BCP Treatment Research Articles

β-Caryophyllene mitigates ischemic stroke-induced white matter lesions by inhibiting pyroptosis

β-Caryophyllene (BCP), a selective agonist for cannabinoid receptor 2 (CB2R), has demonstrated promising protective effects in various pathological conditions. However, the neuroprotective effects of BCP on white matter damage induced by ischemic stroke have not been elucidated previously. In this study, we find that BCP not only improves sensorimotor and cognitive function via CB2R but also mitigates white matter lesions in mice following ischemic stroke. Furthermore, BCP enhances the viability of MO3.13 oligodendrocytes after oxygen-glucose deprivation and reoxygenation (OGD/R), attenuating OGD/R-induced cellular damage and pyroptosis. Notably, these protective effects of BCP are partially enhanced by the NLRP3 inhibitor MCC950 and counteracted by the NLRP3 activator nigericin. In addition, nigericin significantly exacerbates neurological outcomes and increases white matter lesions following BCP treatment in middle cerebral artery occlusion (MCAO) mice. These results suggest that BCP may ameliorate neurological deficits and white matter damage induced by cerebral ischemia through inhibiting NLRP3-mediated pyroptosis.

Cannabinoid 2 Receptor Activation Protects against Diabetic Cardiomyopathy through Inhibition of AGE/RAGE-Induced Oxidative Stress, Fibrosis, and Inflammasome Activation.

Oxidative stress, fibrosis, and inflammasome activation from advanced glycation end product (AGE)-receptor of advanced glycation end product (RAGE) interaction contribute to diabetic cardiomyopathy (DCM) formation and progression. Our study revealed the impact of β-caryophyllene (BCP) on activating cannabinoid type 2 receptors (CB2Rs) against diabetic complication, mainly cardiomyopathy and investigated the underlying cell signaling pathways in mice. The murine model of DCM was developed by feeding a high-fat diet with streptozotocin injections. After the development of diabetes, the animals received a 12-week oral BCP treatment at a dose of 50 mg/kg/body weight. BCP treatment showed significant improvement in glucose tolerance and insulin resistance and enhanced serum insulin levels in diabetic animals. BCP treatment effectively reversed the heart remodeling and restored the phosphorylated troponin I and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a expression. Ultrastructural examination showed reduced myocardial cell injury in DCM mice treated with BCP. The preserved myocytes were found to be associated with reduced expression of AGE/RAGE in DCM mice hearts. BCP treatment mitigated oxidative stress by inhibiting expression of NADPH oxidase 4 and activating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. Also, BCP suppressed cardiac fibrosis and endothelial-to-mesenchymal transition in DCM mice by inhibiting transforming growth factor β (TGF-β)/suppressor of mothers against decapentaplegic (Smad) signaling. Further, BCP treatment suppressed nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in DCM mice and alleviated cellular injury to the pancreatic tissues evidenced by significant elevation of the number of insulin-positive cells. To demonstrate a CB2R-dependent mechanism of BCP, another group of DCM mice were pretreated with AM630, a CB2R antagonist. AM630 was observed to abrogate the beneficial effects of BCP in DCM mice. Taken together, BCP demonstrated the potential to protect the myocardium and pancreas of DCM mice mediating CB2R-dependent mechanisms. SIGNIFICANCE STATEMENT: BCP, a CB2R agonist, shows protection against DCM. BCP attenuates oxidative stress, inflammation, and fibrosis in DCM via activating CB2Rs. BCP mediating CB2R activation favorably modulates AGE/RAGE, PI3K/AKT/Nrf2β and TGF-β/Smad and (NLRP3) inflammasome in diabetic cardiomyopathy.

Soil Fertility and Bacterial Community Composition in Response to the Composting of Biochar-Amended Chicken Manure

Amidst the burgeoning expanse of the poultry sector, the escalation of chicken manure production has ensued, potentially exacerbating ecological contamination. However, the application of chicken manure is bound to transmute the habitat of edaphic microorganisms, precipitating an alteration in the soil’s microbial consortium. The composting of biochar-amended chicken manure and wood chips (biochar composting products, BCPs) was used to improve Chinese cabbage (Brassica campestris L.) production and regulate soil properties and bacterial community structure. On the 40th day of Chinese cabbage growth, soil and Chinese cabbage were collected for laboratory analysis. The effects of different proportions of BCPs (0, 1%, 3%, 5% and 7% biochar) on soil fertility, enzyme activity, the microbial community and the growth of Chinese cabbage were studied under facility conditions. The results showed that the growth performance and quality of Chinese cabbage were significantly increased with increasing BCP ratios. Soil fertility indicators including pH, AN, AP, AK and SOM were significantly increased, except for the pH value in the 1% BCP group. The activities of phosphatase, catalase and urease were increased for all groups of BCP treatment. The soil microbial community response was significantly different, and the application of 5% and 7% BCPs reduced the abundance, diversity and evenness of soil bacteria. Chinese cabbage growth performance was positively correlated with an increase in BCP supplemental levels in the range of 3–5%. Also, the abundance, diversity and uniformity of the soil microbial community were improved in the 3% BCP treatment group. Therefore, the dominant bacterial phyla were Bacteroidetes, Actinobacteriota, Gemmatimonadota, Myxococcota, Bdellovibrionota and Firmicutes, especially the Bradyrhizobium of Proteobacteria. BCP treatment reduced the degradation of soil organic matter. In addition, it also improved the relative abundance of sequences associated with improving soil fertility. Collectively, these findings offer insights for the re-evaluation of application management strategies for BCP organic fertilizers.

