO, reperfusion was initiated by withdrawing the nylon monofilament. Sham-operated mice underwent identical procedure but the filament was not inserted. During the surgical procedure, rectal temperature was maintained at 37 ± 5◦C using a thermostatically controlled infrared lamp. At 24 h of reperfusion, neurological function deficits were scored, and animals that scored from 1 to 4 were chosen for further experiment. Those animals that showed brain hemorrhage or with no ischemia (three mice) were exclude from the study. The mortality rate was 0.3%. Animal administration and cell administration Mice were given three days of continuous BCP (72mg/kg) administration, and the body weight changes were recorded once a day. The fourth day, the model was established, the ischemia was 1 hour, and the next experiment was carried out after 24 hours of reperfusion. BCP (10nM) was given to primary nerve cells on the sixth day and a half after liquid exchange. 24 hours after administration, OGD1 hours and reoxygenation 24 hours later, the next experiment was continued. Detection of brain injury appearance Neurological score–After 24 hours of reperfusion, the neurobehavioral scores of mice in different treatment groups were evaluated by the Longa score (Longa, et al. 1989) . Cerebral infarct volume–The mice were given deep anesthesia after reperfusion for 24 h and decapitated for 15 min at -20 ℃, and then cut into 5 pieces (1 mm) and then incubated at 37 ℃ in 2% TTC staining solution for 30 min. The brain slices were put into 4% paraformaldehyde for 24 hours, and transferred to 4% paraformaldehyde overnight. The brain slices was removed by filter paper and placed in a black background. The normal brain tissue was red after staining, and the area of the infarct was white. The digital camera was photographed and the volume of cerebral infarction was determined by image-Pro Plus 5 software. Mice brains were infused with 4% neutral-buffered formaldehyde at indicated time, fixated for 24 h. Ethanol in graded concentrations and xylene were then used to dehydrate the brain tissue, and then they were embedded into paraffin. Hematoxylin and eosin (H&E) were used to stain the paraffin sections (5µm), according to the standard protocol. Histological analysis of the same region in each experiment was performed with a light microscope. Paraffin sections were stained with toluene blue. The Nissan bodies in the same area were observed under light microscope. Immunohistochemical/Immunofluorescence/Fluorescence probe in situ hybridization Immunohistochemical–After paraffin section dewaxing, the antigen was repaired in microwave oven, and then the endogenous peroxide was blocked with 3% hydrogen peroxide. The sections were sealed with serum for half an hour, then the first antibody and the second antibody were added, and then the nucleus was stained with DBA and restained with hematoxylin. After sealing the film, it was examined by microscope. Immunofluorescence–After dewaxing, the antigen was repaired in microwave oven, the spontaneous fluorescence quenching agent was added, the serum was blocked for half an hour, and the first antibody and the second antibody were added respectively. Finally, the nucleus was restained with DAPI. After sealing the film, it was examined by microscope. Western Blot Analysis Mouse ischemic brain tissues were harvested at 24h post-reperfusion, and then homogenized in RIPA lysis buffer (P00113D; Beyotime, Shanghai, China). The whole ischemic brain tissues were used to determine HMGB1 and TLR4 and RAGE protein levels. The protein was separated using sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE; P0012A, Beyotime, China) with a 12% polyacrylamide gel and a 10% polyacrylamide gel, and then transferred to polyvinylidene fluoride (PVDF) membrane. Then the membranes were blocked with non-fat milk (5%) and incubated overnight at 4◦C with the following primary antibodies: rabbit polyclonal antibody against HMGB1 (10829-1-AP, Proteintech, 1:250), TLR4 (19811-1-AP, Proteintech, 1:250), RAGE (AF5309, Affinity, 1:1000), and mouse internal ginseng antibody (beta-actin; 10829-1-AP, Proteintech, 1:1000). After three washes, secondary goat anti-rabbit/mouse (Bostor, China, 1:3,000) was performed to conjugate with alkaline phosphatase for 1 h at room temperature. Enhanced chemiluminescence was used to determine the immune reactivity. Gel imaging apparatus (Bio- Rad, Hercules, CA, USA) and Image Lab (Bio-Rad, Hercules, CA, USA) were used to scan and analyze the bands. PCR Real Time Quantitative Polymerase Chain Reaction Analysis Total RNA of cortex of ischemic brain were extracted using a Trizol kit (Sangon Biotech, Shanghai) and cDNA was prepared via using the AMV first chain cDNA synthesis kit (Sangon Biotech. Shanghai), according to manufacturer’s protocol. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a 10 µL volume using SYBR Premix (Bimake). The following cycling conditions were used: 30 s at 95◦C followed by 40 cycles of 5 s at 95◦C and 30 s at 60◦C. Enzyme-Linked Immunosorbent Assay (ELISA) TNF-a, IL-1β, IL-6 levels in ischemic brain tissue homogenate were detected using an ELISA kit according the manufacturer’s instructions. Statistical analysis All data are presented as the means ± SD. Graphpad software was used for statistical analysis. Differences between groups were assessed by the t-test and a value of P < 0.05 was considered statistically significant. Results 1 Inflammation injury occurred and HMGB1 increased in mouse with MCAO Male mice were prematurely fasted for 12 hours followed by right middle cerebral artery occlusion (MCAO) surgery, in which the sham group isolated vessels without plug line insertion (Fig. 1A). After an hour of clipping, the blood flow was restored. Mice status were examined 24 h later, and subsequent experiments were performed. The neurobehavioral score of mice with MCAO was higher than that of sham group (Fig. 1B). Consecutive brain sections stained with TTC indicate mouse with MCAO had cerebral infarction (Fig. 1C). HE staining showed injured brain tissue in MCAO group (Fig. 1D). Nissl staining showed that the injury of Nissl corpuscles was serious in the injury group (Fig. 1E). As can be seen from the above results, the MCAO model was successful, and the brain injury occurred. HMGB 1 has been shown to play a critical role in the development of late stages of inflammation(Zhang, et al. 2020). To further determine the mechanism of injury, we examined the expression of HMGB 1 after the onset of mouse brain injury. Brain tissues from mice were collected to detect level of HMGB1 in each group. The HMGB1 expression level in the brain was measured by western blot and Q-PCR assay. HMGB1 level was increased in injured group in vivo (Fig. 1F-G). The results of immunohistochemistry showed that HMGB1 was highly expressed in the injured group (Fig. 1H). The HMB1 expression level also measured by immune-fluorescence. It was found that the expression of HMGB1 raised sharply in I/R group compared with sham group (Fig. 1I). Finally, we examined the level of proinflammatory factors in mouse brain homogenate grinding fluid and found that the proinflammatory factor expression level was increased after MCAO surgery (Fig. 1J). At this point, we basically determined that inflammation occurred after cerebral ischemia and reperfusion, and HMGB 1 expression was elevated. 2 Inflammation injury occurred and HMGB1 increased in primary neurons with OGD After verifying the reperfusion damage in vivo as well as HMGB1 expression, we continued to try to explore it in vitro. Primary neurons were extracted from 24-h neonatal mice for subsequent detection. First, we performed oxygen glucose deprivation (OGD) experiments on the cells, and then collected the cell supernatant to detect the expression levels of proinflammatory factors. The results showed that neurons secreted pro-inflammatory factors after OGD (Fig. 2A). The HMGB1 expression level was measured by western blot and Q-PCR assay. HMGB1 level was increased in OGD group in vitro (Fig. 2B-C). Immunofluorescence measurement of neuronal cells also indicated that OGD group had a higher level of HMGB1 (Fig. 2D). 