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Figure 1. In vitro assays. (A) Viability of L02 cells with different concentrations of H2O2. (B) Viability of L02 cells damaged by H2O2 after 24 h incubation of different concentration of peptide. (CâG) The cell cycle distribution in L02 cells with flow cytometry. (H) Quantitative analysis of cell cycle distribution in each group. (IâP) The apoptosis ratios of L02 cells in different groups with flow cytometry. (Q) Quantitative analysis of the percentage of cell apoptosis in each group. Comparisons were made with one-way ANOVA. Values were expressed as the percentage of the control and represented as mean ± SD. (n = 3) (compared to Ctrl, # p < 0.05; compared to 0, * p < 0.05) (n = 3).
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Figure 2. The ROS levels of L02 cells. (AâE) The mean DCF fluorescence intensity of L02 cells in different groups with flow cytometry. (F) Histogram overlay of each group. (G) Quantitative analysis of ROS relative units in each group. Comparisons were made with one-way ANOVA. Values were expressed as the ratio of the control and represented as mean ± SD (n = 3) (compared to Ctrl, #
p < 0.05; compared to H2O2, * p < 0.05) (n = 3).
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Figure 3. Peptide P03 attenuated hepatic injury induced by binge alcohol exposure. (A) Schematic diagram depicting the experimental approach to evaluate the effect of P03 supplementation on mice with binge alcohol exposure. (B) Body weight change. The data in week 0 represent the body weight of mice during acclimation. (C) Organ coefficients of acute ethanol-intoxicated mice in different groups. The organ coefficientâ = âorgan weight/body weight. (DâG) Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (γ-GGT) levels and ratios of AST vs. ALT in different groups. Values are expressed as mean ± SD. (Compared to Ctrl, #
p < 0.05. Compared to ethanol, * p < 0.05) (n = 5).
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Figure 4. Alterations in antioxidant enzymes in rodent livers and immunoblots of HO-1, NQO-1 and Nrf2. (A,B) Quantification of GPx and SOD in different groups (n = 5). (C,D) Hepatic levels of NQO-1 and HO-1 protein. (EâG) Relative protein levels of Nrf2 in nucleus and cytoplasm in each group (n = 3). Densitometric quantification of HO-1, NQO-1, cytoplasmic and nuclear Nrf2 were performed and normalized to β-actin levels (for the cytosolic factions) or Lamin B1 (for the nuclear fractions). (H) Western blot analysis of indicated proteins in liver lysates of different groups. Values are represented as mean ± SD and plotted as fold-change. (Compared to Ctrl, #
p < 0.05. Compared to ethanol, * p < 0.05).
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Figure 5. Alterations in lipid metabolism in mice with binge alcohol exposure. (AâE) Representative images of histological liver sections after Oil Red O staining. Sections were visualized using Bioquant Osteo (20Ã) and quantified using Image Pro plus software. (F) The corresponding quantification of Oil Red O staining in different groups. (G,H) Serum triglycerides and total cholesterol levels in different groups. (I) The levels of hepatic malondialdehyde (MDA) were detected colorimetrically in different groups. Comparisons were made with one-way ANOVA. Values are represented as mean ± SD. (Compared to Ctrl, #
p < 0.05. Compared to ethanol, * p < 0.05) (n = 5).
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Figure 6. Anti-inflammatory effects of P03 in mice with binge alcohol exposure. (AâE) Representative images of histological liver sections after H&E staining. Sections were visualized using Bioquant Osteo (20Ã). Yellow arrows represent neutrophil infiltration surrounding the central vein (CV); blue arrows represent microvesicular steatosis. Scale bar = 50 μM. (FâI) Serum levels of inflammatory cytokines and LPS in different groups. Comparisons were made with one-way ANOVA. Values are represented as mean ± SD. (Compared to Ctrl, #
p < 0.05. Compared to ethanol, * p < 0.05) (n = 5).
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Figure 7. Immunoblots of nuclear and cytoplasmic NF-κB. (A) Western blot analysis of NF-κB in different groups. (BâD) Relative protein levels of NF-κB in nucleus and cytoplasm in each group. Densitometric quantification of nuclear and cytoplasmic NF-κB were performed and normalized to β-actin levels (for the cytosolic factions) or Lamin B1 (for the nuclear fractions). The values are represented as mean ± SD and plotted as fold-change. (Compared to Ctrl, #
p < 0.05. Compared to ethanol, * p < 0.05).
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Figure 8. Immunoblots of Bcl-2, Bax, Cytochrome C, Caspase-3 and PARP. (A) Immunoblots of indicated proteins. (BâF) Densitometric quantification of Bcl-2, Bax, Cytochrome C, Caspase-3 and PARP were performed and normalized to β-actin levels. (G) The ratios of Bcl-2 vs. Bax were calculated from densitometric analysis. The values represent mean ± SD and are plotted as fold-change. (Compared to Ctrl, #
p < 0.05. Compared to ethanol, * p < 0.05) (n = 3).
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Figure 9. Immunoblots of PGC-1α, OPA-1, Mfn-2, and Drp-1. (A) Immunoblots of indicated proteins. (BâE) Densitometric quantification of PGC-1α, OPA-1, Mfn-2, and Drp-1 were performed and normalized to β-actin levels. The values are represented as mean ± SD and plotted as fold-change. (Compared to Ctrl, #
p < 0.05. Compared to ethanol, * p < 0.05) (n = 3).
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Figure 10. Immunoblots of mitophagy-related proteins. (A) Immunoblots of indicated proteins. (BâH) Densitometric quantification of p62, Beclin-1, Parkin, PINK1 and LC3 (I, II) were performed and normalized to β-actin levels. The values are represented as triplicate experiments ± SD and plotted as fold-change. (Compared to Ctrl, #
p < 0.05. Compared to ethanol, * p < 0.05) (n = 3).
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