Preventive effects of indole-3-carbinol against alcohol-induced liver injury in mice via antioxidant, anti-inflammatory, and anti-apoptotic mechanisms: Role of gut-liver-adipose tissue axis

Abstract

Indole-3-carbinol (I3C), found in Brassica family vegetables, exhibits antioxidant, anti-inflammatory, and anti-cancerous properties. Here, we aimed to evaluate the preventive effects of I3C against ethanol (EtOH)-induced liver injury and study the protective mechanism(s) by using the well-established chronic-plus-binge alcohol exposure model. The preventive effects of I3C were evaluated by conducting various histological, biochemical, and real-time PCR analyses in mouse liver, adipose tissue, and colon, since functional alterations of adipose tissue and intestine can also participate in promoting EtOH-induced liver damage. Daily treatment with I3C alleviated EtOH-induced liver injury and hepatocyte apoptosis, but not steatosis, by attenuating elevated oxidative stress, as evidenced by the decreased levels of hepatic lipid peroxidation, hydrogen peroxide, CYP2E1, NADPH-oxidase, and protein acetylation with maintenance of mitochondrial complex I, II, and III protein levels and activities. I3C also restored the hepatic antioxidant capacity by preventing EtOH-induced suppression of glutathione contents and mitochondrial aldehyde dehydrogenase-2 activity. I3C preventive effects were also achieved by attenuating the increased levels of hepatic proinflammatory cytokines, including IL1β, and neutrophil infiltration. I3C also attenuated EtOH-induced gut leakiness with decreased serum endotoxin levels through preventing EtOH-induced oxidative stress, apoptosis of enterocytes, and alteration of tight junction protein claudin-1. Furthermore, I3C alleviated adipose tissue inflammation and decreased free fatty acid release. Collectively, I3C prevented EtOH-induced liver injury via attenuating the damaging effect of ethanol on the gut-liver-adipose tissue axis. Therefore, I3C may also have a high potential for translational research in treating or preventing other types of hepatic injury associated with oxidative stress and inflammation.

Keywords: Alcohol; Apoptosis; Indole-3-carbinol; Inflammation; Liver; Oxidative stress.

Fig. 1. Histological and biochemical analyses for the beneficial effects of I3C on ethanol-induced hepatic injury

Representative photomicrographs (200×) following (A) H&E, and (B) Oil Red O staining are shown for the indicated mouse groups. The levels of (C) hepatic triglyceride (TG), (D) serum ALT, (E) serum AST, (F) serum ethanol, (G) serum FFA, (H) serum adiponectin, (I) serum leptin and (J) serum endotoxin in different groups are presented. Significance was determined by one-way ANOVA followed by Tukey’s analysis (P<0.05). Labeled characters without a common letter represent significant differences from the other group(s).

Fig. 2. Preventive effects of I3C on ethanol-induced hepatocyte apoptosis

(A) TUNEL-positive hepatocytes were marked by black arrows and (B) quantified in high-power fields (×200) for the indicated mouse groups. (C) Caspase 3-activity, (D) representative results of immunoblot and (E) densitometric analysis for cleaved PARP-1 normalized to β-actin are shown. Significance was determined by one-way ANOVA followed by Tukey’s analysis (P<0.05). Labeled characters without a common letter represent significant differences from the other group(s).

Fig. 3. Preventive effects of I3C against the increased amounts of lipid peroxidation and H₂O₂ with decreased GSH levels and ALDH2 activity following ethanol exposure

(A) Representative photomicrographs (200×) of immunohistochemistry for 4-HNE-protein adducts (marker for lipid peroxidation), (B) the amounts of MDA+HAE, (C) H₂O₂, (D) reduced GSH are presented for the indicated mouse groups. (E) Representative results of immunoblot and densitometric analyses for mitochondrial ALDH2 normalized to ATP5B, and (F) ALDH2 activity are shown. Significance was determined by one-way ANOVA followed by Tukey’s analysis (P<0.05). Labeled characters without a common letter represent significant differences from the other group(s).

Fig. 4. Preventive effects of I3C on increased oxidative stress-producing proteins following ethanol exposure

(A) Representative photomicrographs (200×) of immunohistochemistry for CYP2E1 and (B) representative results of immunoblot and (C,D,E) densitometric analysis for CYP2E1, NADPH-oxidase, and iNOS, respectively, normalized to β-actin, are shown for the indicated mouse groups. Significance was determined by one-way ANOVA followed by Tukey’s analysis (P<0.05). Labeled characters without a common letter represent significant differences from the other group(s).