3 High expression of HMGB1 aggravated inflammation in MACO mice To further explore the effect of HMGB1 on inflammation in MCAO mice, we performed overexpression or silencing of HMGB 1 in mice with lateral ventricles. The results showed that after silencing HMGB 1, the neurobehavioral score decreased significantly after cerebral ischemia and reperfusion, suggesting reduced nerve injury in mice (Fig. 3A). More intuitively, in the staining experiment of serial brain sections of mice, mice in the HMGB1 silenced group had a significant reduction in cerebral infarction volume (Fig. 3B). As before, in the HE-stained and Ni-stained sections, we also observed that the brain damage was largely relieved after HMGB1 silencing (Fig. 3C-D). After observing a series of representations, we further examined the proportion of proinflammatory factor expression in extracts from mouse brain homogenates. In the HMGB1 overexpression group, the expression of proinflammatory factors was significantly higher than that in the other groups, while the proinflammatory factor level was significantly decreased in the RNAi-HMGB 1 group (Fig. 3E). These results surface, HMGB1 is one of the key factors in the development of inflammation after ischemia and reperfusion injury in mice. 4 High expression of HMGB1 aggravated inflammation in vivo and vitro via RAGE/TLR4 In this part of the study, we sought to further find the mechanism by which HMGB 1 regulates inflammation. We first adopted the network pharmacological induction method and found that RAGE and TLR 4 were the most highly associated with HMGB 1 in inflammatory diseases (Fig. 4A). For further validation, we examined the mRNA and protein content of RAGE and TLR 4 in mouse brain tissues. We found that RAGE and TLR 4 were elevated after injury, and they were highest in injured mice overexpressing HMGB1. Also, evidence that HMGB1 may regulate inflammation through RAGE/TLR4 is that RAGE/TLR4 expression is significantly reduced after silencing HMGB1 in injured mice (Fig. 4B-C). Similarly, this result was again on subsequent primary mouse neurons (Fig. 4D-E). Finally, in this part, we examined the expression of proinflammatory factors in the culture supernatant of mouse primary neuronal cells. What we can know is the fact that overexpression of HMGB1 brings neurons to higher levels of proinflammatory factors after OGD, and silencing of HMGB1 partly reduces proinflammatory factor expression (Fig. 4F). The results of this part basically suggest that HMGB 1 may affect the inflammatory process by regulating RAGE/TLR4 expression and thus affecting the expression of proinflammatory factors. 5 BCP protects mice from cerebral ischemia-reperfusion injury It has long been a consensus that drugs to treat brain diseases must be able to cross the blood-brain barrier. Our group found that BCP can cross the blood-brain barrier, and it was reported to have anti-inflammatory effects. In our study, we gave mice 72mg / kg BCP daily one week earlier and performed MCAO surgery one week later (Fig. 5A). The mice were scored at the experimental endpoint and found that the mice in the BCP group significantly improved stroke (Fig. 5B). Similarly, the cerebral infarction volume was significantly reduced in mice fed with BCP a week earlier after MCAO surgery (Fig. 5C). And the results of Ni staining and HE staining also proved again that BCP has a better neuroprotective effect (Fig. 5D-E). 6 BCP protects mice from cerebral ischemia-reperfusion injury by regulating HMGB1 In the previous study, we found that BCP has a promising therapeutic effect of neuroinflammation in mice. BCP has been shown to have anti-inflammatory effects, and HMGB 1 in turn is a key molecule associated with inflammation, so we went on to detect HMGB 1 changes in mouse brain as well as in mouse primary neurons after BCP treatment. We found that both in the mRNA and protein levels, brain HMGB1 levels were significantly lower than those in the BCP group (Fig. 6A-B). This suggests that BCP exerting neuroprotective effects may be related to HMGB1. This result is consistent with the experimental results of primary mouse neurons (Fig. 6C-D). Finally, the anti-inflammatory and protective effects of BCP were also effectively reduced in vivo and in vitro (Fig. 6E-F), which further demonstrated the levels of BCP. Discussion Our study revealed a huge association between HMGB 1 expression and stroke. After ischemic stroke, there will be a series of serious inflammatory cascade reactions. Stroke is a disease caused by insufficient or abnormal blood supply to the brain due by sudden obstruction or rupture of the cerebral blood vessels(Tu, et al. 2023), and its pathogenesis involves dysregulation of multiple inflammatory responses and neuroprotective mechanisms. As a pro-inflammatory molecule, HMGB 1 can play a role in the development and progression of ischemic stroke. Some studies have shown that cell death occurs with the release of HMGB1, and the role of HMGB1 in necrotizing inflammation has been studied(Kaczmarek, et al. 2013; Scaffidi, et al. 2002; Yanai, et al. 2009). HMGB1 has a close relationship with inflammation. It is a nuclear protein that can be released from the nucleus outside the cell during cell damage or inflammation and acts as a pro-inflammatory signaling molecule involved in the regulation of the inflammatory response(Treutiger, et al. 2003). HMGB1 is able to promote the development and maintenance of the inflammatory response through several mechanisms. Moreover, HMGB1 can also recruit and activate immune cells and enhance the activity during inflammatory cell infiltration and tissue damage repair. Therefore, HMGB1 plays an important role in the regulation of inflammation, participating not only in the initiation phase of the inflammatory response, but also in the persistence and chronicity of inflammation. We found it can bind to RAGE and TLR4, and activate downstream inflammatory signaling pathways, leading to the release of inflammatory factors such as TNF-α, IL-1β and IL-6, and then trigger inflammatory response. These results were verified in animal experiments and in cell experiments in primary neurons in our study. To make this result more plausible, we overexpressed and silenced HMGB 1 both in vivo and in vitro. The results of this part are also very strong and direct evidence that the high expression of HMGB 1 can aggravate the nerve injury and inflammation after stroke. At a time when stroke treatment is in a bottleneck, it is urgent and necessary to conduct drug development for key factors affecting disease occurrence and development. Our finding is important and meaningful: drugs that reduce HMGB 1 expression may be potential drugs for the treatment of stroke. An important principle for drug search for brain diseases is to pass through the blood-brain barrier. BCP is a naturally occurring terpene compound widely found in many plants, especially at high levels in cannabis. It has several biological activities, including anti-inflammatory, antioxidant, antibacterial, and analgesic effects. In pharmacological studies, BCP has been considered to have therapeutic potential for several diseases, including inflammatory diseases, neurodegenerative diseases and pain management. In addition, it has been studied for antifungal and insect resistance effects in plants. Some previous basic studies by our team support the potential of BCP in neuroprotection, but the specific application in stroke treatment still needs more clinical experiments and in-depth research to validate its safety and efficacy. Therefore, the current research on β -bamboo in stroke treatment is in its infancy and requires further scientific exploration and validation. We also verified the effect of BCP on HMGB1 in vitro and in vivo, and found that it could effectively reduce the effect of HMGB1. This suggests that the anti-inflammatory and neuroprotective effects of BCP may coincide with those of HMGB1. Conclusion In a word, this study confirmed for the first time that BCP may affect the neuroprotective effect of downstream HMGB1 and other inflammatory factors However, the specific mechanism of BCP and HMGB1 needs to be further explored. It provides a new potential therapeutic target and research direction for the treatment of stroke. Abbreviations I/R Ischemia/Reperfusion NF-κB Nuclear Factor Kappa B IL interleukin OGD/R Oxygen-Glucose Deprivation/Reoxygenation MCAO Middle Cerebral Artery Occlusion HMGB1 High-mobility group box-1 RAGE receptor for advanced glycation