Abstract CT235: RNA-seq molecular subtypes and gene expression signatures in the phase 3 IMpower151 study of 1L bevacizumab + pemetrexed/paclitaxel ± atezolizumab in metastatic NSCLC

Abstract Introduction IMpower151 (NCT04194203) was a phase 3 study to evaluate the efficacy and safety of atezolizumab, bevacizumab, carboplatin, plus paclitaxel or pemetrexed (ABCP) vs BCP, as 1L treatment for metastatic nsqNSCLC. IMpower151 did not meet its primary endpoint of INV-PFS. The objectives of this biomarker analysis were to characterize the molecular subtypes of metastatic nsqNSCLC and to explore the relationship between molecular subtype, predefined gene expression signatures, PD-L1 status and clinical outcomes with ABCP and BCP. Methods RNA sequencing of 250 nsqNSCLC tumor samples from IMpower151 was analyzed in the ACBP (n=123) and BCP (n=127) treatment arms. Non-negative matrix factorization (NMF) was applied to variably expressed genes to define molecular subtypes. Gene expression signatures were defined based on previously published associations with their respective biology. Tumor PD-L1 levels were determined by SP263 immunohistochemistry (Ventana). Results No PD-L1 dependent difference of survival benefit of ABCP compared to BCP was observed at the cutoff of TC 1% or TC 50%. Four molecular subtypes (NMF1-NMF4) were identified based on RNA sequencing. NMF1 (n=78) were immune-depleted and had low tumor proliferation. NMF2 (n=73) were characterized by low level of anti-tumor immune cells, increased expression of stromal signatures and high tumor proliferation. NMF3 (n=65) had the most inflamed transcriptomic profile with high enrichment of immune cells, and increased expression of stromal signatures as well. NMF4 (n=34) had the enrichment of granulocytes, a less inflamed immunophenotype and decreased expression of stromal signatures. The positive rate of PD-L1 expression with TC>=1% was significantly lower in NMF1 compared to other subtypes. None of patients with the four NMF subtypes showed significantly longer PFS or OS with ABCP than BCP. Overall, patients with NMF2 showed numerically shorter PFS and patients with NMF3 showed numerically longer PFS than other subtypes. Gene expression signatures of antitumor cytokines are correlated with survival benefit of ABCP compared to BCP. Patients with high expression level of antitumor cytokines showed substantially improved PFS and OS benefit from ABCP than BCP in both ITT population and EGFR mutation population. Conclusion Four molecular subtypes of nsqNSCLC with different immune-cell and tumor microenvironmental features were characterized. NMF2 may identify a fibrotic and immune-depleted subgroup correlated with worse prognosis. NMF3 may identify an immune-inflamed subgroup correlated with better prognosis. Neither NMF subtypes nor PD-L1 IHC level showed a correlation with survival benefit of ABCP controlled by BCP. The gene expression signature of antitumor cytokines could serve as a potential predictive biomarker for survival benefit of ABCP in metastatic nsqNSCLC including EGFR mutant patients. This finding is based on the exploratory analysis of a single clinical trial with limited sample size. It needs further investigation in other cohorts of tumor tissues and with larger clinical potential in blood samples as well. Citation Format: Caicun Zhou, Charlie Sun, Stacey Huang, Xiaorong Dong, Gongyan Chen, Zhehai Wang, Xianghua Wu, Yu Yao, Yiping Zhang, Ying Cheng, Hongming Pan, Xiaodong Zhang, Jiuwei Cui, Lifeng Wang, Xi Chen, Xiaoling Li, Ziping Wang, Qiming Wang, Jianxing He, Mengzhao Wang, Li Qian, Habib Hamidi, Shanshan Li, Marcus Ballinger, David S. Shames, Minu Srivastava. RNA-seq molecular subtypes and gene expression signatures in the phase 3 IMpower151 study of 1L bevacizumab + pemetrexed/paclitaxel ± atezolizumab in metastatic NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT235.

