Cannabinoid Receptor-2 agonist AM1241 Attenuates Myocardial Ischemia-Reperfusion-Induced Oxidative Stress in Rats via Nrf2/HO-1 Pathway.

OBJECTIVE: The cannabinoid receptor-2 agonist AM1241 exhibits notable cardioprotective effects against myocardial infarction, positioning it as a promising therapeutic candidate for cardiovascular disease. This study explores AM1241's protective role in myocardial ischemia-reperfusion (IR) injury and its association with the Nrf2/HO-1 pathway. METHODS: In an established Sprague-Dawley rat IR model, AM1241's impact on cardiac injury was assessed through echocardiography, 2,3,5-triphenyl tetrazolium chloride staining, and histological analysis. H9c2 cells underwent hypoxia-reoxygenation, with AM1241's influence on cell viability determined by the CCK-8 assay. Reactive oxygen species (ROS) production was measured using the DCFH-DA assay, and Nrf2 and HO-1 protein expressions were evaluated through immunofluorescence and Western blot. RESULTS: Myocardial ischemia-reperfusion injury (MIRI) increased infarct size, inflammatory cell presence, oxidative and nitrosative stress, impaired cardiac function, and elevated apoptosis rates. AM1241 mitigated these effects, enhancing cell viability, reducing ROS production, and upregulating Nrf2 and HO-1 expression. The antioxidant effect of AM1241 was inhibited by ML385 intervention. CONCLUSIONS: AM1241 attenuates oxidative stress, alleviates MIRI, and activates the Nrf2/HO-1 signaling pathway, underscoring its potential as a therapeutic strategy for MIRI.

Treatment of Diet-Induced Obese Rats with CB2 Agonist AM1241 or CB2 Antagonist AM630 Reduces Leptin and Alters Thermogenic mRNA in Adipose Tissue.

Diet-induced obesity (DIO) is a contributor to co-morbidities, resulting in alterations in hormones, lipids, and low-grade inflammation, with the cannabinoid type 2 receptor (CB2) contributing to the inflammatory response. The effects of modulating CB2 with pharmacological treatments on inflammation and adaptations to the obese state are not known. Therefore, we aimed to investigate the molecular mechanisms in adipose tissue of CB2 agonism and CB2 antagonism treatment in a DIO model. Male Sprague Dawley rats were placed on a high-fat diet (HFD) (21% fat) for 9 weeks, then received daily intraperitoneal injections with a vehicle, AM630 (0.3 mg/kg), or AM1241 (3 mg/kg), for a further 6 weeks. AM630 or AM1241 treatment in DIO rats did not alter their body weight, food intake, or liver weight, and it had no effect on their numerous circulating cytokines or peri-renal fat pad mass. AM1241 decreased heart weight and BAT weight; both treatments (AM630 or AM1241) decreased plasma leptin levels, while AM630 also decreased plasma ghrelin and GLP-1 levels. Both treatments decreased Adrb3 and TNF-α mRNA levels in eWAT and TNF-α levels in pWAT. AM630 treatment also decreased the mRNA levels of Cnr2, leptin, and Slc2a4 in eWAT. In BAT, both treatments decreased leptin, UCP1, and Slc2a4 mRNA levels, with AM1241 also decreasing Adrb3, IL1ß, and PRDM16 mRNA levels, and AM630 increasing IL6 mRNA levels. In DIO, CB2 agonist and CB2 antagonist treatment reduces circulating leptin in the absence of weight loss and modulates the mRNA responsible for thermogenesis.

Activation of CB2R with AM1241 ameliorates neurodegeneration via the Xist/miR-133b-3p/Pitx3 axis.

