Cannabidiol protects liver from binge alcohol-induced steatosis by mechanisms including inhibition of oxidative stress and increase in autophagy

Abstract

Acute alcohol drinking induces steatosis, and effective prevention of steatosis can protect liver from progressive damage caused by alcohol. Increased oxidative stress has been reported as one mechanism underlying alcohol-induced steatosis. We evaluated whether cannabidiol, which has been reported to function as an antioxidant, can protect the liver from alcohol-generated oxidative stress-induced steatosis. Cannabidiol can prevent acute alcohol-induced liver steatosis in mice, possibly by preventing the increase in oxidative stress and the activation of the JNK MAPK pathway. Cannabidiol per se can increase autophagy both in CYP2E1-expressing HepG2 cells and in mouse liver. Importantly, cannabidiol can prevent the decrease in autophagy induced by alcohol. In conclusion, these results show that cannabidiol protects mouse liver from acute alcohol-induced steatosis through multiple mechanisms including attenuation of alcohol-mediated oxidative stress, prevention of JNK MAPK activation, and increasing autophagy.

Keywords: Alcohol; Autophagy; Cannabidiol; Free radicals; Oxidative stress; Steatosis.

Fig. 1

Binge ethanol induces liver injury and accumulation of lipids in mice, and CBD reverses these effects. (A) Serum AST levels; (B) TG levels in liver; (C) ATP levels in liver. Results are from four to six mice in each group. *p < 0.05 and **p < 0.01.

Fig. 2

(A) H&E staining and (B) oil red O staining showing increased lipid accumulation in mouse liver after binge alcohol treatment. CBD decreases this lipid accumulation (image 4 compared to image 3 in (A) and (B)).

Fig. 3

(A) Binge alcohol drinking increases 4-HNE staining in mouse liver (image 3 compared to image 1), and CBD reverses this increase (image 4 compared to image 3). Black arrows point to areas of positive staining of 4-HNE. (B) Binge alcohol, CBD, or ethanol/CBD do not change 3-NT staining in mouse liver (image 2, 3, or 4 compared to image 1).

Fig. 4

Binge alcohol drinking increases phosphorylation of c-Jun N-terminal kinase (JNK) as shown by (A) immunohistochemistry (image 3 compared to image 1) and (B) Western blot. Livers were collected 18 h after the last ethanol gavage. CBD partially blocked the activation of JNK by binge alcohol. *p < 0.05 comparing ethanol/vehicle to vehicle/vehicle. &p < 0.05 comparing ethanol/CBD to ethanol/vehicle. (C) P38 MAPK phosphorylation is not increased by binge alcohol consumption.

Fig. 5

(A) In cytochrome P450 2E1 (CYP2E1)-expressing HepG2 cells, either 50 or 100 mM ethanol treatment increased ROS production (graphs 2 and 4), and CBD partially prevented the increase in ROS (graphs 3 and 5 compared to 2 and 4). ROS were assayed using the Total ROS Detection Kit. The distribution of fluorescence intensity is shown as a histogram, with the y axis indicating the percentage of cells in the total population displaying the specific fluorescence intensity shown on the x axis. (B and C) In vitro 100 mM ethanol treatment increased lipid accumulation in CYP2E1-expressing HepG2 cells. (B) TG levels, expressed as mg/mg protein; *p < 0.05, **p < 0.01. (C) Oil red O staining showing that CBD prevents this lipid accumulation. (D) Quantification of oil red O staining was carried out using the ImageJ program. Staining in the vehicle/vehicle group was taken as 100, and staining of all other groups was expressed relative to the vehicle/vehicle group. *p < 0.05 compared to vehicle/vehicle group. (E and F) CYP2E1 protein expression and catalytic activity were assayed 48 h after the last treatment with CBD. CBD itself does not have any effect on either CYP2E1 protein expression or CYP2E1 activity.

Fig. 6

Effects of CBD and CBD + ethanol on autophagy. LC3 is microtubule-associated protein 1 A/1B light-chain 3. During autophagy, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine (lipidation) to form LC3–phosphatidylethanolamine (LC3-II), which is recruited to autophagosomal membranes. The LC3-II/LC3-I ratio is often used to assay autophagy and, when assayed in the presence of lysosomal inhibitors such as chloroquine, it indicates autophagic flux. (A) E47 HepG2 cells were incubated with 5 μM CBD in the absence or presence of 10 mM chloroquine (CQ) for up to 8 h and immunoblots were carried out to assay for LC3-II, LC3-I, and actin. (B) Binge alcohol drinking decreases autophagy in mouse liver. Pretreatment of mice with 5 mg/kg CBD prevents the decrease in autophagy by alcohol. *p < 0.05 compared to vehicle/vehicle group. #p < 0.05 compared to ethanol/vehicle group. &p < 0.05 compared to vehicle/CBD group.