Mangiferin Ameliorates CCl4-Triggered Acute Liver Injury by Inhibiting Inflammatory Response and Oxidative Stress: Involving the Nrf2-ARE Pathway

Abstract

Purpose: Acute liver injury (ALI) is characterized by inflammation and oxidative stress (OS). Although mangiferin (MGF) has antioxidant and anti-inflammatory effects, its role in ALI remains unclear. Accordingly, we investigated the MGF molecular mechanism in carbon tetrachloride (CCl₄)-induced ALI in vivo and in vitro.

Materials and methods: The CCl₄ was utilized to induce ALI in mice. In vivo, the therapeutic effects of MGF on CCl₄-induced liver injury were evaluated through biochemical assays and histomorphological analysis. Additionally, immunohistochemistry, immunofluorescence, ELISA and Western blotting were further applied to explore the mechanism. In vitro, The CCK-8 assay and flow cytometry were employed to investigate the protective effects of MGF against CCl₄-induced toxicity in HepG2 cells, while mitochondrial reactive oxygen species levels and Western blotting were used to explore the biological effects and molecular mechanisms.

Results: MGF treatment resulted in a reduction in serum levels of AST and ALT, diminished concentrations of TNF-α, IL-6, and IL-1β in liver tissue, and concurrently decreased cellular apoptosis. Furthermore, MGF pretreatment enhanced the activity of SOD and GSH while concurrently diminishing the MDA production. This study further demonstrated the upregulation of Nrf2, NQO1, and HO-1 protein expression levels, as well as the downregulation of p-p65 protein expression levels. In vitro investigations revealed that the mitigation of CCl₄-induced inflammation and OS by MGF was mediated via the Nrf2- antioxidant response element (ARE) pathway, which was disrupted by ML385 in HepG2 cells.

Conclusion: CCl₄ can induce liver injury, while treatment with MGF mitigates ALI by inhibiting oxidative stress, inflammation, and apoptosis. The protective mechanism of MGF is mediated by the Nrf2-ARE pathway activation.

Keywords: Nrf2; inflammation; liver injury; mangiferin; oxidative stress.

Figure 1

MGF protects against CCl₄-triggered liver injury. (A) MGF chemical structure. (B-C) AST and ALT levels after the CCl₄ challenge. (D) Liver tissue representative image. (E) Changes in liver index in the four groups. (F) H&E-stained liver section representative image (magnification: 100×), scale bar: 200 μm. (G) Quantifying liver tissue necrosis area. Data are expressed as mean ± SD (n = 6). **P < 0.01, ***P < 0.001 versus control group. ^#P < 0.05, ^##P < 0.01 versus CCl₄ group.

Figure 2

MGF effect on CCl₄-triggered cell apoptosis in liver tissues. (A) Representative TUNEL-stained sections demonstrating apoptosis in mice liver tissue (magnification: 200×). (B) Statistical analysis of TUNEL-positive cells was performed. (C) Determination of Bcl-2, Bax, and cleaved Caspase-3 expression in the liver by immunohistochemistry (original magnification, 200×). (D–G) Bcl-2, Bax, Bcl-2/Bax ratio, and cleaved Caspase-3 densitometric analysis. Data are expressed as mean ± SD (n = 6). **P < 0.01, ***P < 0.001 versus control group. ^#P < 0.05, ^##P < 0.01 versus CCl₄ group.

Figure 3

MGF ameliorates CCl₄ exposure-caused OS and inflammatory responses in the mice liver tissues. (A) MDA levels (B) SOD and (C) GSH activities in the mice liver tissues. (D) TNF-α, (E) IL-1β, and (F) IL-6 levels in mice liver tissues. (G) Indicating Nrf2, NQO1, HO-1, p-p65 and p65 protein expression in different groups through Western blotting. (H–K) Quantification of the Western blotting data for Nrf2, NQO1, HO-1, and p-p65/p65. Data are expressed as mean ± SD (n = 6). **P < 0.01, ***P < 0.001 versus control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus CCl₄ group.

Figure 4

MGF impact on the Nrf2 pathway in CCl₄-treated mice. Immunofluorescence of (A) Nrf2, (B) NQO1, and (C) HO-1 in liver tissues. (D–F) Mean integrated optical density of immunofluorescence staining for Nrf2, NQO1, and HO-1. Data are expressed as mean ± SD (n = 6). **P < 0.01, ***P < 0.001 versus control group. ^#P < 0.05, ^##P < 0.01 versus CCl₄ group.

Figure 5

Effect of MGF on CCl₄-induced cytotoxicity and apoptosis in HepG2 cells. (A) Cell viability was evaluated using the CCK-8 assay. (B) ALT and (C) AST levels in the supernatants of HepG2 cells. (D) Flow plots of apoptosis in the different groups. (E) Quantification of apoptotic cells. (F) Indicating Bcl-2, Bax, and cleaved Caspase-3 protein expression in different groups through Western blotting. (G–J) Quantitative results for Bcl-2, Bax, Bcl-2/Bax ratio, and cleaved Caspase-3. Data are expressed as mean ± SD (n = 6). ***P < 0.001 versus control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus CCl₄ group. ^&P < 0.05, ^&&P < 0.01 versus CCl₄+MGF group.

Figure 6

MGF relieves CCl₄-induced inflammatory response and OS in HepG2 cells. (A) TNF-α, (B) IL-6, and (C) IL-1β levels in different groups. (D) MDA level, (E) SOD, and (F) GSH activities in each group. (G-H) Mitochondrial superoxide was detected through immunofluorescence using MitoSox Red and MitoTracker Green staining. (I) ATP content in different groups. Data are expressed as mean ± SD (n = 6). ***P < 0.001 versus the control group. ^#P < 0.05, ^##P < 0.01 versus CCl₄ group. ^&P < 0.05, ^&&P < 0.01 versus CCl₄+MGF group.

Figure 7

MGF impact on Nrf2 pathway in CCl₄-treated HepG2 cells. (A) Indicating Nrf2, NQO1, and HO-1 protein expression in different groups through Western blotting. (B–D) Quantifying Western blotting data for Nrf2, NQO1, and HO-1. (E) Indicating p-p65 and p65 protein expression in different groups through Western blotting. (F) Quantifying Western blotting data for p-p-65/p65. Data are expressed as mean ± SD (n = 6). **P < 0.01, ***P < 0.001 versus control group. ^#P < 0.05, ^###P < 0.001 versus CCl₄ group. ^&P < 0.05 versus CCl₄+MGF group.

Figure 8

Mechanism of MGF protective impact against hepatotoxicity induced by CCl₄.