5-O-Demethylnobiletin Alleviates CCl4-Induced Acute Liver Injury by Equilibrating ROS-Mediated Apoptosis and Autophagy Induction

Abstract

Polymethoxyflavanoids (PMFs) have exhibited a vast array of therapeutic biological properties. 5-O-Demethylnobiletin (5-DN) is one such PMF having anti-inflammatory activity, yet its role in hepatoprotection has not been studied before. Results from in vitro study revealed that 5-DN did not exert a high level of cytotoxicity on HepG2 cells at 40 μM, and it was able to rescue HepG2 cell death induced by carbon tetrachloride (CCl₄). Subsequently, we investigated acute liver injury on BALB/c mice induced by CCl₄ through the intraperitoneal injection of 1 mL/kg CCl₄ and co-administration of 5-DN at (1 and 2 mg/kg) by oral gavage for 15 days. The results illustrated that treatment with 5-DN attenuated CCl₄-induced elevated serum aminotransferase (AST)/alanine aminotransferase (ALT) ratio and significantly ameliorated severe hepatic damage such as inflammation and fibrosis evidenced through lesser aberrations in the liver histology of 5-DN dose groups. Additionally, 5-DN efficiently counteracted and equilibrated the production of ROS accelerated by CCl₄ and dramatically downregulated the expression of CYP2E1 vitally involved in converting CCl₄ to toxic free radicals and also enhanced the antioxidant enzymes. 5-DN treatment also inhibited cell proliferation and inflammatory pathway abnormally regulated by CCl₄ treatment. Furthermore, the apoptotic response induced by CCl₄ treatment was remarkably reduced by enhanced Bcl-2 expression and noticeable reduction in Bax, Bid, cleaved caspase 3, caspase 9, and apaf-1 expression. 5-DN treatment also induced the conversion of LC3 and promoted the autophagic flux. Conclusively, 5-DN exhibited hepatoprotective effects in vitro and in vivo and prevented liver fibrosis induced by CCl₄.

Keywords: 5-O-demethylnobiletin (5-DN); MAP kinase; apoptosis; autophagy; cytochrome P450; fibrosis; inflammation; reactive oxygen species (ROS).

Figure 1

5-O-Demethylnobiletin (5-DN) rescued HepG2 cells from carbon tetrachloride (CCl₄)-induced cell death. (A) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points after varying doses of CCl₄ (5, 10, 20 mM). (B) MTT assay after 24 h treatment to HepG2 cells with different doses of 5-DN (10, 20, 40 μM). (C) MTT assay after co-treatment with CCl₄ (20 mM) and 5-DN (10, 20, 40 μM). (D) Estimation of LDH, malondialdehyde (MDA), and GSH from HepG2 cell homogenates. (E) Cell morphology of HepG2 cells after 24 h co-treatment with CCl₄ and 5-DN. (F) H₂DCFDA staining of HepG2 cells. (G) AO-EtBr staining of HepG2 cells after 24 h co-treatment with CCl₄ and 5-DN. (H) JC-1 staining of HepG2 cells after 24 h co-treatment with CCl₄ and 5-DN. Magnification: 200×, Scale bar (100 μM). Images were captured using Olympus BX51 fluorescence microscope. The data represented here are the mean ± S.D. from three independent experiments where * p < 0.05, ** p < 0.01, *** p < 0.001, Control vs CCl4, CCl₄ vs. 5–DN (1–2 mg/kg). Statistical significance analysis was carried out through one-way analysis of variance (ANOVA) prism.

Figure 2

5-DN attenuated CCl₄-induced severe hepatic damage in BALB/c mice (mice, n = 6/group). (A) Assessment of the body weight from different mice groups over a period of 15 days. (B) Liver weight/ body weight ratio in percentage of different mice groups. (C) H&E staining on liver tissues. Asterisk (*): represents the regenerative nodules, Arrow (→): represents the fibrous septa extending from the veins, and zoomed images represent infiltration of inflammatory cells from central veins. (D) Serum ALT analysis. (E) Serum AST analysis. Magnification: 200×, Scale bar (100 μM). Images were captured using Olympus BX51 fluorescence microscope. The data are represented here are the mean ± S.D. from three independent experiments where * p < 0.05, *** p < 0.001, Control vs. CCl₄, CCl₄ vs. 5–DN (1–2 mg/kg). Statistical significance analysis was carried out through one-way analysis of variance (ANOVA) prism.

