CB1R Promotes Chronic Alcohol-Induced Neuronal Necroptosis in Mice Prefrontal Cortex.

AIMS: Alcohol abuse induces multiple neuropathology and causes global burden to human health. Prefrontal cortex (PFC) is one of the most susceptible regions to alcohol-induced neuropathology. However, precise mechanisms underlying these effects on PFC remain to be elucidated. Herein, we investigated whether RIP1/RIP3/MLKL-mediated necroptosis was involved in the alcohol-induced PFC injury, and explored the effect that cannabinoid receptors (CBRs) exerted on the neurotoxicity of alcohol. METHODS: In this study, dynamic development of neuronal necroptosis in the PFC region was monitored after 95% (v/v) alcohol vapor administration for 15 and 30 days, respectively. Selective CBRs agonists or inverse agonists were pretreated according to the experimental design. All the PFC tissues were isolated and further examined by biochemical and histopathological analyses. RESULTS: It was found that chronic alcohol exposure increased the protein level of MLKL and also the phosphorylated levels of RIP1, RIP3 and MLKL in a time-dependent manner, all of which indicated the activation of necroptosis signaling. Particularly, compared to astrocytes, neurons from the PFC showed more prototypical necrotic morphology in response to alcohol insults. In parallel, an increased protein level of CB1R was also found after 15 and 30 days alcohol exposure. Administration of specific inverse agonists of CB1R (AM251 and AM281), but not its agonists or CB2R modulators, significantly alleviated the RIP1/RIP3/MLKL-mediated neuronal necroptosis. CONCLUSION: We reported the involvement of RIP1/RIP3/MLKL-mediated necroptosis in alcohol-induced PFC neurotoxicity, and identified CB1R as a critical regulator of neuronal necroptosis that enhanced our understanding of alcohol-induced neuropathology in the PFC.

Abnormally High Blood Acetaldehyde Concentrations Suggest Potential Postmortem Ethanol Generation.

Ethanol is one of the most commonly used and abused substances worldwide. Identifying whether the source of ethanol detected in corpses is antemortem ingestion or postmortem generation is especially important for determining the cause of death, which remains a vibrant field of research. During the synthesis of ethanol in the putrefaction process of corpses, other small molecules such as acetaldehyde and n-propanol could also be produced. According to our prospective statistical analysis based on authentic samples from forensic cases, it is rational to suspect ethanol generation after death when the concentration of acetaldehyde detected in blood exceeds 0.014 g/dL. Through in vitro simulation experiments, in addition to confirming that ethyl glucuronide and ethyl sulfate are the reliable biomarkers of antemortem ingestion of ethanol, we propose that acetaldehyde is far more sensitive than n-propanol as a potential marker in the blood of corpses for postmortem ethanol formation.

Simultaneous Quantification of 38 Psychotropic Drugs and Relevant Metabolites in Blood using LC-MS-MS.

The trend for the concomitant prescription of antidepressants and antipsychotics is increasing. This calls for a veracious screening and quantifying method for forensic and clinical use. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the simultaneous determination and quantification of 38 antidepressants, antipsychotics and relevant metabolites in small volumes (200 µL) of human whole blood. Analytes and deuterated internal standards were extracted using liquid-liquid extraction. The separation, determination and quantification of the analytes were performed using an LC-MS-MS system equipped with an ACQUITY UPLC® BEH Phenyl Column under a positive electrospray ionization mode. After validation, the analytical procedure was proved to be highly sensitive, with a limit of detection ranging from 0.0005 to 1 ng/mL and a lower limit of quantification ranging from 0.002 to 2 ng/mL. Bias and within- and between-run precision were within 14.7% for all analytes. Recoveries were reproducible and those of 35 analytes were >50%. Dilution integrity was evaluated to ensure that the therapeutic and toxic blood concentration ranges of target compounds were fully covered. Finally, this method was applied to authentic whole blood samples collected from two forensic cases, which demonstrated its practical usefulness of providing accurate and comprehensive information concerning the previous medication of the deceased.

Spatio-temporal effects of acute alcohol intoxication on endocannabinoid system in rat brain and blood.

The endocannabinoid system (ECS) has been shown to play a critical role in the regulation of alcohol intake and alcohol-related behaviors. However, there are discrepancies between studies examining the interaction of the ECS and alcohol administration due to different experimental procedures. The present study aims at clarifying the time course effects of acute alcohol consumption on the ECS in the peripheral circulatory systems and central nervous systems of the same cohort of subjects. We have closely monitored the critical indicators reflecting changes of the ECS during the entire process from alcohol absorption to its metabolization, after acute alcohol (4.5 g/kg) intake by intragastric administration, including two key endocannabinoids (arachidonoylethanolamide and 2-arachidonoylglycerol) and their hydrolytic enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) in blood and three brain regions, as well as a crucial and abundant receptor (cannabinoid 1 receptor) of the ECS in the three brain regions. Our results indicate that acute alcohol consumption inhibits endocannabinoid (eCB) production in the blood and in the prefrontal cortex of the brain, whereas the reverse was observed in the brain regions of the hippocampus and striatum. The variation between levels of two hydrolytic enzymes in the blood and in the three brain regions failed to reach statistical significance. After acute alcohol consumption, CB1R levels in striatum, hippocampus, and prefrontal cortex showed a similar trend of increasing, while the significant changes occurred at different time points. The present findings reveal different ligand-receptor changing patterns in the blood and in different brain regions, supporting the notion that the ECS plays a vital role in acute alcohol intoxication. Additionally, the temporal effects of alcohol on key elements of the ECS of blood and different brain nuclei were different. Our investigation may lead to a deeper understanding of the effect of acute alcohol consumption on the ECS.