Activation of cannabinoid receptor type II (CB2R) by AM1241 has been demonstrated to protect dopaminergic neurons in Parkinson's disease (PD) animals. However, the specific mechanisms of the action of the CB2R agonist AM1241 for PD treatment have not been characterized. Wild-type (WT), CB1R knockout (CB1-KO), and CB2R knockout (CB2-KO) mice were exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 1 week to obtain a PD mouse model. The therapeutic effects of AM1241 were evaluated in each group. Behavioral tests, analysis of neurotransmitters, and immunofluorescence results demonstrated that AM1241 ameliorated PD in WT animals and CB1-KO animals. However, AM1241 did not ameliorate PD symptoms in CB2-KO mice. RNA-seq analysis identified the lncRNA Xist as an important regulator of the protective actions of AM1241. Specifically, AM1241 allowed WT and CB1-KO animals treated with MPTP to maintain normal expression of Xist, which affected the expression of miR-133b-3p and Pitx3. In vitro, overexpression of Xist or AM1241 protected neuronal cells from death induced by 6-hydroxydopamine and increased Pitx3 expression. The CB2 receptor agonist AM1241 alleviated PD via regulation of the Xist/miR-133b-3p/Pitx3 axis, and revealed a new approach for PD treatment.

[Effect of cannabinoid receptor-2 agonist AM1241 on platelet-derived growth factor expression in the liver tissue of mice with hepatic fibrosis].

Objective: To investigate the effect of cannabinoid receptor-2 (CB2) agonist AM1241 on the mRNA and protein expression of platelet-derived growth factor (PDGF) and collagen-III (Col-III) in the liver tissue of mice with experimental liver fibrosis induced by carbon tetrachloride (CCl(4)). Methods: Totally 38 8-week-old male C57BL/6J mice were randomly divided into control group, model group, 3 mg/kg CB2 receptor agonist (AM1241) group, and 9 mg/kg AM1241 group. All mice, except for the control group, were treated with 30% CCl(4) (three times a week, 5 ml/kg body weight, 16 weeks) to establish a liver fibrosis model. Meanwhile, 3 and 9 mg/kg AM1421 was intraperitoneally injected for daily intervention, respectively. The dosage was adjusted according to actual body weight. The same solvent was given in the control group. The serum level of aspartate aminotransferase (AST) was measured by serum enzyme digestion. The liver inflammation and fibrosis were observed by HE staining of tissue slices. The mRNA and protein expression of PDGF and Col-III in hepatic tissue was determined by real-time PCR and immunohistochemistry. Results: Compared with the control group, the mice in model group showed severe liver fibrosis, significantly elevated serum AST level (742 ± 300.8 U/L vs 118.1 ± 31.1 U/L, P < 0.05), and significantly increased mRNA and protein expression of PDGF and Col-III in liver tissue (P < 0.05). Compared with the model group, the mice in 3 mg/kg AM1241 group and 9 mg/kg AM1241 group had less severe liver fibrosis, and significantly reduced serum AST levels (116.6 ± 13.68 U/L vs 742 ± 300.8 U/L, P < 0.05; 113.8 ± 16.01 U/L vs 742 ± 300.8 U/L, P < 0.05) and mRNA and protein expression of PDGF and Col-III in liver tissue (P < 0.05). Conclusion: CB2 receptor agonist AM1241 can inhibit the mRNA and protein expression of PDGF in the liver tissue of mice with hepatic fibrosis, and reduce extracellular matrix synthesis.

The effects and mechanisms of AM1241 in alleviating cerebral ischemia-reperfusion injury.