Beta-Caryophyllene, a Plant-Derived CB2 Receptor Agonist, Protects SH-SY5Y Cells from Cadmium-Induced Toxicity.

Cadmium (Cd) is a transition heavy metal that is able to accumulate in the central nervous system and may induce cell death through reactive oxygen species (ROS)-mediated mechanisms and inactivating the antioxidant processes, becoming an important risk factor for neurodegenerative diseases. The antioxidant effects of cannabinoid receptor modulation have been extensively described, and, in particular, β-Caryophyllene (BCP), a plant-derived cannabinoid 2 receptor (CB2R) agonist, not only showed significant antioxidant properties but also anti-inflammatory, analgesic, and neuroprotective effects. Therefore, the aim of the present study was to evaluate BCP effects in a model of Cd-induced toxicity in the neuroblastoma SH-SY5Y cell line used to reproduce Cd intoxication in humans. SH-SY5Y cells were pre-treated with BCP (25, 50, and 100 μM) for 24 h. The day after, cells were challenged with cadmium chloride (CdCl2; 10 μM) for 24 h to induce neuronal toxicity. CdCl2 increased ROS accumulation, and BCP treatment significantly reduced ROS production at concentrations of 50 and 100 μM. In addition, CdCl2 significantly decreased the protein level of nuclear factor erythroid 2-related factor 2 (Nrf2) compared to unstimulated cells; the treatment with BCP at a concentration of 50 μM markedly increased Nrf2 expression, thus confirming the BCP anti-oxidant effect. Moreover, BCP treatment preserved cells from death, regulated the apoptosis pathway, and showed a significant anti-inflammatory effect, thus reducing the pro-inflammatory cytokines increased by the CdCl2 challenge. The results indicated that BCP preserved neuronal damage induced by Cd and might represent a future candidate for protection in neurotoxic conditions.

β-Caryophyllene Inhibits Oxaliplatin-Induced Peripheral Neuropathy in Mice: Role of Cannabinoid Type 2 Receptors, Oxidative Stress and Neuroinflammation.

Peripheral neuropathy is an important adverse effect caused by some chemotherapeutic agents, including oxaliplatin (OXA). OXA-induced peripheral neuropathy (OIPN) is a challenging condition due to diagnostic complexities and a lack of effective treatment. In this study, we investigated the antiallodynic effect of β-caryophyllene (BCP), a cannabinoid type 2 (CB2) receptor agonist, in a mouse model of OIPN. BCP treatment inhibited OXA-induced mechanical and cold allodynia in both preventive and therapeutic drug treatment regimens. Experiments with the CB2 receptor agonist GW405833 confirmed the role of CB2 receptors in OIPN. The CB2 antagonist SR144528 abrogated the anti-nociceptive effect of BCP on mechanical allodynia, without impacting OXA-induced sensitivity to cold. BCP decreased neuroinflammation, as inferred from TNF, IL-1β, IL-6, and IL-10 profiling, and also reduced ROS production, lipid peroxidation, and 4-hydroxynonenal protein adduct formation in the spinal cords of OXA-treated mice. BCP did not affect the antitumor response to OXA or its impact on blood cell counts, implying that the cytotoxicity of OXA was preserved. These results underscore BCP as a candidate drug for OIPN treatment via CB2 receptor-dependent mechanisms, and anti-inflammatory and antioxidant responses in the spinal cord.

β-Caryophyllene, a Dietary Phytocannabinoid, Alleviates Diabetic Cardiomyopathy in Mice by Inhibiting Oxidative Stress and Inflammation Activating Cannabinoid Type-2 Receptors.