Pharmacological activation of CB2 receptor protects against ethanol-induced myocardial injury related to RIP1/RIP3/MLKL-mediated necroptosis.

Chronic ethanol abuse can lead to harmful consequences for the heart, resulting in systolic dysfunction, variability in the heart rate, arrhythmia, and cardiac remodelling. However, the precise molecular mechanism responsible for ethanol-induced cardiomyopathy is poorly understood. In this regard, the present study aimed to describe the RIP1/RIP3/MLKL-mediated necroptotic cell death that may be involved in ethanol-induced cardiomyopathy and characterize CBR-mediated effects on the signalling pathway and myocardial injury. We performed an ethanol vapour administration experiment to analyse the effects of ethanol on cardiac structure and function in male C57BL/6J mice. Ethanol induced a significant decline in the cardiac structure and function, as evidenced by a decline in ejection fraction and fractional shortening, and an increase in serum Creatine Kinase levels, myocardial collagen content, and inflammatory reaction. Furthermore, ethanol also upregulated the expression levels of necroptosis-related markers such as p-RIP1, p-RIP3, and p-MLKL in the myocardium. Nec-1 treatment exerted significant cardioprotective effects by salvaging the heart tissue, improving the cardiac function, and mitigating inflammation and necroptosis. In addition, ethanol abuse caused an imbalance in the endocannabinoid system and regulated two cannabinoid receptors (CB1R and CB2R) in the myocardium. Treatment with selective CB2R agonists, JWH-133 or AM1241, markedly improved the cardiac dysfunction and reduced the ethanol-induced necroptosis in the myocardium. Altogether, our data provide evidence that ethanol abuse-induced cardiotoxicity can possibly be attributed to the RIP1/RIP3/MLKL-mediated necroptosis. Moreover, pharmacological activation of CB2R may represent a new cardioprotective strategy against ethanol-induced cardiotoxicity.

Cannabinoid 1 Receptor Antagonists Play a Neuroprotective Role in Chronic Alcoholic Hippocampal Injury Related to Pyroptosis Pathway.

BACKGROUND: Alcohol use disorders affect millions of people worldwide, and there is growing evidence that excessive alcohol intake causes severe damage to the brain of both humans and animals. Numerous studies on chronic alcohol exposure in animal models have identified that many functional impairments are associated with the hippocampus, which is a structure exhibiting substantial vulnerability to alcohol exposure. However, the precise mechanisms that lead to structural and functional impairments of the hippocampus are poorly understood. Herein, we report a novel cell death type, namely pyroptosis, which accounts for alcohol neurotoxicity in mice. METHODS: For this study, we used an in vivo model to induce alcohol-related neurotoxicity in the hippocampus. Adult male C57BL/6 mice were treated with 95% alcohol vapor either alone or in combination with selective cannabinoid receptor antagonists or agonists, and VX765 (Belnacasan), which is a selective caspase-1 inhibitor. RESULTS: Alcohol-induced in vivo pyroptosis occurs because of an increase in the levels of pyroptotic proteins such as nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), caspase-1, gasdermin D (GSDMD), and amplified inflammatory response. Our results indicated that VX765 suppressed the expression of caspase-1 and inhibited the maturation of the proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-18. Additionally, chronic alcohol intake created an imbalance in the endocannabinoid system and regulated 2 cannabinoid receptors (CB1R and CB2R) in the hippocampus. Specific antagonists of CB1R (AM251 and AM281) significantly ameliorated alcohol-induced pyroptosis signaling and inactivated the inflammatory response. CONCLUSIONS: Alcohol induces hippocampal pyroptosis, which leads to neurotoxicity, thereby indicating that pyroptosis may be an essential pathway involved in chronic alcohol-induced hippocampal neurotoxicity. Furthermore, cannabinoid receptors are regulated during this process, which suggests promising therapeutic strategies against alcohol-induced neurotoxicity through pharmacologic inhibition of CB1R.

Proteomic Analysis of Brain Regions Reveals Brain Regional Differences and the Involvement of Multiple Keratins in Chronic Alcohol Neurotoxicity.