end products TLR4 Toll-like receptors 4 BCP β-caryophyllene ELISA Enzyme-Linked Immunosorbent Assay RT-qPCR Real-Time Quantitative Polymerase Chain Reaction TTC 2,3,5-Triphenyltetrazolium Chloride RNAi RNA interference MAP2 Microtubule-Associated Protein 2 DAPI 4’,6-diamidino-2-phenylindole PBS Phosphate-Buffered Saline FBS Fetal Bovine Serum DMEM Dulbecco’s Modified Eagle Medium EDTA Ethylenediaminetetraacetic Acid BCA Bicinchoninic Acid PVDF Polyvinylidene Difluoride GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Declarations Ethics approval and consent to participate All animal procedures were approved by the Experimental Ethics Committee of Chongqing Medical University (Reference Number: 2015027) and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Consent for publication Not applicable. Availability of data and materials The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by Chongqing Science and Technology Commission (General subjects of basic Research and Frontier Exploration in Chongqing, cstc2018jcyjAX0378). This work was financially supported by the Chongqing science technology commission of China (CSTB2023NSCQ-MSX0267) Authors’ contribution WYC, YY and DZ envisioned the experiment. MT, WYC, CS, YY and LJD designed the experiment. WYC and LSW performed bioinformatics analysis. WYC and LDH contributed to the construction of animal model. WYC and YY performed stereotactic surgery. WYC and Laxman contributed to in situ hybrid experiment. WYC, YY and DZ analyzed the results and WYC wrote a paper. All authors reviewed the manuscript. 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Cite Share Download PDF Status: Published Journal Publication published 24 Mar, 2025 Read the published version in Molecular Medicine → Version 1 posted Editorial decision: Revision requested 18 Jan, 2025 Reviews received at journal 08 Jan, 2025 Reviewers agreed at journal 30 Dec, 2024 Reviews received at journal 07 Oct, 2024 Reviewers agreed at journal 07 Oct, 2024 Reviewers invited by journal 18 Aug, 2024 Editor assigned by journal 14 Aug, 2024 Submission checks completed at journal 14 Aug, 2024 First submitted to journal 12 Aug, 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-4898492","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":344187939,"identity":"83e7d182-faec-4c88-98f2-3dd9489d54e1","order_by":0,"name":"Yuchun Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABF0lEQVRIie3PMUsDMRTA8SfCnUPw1tfB+hUCLgctfpYEoTfYdnG5oUOKkNvsar/FidD5HYFOObtWOomzcJObxbRy4HD2HAXzJ4SE5Dc8AJ/vDxaEU0XI+2J3IYC4fjj+kZwyM6U4HXwRAmwnXUxuKbVmT+BXJECp6FmvxlFUFqaaIETZ9QAh7UkVltRI2Isq5npz07kfC6IlAtq3BYJNpGJj0UhCqUxHb2S+ZpwoQOA4WuCRNlIh440EHPnQTzJfWUe2NdkeICduFrQkcxpyKnRN1AHCCkfSKznfzVLeIXOzPMZimVxoNmwk51n2WiG/lLOoNFX63u9G2ehhXU16Z7PQNpJvsXoX++XGbI3VB9H+1+fz+f5Vn/vaavmetr0JAAAAAElFTkSuQmCC","orcid":"","institution":"Chongqing Medical University, The Key Laboratory of Biochemistry and Molecular Pharmacology","correspondingAuthor":true,"prefix":"","firstName":"Yuchun","middleName":"","lastName":"Wang","suffix":""},{"id":344187940,"identity":"bf648521-b0a1-4843-8c98-2761cc0707e8","order_by":1,"name":"Yang Yang","email":"","orcid":"","institution":"Department of Pharmacy, Chongqing Health Center for Women and Children, Women and Children's Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yang","suffix":""},{"id":344187941,"identity":"59586f12-e379-451c-a8cf-4264e2c5c159","order_by":2,"name":"Tuo Meng","email":"","orcid":"","institution":"Department of child health in Children's Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tuo","middleName":"","lastName":"Meng","suffix":""},{"id":344187942,"identity":"ced263aa-a795-432b-95df-f04a0979b3a9","order_by":3,"name":"Shengwei Liu","email":"","orcid":"","institution":"Department of Pharmacy, Yongchuan Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shengwei","middleName":"","lastName":"Liu","suffix":""},{"id":344187943,"identity":"38bcba54-9b57-4ef6-a0b3-31dd7817bfbc","order_by":4,"name":"Jingdong Liu","email":"","orcid":"","institution":"Chongqing Medical University, The Key Laboratory of Biochemistry and Molecular Pharmacology","correspondingAuthor":false,"prefix":"","firstName":"Jingdong","middleName":"","lastName":"Liu","suffix":""},{"id":344187944,"identity":"13887462-6372-4ee3-8360-a86f67b613d1","order_by":5,"name":"Daohang Liu","email":"","orcid":"","institution":"Chongqing Medical University, The Key Laboratory of Biochemistry and Molecular Pharmacology","correspondingAuthor":false,"prefix":"","firstName":"Daohang","middleName":"","lastName":"Liu","suffix":""},{"id":344187945,"identity":"c709b550-ee21-4ab4-a402-d8413a4afb41","order_by":6,"name":"Bharati Laxman","email":"","orcid":"","institution":"Chongqing Medical University, The Key Laboratory of Biochemistry and Molecular Pharmacology","correspondingAuthor":false,"prefix":"","firstName":"Bharati","middleName":"","lastName":"Laxman","suffix":""},{"id":344187946,"identity":"3b52116a-7047-4692-bee5-fb4f356f2572","order_by":7,"name":"Sha Chen","email":"","orcid":"","institution":"Chongqing Medical University, The Key Laboratory of Biochemistry and Molecular Pharmacology","correspondingAuthor":false,"prefix":"","firstName":"Sha","middleName":"","lastName":"Chen","suffix":""},{"id":344187947,"identity":"40457b8d-8a58-4af9-9704-5134d0422885","order_by":8,"name":"Zhi Dong","email":"","orcid":"","institution":"Chongqing Medical University, The Key Laboratory of Biochemistry and Molecular Pharmacology","correspondingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2024-08-12 07:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4898492/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4898492/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s10020-025-01171-z","type":"published","date":"2025-03-24T15:57:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66378159,"identity":"0b62d4d9-c820-4847-aaec-b5bb5dc71c41","added_by":"auto","created_at":"2024-10-11 06:38:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2194433,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAfter cerebral ischemia and reperfusion, the mice developed brain damage, and the expression of HMGB1 was elevated.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Timeline of the mouse experiments. \u003cstrong\u003eB\u003c/strong\u003eNeurobehavioral evaluation of mice with or without MCAO. Higher scores indicate more severe neurological damage. \u003cstrong\u003eC \u003c/strong\u003eTTC staining of serial brain sections of the mice. White represents the infarcted fraction.\u003cstrong\u003e D\u003c/strong\u003e HE staining of the cortical portion of the mouse brain. Nuclei are colored in blue and the cytoplasm is colored in red. \u003cstrong\u003eE\u003c/strong\u003e Partial Nic staining of mouse cerebral cortex. The large and large number of Nissl bodies indicates that nerve cells have a strong function in protein synthesis; on the contrary, Nissl bodies will decrease or even disappear when nerve cells are damaged. \u003cstrong\u003eF\u003c/strong\u003e HMGB1 mRNA levels in ipsilateral brain tissues from control and MCAO mice. \u003cstrong\u003eG\u003c/strong\u003e Protein levels of HMGB1 relative to internal reference in ipsilateral brain tissue from control and MCAO mice. \u003cstrong\u003eH\u003c/strong\u003e Immunohistochemical staining of HMGB1 in ipsilateral brains of control mice and MCAO mice. Blue represents negative, and brown is positive. \u003cstrong\u003eI\u003c/strong\u003e Immunofluorescence staining of HMGB1 in the ipsilateral brain of control and MCAO mice. DAPI localized the nucleus and MAP 2 as a neural cell marker. \u003cstrong\u003eJ\u003c/strong\u003e Detection of proinflammatory factors in ipsilateral brain tissue of control mice and MCAO mice. n =6.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4898492/v1/8961c4c668daaac7c2474235.png"},{"id":66378162,"identity":"65ca1617-23e1-421a-8b1f-248f8cc5d4d6","added_by":"auto","created_at":"2024-10-11 06:38:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":433502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAfter hypoxia reoxygenation experiments, mouse primary cortical neurons were tested for proinflammatory factors and HMGB 1 expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Proinflammatory factor levels in primary cortical neurons in control and hypoxic reoxygenation group. \u003cstrong\u003eB\u003c/strong\u003e HMGB1 mRNA levels in primary cortical neurons in the control and hypoxic reoxygenation groups. \u003cstrong\u003eC \u003c/strong\u003eThe HMGB1 protein levels in primary cortical neurons in control and hypoxia relative to the internal parameters.