OBJECTIVE: Research has shown that cerebral ischemia-reperfusion injury (CIRI) involves a series of physiological and pathological mechanisms, including inflammation, oxidative stress, and cell apoptosis. The cannabinoid receptor 2 agonist AM1241 has been found to have anti-inflammatory and anti-oxidative stress effects. However, it is unclear whether AM1241 has a protective effect against brain ischemia-reperfusion injury, and its underlying mechanisms are not yet known. METHODS: In this study, we investigated the anti-inflammatory, anti-oxidative stress, and anti-apoptotic effects of AM1241 and its mechanisms in BV2 cells stimulated with H2O2 and in a C57BL/6 mouse model of CIRI in vitro and in vivo, respectively. RESULTS: In vitro, AM1241 significantly inhibited the release of pro-inflammatory cytokines TNF-α and IL-6, reactive oxygen species (ROS), and the increase in Toll-like receptor 4/myeloid differentiation protein 2 (MD2/TLR4) complex induced by H2O2. Under H2O2 stimulation, MD2 overexpression resulted in increased levels of MD2/TLR4 complex, TNF-α, IL-6, NOX2, BAX, and Cleaved-Caspase3 (C-Caspase3), as well as the activation of the MAPK pathway and NF-κB, which were reversed by AM1241. In addition, molecular docking experiments showed that AM1241 directly interacted with MD2. Surface Plasmon Resonance (SPR) experiments further confirmed the binding of AM1241 to MD2. In vivo, AM1241 significantly attenuated neurofunctional impairment, brain edema, increased infarct volume, oxidative stress levels, and neuronal apoptosis in CIRI mice overexpressing MD2. CONCLUSION: Our study demonstrates for the first time that AM1241 alleviates mouse CIRI by inhibiting the MD2/TLR4 complex, exerting anti-inflammatory, anti-oxidative stress and anti-apoptotic effects.

Cannabinoid CB2 receptor agonist reduces local and systemic inflammation associated with pneumonia-induced sepsis in mice.

Sepsis is a severe condition secondary to dysregulated host response to infection leading to tissue damage and organ dysfunction. Cannabinoid CB2 receptor has modulatory effects on the immune response. Therefore, this study investigated the effects of a cannabinoid CB2 receptor agonist on the local and systemic inflammatory process associated with pneumonia-induced sepsis. Pneumonia-induced sepsis was induced in mice by intratracheal inoculation of Klebsiella pneumoniae. Tissue and bronchoalveolar lavage (BAL) were collected 6, 24, or 48 h after surgery. Mice were treated with CB2 agonist (AM1241, 0.3 and 3 mg/kg, i.p.) and several parameters of inflammation were evaluated 24 h after sepsis induction. Polymorphonuclear cell migration to the infectious focus peaked 24 h after pneumonia-induced sepsis induction in male and female animals. Septic male mice presented a significant reduction of cannabinoid CB2 receptor density in the lung tissue after 24 h, which was not observed in females. CB2 expression in BAL macrophages was also reduced in septic animals. Treatment of septic mice with AM1241 reduced cell migration, local infection, myeloperoxidase activity, protein extravasation, and NOS-2 expression in the lungs. In addition, the treatment reduced plasma IL-1ß, increased IL-10 and reduced the severity and mortality of septic animals. These results suggest that AM1241 promotes an interesting balance in the inflammatory response, maintaining lung function and preventing organ injury. Therefore, cannabinoid CB2 receptors are potential targets to control the excessive inflammatory process that occurs in severe conditions, and agonists of these receptors can be considered promising adjuvants in pneumonia-induced sepsis treatment.

AM1241-Loaded Poly(ethylene glycol)-Dithiothreitol Hydrogel Repairs Cranial Bone Defects by Promoting Vascular Endothelial Growth Factor and COL-1 Expression.

Objective: To explore the repair effect of the prepared drug-loaded AM1241 poly(ethylene glycol)-dithiothreitol (PEG-DTT) hydrogel on cranial bone defects in SD rats. Methods: The PEG-DTT hydrogel under borax catalysis was quickly prepared, and the characterization of the material was observed by a scanning electron microscope. The effect of AM1241 on cell activity and bone tissue differentiation was tested. The SD rat model of cranial bone defect was established, and the defect was repaired by injecting the prepared hydrogel into the defect. The defect was divided into four groups, namely, sham group, blank group, PEG-DTT group, and PEG-DTT + AM1241 group. The rats were euthanized, and whole cranial bone was taken out for micro-CT and histological observation. Results: The prepared hydrogel is porous; it is liquid when heated to 80°C and a hydrogel when cooled to 25°C. 5-10 µM AM1241 increased osteoblast activity. A moderate amount of AM1241 can promote osteogenic differentiation. Both the PEG-DTT group and PEG-DTT + AM1241 group showed obvious new bone tissue formation, but the PEG-DTT + AM1241 group had a better effect. In addition, the new bone tissue in the PEG-DTT + AM1241 group was significantly more than that in the other groups. Conclusion: The prepared AM1241-loaded PEG-DTT hydrogel showed a good repair effect on SD rats with cranial bone defects. It can be used as materials for cranial bone repair in SD rats with cranial bone defects, but the repair effect is weaker than that of normal bone. These results provide a theoretical and practical basis for its further clinical application.