AIMS: Alcohol abuse has attracted public attention and chronic alcohol exposure can result in irreversible structural changes in the brain. The molecular mechanisms underlying alcohol neurotoxicity are complex, mandating comprehensive mining of spatial protein expression profile. METHODS: In this study, mice models of chronic alcohol intoxication were established after 95% alcohol vapor administration for 30 consecutive days. On Day 30, striatum (the dorsal and ventral striatum) and hippocampus, the two major brain regions responsible for learning and memorizing while being sensitive to alcohol toxicity, were collected. After that, isobaric tags for relative and absolute quantitation -based quantitative proteomic analysis were carried out for further exploration of the novel mechanisms underlying alcohol neurotoxicity. RESULTS: Proteomic results showed that in the striatum, 29 proteins were significantly up-regulated and 17 proteins were significantly down-regulated. In the hippocampus, 72 proteins were significantly up-regulated, while 2 proteins were significantly down-regulated. Analysis of the overlay proteins revealed that a total of 102 proteins were consistently altered (P < 0.05) in both hippocampus and striatum regions, including multiple keratins such as Krt6a, Krt17 and Krt5. Ingenuity pathway analysis revealed that previously reported diseases/biofunctions such as dermatological diseases and developmental disorders were enriched in those proteins. Interestingly, the glucocorticoid receptor (GR) signaling was among the top enriched pathways in both brain regions, while multiple keratins from the GR signaling such as Krt1 and Krt17 exhibited significantly opposite expression patterns in the two brain nuclei. Moreover, there are several other involved pathways significantly differed between the hippocampus and striatum. CONCLUSIONS: Our data revealed brain regional differences upon alcohol consumption and indicated the critical involvement of keratins from GR signaling in alcohol neurotoxicity. The differences in proteomic results between the striatum and hippocampus suggested a necessity of taking into consideration brain regional differences and intertwined signaling pathways rather than merely focusing on single nuclei or molecule during the study of drug-induced neurotoxicity in the future.

Opposite effects of cannabinoid CB1 and CB2 receptors on antipsychotic clozapine-induced cardiotoxicity.

BACKGROUND AND PURPOSE: Clozapine is an atypical antipsychotic drug that is very efficacious in treating psychosis, but the risk of severe cardiotoxicity limits its clinical use. The present study investigated the harmful effects of clozapine on myocardium and assessed the involvement of cannabinoid receptors in its cardiotoxicity. EXPERIMENTAL APPROACH: Clozapine alone or in combination with selective cannabinoid receptor antagonists or agonists were used to treat mice and cardiomyocytes. KEY RESULTS: Clozapine induced myocardial inflammation and infiltration 7 days after i.p. injection. Mice survival rate and myocardial infiltration, and fibrotic lesions were dose-dependently worsened by clozapine. Clozapine decreased major endocannabinoid levels in sera and cultured cardiomyocytes. Cannabinoid CB1 receptors decreased in clozapine-treated hearts and were translocated from cytomembranes to cytoplasm and nuclei, whereas CB2 receptors increased in clozapine-treated hearts and inversely translocated from nuclei to the cytomembrane. Selective antagonists of CB1 receptors, rimonabant and AM281, but not its selective agonist arachidonyl-2'-chloroethylamide, ameliorated clozapine-induced myocardial inflammatory infiltration and fibrotic lesions. In contrast, selective agonists of CB2 receptors, AM1241 and JWH-133, but not its selective antagonist AM630, blunted clozapine-mediated cardiotoxicity in mice. In cultured cardiomyocytes, clozapine increased the pro-inflammatory factor IL-1ß and the concentrations of myocardial injury markers (LDH and aspartate aminotransferase); these effects were reversed by either a CB1 antagonist or CB2 agonist and further prevented by combined pretreatments. CONCLUSIONS AND IMPLICATIONS: Our data provide evidence that cannabinoid CB1 and CB2 receptors have opposite effects and selective antagonists of CB1 or agonists of CB2 receptors might confer protective effects against clozapine in myocardium.

Surrogate analyte-based quantification of main endocannabinoids in whole blood using liquid chromatography-tandem mass spectrometry.

Endocannabinoids (eCBs) are endogenous ligands of the endocannabinoid system that are known to regulate several physiological and behavioral processes. Previous studies have developed methods for the detection of main eCBs including arachidonylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), mostly in serum or plasma. Whole blood is a superior biomaterial for eCBs analysis owing to the nature of the shortened isolation procedure and decreased risk of 2-AG isomerization during preparation. In this study, a surrogate analyte-based liquid chromatography-tandem mass spectrometry assay was developed for the measurement of AEA, 2-AG and its isomer 1-arachidonoylglycerol (1-AG) using a maximum of 100 µL whole blood. Chromatographic separation was achieved using a reverse-phase column and a gradient elution. Detection was performed in selected reaction monitoring mode with an electrospray ionization source. The limits of detection of three eCBs were 0.05-0.1 ng/mL. Good linearity was observed over the concentration range. Intra- and inter-assay accuracy and precision were ≤10.9 and ≤8.7% at four quality control levels. The response factor and parallelism experiment illustrated that the surrogate analytes were suitable for accurate quantification of the main eCBs in whole blood. This surrogate analyte approach was successfully applied to authentic blood samples obtained from alcohol negative drivers and those under the influence of alcohol.