\u003cstrong\u003e D\u003c/strong\u003e HMGB1 immunofluorescence levels in primary cortical neurons in control and hypoxia reoxygenation groups. DAPI localized the nucleus and MAP 2 as a neuronal marker. n=4\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4898492/v1/12671f6feff99d52cbebe46e.png"},{"id":66378161,"identity":"95c7fa81-aae9-4f6a-a003-9c400cfa24ec","added_by":"auto","created_at":"2024-10-11 06:38:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3100968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of HMGB1 exacerbates cerebral ischemia-reperfusion injury in mice, and silencing of HMGB1 reduces the injury.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Neurobehavioral evaluation of mice in different groups. Higher scores indicate more severe neurological damage. \u003cstrong\u003eB \u003c/strong\u003eTTC staining of serial brain sections of the mice. White represents the infarcted fraction. \u003cstrong\u003eC\u003c/strong\u003e HE staining of the cortical portion of the mouse brain. Nuclei are colored in blue and the cytoplasm is colored in red. \u003cstrong\u003eD\u003c/strong\u003e Partial Nic staining of mouse cerebral cortex. The large and large number of Nissl bodies indicates that nerve cells have a strong function in protein synthesis; on the contrary, Nissl bodies will decrease or even disappear when nerve cells are damaged. \u003cstrong\u003eE\u003c/strong\u003e Detection of proinflammatory factors in ipsilateral brain tissue of control mice and MCAO mice. n =6.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4898492/v1/238e87f465b544ea49aed13c.png"},{"id":66378164,"identity":"bdd70419-5b94-4a94-9d51-59b6ac9990f2","added_by":"auto","created_at":"2024-10-11 06:38:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1248578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn cerebral ischemia and reperfusion, HMGB 1 affects the expression of downstream RAGE and TLR 4 in vivo and in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003ePPI network in HMGB 1 in inflammatory diseases in the brain. Different colors represent different molecules, and the connecting lines represent the existence of mutual relations. Shorter the connecting line, fewer nodes pass through, indicating stronger interrelationships. \u003cstrong\u003eB\u003c/strong\u003e HMGB1 mRNA levels in ipsilateral brain tissues in different groups. \u003cstrong\u003eC\u003c/strong\u003e Protein levels of HMGB1 relative to internal reference in ipsilateral brain tissue from different groups mice. \u003cstrong\u003eD\u003c/strong\u003eHMGB1 mRNA levels in primary cortical neurons in different groups. \u003cstrong\u003eE \u003c/strong\u003eThe HMGB1 protein levels in primary cortical neurons in different groups. \u003cstrong\u003eF\u003c/strong\u003e Detection of proinflammatory factors in culture supernatant of primary neuronal cells. n =6 in vivo experiments and n=4 in vitro experiments.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4898492/v1/6af2a2f83b363d1f265d8104.png"},{"id":66378163,"identity":"e40f4976-297e-48cb-a9b2-2df768d68b2b","added_by":"auto","created_at":"2024-10-11 06:38:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1986334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-caryophyllene can play a protective role in cerebral ischemia and reperfusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Timeline of the mouse experiments. \u003cstrong\u003eB\u003c/strong\u003eNeurobehavioral evaluation of mice with or without MCAO. Higher scores indicate more severe neurological damage. \u003cstrong\u003eC \u003c/strong\u003eTTC staining of serial brain sections of the mice. White represents the infarcted fraction.\u003cstrong\u003e D\u003c/strong\u003e HE staining of the cortical portion of the mouse brain. Nuclei are colored in blue and the cytoplasm is colored in red. \u003cstrong\u003eE\u003c/strong\u003e Partial Nic staining of mouse cerebral cortex. The large and large number of Nissl bodies indicates that nerve cells have a strong function in protein synthesis; on the contrary, Nissl bodies will decrease or even disappear when nerve cells are damaged.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4898492/v1/0d76f72a2d410f9e4179ee75.png"},{"id":66378160,"identity":"cf954c62-179b-4396-958e-d452c9e4df66","added_by":"auto","created_at":"2024-10-11 06:38:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":487610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-caryophyllene play a protective role in cerebral ischemia and reperfusion maybe related to the effect on HMGB1 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e HMGB1 mRNA levels in ipsilateral brain tissues in different groups. \u003cstrong\u003eB\u003c/strong\u003e Protein levels of HMGB1 relative to internal reference in ipsilateral brain tissue from different groups mice. \u003cstrong\u003eC\u003c/strong\u003e HMGB1 mRNA levels in primary cortical neurons in different groups. \u003cstrong\u003eD \u003c/strong\u003eThe HMGB1 protein levels in primary cortical neurons in different groups. \u003cstrong\u003eE\u003c/strong\u003e Detection of proinflammatory factors in ipsilateral brain tissue of different group mice.\u003cstrong\u003eF\u003c/strong\u003e Detection of proinflammatory factors in culture supernatant of primary neuronal cells. n =6 in vivo experiments and n=4 in vitro experiments.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4898492/v1/86950e38d90e3ea0ddbdf052.png"},{"id":79605575,"identity":"38f6e651-0564-4ca1-9112-a8c7b316b4fa","added_by":"auto","created_at":"2025-03-31 16:11:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13353799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4898492/v1/d27228b5-6ae8-4fb9-93df-0b002fe52fc3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"β-caryophyllene to relieve inflammation by inhibiting HMGB1 signaling in ischemic stroke mice","fulltext":[{"header":"Background","content":"\u003cp\u003eStroke is a local or global brain injury caused by complex causes, mainly including ischemic stroke and hemorrhagic stroke, among which ischemic stroke accounts for the vast majority(Ajoolabady, et al. 2021). Since the 1990s, the incidence of stroke has increased year by year, and the onset age has decreased year by year(Rosengren, et al. 2013). In some countries, about 10% of all deaths die from a stroke(Goyal, et al. 2015). With high morbidity and mortality, ischemic stroke can affect people of all ages, accounts for 88% of stroke(Fu, et al. 2019). All of these seriously affect the normal life of stroke patients and their families. At present, the clinical treatment mode for stroke patients is mainly surgical treatment and drug treatment(Jovin, et al. 2022). There is no specific drug for drug treatment in ischemic stroke. While thrombolysis by surgery is usually used in the treatment of acute ischemic stroke, which can lead to serious ischemia reperfusion injury again. In ischemia stroke and reperfusion process, the injury leads to a severe inflammatory reaction and neuronal death. Therefore, reducing inflammatory response and protecting neuron cells has become an important purpose before and especially after thrombolysis in patients with ischemic stroke. New and more effective drugs need to be found.\u003c/p\u003e \u003cp\u003eHigh-mobility group box-1 (HMGB1) is a nuclear protein with late proinflammatory effects (Zhang, et al. 2020)that is widely distributed in mammalian cells. Recent studies have found that necrotic cells can release HMGB1(Scaffidi, et al. 2002; Zhao, et al. 2023) from outside the nucleus to induce an inflammatory response. When damaged cells from HMGB1 mice were cultured with macrophages(Xue, et al. 2020), the intensity of inflammatory response was significantly reduced. It can be known that the passive release of HMGB1 has an important role in the tissue necrotizing inflammatory response. To reduce HMGB1 expression may be important for suppressing the inflammatory response in ischemic stroke.