The protective effect of cannabinoid type II receptor agonist AM1241 on ConA-induced liver injury in mice via mitogen-activated protein kinase signalling pathway.

INTRODUCTION: The endocannabinoid system plays an important role in regulating the immune responses in inflammation. At present, there are no good clinical drugs for many immune liver diseases. METHODS: We explored the protective effect of the cannabinoid type II (CB2) receptor agonist AM1241 on the liver of mice with acute liver injury caused by concanavalin from the perspective of inflammation and immunity. Pathological evaluation in hepatic tissue was examined by haematoxylin and eosin (HE) staining and the levels of biochemical parameters in the serum were measured by automatic biochemical analysis. The content of inflammatory factors was measured by enzyme-linked immunosorbent assay and real-time quantitative reverse transcription polymerase chain reaction (real-time PCR). The liver apoptosis-related proteins were observed by immunohistochemistry. The expression of liver injury-related proteins was analysed by Western blot. Immune cells were isolated from the liver of mice and studied in vitro. RESULTS: Reduced levels of alanine transaminase and aspartate transaminase were observed in ConA-induced liver injury mice treated with AM1241, together with attenuated liver damage evidenced by H&E staining. Moreover, AM1241 inhibited the protein and gene expression levels of TNF-α, IL-6 and IFN-γ in the livers of mice. The phosphorylation levels of p38, JNK, ERK1/2, P65 and cAMP response element-binding protein (CREB) in the mouse were significantly reduced in AM1241 pretreatment, while the level of p-JNK increased. In addition, the P/T-P65 and P/T-CREB of the AM1241 pretreatment group were significantly reduced. The results of immunohistochemistry measurement are consistent with those of Western blotting. The CB2-mediated effect is through macrophage-like Kupffer cells. CONCLUSION: Our study suggests that the ConA-induced liver injury model in mice is protected by CB2 agonist AM1241 by modulation of CB2 receptor-rich immune cells, for example, Kupffer cells. Reduced inflammatory responses regulate apoptosis/cell death in the liver particularly hepatocytes and other parenchymal cells.

AM1241 alleviates myocardial ischemia-reperfusion injury in rats by enhancing Pink1/Parkin-mediated autophagy.

AIMS: The purpose of this study was to reveal the therapeutic efficacy and underlying mechanism of cannabinoid type 2 receptor agonist (AM1241) on myocardial ischemia-reperfusion injury (MIRI) in rats. MAIN METHODS: We established a rat myocardial ischemia/reperfusion (I/R) model and H9c2 hypoxia/reoxygenation (H/R) model. ELISA was used to determine the concentrations of cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in plasma. EB/TTC staining was performed to observe the myocardial infarct size. Besides, the pathological changes of myocardial tissue were identified via H&E staining and Masson's trichrome staining. TUNEL assay was performed to examine myocardial apoptosis. Then, the protein expression of Pink1, Parkin and autophagy-related markers (Beclin-1, P62 and LC3) were detected by Western blot, and autophagy was evaluated by Mitotracker staining. KEY FINDINGS: The results of EB/TTC staining, H&E staining, Masson's trichrome staining and cardiac enzymes measuring showed that AM1241 treatment significantly diminished infarct size, the structural abnormalities and the activities of cardiac enzymes (cTnI, CK-MB, AST and LDH). AM1241 also significantly reduced the number of TUNEL-positive cells induced by I/R in a dose-dependent manner. Furthermore, AM1241 activated Pink1/Parkin signaling pathway and upregulated autophagy level. SIGNIFICANCE: AM1241 exerts a protective effect against MIRI in rats by inducing autophagy through the activation of Pink1/Parkin pathway.

Effects of cannabinoid receptor 2 synthetic agonist, AM1241, on bleomycin induced pulmonary fibrosis.

