Selective Intestinal Decontamination Mitigates Binge Alcohol-Induced Cardiovascular Dysfunction via Reduced Anandamide Production
Binge drinking contributes to a rising number of emergency room visits in the United States. Our previous work demonstrated that binge drinking impairs cardiac performance and exerts complex hemodynamic alterations through activation of the endocannabinoid–CB1 receptor (CB1R) signaling pathway. Anandamide, an endogenous CB1R agonist, is produced in response to stimuli such as endotoxin challenge and oxidative stress; however, the effect of binge drinking on myocardial anandamide production and subsequent CB1R-mediated cardiodepression remains unclear. Here, we studied the impact of endotoxins derived from intestinal Gram-negative bacteria on myocardial anandamide formation and CB1R-dependent cardiac dysfunction following acute alcohol intoxication. Using a mouse model of binge drinking (5 g/kg orally), we observed a reduced rate of mesenteric microcirculation concurrent with elevated circulating endotoxin levels. Selective intestinal decontamination significantly lowered circulating endotoxin levels, coinciding with reduced cardiac anandamide levels at 3 hours post-binge. These changes were paralleled with improved left ventricular performance, vascular tone, and reduced myocardial oxidative stress. Moreover, cardiac RNA sequencing revealed marked alterations in gene expression following alcohol gavage that were absent in mice with prior selective intestinal decontamination. These findings provide novel mechanistic insights that highlight the crucial role of intestinal Gram-negative bacteria in modulating cardiac anandamide production after binge drinking, thereby exacerbating cardiovascular function and blood redistribution. Cardiac tissues were homogenized in Trizol (Invitrogen, Carlsbad, CA, USA), and total RNA was isolated with a Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. RNA concentrations were determined using a NanoDrop 2000 Spectrophotometer (Thermo Scientific). AmpliSeq libraries were prepared using the Ion AmpliSeq™ Library Kit Plus (Thermo Fisher Scientific, 4488990) per the manufacturer's instructions. Library quality was determined using the Agilent High Sensitivity DNA Kit (Agilent Technologies, 5067-4626), and library concentrations were quantified using the Ion Library TaqMan™ Quantitation Kit (Life Technologies, 4468802), per manufacturer's instructions. Barcoded AmpliSeq libraries were loaded on the sequencing chips using the Ion 540TM Chip Kit (ThermoFisher, A27766) and Ion Chef™ Instrument. Libraries were sequenced on the Ion Torrent S5 Sequencing System via the Ion Torrent 540-OT2 kit (ThermoFisher, A27753). 8 to 9 libraries were loaded per sequencing chip, and 2 sequencing ships were loaded and sequenced at a time. An average of 8.5 million counts per sample were sequenced. Alignment and gene expression count were performed using Ion Torrent AmpliSeq RNA Plugin v0.5.4.0 (Thermo Fisher) using mm10 genome. Overall design: In the first set of experiments, mice were exposed to a single alcohol gavage at a dose of 5g/kg body weight following overnight fasting . As for the control group, mice were administered saline similarly. Intestinal microcirculation, morphology, and levels of systemic circulating endotoxins were evaluated at different time points following alcohol gavage. In the second cohort, mice were treated orally with a mixture of antibiotics (Sigma-Aldrich, St. Louis, MO, USA)  before alcohol gavage to selectively eliminate intestinal Gram-negative flora, as previously described, with minor modifications.20,21 Briefly, mice were randomly assigned to control or antibiotic treatment groups, where they either received water or non-absorbable antibiotics (neomycin, polymyxin-B, and nystatin; 200 mg/kg, 150 mg/kg, and 1 mg/kg, respectively) for a 14-day pre-treatment period prior to alcohol gavage. After selective intestinal decontamination, mice were exposed to alcohol, as described above. Endpoints were collected 3 hours after alcohol gavage.