\u003c/p\u003e \u003cp\u003eβ-caryophyllene (BCP) is a kind of sesquiterpenoids, which has strong anti-inflammatory activity and inhibits inflammatory cascade reaction(Agnes, et al. 2023). Our previous studies have shown that BCP can reduce the volume of cerebral infarction after CIR injury in rats. While the effect of BCP on HMGB1 in cerebral ischemia has not been studied.\u003c/p\u003e \u003cp\u003eIt has been confirmed that the pro-inflammatory effect of HMGB1 is related to cell surface receptors, including Toll-like receptors (TLR4) and advanced glycosylation end product receptors (RAGE)(Yang, et al. 2017). Bioinformatics analysis also found that HMGB1, TLR4 and RAGE receptors were closely related, or even directly related. Therefore, this study also wants to explore whether there is a relationship between the core of HMGB1 and downstream TLR4/RAGE. And hope to find the potential drug on this basis.\u003c/p\u003e \u003cp\u003eIn this study, we explore the relationship between HMGB1/TLR4/RAGE and the effect of BCP in vivo and in vitro to provide a theoretical basis for the application of BCP in stroke.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eβ-caryophyllene, 2,3,5-triphenyltetrazolium chloride (TTC), cytosine arabinoside (Ara-C), L-glutamine, and poly-L-lysine (0.1%) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). All cell culture medium and fetal bovine serum (FBS) were obtained from GIBCO (Life Technologies, Grand Island, NY, USA). ELISA kit against interleukin-1b (IL-1b), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) were obtained from USCN (Life Science Inc., Harrington Oakland,CA, USA). RNAi-HMGB1 and GV-HMGB1 were from Gene (Shanghai).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eNewborn C57BL/6 mice in 24 hours and adult male mice C57BL/6 (20\u0026ndash;25 g) in a specific pathogen-free (SPF) grade were obtained from the Experimental Animal Center, Chongqing Medical University (Chongqing, China). All animal procedures were approved by the Experimental Ethics Committee of Chongqing Medical University (Reference Number: 2015027) and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All surgeries were performed under anesthesia, and all efforts were made to minimize the animals\u0026rsquo; suffering.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003ePrimary Neuron Cultures\u003c/h2\u003e \u003cp\u003ePrimary cortical neurons were prepared from newborn mice (in 24 hours) as described previously. Cortex were minced and dissected with trypsin-EDTA (0.125mg/mL) in Hank\u0026rsquo;s balanced salt solution (HBSS). Neurons were cultured in plates, precoated with 0.01%poly-L-lysine, with Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium medium containing 10% FBS. After 4 h of incubation, neurons were maintained in neurobasal A medium supplemented with 2% B27 and 1% glutamine (2mM). Every 3 days, 50% of the culture medium was changed. Microtubule-associated protein-2 (MAP 2), the specific marker of neuron, was used to identify the purity of primary neurons by immunofluorescence. The primary cells, which commonly consist of \u0026gt;\u0026thinsp;95% neurons, were used in the experiments on the 7th day in vitro.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eThe experimental design consists of three parts.\u003c/p\u003e \u003cp\u003eIn the first part, the animals were divided into the sham group and the I/ R group, and the cells were divided into normal and OGD groups, to explore and confirm the occurrence of injury and the change of the key factors in the ischemic model.\u003c/p\u003e \u003cp\u003eIn the second part, the animals were divided into four groups: sham group, I/R group, RNAi-HMGB1 group, GV-HMGB1 group. The cells were divided into four groups: normal group, OGD group, RNAi-HMGB1 group, GV-HMGB1 group.\u003c/p\u003e \u003cp\u003eThe third part is to explore the relationship between BCP and HMGB1 pathway. The animals were divided into sham group, I/R group, and I/R BCP (72mg/kg) groups, while the cells were divided into normal group, OGD group, and OGD BCP (10\u0026micro;M) groups.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eOxygen-Glucose Deprivation and Re-oxygenation Treatments in vitro/ Transient Focal Cerebral Ischemia in vivo\u003c/h2\u003e \u003cp\u003eOxygen-glucose deprivation and re-oxygenation (OGD/R) were used as an in vitro model for ischemia (Zhang, et al. 2007). Briefly, at the seventh in vitro, neurons were washed and incubated with glucose-free medium, subsequently transferred to an anaerobic incubator equilibrated with 94% N2, 5% CO2, and 1% O2 at 37◦C for 1 h. The cells were then returned to the normoxic incubator with 25mM glucose without serum for 24h. Control neurons were cultured in the same medium supplemented with 25mM glucose in a normoxic incubator.\u003c/p\u003e \u003cp\u003eMale mice underwent procedures to cause transient focal cerebral ischemia via right middle cerebral artery occlusion (MCAO)\u003csup\u003e(Sugo, et al. 2002)\u003c/sup\u003e. Briefly, mice were anesthetized with isoflurane (induced with 3% and maintained by 1.0\u0026ndash;1.5%) mixed with oxygen and nitrogen using a facemask. The right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were separated carefully under an operating microscope. A 6\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon monofilament (Guangzhou Jialing Biotechnology Co., Ltd., Guangzhou, China) was inserted through the stump of ECA into the ICA and advanced into the middle cerebral artery until light resistance was felt (\u0026sim;8\u0026ndash;12mm). After 1 h of MCAO, reperfusion was initiated by withdrawing the nylon monofilament. Sham-operated mice underwent identical procedure but the filament was not inserted. During the surgical procedure, rectal temperature was maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;5◦C using a thermostatically controlled infrared lamp. At 24 h of reperfusion, neurological function deficits were scored, and animals that scored from 1 to 4 were chosen for further experiment. Those animals that showed brain hemorrhage or with no ischemia (three mice) were exclude from the study. The mortality rate was 0.3%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimal administration and cell administration\u003c/h2\u003e \u003cp\u003eMice were given three days of continuous BCP (72mg/kg) administration, and the body weight changes were recorded once a day. The fourth day, the model was established, the ischemia was 1 hour, and the next experiment was carried out after 24 hours of reperfusion.\u003c/p\u003e \u003cp\u003eBCP (10nM) was given to primary nerve cells on the sixth day and a half after liquid exchange. 24 hours after administration, OGD1 hours and reoxygenation 24 hours later, the next experiment was continued.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetection of brain injury appearance\u003c/h2\u003e \u003cp\u003eNeurological score\u0026ndash;After 24 hours of reperfusion, the neurobehavioral scores of mice in different treatment groups were evaluated by the Longa score\u003csup\u003e(Longa, et al. 1989)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCerebral infarct volume\u0026ndash;The mice were given deep anesthesia after reperfusion for 24 h and decapitated for 15 min at -20 ℃, and then cut into 5 pieces (1 mm) and then incubated at 37 ℃ in 2% TTC staining solution for 30 min. The brain slices were put into 4% paraformaldehyde for 24 hours, and transferred to 4% paraformaldehyde overnight. The brain slices was removed by filter paper and placed in a black background. The normal brain tissue was red after staining, and the area of the infarct was white. The digital camera was photographed and the volume of cerebral infarction was determined by image-Pro Plus 5 software.\u003c/p\u003e \u003cp\u003eMice brains were infused with 4% neutral-buffered formaldehyde at indicated time, fixated for 24 h. Ethanol in graded concentrations and xylene were then used to dehydrate the brain tissue, and then they were embedded into paraffin. Hematoxylin and eosin (H\u0026amp;E) were used to stain the paraffin sections (5\u0026micro;m), according to the standard protocol. Histological analysis of the same region in each experiment was performed with a light microscope. Paraffin sections were stained with toluene blue. The Nissan bodies in the same area were observed under light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical/Immunofluorescence/Fluorescence probe in situ hybridization\u003c/h2\u003e \u003cp\u003eImmunohistochemical\u0026ndash;After paraffin section dewaxing, the antigen was repaired in microwave oven, and then the endogenous peroxide was blocked with 3% hydrogen peroxide. The sections were sealed with serum for half an hour, then the first antibody and the second antibody were added, and then the nucleus was stained with DBA and restained with hematoxylin. After sealing the film, it was examined by microscope.