Ivabradine Attenuates Experimental Hepatic Fibrosis by Modulating Inflammatory and Apoptotic Signaling Pathways

Figures

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  Figure 1. Impact of ivabradine on hepatic H&E changes. Microscopic pictures of H&E-stained liver sections. (A,B) Normal group showing normal arrangement of hepatocytes, normal central veins, portal areas, and sinusoids. (C,D) TAA group showing severe portal lesions, including fibrosis (thick black arrow), congestion (red arrow), edema (blue arrow), mononuclear cell infiltration (arrowhead), and bile ductule hyperplasia (thin black arrow). Daily administration of low (E,F) and high doses of ivabradine (G,H) induced a significant amelioration of TAA-induced histopathological changes in the liver in a dose-dependent manner.

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  Figure 2. Impact of ivabradine on hepatic collagen deposition evaluated by Masson’s trichrome staining. Hepatic specimen stained with Masson’s trichrome stain for the evaluation of liver fibrosis. (A–D) Normal group showing normal deposition of collagen around blood vessels (portal or central vein). (E–H) TAA group showing dense bluish portal collagen deposition extending and separating the hepatic parenchyma. Administration of low dose of ivabradine (I–L) showed either fine bridging collagen fibrils or mild periportal collagen deposition focally extending and replacing the surrounding hepatocytes, while administering high dose of ivabradine (M–P) revealed low perivascular collagen deposition. Arrows = positive collagen deposition. Image magnification: 100× = bar 100 μm and 400× = bar 50 μm. (Q) effects of ivabradine on collagen deposition in the liver. Data are presented as mean ± SEM (n = 10). a, b, and c p < 0.05 significantly different compared to normal, TAA, and ivabradine 10 + TAA, respectively, using one-way ANOVA followed by the Tukey–Kramer multiple comparisons post hoc test.

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  Figure 3. Impact of ivabradine on hepatic TGF-β expression. Representative TGF-β expression and localization in hepatic sections of different treatment groups. (A–D) Control group (low- and high-power view) showing negative-to-low cytoplasmic expression in hepatocytes. (E–H) TAA group (low- and high-power view) showing diffuse moderate-to-high cytoplasmic expression in hepatocytes and invading inflammatory cells. (I–L) Low ivabradine (low- and high-power view) showing low-to-mild faint cytoplasmic expression in hepatocytes and invading inflammatory cells. (M–P) High ivabradine (low- and high-power view) showing mild faint cytoplasmic expression in hepatocytes with scattered positivity in invading inflammatory cells. Thin arrows = positive hepatocytes, thick arrows = positive inflammatory cells. Image magnification: 100× = bar 100 μm, 400× = bar 50 μm; (Q) TGF-β percentage expression in the hepatic sections of different groups. Data are presented as mean ± SEM (n = 10). a, b, and c p < 0.05 significantly different compared to normal, TAA, and ivabradine 10 + TAA, respectively, using one-way ANOVA followed by the Tukey–Kramer multiple comparisons post hoc test.

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  Figure 4. Impact of ivabradine on hepatic α-SMA expression. Representative α-SMA expression and localization in hepatic sections of different treatment groups. (A–D) Control group (low- and high-power view) showing preserved hepatic architecture without nodule formation (black star). Negative cytoplasmic expression of α-SMA is observed in hepatocytes, with positive brownish immunostaining restricted to the blood vessel walls (thick arrows). (E–H) TAA group (low- and high-power view) showing significant loss of hepatic architecture and extensive nodule formation (yellow star). Marked bridging fibrosis with intense brownish staining is evident (red star), along with diffuse cytoplasmic expression of α-SMA in activated stellate cells (thin arrows). (I–L) Low ivabradine (low- and high-power view) showing improvement and protective antifibrotic effects; the hepatic architecture is restored, showing only low scattered cytoplasmic expression in the pericentral area while maintaining normal staining of blood vessel walls. (M–P) High ivabradine (low- and high-power view) showing near-normal restoration of hepatic architecture with a significant reduction in α-SMA expression, which is primarily…