Diabetes mellitus (DM) and its associated complications are considered one of the major health risks globally. Among numerous complications, diabetic cardiomyopathy (DCM) is characterized by increased accumulation of lipids and reduced glucose utilization following abnormal lipid metabolism in the myocardium along with oxidative stress, myocardial fibrosis, and inflammation that eventually result in cardiac dysfunction. The abnormal metabolism of lipids plays a fundamental role in cardiac lipotoxicity following the occurrence and development of DCM. Recently, it has been revealed that cannabinoid type-2 (CB2) receptors, an essential component of the endocannabinoid system, play a crucial role in the pathogenesis of obesity, hyperlipidemia, and DM. Provided the role of CB2R in regulating the glucolipid metabolic dysfunction and its antioxidant as well as anti-inflammatory activities, we carried out the current study to investigate the protective effects of a selective CB2R agonist, β-caryophyllene (BCP), a natural dietary cannabinoid in the murine model of DCM and elucidated the underlying pharmacological and molecular mechanisms. Mice were fed a high-fat diet for 4 weeks followed by a single intraperitoneal injection of streptozotocin (100 mg/kg) to induce the model of DCM. BCP (50 mg/kg body weight) was given orally for 12 weeks. AM630, a CB2R antagonist, was given 30 min before BCP treatment to demonstrate the CB2R-dependent mechanism of BCP. DCM mice exhibited hyperglycemia, increased serum lactate dehydrogenase, impaired cardiac function, and hypertrophy. In addition, DCM mice showed alternations in serum lipids and increased oxidative stress concomitant to reduced antioxidant defenses and enhanced cardiac lipid accumulation in the diabetic heart. DCM mice also exhibited activation of TLR4/NF-κB/MAPK signaling and triggered the production of inflammatory cytokines and inflammatory enzyme mediators. However, treatment with BCP exerted remarkable protective effects by favorable modulation of the biochemical and molecular parameters, which were altered in DCM mice. Interestingly, pretreatment with AM630 abrogated the protective effects of BCP in DCM mice. Taken together, the findings of the present study demonstrate that BCP possesses the capability to mitigate the progression of DCM by inhibition of lipotoxicity-mediated cardiac oxidative stress and inflammation and favorable modulation of TLR4/NF-κB/MAPK signaling pathways mediating the CB2R-dependent mechanism.

Antinociceptive and chondroprotective effects of prolonged β-caryophyllene treatment in the animal model of osteoarthritis: Focus on tolerance development

Osteoarthritis (OA) is a chronic joint disease in which cartilage degeneration leads to chronic pain. The endocannabinoid system has attracted attention as an emerging drug target for OA. However, the therapeutic potential of cannabinoids is limited by psychoactive side-effects related to CB1 activation and tolerance development for analgesic effects. β-Caryophyllene (BCP) is a low-efficacy natural agonist of CB2 and a common constituent of human diet with well-established anti-inflammatory properties. The results presented herein show the anti-nociceptive and chondroprotective potential of BCP in an animal model of OA induced by intra-articular injection of monoiodoacetate (MIA). Behavioural assessment included pressure application measurement and kinetic weight bearing tests. Histological assessment of cartilage degeneration was quantified using OARSI scoring. Experiments established the dose-response effects of BCP and pharmacological mechanisms of the antinociceptive action dependent on CB2 and opioid receptors. Chronic BCP treatment was able to hamper cartilage degeneration without producing tolerance for the analgesic effects. The data presented herein show that BCP is able to produce both acute and prolonged antinociceptive and chondroprotective effects. Together with the safety profile and legal status of BCP, these results indicate a novel and promising disease-modifying strategy for treating OA.This article is part of the Special Issue on ‘Advances in mechanisms and therapeutic targets relevant to pain’.

β-Caryophyllene induces apoptosis and inhibits cell proliferation by deregulation of STAT-3/mTOR/AKT signaling in human bladder cancer cells: An in vitro study.

The current studyaimed to explore the antitumor effect of β-caryophyllene (BCP) on two different cell lines ofT24 and 5637 human bladder cancer (BC) cells and its potential molecular mechanisms in inhibition of STAT-3/mTOR/AKT signaling pathways and the inductive process of apoptosis mechanism. The results indicatedthat BCP showedsignificant cytotoxicity in BC T24 and 5637 cells ina dose- and time-dependent manner, and IC50 values were 40 µg/ml in the BC cells T24 and 5637. Reactive oxygen species (ROS) synthesis and apoptosis induction were significantly developed, but the mitochondrial membrane potential (Δψm) decreased on BCP treatment as detected by the fluorescence method. Moreover, cell migration was markedly reduced in BCP and Bax, Bcl-2 mRNA expression was modified. Finally, it was found that the STAT-3, mTOR, and AKT protein expressions were suppressed via inhibition of cytotoxicity in T24 and 5637 cells. Therefore, we finally concluded that BCP isan effective treatment against BC T24 and 5637 cells, and it hasgreat chemotherapeutic potentialfor further bladder carcinoma treatment.

β-Caryophyllene, a natural bicyclic sesquiterpene attenuates β-adrenergic agonist-induced myocardial injury in a cannabinoid receptor-2 dependent and independent manner