\u003c/p\u003e \u003cp\u003eImmunofluorescence\u0026ndash;After dewaxing, the antigen was repaired in microwave oven, the spontaneous fluorescence quenching agent was added, the serum was blocked for half an hour, and the first antibody and the second antibody were added respectively. Finally, the nucleus was restained with DAPI. After sealing the film, it was examined by microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eMouse ischemic brain tissues were harvested at 24h post-reperfusion, and then homogenized in RIPA lysis buffer (P00113D; Beyotime, Shanghai, China). The whole ischemic brain tissues were used to determine HMGB1 and TLR4 and RAGE protein levels. The protein was separated using sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE; P0012A, Beyotime, China) with a 12% polyacrylamide gel and a 10% polyacrylamide gel, and then transferred to polyvinylidene fluoride (PVDF) membrane. Then the membranes were blocked with non-fat milk (5%) and incubated overnight at 4◦C with the following primary antibodies: rabbit polyclonal antibody against HMGB1 (10829-1-AP, Proteintech, 1:250), TLR4 (19811-1-AP, Proteintech, 1:250), RAGE (AF5309, Affinity, 1:1000), and mouse internal ginseng antibody (beta-actin; 10829-1-AP, Proteintech, 1:1000). After three washes, secondary goat anti-rabbit/mouse (Bostor, China, 1:3,000) was performed to conjugate with alkaline phosphatase for 1 h at room temperature. Enhanced chemiluminescence was used to determine the immune reactivity. Gel imaging apparatus (Bio- Rad, Hercules, CA, USA) and Image Lab (Bio-Rad, Hercules, CA, USA) were used to scan and analyze the bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePCR Real Time Quantitative Polymerase Chain Reaction Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA of cortex of ischemic brain were extracted using a Trizol kit (Sangon Biotech, Shanghai) and cDNA was prepared via using the AMV first chain cDNA synthesis kit (Sangon Biotech. Shanghai), according to manufacturer\u0026rsquo;s protocol. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a 10 \u0026micro;L volume using SYBR Premix (Bimake). The following cycling conditions were used: 30 s at 95◦C followed by 40 cycles of 5 s at 95◦C and 30 s at 60◦C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eTNF-a, IL-1β, IL-6 levels in ischemic brain tissue homogenate were detected using an ELISA kit according the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Graphpad software was used for statistical analysis. Differences between groups were assessed by the t-test and a value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e1 Inflammation injury occurred and HMGB1 increased in mouse with MCAO\u003c/h2\u003e\n \u003cp\u003eMale mice were prematurely fasted for 12 hours followed by right middle cerebral artery occlusion (MCAO) surgery, in which the sham group isolated vessels without plug line insertion (Fig. 1A). After an hour of clipping, the blood flow was restored. Mice status were examined 24 h later, and subsequent experiments were performed. The neurobehavioral score of mice with MCAO was higher than that of sham group (Fig. 1B). Consecutive brain sections stained with TTC indicate mouse with MCAO had cerebral infarction (Fig. 1C). HE staining showed injured brain tissue in MCAO group (Fig. 1D). Nissl staining showed that the injury of Nissl corpuscles was serious in the injury group (Fig. 1E). As can be seen from the above results, the MCAO model was successful, and the brain injury occurred. HMGB 1 has been shown to play a critical role in the development of late stages of inflammation(Zhang, et al. 2020). To further determine the mechanism of injury, we examined the expression of HMGB 1 after the onset of mouse brain injury. Brain tissues from mice were collected to detect level of HMGB1 in each group. The HMGB1 expression level in the brain was measured by western blot and Q-PCR assay. HMGB1 level was increased in injured group in vivo (Fig. 1F-G). The results of immunohistochemistry showed that HMGB1 was highly expressed in the injured group (Fig. 1H). The HMB1 expression level also measured by immune-fluorescence. It was found that the expression of HMGB1 raised sharply in I/R group compared with sham group (Fig. 1I). Finally, we examined the level of proinflammatory factors in mouse brain homogenate grinding fluid and found that the proinflammatory factor expression level was increased after MCAO surgery (Fig. 1J). At this point, we basically determined that inflammation occurred after cerebral ischemia and reperfusion, and HMGB 1 expression was elevated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003e2 Inflammation injury occurred and HMGB1 increased in primary neurons with OGD\u003c/h2\u003e\n \u003cp\u003eAfter verifying the reperfusion damage in vivo as well as HMGB1 expression, we continued to try to explore it in vitro. Primary neurons were extracted from 24-h neonatal mice for subsequent detection. First, we performed oxygen glucose deprivation (OGD) experiments on the cells, and then collected the cell supernatant to detect the expression levels of proinflammatory factors. The results showed that neurons secreted pro-inflammatory factors after OGD (Fig. 2A). The HMGB1 expression level was measured by western blot and Q-PCR assay. HMGB1 level was increased in OGD group in vitro (Fig. 2B-C). Immunofluorescence measurement of neuronal cells also indicated that OGD group had a higher level of HMGB1 (Fig. 2D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e3 High expression of HMGB1 aggravated inflammation in MACO mice\u003c/h2\u003e\n \u003cp\u003eTo further explore the effect of HMGB1 on inflammation in MCAO mice, we performed overexpression or silencing of HMGB 1 in mice with lateral ventricles. The results showed that after silencing HMGB 1, the neurobehavioral score decreased significantly after cerebral ischemia and reperfusion, suggesting reduced nerve injury in mice (Fig. 3A). More intuitively, in the staining experiment of serial brain sections of mice, mice in the HMGB1 silenced group had a significant reduction in cerebral infarction volume (Fig. 3B). As before, in the HE-stained and Ni-stained sections, we also observed that the brain damage was largely relieved after HMGB1 silencing (Fig. 3C-D). After observing a series of representations, we further examined the proportion of proinflammatory factor expression in extracts from mouse brain homogenates. In the HMGB1 overexpression group, the expression of proinflammatory factors was significantly higher than that in the other groups, while the proinflammatory factor level was significantly decreased in the RNAi-HMGB 1 group (Fig. 3E). These results surface, HMGB1 is one of the key factors in the development of inflammation after ischemia and reperfusion injury in mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e4 High expression of HMGB1 aggravated inflammation in vivo and vitro via RAGE/TLR4\u003c/h2\u003e\n \u003cp\u003eIn this part of the study, we sought to further find the mechanism by which HMGB 1 regulates inflammation. We first adopted the network pharmacological induction method and found that RAGE and TLR 4 were the most highly associated with HMGB 1 in inflammatory diseases (Fig. 4A). For further validation, we examined the mRNA and protein content of RAGE and TLR 4 in mouse brain tissues. We found that RAGE and TLR 4 were elevated after injury, and they were highest in injured mice overexpressing HMGB1. Also, evidence that HMGB1 may regulate inflammation through RAGE/TLR4 is that RAGE/TLR4 expression is significantly reduced after silencing HMGB1 in injured mice (Fig. 4B-C). Similarly, this result was again on subsequent primary mouse neurons (Fig. 4D-E). Finally, in this part, we examined the expression of proinflammatory factors in the culture supernatant of mouse primary neuronal cells. What we can know is the fact that overexpression of HMGB1 brings neurons to higher levels of proinflammatory factors after OGD, and silencing of HMGB1 partly reduces proinflammatory factor expression (Fig. 4F). The results of this part basically suggest that HMGB 1 may affect the inflammatory process by regulating RAGE/TLR4 expression and thus affecting the expression of proinflammatory factors.