Bleomycin (BLM) is a chemotherapeutic agent that can cause pulmonary fibrosis. Little is known about the possible protective role of the CB2 receptor agonist, AM1241. We investigated the effects of CB2 receptor activation by AM1241 on BLM induced lung fibrosis in a rat model. BLM was administered via the trachea. Adult female Wistar rats were divided into five groups: saline (control group), BLM (BLM group), CB2 agonist (AM1241) + BLM (BLMA group), CB2 antagonist (AM630) and CB2 agonist (AM1241) + BLM (BLMA + A group), and vehicle (dimethylsulfoxide) + BLM (BLM + vehicle group). Hydroxyproline, collagen type 1, total protein, glutathione (GSH), malondialdehyde (MDA), interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels were measured in lung fibrosis and control tissue using standard methods. We investigated the histopathology of lung tissue to determine the extent of fibrosis. We found significantly higher levels of hydroxyproline, TNF-α, IL-6 and total protein in the BLM group compared to the BLMA group. The level of GSH also was higher in the BLMA group compared to the BLM group. Inflammation and fibrotic changes were significantly reduced in the BLMA group. Our findings suggest that CB2 receptor activation provided protection against BLM induced pulmonary fibrosis by suppressing oxidative stress and increasing cytokines.

CB2 Agonist (AM1241) Improving Effect on Ovalbumin-Induced Asthma in Rats.

Asthma is a disease characterized by spontaneous contraction of the airways in response to a wide variety of endogenous and exogenous stimuli. Many asthma models are used to mimic the human asthma model in the literature. In order to better understand the role of the cannabinoid (CB) 2 receptor in the ovalbumin (OVA)-induced asthma model, a combination of both selective CB2 agonist (AM1241) and antagonist (AM630) was used to improve inflammatory hypersensitivity and edema in rats. In the present study, it was found that OVA decreased body weight (p < 0.05), increased lung weights (p < 0.05), increased diastolic and systolic blood pressure (p < 0.001), and caused irregularity in pulmonary functions (p < 0.001). Moreover, CB2 agonist was found not to reduce body weight, cause blood pressure and respiratory irregularities (p < 0.05). OVA led to increase in IgE, TNF-α, IL-4, MDA level (p < 0.001), and total WBC count (p < .05). CB2 treatment caused to reduce the number of total WBC and the level of total protein in BALF, to hinder to increase level of MDA, IgE, TNF-α, and IL-4 (p < 0.05) in BALF or serum or lung tissue. But CB2-antagonist treatment prevented the protective effect of CB2 agonist. The aim of this study was to study the role of the CB2 receptor in the OVA induced asthma model, to improve inflammatory hypersensitivity, and edema in the rats. The results suggested that CB2 agonist administration to OVA induced asthmatic rats via anti-asthmatic potential through inhibition of parameters such as IgE, IL-4, TNF-α, microvascular escape, and oxidative stress.

Activation of cannabinoid type 2 receptor protects skeletal muscle from ischemia-reperfusion injury partly via Nrf2 signaling.

AIMS: Cannabinoid type 2 (CB2) receptor activation has been shown to attenuate IRI in various organs. NF-E2-related factor (Nrf2) is an anti-oxidative factor that plays multiple roles in regulating cellular redox homeostasis and modulating cell proliferation and differentiation. The protective effects of CB2 receptor activation on skeletal muscle IRI and the underlying mechanism that involves Nrf2 signaling remain unknown. MAIN METHODS: We evaluated the in vivo effect of CB2 receptor activation by the CB2 receptor agonist AM1241 on IR-induced skeletal muscle damage and early myogenesis. We also assessed the effects of CB2 receptor activation on C2C12 myoblasts differentiation and H2O2-induced C2C12 myoblasts damage in vitro, with a focus on the mechanism of Nrf2 signaling. KEY FINDINGS: Our results showed that CB2 receptor activation reduced IR-induced histopathological lesions, edema, and oxidative stress 1 day post-injury and accelerated early myogenesis 4 days post-injury in mice. Nrf2 knockout mice that were treated with AM1241 exhibited deteriorative skeletal muscle oxidative damage and myogenesis. In vitro, pretreatment with AM1241 significantly increased the expression of Nrf2 and its nuclear translocation, attenuated the decrease in H2O2-induced C2C12 cell viability, and decreased reactive oxygen species generation and apoptosis. CB2 receptor activation also significantly enhanced C2C12 myoblasts differentiation, which was impaired by silencing Nrf2. SIGNIFICANCE: Overall, CB2 receptor activation protected skeletal muscle against IRI by ameliorating oxidative damage and promoting early skeletal muscle myogenesis, which was partly via Nrf2 signaling.