The downregulation of cannabinoid type-2 receptors (CB2R) have been reported in numerous diseases including cardiovascular diseases (CVDs). The activation of CB2R has recently emerged as an important therapeutic target to mitigate myocardial injury. We examined whether CB2R activation can protect against isoproterenol (ISO)-induced myocardial injury (MI) in rats. In the present study, we investigated the cardioprotective effect of β-caryophyllene (BCP), a naturally occurring dietary cannabinoid in rat model of MI. Rats were pre- and co-treated with BCP (50 mg/kg, orally) twice daily for 10 days along with subcutaneous injection of ISO (85 mg/kg) at an interval of 24 h for two days (9th and 10th days). AM630 (1 mg/kg), a CB2 receptor antagonist, was injected intraperitoneal as a pharmacological challenge prior to BCP treatment to reveal CB2R-mediated cardioprotective mechanisms of BCP. Desensitization of beta-adrenergic receptor (β-AR) signaling, receptor phosphorylation and recruitment of adapter β-arrestins were observed in ISO-induced MI in rats. ISO injections caused impaired cardiac function, elevated the levels of serum cardiac marker enzymes, and enhanced oxidative stress markers along with altered PI3K/Akt and NrF2/Keap1/HO-1 signaling pathways. ISO also promoted lysosomal dysfunction along with activation of NLRP3 inflammasomes and TLR4-NFκB/MAPK signaling and triggered rise in proinflammatory cytokines. There was a concomitant mitochondrial dysfunction followed by the activation of endoplasmic reticulum (ER) stress-mediated Hippo signaling and intrinsic pathway of apoptosis as well as altered autophagic flux/mTOR signaling in ISO-induced MI. Furthermore, ISO also triggered dyslipidemia evidenced by altered lipids, lipoproteins and lipid marker enzymes along with ionic homeostasis malfunction. However, treatment with BCP resulted in significant protective effects on all biochemical and molecular parameters analyzed. The cardioprotective effects were further strengthened by preservation of cardiomyocytes and cell organelles as observed in histopathological and ultrastructural studies. Interestingly, treatment with AM630, a CB2R antagonist was observed to abrogate the protective effects of BCP on the biochemical and molecular parameters except hyperlipidemia and ionic homeostasis in ISO-induced MI in rats. The present study findings demonstrate that BCP possess the potential to protect myocardium against ISO-induced MI in a CB2-dependent and independent manner.

PKA-Dependent Membrane Surface Recruitment of CI-AMPARs Is Crucial for BCP-Mediated Protection Against Post-acute Ischemic Stroke Cognitive Impairment.

Post-acute ischemic stroke cognitive impairment frequently occurs and seriously affects patients daily activities. Recruitment of GluA2-containing Ca2+-impermeable AMPA receptors (CI-AMPARs) to hippocampal synaptic membrane surfaces was shown to trigger synaptic plasticity. Currently, the effect of CI-AMPAR trafficking on acute ischemic stroke remains poorly understood. β-Caryophyllene (BCP) has been shown to ameliorate cognitive impairment. However, the mechanism has not been characterized. In this study, a 60-min temporary middle cerebral artery occlusion (MCAO) model was established to simulate the pathology of acute ischemic stroke. BCP reduced neurologic deficits, cerebral infarct volume, and pathological damage in MCAO mice and caused CI-AMPARs to translocate to synaptic membranes in the hippocampus; surface expression of CI-AMPARs was also decreased in MCAO mice. Furthermore, this study also showed that BCP treatment significantly activated the cAMP/PKA pathway, which is consistent with the synaptic membrane expression of CI-AMPARs. To better understand the underlying mechanisms, the PKA inhibitor H-89 was used to study the role of BCP in MCAO mice. Interestingly, H-89 treatment significantly disrupted the BCP-mediated facilitation of CI-AMPAR translocation to the synaptic membrane surface and substantially attenuated BCP-induced protection against acute ischemic stroke. Additionally, inhibition the cAMP/PKA pathway not only reduced BCP-induced inhibition of AMPAR-mediated excitatory postsynaptic currents in the hippocampal CA1 region but also decreased the effect of BCP-mediated protection against post-acute ischemic stroke cognitive impairment. Taken together, these data indicate that PKA-dependent synaptic membrane surface recruitment of CI-AMPARs is crucial for the neuroprotective effect of BCP against acute ischemic stroke and protection against post-acute ischemic stroke cognitive impairment.

β-Caryophyllene exerts protective antioxidant effects through the activation of NQO1 in the MPTP model of Parkinson’s disease

Parkinson's disease (PD) is a neurodegenerative disorder, caused by the selective death of dopaminergic neurons in the substantia nigra pars compacta. β-caryophyllene (BCP) is a phytocannabinoid with several pharmacological properties, producing anti-inflammatory and antihypertensive effects. In addition, BCP protects dopaminergic neurons from neuronal death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), yet it remains unclear if this effect is due to its antioxidant activity. To assess whether this is the case, the effect of BCP on the expression and activity of NAD(P)H quinone oxidoreductase (NQO1) was evaluated in mice after the administration of MPTP. Male C57BL/6 J mice were divided into four groups, the first of which received saline solution i.p. in equivalent volume and served as a control group. The second group received MPTP. The second group received MPTP hydrochloride (5 mg/kg, i.p.) daily for seven consecutive days. The third group received BCP (10 mg/kg) for seven days, administered orally and finally, the fourth group received MPTP as described above and BCP for 7 days from the fourth day of MPTP administration. The results showed that BCP inhibits oxidative stress-induced cell death of dopaminergic neurons exposed to MPTP at the same time as it enhances the expression and enzymatic activity of NQO1. Also, the BCP treatment ameliorated motor dysfunction and protected the dopaminergic cells of the SNpc from damage induced by MPTP. Hence, BCP appears to achieve at least some of its antioxidant effects by augmenting NQO1 activity, which protects cells from MPTP toxicity. Accordingly, this phytocannabinoid may represent a promising pharmacological option to safeguard dopaminergic neurons and prevent the progression of PD.