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e5 BCP protects mice from cerebral ischemia-reperfusion injury\u003c/h2\u003e\n \u003cp\u003eIt has long been a consensus that drugs to treat brain diseases must be able to cross the blood-brain barrier. Our group found that BCP can cross the blood-brain barrier, and it was reported to have anti-inflammatory effects. In our study, we gave mice 72mg / kg BCP daily one week earlier and performed MCAO surgery one week later (Fig. 5A). The mice were scored at the experimental endpoint and found that the mice in the BCP group significantly improved stroke (Fig. 5B). Similarly, the cerebral infarction volume was significantly reduced in mice fed with BCP a week earlier after MCAO surgery (Fig. 5C). And the results of Ni staining and HE staining also proved again that BCP has a better neuroprotective effect (Fig. 5D-E).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e6 BCP protects mice from cerebral ischemia-reperfusion injury by regulating HMGB1\u003c/h2\u003e\n \u003cp\u003eIn the previous study, we found that BCP has a promising therapeutic effect of neuroinflammation in mice. BCP has been shown to have anti-inflammatory effects, and HMGB 1 in turn is a key molecule associated with inflammation, so we went on to detect HMGB 1 changes in mouse brain as well as in mouse primary neurons after BCP treatment. We found that both in the mRNA and protein levels, brain HMGB1 levels were significantly lower than those in the BCP group (Fig. 6A-B). This suggests that BCP exerting neuroprotective effects may be related to HMGB1. This result is consistent with the experimental results of primary mouse neurons (Fig. 6C-D). Finally, the anti-inflammatory and protective effects of BCP were also effectively reduced in vivo and in vitro (Fig. 6E-F), which further demonstrated the levels of BCP.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study revealed a huge association between HMGB 1 expression and stroke. After ischemic stroke, there will be a series of serious inflammatory cascade reactions. Stroke is a disease caused by insufficient or abnormal blood supply to the brain due by sudden obstruction or rupture of the cerebral blood vessels(Tu, et al. 2023), and its pathogenesis involves dysregulation of multiple inflammatory responses and neuroprotective mechanisms. As a pro-inflammatory molecule, HMGB 1 can play a role in the development and progression of ischemic stroke. Some studies have shown that cell death occurs with the release of HMGB1, and the role of HMGB1 in necrotizing inflammation has been studied(Kaczmarek, et al. 2013; Scaffidi, et al. 2002; Yanai, et al. 2009). HMGB1 has a close relationship with inflammation. It is a nuclear protein that can be released from the nucleus outside the cell during cell damage or inflammation and acts as a pro-inflammatory signaling molecule involved in the regulation of the inflammatory response(Treutiger, et al. 2003). HMGB1 is able to promote the development and maintenance of the inflammatory response through several mechanisms. Moreover, HMGB1 can also recruit and activate immune cells and enhance the activity during inflammatory cell infiltration and tissue damage repair. Therefore, HMGB1 plays an important role in the regulation of inflammation, participating not only in the initiation phase of the inflammatory response, but also in the persistence and chronicity of inflammation.\u003c/p\u003e \u003cp\u003eWe found it can bind to RAGE and TLR4, and activate downstream inflammatory signaling pathways, leading to the release of inflammatory factors such as TNF-α, IL-1β and IL-6, and then trigger inflammatory response. These results were verified in animal experiments and in cell experiments in primary neurons in our study. To make this result more plausible, we overexpressed and silenced HMGB 1 both in vivo and in vitro. The results of this part are also very strong and direct evidence that the high expression of HMGB 1 can aggravate the nerve injury and inflammation after stroke.\u003c/p\u003e \u003cp\u003eAt a time when stroke treatment is in a bottleneck, it is urgent and necessary to conduct drug development for key factors affecting disease occurrence and development. Our finding is important and meaningful: drugs that reduce HMGB 1 expression may be potential drugs for the treatment of stroke.\u003c/p\u003e \u003cp\u003eAn important principle for drug search for brain diseases is to pass through the blood-brain barrier. BCP is a naturally occurring terpene compound widely found in many plants, especially at high levels in cannabis. It has several biological activities, including anti-inflammatory, antioxidant, antibacterial, and analgesic effects. In pharmacological studies, BCP has been considered to have therapeutic potential for several diseases, including inflammatory diseases, neurodegenerative diseases and pain management. In addition, it has been studied for antifungal and insect resistance effects in plants.\u003c/p\u003e \u003cp\u003eSome previous basic studies by our team support the potential of BCP in neuroprotection, but the specific application in stroke treatment still needs more clinical experiments and in-depth research to validate its safety and efficacy. Therefore, the current research on β -bamboo in stroke treatment is in its infancy and requires further scientific exploration and validation.\u003c/p\u003e \u003cp\u003eWe also verified the effect of BCP on HMGB1 in vitro and in vivo, and found that it could effectively reduce the effect of HMGB1. This suggests that the anti-inflammatory and neuroprotective effects of BCP may coincide with those of HMGB1.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn a word, this study confirmed for the first time that BCP may affect the neuroprotective effect of downstream HMGB1 and other inflammatory factors However, the specific mechanism of BCP and HMGB1 needs to be further explored. It provides a new potential therapeutic target and research direction for the treatment of stroke.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eI/R Ischemia/Reperfusion\u003c/p\u003e\n\u003cp\u003eNF-\u0026kappa;B Nuclear Factor Kappa B\u003c/p\u003e\n\u003cp\u003eIL interleukin\u003c/p\u003e\n\u003cp\u003eOGD/R Oxygen-Glucose Deprivation/Reoxygenation\u003c/p\u003e\n\u003cp\u003eMCAO Middle Cerebral Artery Occlusion\u003c/p\u003e\n\u003cp\u003eHMGB1\u0026nbsp;High-mobility group box-1\u003c/p\u003e\n\u003cp\u003eRAGE receptor for advanced glycation end products\u003c/p\u003e\n\u003cp\u003eTLR4\u0026nbsp;Toll-like receptors 4\u003c/p\u003e\n\u003cp\u003eBCP\u0026nbsp;\u0026beta;-caryophyllene\u003c/p\u003e\n\u003cp\u003eELISA Enzyme-Linked Immunosorbent Assay\u003c/p\u003e\n\u003cp\u003eRT-qPCR Real-Time Quantitative Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eTTC 2,3,5-Triphenyltetrazolium Chloride\u003c/p\u003e\n\u003cp\u003eRNAi RNA interference\u003c/p\u003e\n\u003cp\u003eMAP2 Microtubule-Associated Protein 2\u003c/p\u003e\n\u003cp\u003eDAPI 4\u0026rsquo;,6-diamidino-2-phenylindole\u003c/p\u003e\n\u003cp\u003ePBS Phosphate-Buffered Saline\u003c/p\u003e\n\u003cp\u003eFBS Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eDMEM Dulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e\n\u003cp\u003eEDTA Ethylenediaminetetraacetic Acid\u003c/p\u003e\n\u003cp\u003eBCA Bicinchoninic Acid\u003c/p\u003e\n\u003cp\u003ePVDF Polyvinylidene Difluoride\u003c/p\u003e\n\u003cp\u003eGAPDH Glyceraldehyde 3-Phosphate Dehydrogenase\u003c/p\u003e\n\u003cp\u003eSDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Experimental Ethics Committee of Chongqing Medical University (Reference Number: 2015027) and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Chongqing Science and Technology Commission (General subjects of basic Research and Frontier Exploration in Chongqing,\u0026nbsp;cstc2018jcyjAX0378).