Fig. 5. Preventive effects of I3C against altered levels of mitochondrial complex proteins and activities induced by ethanol

(A, B) Representative results of immunoblot and densitometric analysis for CI subunit NDUFB8, (A, C) CII-SDHB, (A, D) CIII-core protein 2 (UQCR2), (A, E) CIV subunit 1 (MTCO1), and (A, F) CV alpha subunit (ATP5A) using the specific total OXPHOS antibody are presented for the indicated mouse groups. ATP5B protein was used for normalization of the mitochondrial proteins. The relative activities of (G) complex I, (H) complex II, and (I) complex III are shown for the different mouse groups. Significance was determined by one-way ANOVA followed by Tukey’s analysis (P<0.05). Labeled characters without a common letter represent significant differences from the other group(s).

Fig. 6. Preventive effects of I3C on ethanol-induced protein acetylation

(A) Representative results of immunoblot and (B) densitometric analysis for acetyl-lysine proteins normalized to β-actin are presented for the indicated mouse groups. Significance was determined by one-way ANOVA followed by Tukey’s analysis (P<0.05). Labeled characters without a common letter represent significant differences from the other group(s).

Fig. 7. Protective effects of I3C against ethanol-induced hepatic inflammation

(A and B) Representative results of immunoblot and densitometric analysis for cleaved IL1β, (A and C) cleaved osteopontin (OPN) and (D) Caspase-1 activities are presented for the indicated mouse groups. β-Actin was used as a loading control for protein normalization. (E and F) Representative photomicrographs (200×) of immunohistochemistry for Ly6g and MPO, respectively, for the different groups. Significance was determined by one-way ANOVA followed by Tukey’s analysis (P<0.05). Labeled characters without a common letter represent significant differences from the other group(s).

Fig. 8. Histological and biochemical analyses for the preventive effects of I3C on ethanol-induced colon injury

(A) Representative photomicrographs (200×) of H&E staining of colon are presented for the indicated mouse groups. (B) Serum endotoxin, (C) Representative photomicrographs (200×) of immunohistochemistry for 4-HNE-protein adducts, (D) MDA+HAE levels (as markers for lipid peroxidation), (E) TUNEL-positive colon enterocytes and (F) quantification in high-power fields (×200) are shown for the indicated mouse groups. Colon levels of (G) claudin-1 were determined by immunofluorescent microscopy after labelling the protein with anti-claudin-1 antibody followed by green fluorescence-labeled secondary antibody. Nuclei were counter-stained with DAPI (blue). (H) Representative immunoblot and densitometric analyses of claudin-1 in the different groups are shown. Significance was determined by one-way ANOVA followed by Tukey’s analysis (P<0.05). Labeled characters without a common letter represent significant differences from the other group(s).

Fig. 9. Beneficial effects of I3C against ethanol-induced epididymal adipose tissue inflammation and adipocyte apoptosis

(A) The mRNA levels of the indicated genes involved in inflammation and macrophage infiltration and relative to cyclophilin transcript are shown for the different mouse groups. (B) TUNEL positive adipocytes identified by black arrows and (C) quantification in high-power fields are shown. Data are presented as mean ± SEM. Significance was determined by one-way ANOVA followed by Tukey’s analysis (P<0.05). Labeled characters without a common letter represent significant differences from the other group(s).

Fig. 10. Schematic diagram for the protective effects of I3C against inflammatory liver injury in the chronic-binge ethanol exposure model

Ethanol increased the ROS levels by the combined effects of elevated amounts of CYP2E1 and NADPH-oxidase with altered mitochondrial complex proteins and by decreased the cellular antioxidant defense such as GSH and mitochondrial ALDH2 activity. Increased ROS may directly damage and/or stimulate apoptosis/necrosis of hepatocytes which might then activate immune cells to produce proinflammatory cytokines, including IL1β, and chemokines. Chronic-binge alcohol exposure also increased gut leakiness with elevated serum endotoxin which reaches the liver via portal vein, contributing to activation of hepatic Kupffer cells to produce large amounts of proinflammatory cytokines. In addition, chronic-binge alcohol feeding stimulated adipocyte apoptosis and inflammation to produce cytokines/adipokines and release FFAs which reach the liver via the hepatic artery. Thus, oxidative stress and inflammation from multiple sources such as liver, gut and adipose tissue are likely to exert ethanol-mediated inflammatory liver injury in a cooperative manner. On the other hand, I3C significantly prevented ethanol-mediated hepatic injury via its antioxidant and anti-inflammatory effects through the gut-liver-adipose tissue axis.