β-Caryophyllene inhibits high glucose-induced oxidative stress, inflammation and extracellular matrix accumulation in mesangial cells

β-Caryophyllene (BCP) is a bicyclic sesquiterpene compound that has anti-diabetic activity. However, the effect of BCP on diabetic nephropathy (DN) remains unclear. Here, we aimed to evaluate the potential role of BCP in high glucose (HG)-induced glomerular mesangial cells (MCs). MCs were maintained under HG condition to simulate DN in vitro. Our results showed that BCP inhibited HG-induced cell proliferation, ROS production and NADPH oxidase (NOX) 2/4 expression. BCP exhibited anti-inflammatory activity with decreased levels of TNF-α, IL-1β, IL-6 in HG-induced MCs. Moreover, BCP treatment suppressed the HG-induced secretion of fibronectin (FN) and collagen IV (Col IV) in MCs. Furthermore, BCP suppressed the NF-κB activation and enhanced the Nrf2 activation in HG-induced MCs. However, inhibition of Nrf2 attenuated the protective effects of BCP on HG-induced MCs, while inhibition of NF-κB enhanced the nephro-protective effects of BCP on MCs. In conclusion, these findings demonstrated that BCP executed protective effects on HG-induced MCs via regulating NF-κB and Nrf2 signaling pathways.

JAK1/STAT3 regulatory effect of β-caryophyllene on MG-63 osteosarcoma cells via ROS-induced apoptotic mitochondrial pathway by DNA fragmentation.

Currently, available treatment for osteosarcoma is combinational chemotherapy of doxorubicin, cisplatin, and methotrexate before and after surgery with overall 5-year survival rate of less than 40%. The present study was aimed to assess the anticancer effects of a phytochemical named β-caryophyllene (BCP) in treating osteosarcoma. We assessed the effect of (BCP) on oxidative stress, proliferation, apoptosis, and inflammation in human bone cancer cells MG-63. Our results showed that BCP induced reactive oxygen species (ROS) generation at 20 µM concentration in MG-63 cells. The same dose was also shown to exhibit proapoptotic and antiproliferative effects in bone cancer cells MG-63. We demonstrated that the treatment of MG-63 cells with BCP prompted mitochondrial apoptosis via upregulation of Bax and caspase-3 and downregulation of Bcl-2 as well as prompted mitochondrial membrane potential. Our results also showed stimulation of Janus kinase 1/signal transducer and activator of transcription 3 (JAK1/STAT3) signaling pathway in bone cancer MG-63 cells upon BCP treatment along with the induction of proinflammatory genes at the messenger RNA level. Overall results suggest that the treatment of MG-63 cells with BCP promotes apoptosis and inflammation via ROS and JAK1/STAT3 signaling pathway.

β-Caryophyllene Mitigates Collagen Antibody Induced Arthritis (CAIA) in Mice Through a Cross-Talk between CB2 and PPAR-γ Receptors.

β-caryophyllene (BCP) is a cannabinoid receptor 2 (CB2) agonist that tempers inflammation. An interaction between the CB2 receptor and peroxisome proliferator-activated receptor gamma (PPAR-γ) has been suggested and PPAR-γ activation exerts anti-arthritic effects. The aim of this study was to characterize the therapeutic activity of BCP and to investigate PPAR-γ involvement in a collagen antibody induced arthritis (CAIA) experimental model. CAIA was induced through intraperitoneal injection of a monoclonal antibody cocktail and lipopolysaccharide (LPS; 50 μg/100 μL/ip). CAIA animals were then randomized to orally receive either BCP (10 mg/kg/100 μL) or its vehicle (100 μL of corn oil). BCP significantly hampered the severity of the disease, reduced relevant pro-inflammatory cytokines, and increased the anti-inflammatory cytokine IL-13. BCP also decreased joint expression of matrix metalloproteinases 3 and 9. Arthritic joints showed increased COX2 and NF-ĸB mRNA expression and reduced expression of the PPARγ coactivator-1 alpha, PGC-1α, and PPAR-γ. These conditions were reverted following BCP treatment. Finally, BCP reduced NF-ĸB activation and increased PGC-1α and PPAR-γ expression in human articular chondrocytes stimulated with LPS. These effects were reverted by AM630, a CB2 receptor antagonist. These results suggest that BCP ameliorates arthritis through a cross-talk between CB2 and PPAR-γ.