\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Chongqing science technology commission of China (CSTB2023NSCQ-MSX0267)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWYC, YY and DZ envisioned the experiment. MT, WYC, CS, YY and LJD designed the experiment. WYC and LSW performed bioinformatics analysis. WYC and LDH contributed to the construction of animal model. WYC and YY performed stereotactic surgery. WYC and Laxman contributed to in situ hybrid experiment. WYC, YY and DZ analyzed the results and WYC wrote a paper. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgnes, J. P., et al. 2023 beta-Caryophyllene Inhibits Oxaliplatin-Induced Peripheral Neuropathy in Mice: Role of Cannabinoid Type 2 Receptors, Oxidative Stress and Neuroinflammation. Antioxidants (Basel) 12(10).\u003c/li\u003e\n\u003cli\u003eAjoolabady, A., et al. 2021 Targeting autophagy in ischemic stroke: From molecular mechanisms to clinical therapeutics. Pharmacol Ther 225:107848.\u003c/li\u003e\n\u003cli\u003eFu, C., et al. 2019 Potential Neuroprotective Effect of miR-451 Against Cerebral Ischemia/Reperfusion Injury in Stroke Patients and a Mouse Model. World Neurosurg 130:e54-e61.\u003c/li\u003e\n\u003cli\u003eGoyal, M., et al. 2015 Randomized assessment of rapid endovascular treatment of ischemic stroke. N Engl J Med 372(11):1019-30.\u003c/li\u003e\n\u003cli\u003eJovin, T. G., et al. 2022 Thrombectomy for anterior circulation stroke beyond 6 h from time last known well (AURORA): a systematic review and individual patient data meta-analysis. Lancet 399(10321):249-258.\u003c/li\u003e\n\u003cli\u003eKaczmarek, A., P. Vandenabeele, and D. V. Krysko 2013 Necroptosis: the release of damage-associated molecular patterns and its physiological relevance. Immunity 38(2):209-23.\u003c/li\u003e\n\u003cli\u003eLonga, E. Z., et al. 1989 Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20(1):84-91.\u003c/li\u003e\n\u003cli\u003eRosengren, A., et al. 2013 Twenty-four-year trends in the incidence of ischemic stroke in Sweden from 1987 to 2010. Stroke 44(9):2388-93.\u003c/li\u003e\n\u003cli\u003eScaffidi, P., T. Misteli, and M. E. Bianchi 2002 Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418(6894):191-5.\u003c/li\u003e\n\u003cli\u003eSugo, N., et al. 2002 Social stress exacerbates focal cerebral ischemia in mice. Stroke 33(6):1660-4.\u003c/li\u003e\n\u003cli\u003eTreutiger, C. J., et al. 2003 High mobility group 1 B-box mediates activation of human endothelium. J Intern Med 254(4):375-85.\u003c/li\u003e\n\u003cli\u003eTu, W. J., L. D. Wang, and Report Special Writing Group of China Stroke Surveillance 2023 China stroke surveillance report 2021. Mil Med Res 10(1):33.\u003c/li\u003e\n\u003cli\u003eXue, J., et al. 2020 Asbestos induces mesothelial cell transformation via HMGB1-driven autophagy. Proc Natl Acad Sci U S A 117(41):25543-25552.\u003c/li\u003e\n\u003cli\u003eYanai, H., et al. 2009 HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses. Nature 462(7269):99-103.\u003c/li\u003e\n\u003cli\u003eYang, M., et al. 2017 Neuroprotective Effect of beta-Caryophyllene on Cerebral Ischemia-Reperfusion Injury via Regulation of Necroptotic Neuronal Death and Inflammation: In Vivo and in Vitro. Front Neurosci 11:583.\u003c/li\u003e\n\u003cli\u003eZhang, F., et al. 2007 Neuroprotective effects of leptin against ischemic injury induced by oxygen-glucose deprivation and transient cerebral ischemia. Stroke 38(8):2329-36.\u003c/li\u003e\n\u003cli\u003eZhang, S., et al. 2020 HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia. J Neuroinflammation 17(1):15.\u003c/li\u003e\n\u003cli\u003eZhao, Z. B., et al.2023 Tubular Epithelial Cell HMGB1 Promotes AKI-CKD Transition by Sensitizing Cycling Tubular Cells to Oxidative Stress: A Rationale for Targeting HMGB1 during AKI Recovery. J Am Soc Nephrol 34(3):394-411.\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":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ischemic stroke, inflammatory, HMGB1, BCP","lastPublishedDoi":"10.21203/rs.3.rs-4898492/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4898492/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Characterized by high mortality and high disability rate, ischemic stroke accounts for the vast majority of current stroke patients. Reperfusion after surgical treatment will cause serious secondary damage to the ischemic stroke patient, but there is still no specific drug for the clinical treatment of ischemic stroke. Anti-inflammatory disease is an important part of ischemia and reperfusion injury, and it is urgent to find new anti-inflammatory targets and drugs. High-mobility group box-1(HMGB1) is abundant in both neuronal cell bodies and axons, and has been found to have late pro-inflammatory effects, becoming one of the hot research topics in critical care medicine recently. The increase of HMGB1 expression leads to the aggravation of inflammatory reaction after ischemia stroke. B-caryophyllene (BCP) is a natural drug with anti-inflammatory effects. Whether the anti-inflammatory mechanism of BCP is related to HMGB1 is still unknown. We aimed to investigate the relationship and potential signaling mechanisms between HMGB1 and BCP in ischemia stroke model in vivo and in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eEstablishment of middle cerebral artery embolism model in mice by thread thrombus and primary neurons were exposed to oxygen-glucose deprivation and re-oxygenation (OGD/R) in vitro. In vitro, transfection of HMGB1 DNA overexpression virus（GV-HMGB1）the same time, transfectionHMGB1 DNA silencing virus（RNAi-HMGB1）the same, in vivo , injection of GV-HMGB1 into the lateral ventricle of mice , injection of RNAi-HMGB1 into another group of mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e It was found that HMGB1 increased after ischemic stroke, and further affected the expression of TLR4, RAGE and other related inflammatory factors, thus reducing the inflammatory response and finally protecting the injury. The results confirmed the effect of HMGB1 in effecting TLR4/RAGE signaling and subsequently regulating inflammation, oxidative stress and apoptosis. Furthermore, BCP alleviates ischemic brain damage potentially by suppressing HMGB1/ TLR4/RAGE signaling, reducing expression of IL-1β/IL-6/TNF-α，inhibiting neuronal death and inflammatory response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e These data indicated that BCP exerted a protective effect against ischemia stroke inflammatory injury by adjusting the HMGB1/TLR4/RAGE signaling pathway, which provided new insights into the mechanisms of this therapeutic candidate for the treatment of ischemia stroke.\u003c/p\u003e","manuscriptTitle":"β-caryophyllene to relieve inflammation by inhibiting HMGB1 signaling in ischemic stroke mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-11 06:37:58","doi":"10.21203/rs.3.rs-4898492/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-18T18:54:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-09T00:03:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329469023738593626110866113133816322553","date":"2024-12-30T15:41:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-07T06:44:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231678850351154122371001852690456932217","date":"2024-10-07T05:59:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-18T12:35:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-14T06:46:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-14T06:46:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Medicine","date":"2024-08-12T07:51:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ca862c7-3cf1-4f8d-be73-1f6b97f68df4","owner":[],"postedDate":"October 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-31T16:08:19+00:00","versionOfRecord":{"articleIdentity":"rs-4898492","link":"https://doi.org/10.1186/s10020-025-01171-z","journal":{"identity":"molecular-medicine","isVorOnly":false,"title":"Molecular Medicine"},"publishedOn":"2025-03-24 15:57:05","publishedOnDateReadable":"March 24th, 2025"},"versionCreatedAt":"2024-10-11 06:37:58","video":"","vorDoi":"10.1186/s10020-025-01171-z","vorDoiUrl":"https://doi.org/10.1186/s10020-025-01171-z","workflowStages":[]},"version":"v1","identity":"rs-4898492","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4898492","identity":"rs-4898492","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}