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

doi:10.21203/rs.3.rs-4898492/v1

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

https://doi.org/10.21203/rs.3.rs-4898492/v1

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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

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.

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.

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.

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.

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.

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

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.

Acknowledgements

Not applicable.

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No competing interests reported.

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

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Abstract

Figures

Background

Methods

Materials

Animals

Primary Neuron Cultures

Experimental design

Oxygen-Glucose Deprivation and Re-oxygenation Treatments in vitro/ Transient Focal Cerebral Ischemia in vivo

Animal administration and cell administration

Detection of brain injury appearance

Immunohistochemical/Immunofluorescence/Fluorescence probe in situ hybridization

Western Blot Analysis

PCR Real Time Quantitative Polymerase Chain Reaction Analysis

Enzyme-Linked Immunosorbent Assay (ELISA)

Statistical analysis

Results

1 Inflammation injury occurred and HMGB1 increased in mouse with MCAO

2 Inflammation injury occurred and HMGB1 increased in primary neurons with OGD

3 High expression of HMGB1 aggravated inflammation in MACO mice

4 High expression of HMGB1 aggravated inflammation in vivo and vitro via RAGE/TLR4

5 BCP protects mice from cerebral ischemia-reperfusion injury

6 BCP protects mice from cerebral ischemia-reperfusion injury by regulating HMGB1

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1