Figure 3

5-DN prevented BALB/c mice from CCl₄-induced severe hepatic fibrotic damage (mice, n = 6/group). (A) Prussian blue staining for the detection of iron deposits in liver tissues. (B) Sirius Red staining for the detection of collagen. (C) Immunohistochemical (IHC) staining of smooth muscle α-actin (αSMA). (D) Percentage of fibrosis evaluated through Sirius red staining (E) Integrated optical density measurement of αSMA through IHC. Magnification: 200×, Scale bar (100 μM). Images were captured using Olympus BX51 fluorescence microscope. The data are represented as mean ± S.D. of three independent experiments, *** p < 0.001 and ns (non-significant). Control vs. CCl₄, CCl₄ vs. 5–DN (1–2 mg/kg). Statistical significance analysis was carried out through one-way analysis of variance (ANOVA) prism.

Figure 4

5-DN inhibited CCl₄-induced inflammatory pathway and normalized upregulated cell proliferation pathway (mice, n = 6/group). (A) Estimation of hepatic IL-6. (B) Estimation of hepatic TNF-α. (C) Western blotting analysis of inflammatory and MAP kinase pathway markers. Densitometry analysis of these respective proteins were normalized by β-actin and evaluated through Image J software. The data are represented as mean ± S.D. of three independent experiments * p < 0.05, ** p < 0.01, *** p < 0.001 and ns (non-significant). Control vs CCl₄, CCl₄ vs. 5-DN (1–2 mg/kg). Statistical significance analysis was carried out through a one-way analysis of variance (ANOVA) prism.

Figure 5

5-DN prevented CCl₄-induced oxidative injury by inhibiting the expression of MDA, CYP2E1 boosted antioxidant enzymes (mice, n = 6/group). (A) H₂DCFDA staining of liver tissues and histogram. (B) Immunohistochemical staining of CYP2E1. (C) Integrated optical density of CYP2E1. (D) Western blotting analysis of CYP2E1. (E) Hepatic malondialdehyde assay analysis. (F) Hepatic Superoxide dismutase assay analysis. (G) Hepatic glutathione assay analysis. Magnification: 200×, Scale bar (100 μM). Images were captured using Olympus BX51 fluorescence microscope. Densitometry analysis of these respective proteins were normalized by β-actin and evaluated through Image J software. The data are represented as mean ± S.D. of three independent experiments * p < 0.05, ** p < 0.01, *** p < 0.001 and ns (non-significant). Control vs. CCl₄, CCl₄ vs. 5–DN (1–2 mg/kg). Statistical significance analysis was carried out through one-way analysis of variance (ANOVA) prism.

Figure 6

5-DN blockaded CCl₄-induced apoptosis. (mice, n = 6/group). (A) Western blotting analysis of apoptosis markers and their quantification. (B) TUNEL assay to determine the occurrence of apoptosis. TUNEL-positive hepatocytes were counted from five different representative fields for all the groups and were evaluated and averaged for making the graph (Magnification: 200×). (C) Immunofluorescence staining of cleaved caspase 3 from BALB/c liver tissue (Magnification: 400×, Scheme 100 μM). Images were captured using Olympus BX51 fluorescence microscope. Densitometry analysis of these respective proteins were normalized by β-actin and evaluated through Image J software. The data are represented as mean ± S.D. of three independent experiments * p < 0.05, ** p < 0.01, *** p < 0.001 and ns (non-significant). Control vs. CCl₄, CCl₄ vs. 5-DN (1–2 mg/kg). Statistical significance analysis was carried out through one-way analysis of variance (ANOVA) prism.

Figure 7

5-DN promoted autophagosome formation in CCl4-treated BALB/c mice (mice, n = 6/group). (A) Western blot-ting analysis of important autophagy markers and their quantification. (B) Immunohistochemical staining of LC3B from BALB/c liver tissue Arrow (→): indicates the formation of punctate structure in liver tissue. (Magnification: 200×, Scale bar (100 μM). Images were captured using an Olympus BX51 fluorescence microscope. Densitometry analysis of these respective proteins were normalized by β-actin and evaluated through Image J software. The data are represented as mean ± S.D. of three independent experiments * p < 0.05, ** p < 0.01, *** p < 0.001 and ns (non-significant). Control vs. CCl4, CCl4 vs. 5–DN (1–2 mg/kg). Statistical significance analysis was carried out through one-way analysis of variance (ANOVA) prism.

Figure 8

Role of 5-DN in counteracting CCl4-induced acute liver injury. (↑): represents upregulation of the spe-cific marker in response to CCl4 or 5-DN. (↓): represents downregulation of the specific marker in response to CCl4 or 5-DN. (┴): represents inhibition after treatment with 5-DN.