The cannabinoid receptor 2 agonist, β-caryophyllene, improves working memory and reduces circulating levels of specific proinflammatory cytokines in aged male mice

Age-related cognitive decline has been associated with proinflammatory cytokines, yet the precise relationship between cognitive decline and cytokine load remains to be elucidated. β-caryophyllene (BCP) is a cannabinoid receptor 2 (CB2) agonist with established anti-inflammatory effects that is known to improve memory and increase lifespan. It is of interest to explore the potential of BCP to reduce age-related cognitive decline and proinflammatory cytokine load. In this study, we assessed changes in circulating cytokines across the lifespan, memory performance in young and aged mice, and the effects of BCP on memory function and cytokine load. The plasma levels of 12 cytokines were assessed in male Swiss-Webster mice at 3, 12, and 18 months of age using multiplexed flow cytometry. Working memory was compared in 3 and 12 month-old mice using spontaneous alternations. A dose-response function (100–300 mg/kg, subchronic administration) for BCP-induced memory restoration was determined in 3- and 12- month-old mice. Finally, the effects on cytokine levels of the peak memory enhancing dose of BCP were assessed in 18- month-old mice. Circulating levels of several cytokines significantly increased with age. Multilinear regression analysis showed that IL-23 levels were most strongly associated with age. Aged mice showed deficits in working memory and higher levels of IL-23, both of which were reversed by BCP treatment. BCP appears to reverse age-associated impairments in memory and modulates cytokine production. IL-23 may play a significant role in the aging process, and future research should determine whether it has utility as a biomarker for novel anti-aging therapeutics.

β-Caryophyllene, a natural bicyclic sesquiterpene attenuates doxorubicin-induced chronic cardiotoxicity via activation of myocardial cannabinoid type-2 (CB2) receptors in rats

The cannabinoid type 2 receptor (CB2) has recently emerged as an important therapeutic target for cancer as well as cardiovascular diseases. The CB2 receptor downregulation has been reported in solid tumors and cardiovascular diseases, therefore the CB2 receptor activation has been considered as a viable strategy for chemotherapy as well as cardioprotection. Doxorubicin (DOX) is an important drug that continues to be the mainstay of chemotherapy in solid tumors, leukemia, and lymphoma. However, the use of DOX is often limited due to its lethal cardiotoxicity. Considering the role of CB2 receptors in cardiovascular diseases and cancer, the activation of CB2 receptors may protect against DOX-induced chronic cardiotoxicity in rats. In the present study, we investigated the cardioprotective effect of a selective CB2 receptor agonist; β-Caryophyllene (BCP), a natural bicyclic sesquiterpene, against DOX-induced chronic cardiotoxicity in rats. AM630, a CB2 receptor antagonist was administered as a pharmacological challenge prior to BCP treatment to demonstrate CB2 receptor mediated cardioprotective mechanism of BCP. DOX (2.5 mg/kg) was injected intraperitoneally once a week for five weeks to induce chronic cardiotoxicity in rats. BCP was also injected into rats six days a week for a total duration of five weeks. DOX induced a significant decline in cardiac function and oxidative stress evidenced by the depletion of antioxidant enzymes, glutathione, and increased lipid peroxidation. DOX also triggered activation of nuclear factor kappa B (NF-κB) and increased the levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and expression of the inflammatory mediators (iNOS and COX-2) in the heart. Furthermore, DOX also upregulated the expression of pro-apoptotic markers such as Bax, p53, cleaved PARP, active caspase-3 and downregulated anti-apoptotic marker Bcl-2 in the myocardium. BCP treatment exerted significant cardioprotective effect by salvaging the heart tissues, improving cardiac function, mitigating oxidative stress, inflammation, and apoptosis. The histological and ultrastructural studies also appear in line with our findings of biochemical and molecular parameters. The CB2 receptor-mediated cardioprotective mechanism was further confirmed by the abrogation of the beneficial effects of BCP with prior administration of the CB2 receptor antagonist; AM630. Our study revealed the novel mechanism of BCP in cardioprotection against DOX-induced chronic cardiotoxicity by the activation of CB2 receptors.