AM-1241 CB2 Receptor Agonist Attenuates Inflammation, Apoptosis and Stimulate Progenitor Cells in Bile Duct Ligated Rats.

BACKGROUND: The cannabinoid receptor 2 (CB2) plays a pleiotropic role in the innate immunity and is considered a crucial mediator of liver disease. Cannabinoid CB2 receptor activation has been reported to attenuate liver fibrosis in CCl4 exposed mice and also plays a potential role in liver regeneration in a mouse model of I/R and protection against alcohol-induced liver injury. AIM: In this study, we investigated the impact of CB2 receptors on the antifibrotic and regenerative process associated with cholestatic liver injury. METHODS: Twenty-six rats had bile duct ligation co-treated with silymarin and AM1241 for 3 consecutive weeks. Serum hepatotoxicity markers were determined, and histopathological evaluation was performed. RESULTS: Following bile duct ligation (BDL) for 3 weeks, there was increased aminotransferase levels, marked inflammatory infiltration and hepatocyte apoptosis with induced oxidative stress, as reflected by increased lipid peroxidation. Conversely, following treatment with the CB2 agonist, AM-1241, BDL rats displayed a reduction in liver injury and attenuation of fibrosis as reflected by expression of hydroxyproline and α-smooth muscle actin. AM1241 treatment also significantly attenuated lipid peroxidation end-products, p53-dependent apoptosis and also attenuated inflammatory process by stimulating IL-10 production. Moreover, AM1241 treated rats were associated with significant expression of hepatic progenitor/oval cell markers. CONCLUSION: In conclusion, this study points out that CB2 receptors reduce liver injury and promote liver regeneration via distinct mechanisms including IL-10 dependent inhibition of inflammation, reduction of p53-reliant apoptosis and through stimulation of oval/progenitor cells. These results suggest that CB2 agonists display potent hepatoregenrative properties, in addition to their antifibrogenic effects.

AM1241 alleviates MPTP-induced Parkinson's disease and promotes the regeneration of DA neurons in PD mice.

The main pathological feature of Parkinson's disease (PD) is the loss of dopaminergic neurons in the substantia nigra. In this study, we investigated the role of cannabinoid receptor 2 (CB2R) agonist AM1241 on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in a mouse model of PD. Upon treatment with AM1241, the decreased CB2R level in the PD mouse brain was reversed and the behavior score markedly elevated, accompanied with a dose-dependent increase of dopamine and serotonin. In addition, western blot assay and immunostaining results suggested that AM1241 significantly activated PI3K/Akt/MEK phosphorylation and increased the expression of Parkin and PINK1, both in the substantia nigra and hippocampus. The mRNA expression analysis further demonstrated that AM1241 increased expression of the CB2R and activated Parkin/PINK1 signaling pathways. Furthermore, the increased number of TH-positive cells in the substantia nigra indicated that AM1241 regenerated DA neurons in PD mice, and could therefore be a potential candidate for PD treatment. The clear co-localization of CB2R and DA neurons suggested that AM1241 targeted CB2R, thus also identifying a novel target for PD treatment. In conclusion, the selective CB2 agonist AM1241 has a significant therapeutic effect on PD mice and resulted in regeneration of DA neurons following MPTP-induced neurotoxicity. The possible mechanisms underlying the neurogenesis effect of AM1241 might be the induction of CB2R expression and an increase in phosphorylation of the PI3K/AKT signaling pathway.