Cannabidiol alleviates carbon tetrachloride-induced liver fibrosis in mice by regulating NF-κB and PPAR-α pathways

Abstract

Liver fibrosis has become a serious public health problem that can develop into liver cirrhosis and hepatocellular carcinoma and even lead to death. Cannabidiol (CBD), which is an abundant nonpsychoactive component in the cannabis plant, exerts cytoprotective effects in many diseases and under pathological conditions. In our previous studies, CBD significantly attenuated liver injury induced by chronic and binge alcohol in a mouse model and oxidative bursts in human neutrophils. However, the effects of CBD on liver fibrosis and the underlying mechanisms still need to be further explored. A mouse liver fibrosis model was induced by carbon tetrachloride (CCl₄) for 10 weeks and used to explore the protective properties of CBD and related molecular mechanisms. After the injection protocol, serum samples and livers were used for molecular biology, biochemical and pathological analyses. The results showed that CBD could effectively improve liver function and reduce liver damage and liver fibrosis progression in mice; the expression levels of transaminase and fibrotic markers were reduced, and histopathological characteristics were improved. Moreover, CBD inhibited the levels of inflammatory cytokines and reduced the protein expression levels of p-NF-κB, NF-κB, p-IκBα, p-p38 MAPK, and COX-2 but increased the expression level of PPAR-α. We found that CBD-mediated protection involves inhibiting NF-κB and activating PPAR-α. In conclusion, these results suggest that the hepatoprotective effects of CBD may be due to suppressing the inflammatory response in CCl₄-induced mice and that the NF-κB and PPAR-α signaling pathways might be involved in this process.

Keywords: NF-κB; PPAR-α; cannabidiol; carbon tetrachloride; liver fibrosis.

FIGURE 1

Effects of CBD treatment on liver function in mice with CCl₄-induced liver fibrosis. (A,B) Serum AST and HA levels in the indicated groups. (C) Representative HE staining of liver sections. (D) Fibrosis deposition was observed by Masson trichrome staining. The values represent the means ± SEM (n = 8). ^* p < 0.05 vs. the control group, ^# p < 0.05 vs. the CCl₄ group, as determined by one-way ANOVA, followed by the LSD multiple comparisons test. (a) Control group; (b) CCl₄ group; (c) 4 mg/kg CBD group; (d) 8 mg/kg CBD group; (e) 0.2 mg/kg colchicine group.

FIGURE 2

Effects of CBD on α-SMA and COL-Ⅰ in mice with CCl₄-induced liver fibrosis. (A,B) Representative immunohistochemical staining of α-SMA and COL-Ⅰ. (C,D) Quantification of positive staining areas was performed by ImageJ software. The values represent the means ± SEM (n = 8). ^* p < 0.05 vs. the control group, ^# p < 0.05 vs. the CCl₄ group, as determined by one-way ANOVA, followed by the LSD multiple comparisons test. (a) Control group; (b) CCl₄ group; (c) 4 mg/kg CBD group; (d) 8 mg/kg CBD group; (e) 0.2 mg/kg colchicine group.

FIGURE 3

Effects of CBD on inflammatory cytokines in mice with CCl₄-induced liver fibrosis. (A–C) Serum levels of IL-6 and TNF-α and liver levels of IL-1β. (D) The mRNA levels of IL-6, IL-1β and TNF-α were measured by q-PCR. The values represent the means ± SEM (n = 8). ^* p < 0.05 vs. the control group, ^# p < 0.05 vs. the CCl₄ group, as determined by one-way ANOVA, followed by the LSD multiple comparisons test.

FIGURE 4

Effects of CBD on TGF-β1, α-SMA, and COL- I in mice with CCl₄-induced liver fibrosis. (A) The effects of CBD on the protein expression of TGF-β1, α-SMA, and COL- I were measured by western blotting. (B–D) ImageJ software was used to measure the grey values of the bands. (E) The effect of CBD on the mRNA expression of TGF-β1, α-SMA, and COL- I was determined by q-PCR. The values represent the means ± SEM (n = 8). ^* p < 0.05 vs. the control group, ^# p < 0.05 vs. the CCl₄ group, as determined by one-way ANOVA, followed by the LSD multiple comparisons test.

FIGURE 5

Effects of CBD on NF-κB- and PPAR-α-related proteins in mice with CCl₄-induced liver fibrosis. (A,B) The effects of CBD on the protein expression levels of NF-κB, p-NF-κB, p-IκBα, IκBα, p-p38 MAPK, p38 MAPK, COX-2, and PPAR-α were measured by western blotting. (C–H) ImageJ software was used to measure the grey values of the bands. The values represent the means ± SEM (n = 8). ^* p < 0.05 vs. the control group, ^# p < 0.05 vs. the CCl₄ group, as determined by one-way ANOVA, followed by the LSD multiple comparisons test.