β-Caryophyllene (BCP) ameliorates MPP+ induced cytotoxicity

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases resulting from the continuous death of dopaminergic neurons in substantia nigra. MPP+ (1-methyl-4-phenylpyridinium) has been reported to be a major neurotoxin causing neurotoxic insults on dopaminergic neurons in humans. β-Caryophyllene (BCP), an important cannabinoid derived from the essential oils of different species, has displayed pharmacological properties in different kinds of tissues and cells. However, neuroprotective effects of BCP in PD haven’t been reported before. Our results indicate that treatment with MPP+ in SH-SY5Y cells led to a significant decrease in cell viability, which was restored by BCP. Additionally, BCP suppressed MPP+-induced release of lactic dehydrogenase (LDH) and the generation of reactive oxygen species (ROS). In contrast, BCP treatment restored the reduction in mitochondrial membrane potential (MMP) induced by MPP+. BCP treatment increased intracellular GSH and GPx activity. Also, we found that the antioxidant effects of BCP against MPP+- induced neurotoxicity are dependent on cannabinoid receptor type 2 (CB2R). Moreover, our results indicated that BCP prevented MPP+-induced apoptosis of SH-SY5Y through inhibiting the up-regulation of cleaved Caspase-3, Bax, and restoring the expression of Bcl-2. Besides, BCP markedly suppressed HO-1 activation and c-Jun N-terminal Kinase (JNK) phosphorylation. We conclude that BCP might act as a promising therapeutic agent against MPP+ toxicity in neuronal cells.

Neuroprotective Effect of β-Caryophyllene on Cerebral Ischemia-Reperfusion Injury via Regulation of Necroptotic Neuronal Death and Inflammation: In Vivo and in Vitro.

Necrotic cell death is a hallmark feature of ischemic stroke and it may facilitate inflammation by releasing intracellular components after cell-membrane rupture. Previous studies reported that β-caryophyllene (BCP) mitigates cerebral ischemia-reperfusion (I/R) injury, but the underlying mechanism remains unclear. We explored whether BCP exerts a neuroprotective effect in cerebral I/R injury through inhibiting necroptotic cell death and inflammation. Primary neurons with and without BCP (0.2, 1, 5, 25 μM) treatment were exposed to oxygen-glucose deprivation and re-oxygenation (OGD/R). Neuron damage, neuronal death type and mixed lineage kinase domain-like (MLKL) protein expression were assessed 48 h after OGD/R. Furthermore, mice underwent I/R procedures with or without BCP (8, 24, 72 mg/kg, ip.). Neurologic dysfunction, cerebral infarct volumes, cell death, cytokine levels, necroptosis core molecules, and HMGB1-TLR4 signaling were determined at 48 h after I/R. BCP (5 μM) significantly reduced necroptotic neurons and MLKL protein expression following OGD/R. BCP (24, 72 mg/kg, ip.) reduced infarct volumes, neuronal necrosis, receptor-interaction protein kinase-1 (RIPK1), receptor-interaction protein kinase-3 (RIPK3) expression, and MLKL phosphorylation after I/R injury. BCP also decreased high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) levels. Thus, BCP alleviates ischemic brain damage potentially by inhibiting necroptotic neuronal death and inflammatory response. This study suggests a novel application for BCP as a neuroprotective agent.

β -Caryophyllene ameliorates the development of experimental autoimmune encephalomyelitis in C57BL/6 mice

Multiple sclerosis is the most common autoimmune inflammatory and demyelinating disease of the central nervous system. The experimental autoimmune encephalomyelitis (EAE) is an appropriate and a well-establish model for studying the pathogenesis of MS. β-caryophyllene (BCP), a natural sesquiterpene found in many plant species, is a potent anti-inflammatory compound. Herein we investigated the in vitro and in vivo immunomodulatory effects of BCP on C57BL/6 mice induced with EAE. BCP was in vitro evaluated (4, 20, and 40μM) on splenocytes obtained from EAE-induced C57BL/6 mice, and in vivo (25 or 50mg/kg/day) orally administered on EAE-mice. The clinical course, body weight, cytokines and oxygen radicals production were investigated in C57BL/6 EAE-mice. In vitro and in vivo immunological responses were evaluated by ELISA, and CNS sections were stained by hematoxylin and eosin methods The in vitro production of H2O2, NO, IFN-γ, and TNF- α was inhibited by BCP (20 and 40μM) in cultured cells from EAE-mice. BCP (25 and 50mg/kg/day) reduced clinical score and severity of EAE and inhibited H2O2, NO, TNF-α, IFN-γ and, IL-17 production. EAE-mice, orally treated with BCP (mainly at 50mg/kg/day), displayed levels of cytokines and clinical signs similar to animals with no EAE disease, demonstrating the therapeutic action of BCP on EAE animals. Histopathological and histomorphometric analysis confirmed that BCP treatment significantly reduced the numbers of inflammatory infiltrates and attenuated neurological damages in the CNS of EAE-mice.