852
BINGE ALCOHOL-INDUCED GUT LEAKINESS, LIVER STEATOSIS, AND NEURODEGENERATION VIA CYP2E1-DEPENDENT OXIDATIVE STRESS AND ACROLEIN PRODUCTION THROUGH THE GUT-LIVER-BRAIN AXIS
Date
May 20, 2024
Background: People with alcohol use disorder (AUD) often suffer from multiple tissue injuries. Cytochrome P450-2E1 (CYP2E1) is the ethanol (EtOH)-inducible enzyme present in many organs and can generate oxidative stress and lipid peroxides. Among the lipid peroxides, acrolein has the longest half-life and can form toxic acrolein-adducts involved in alcohol-associated tissue injury. Here, we investigated the role of CYP2E1 in promoting binge alcohol-induced gut, liver, and brain damage through the gut-liver-brain axis using our 4-day alcohol model. Since NADPH oxidases, especially gp91phox-NOX2, are involved in oxidative stress, the differential effects between CYP2E1 and NOX2 on acrolein-adducts production and tissue damage were also studied.
Method: Young wild-type (WT) and Cyp2e1-KO mice or Nox2-KO mice were exposed to EtOH 5 g/kg/dose orally, 12-h intervals for 1, 3, 5, and 7 doses (~10g/kg/day) or dextrose (control) n≥4~6/group. Gut and liver histology (H&E) and neurodegeneration (FluoroJadeC) were determined. Gut dysbiosis and leakiness were evaluated via 16S metagenomics, serum endotoxin, and gut tight/adherens junction protein (TJ/AJ) levels. Changes in acrolein-protein adducts, oxidative stress, inflammatory, and apoptosis markers in gut, liver, and brain were analyzed by immunoblot and enzymatic assays. In vitro, T84 intestinal, AML12 hepatocyte, and Neuro2A neural cells were pretreated with a CYP2E1 inhibitor (chlormethiazole, CMZ) followed by acrolein 12.5-50 µM prior to further analyses.
Results: Binge alcohol caused gut leakiness by increasing gut structural deformation and serum endotoxin with decreased gut TJ/AJ levels (Claudins-1, -4, ZO1, β-, γ-catenins). EtOH also increased liver steatosis and hippocampal neurodegeneration in a dose-dependent manner in WT but not in Cyp2e1-KO mice. Interestingly, acrolein-protein adducts were greatly elevated in the gut, liver, and brain of EtOH-exposed WT but not in the Cyp2e1-KO mice. Cyp2e1-KO mice exhibited higher abundance of health-promoting bacteria (Bacteroidetes and Akkermansia muciniphila) than those of WT mice. EtOH-treated WT mice also showed higher levels of oxidative stress (CYP2E1), inflammation (TNFα), and apoptosis (Bax) than the Cyp2e1-KO mice. In contrast, EtOH-exposed Nox2-KO mice exhibited similar levels of gut deformation, liver steatosis, neurodegeneration, acrolein-protein adducts, oxidative stress, apoptosis, and serum endotoxin to the WT mice. In vitro mechanistic study, acrolein-induced cell death was observed in the presence of CYP2E1 in T84, AML12, and Neuro2A cells, and these were protected in the absence of CYP2E1 by pretreatment with CMZ.
Conclusion: This study illustrated a novel mechanism of alcohol-induced gut, liver, and brain injuries via increased acrolein-protein adducts in a CYP2E1-dependent manner, suggesting CYP2E1 an important therapeutic target for AUD.
Method: Young wild-type (WT) and Cyp2e1-KO mice or Nox2-KO mice were exposed to EtOH 5 g/kg/dose orally, 12-h intervals for 1, 3, 5, and 7 doses (~10g/kg/day) or dextrose (control) n≥4~6/group. Gut and liver histology (H&E) and neurodegeneration (FluoroJadeC) were determined. Gut dysbiosis and leakiness were evaluated via 16S metagenomics, serum endotoxin, and gut tight/adherens junction protein (TJ/AJ) levels. Changes in acrolein-protein adducts, oxidative stress, inflammatory, and apoptosis markers in gut, liver, and brain were analyzed by immunoblot and enzymatic assays. In vitro, T84 intestinal, AML12 hepatocyte, and Neuro2A neural cells were pretreated with a CYP2E1 inhibitor (chlormethiazole, CMZ) followed by acrolein 12.5-50 µM prior to further analyses.
Results: Binge alcohol caused gut leakiness by increasing gut structural deformation and serum endotoxin with decreased gut TJ/AJ levels (Claudins-1, -4, ZO1, β-, γ-catenins). EtOH also increased liver steatosis and hippocampal neurodegeneration in a dose-dependent manner in WT but not in Cyp2e1-KO mice. Interestingly, acrolein-protein adducts were greatly elevated in the gut, liver, and brain of EtOH-exposed WT but not in the Cyp2e1-KO mice. Cyp2e1-KO mice exhibited higher abundance of health-promoting bacteria (Bacteroidetes and Akkermansia muciniphila) than those of WT mice. EtOH-treated WT mice also showed higher levels of oxidative stress (CYP2E1), inflammation (TNFα), and apoptosis (Bax) than the Cyp2e1-KO mice. In contrast, EtOH-exposed Nox2-KO mice exhibited similar levels of gut deformation, liver steatosis, neurodegeneration, acrolein-protein adducts, oxidative stress, apoptosis, and serum endotoxin to the WT mice. In vitro mechanistic study, acrolein-induced cell death was observed in the presence of CYP2E1 in T84, AML12, and Neuro2A cells, and these were protected in the absence of CYP2E1 by pretreatment with CMZ.
Conclusion: This study illustrated a novel mechanism of alcohol-induced gut, liver, and brain injuries via increased acrolein-protein adducts in a CYP2E1-dependent manner, suggesting CYP2E1 an important therapeutic target for AUD.
CYP2E1 (but not NOX2) is a key driver in promoting binge alcohol-induced gut, liver, and brain injuries via increasing oxidative stress and acrolein-protein adducts.