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  Figure 5. Impact of ivabradine on hepatic α-SMA expression. Representative COL1A1 expression and localization in hepatic sections of different treatment groups. (A–D) Control group (low- and high-power view) showing preserved hepatic architecture without nodule formation (black star). Negative cytoplasmic expression of COL1A1 is observed in hepatocytes, with positive brownish immunostaining restricted to the blood vessel walls (thick arrows). (E–H) TAA group (low- and high-power view) showing significant loss of hepatic architecture and extensive nodule formation (yellow star). Marked bridging fibrosis with intense brownish staining is evident (red star), along with diffuse cytoplasmic expression of COL1A1 in activated stellate cells (thin arrows). (I–L) Low ivabradine (low- and high-power view) showing improvement and protective antifibrotic effects; the hepatic architecture is restored, showing only low scattered cytoplasmic expression in the pericentral area while maintaining normal staining of blood vessel walls. (M–P) High ivabradine (low- and high-power view) showing near-normal restoration of hepatic architecture with a significant reduction in COL1A1 expression, which is primarily…

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  Figure 6. Impact of ivabradine on hepatic oxidative/antioxidant capacity. Data are presented as mean ± SEM (n = 10). a, b, and c p < 0.05 significantly different compared to normal, TAA, and ivabradine 10 + TAA, respectively, using one-way ANOVA followed by the Tukey–Kramer multiple comparisons post hoc test.

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  Figure 7. Impact of ivabradine on the hepatic expressions of NF-κB p65/TNF-α. Representative NF-κB p65 expression and localization in hepatic sections of different treatment groups. (A–D) Normal group (low- and high-power view) showing faint cytoplasmic and nuclear expression in hepatocytes. (E–H) TAA group (low- and high-power view) showing diffuse high cytoplasmic expression in hepatocytes with nuclear expression in surrounding periportal invading inflammatory cells. (I–L) Low ivabradine (low- and high-power view) showing moderate faint-to-strong cytoplasmic expression with scattered nuclear positivity in invading inflammatory cells. (M–P) High ivabradine (low- and high-power view) showing mild-to-moderate cytoplasmic expression in hepatocytes. Thin arrows = positive hepatocytes, thick arrows = positive inflammatory cells. Image magnification: 100× = bar 100 μm, 400× = bar 50 μm; (Q) NF-κB p65 percentage expression in the hepatic sections of different treatment groups; (R) level of TNF-α in hepatic tissue. Data are presented as mean ± SEM (n = 10). a, b, and c p < 0.05 significantly different compared to normal, TAA, and ivabradine 10 + TAA, respectively, using one-way ANOVA followed by…

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  Figure 8. (A–C) Impact of ivabradine on hepatic PI3K, pPI3K, AKT, pAKT, and mTOR expressions. Data are presented as mean ± SEM (n = 3). a, b, and c p < 0.05 significantly different compared to normal, TAA, and ivabradine 10 + TAA, respectively, using one-way ANOVA followed by the Tukey–Kramer multiple comparisons post hoc test.

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  Figure 9. Impact of ivabradine on hepatic levels of Bax and caspase-3. Data are presented as mean ± SEM (n = 10). a, b, and c p < 0.05 significantly different compared to normal, TAA, and ivabradine 10 + TAA, respectively, using one-way ANOVA followed by the Tukey–Kramer multiple comparisons post hoc test.

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  Figure 10. Impact of ivabradine on hepatic Bcl-2 expression. Representative Bcl-2 expression and localization in hepatic sections of different treatment groups. (A–D) Normal group (low- and high-power view) showing extensive cytoplasmic expression in hepatocytes. (E–H) TAA group (low- and high-power view) showing low-to-mild cytoplasmic expression in hepatocytes with low positivity in invading inflammatory cells. (I–L) Low ivabradine (low- and high-power view) showing moderate immunopositive cytoplasmic stained hepatocytes. (M–P) High ivabradine (low- and high-power view) showing moderate-to-high cytoplasmic expression in hepatocytes. Thin arrows = positive hepatocytes, thick arrow = positive inflammatory cells. Image magnification: 100× = bar 100μm, 400× = bar 50 μm; (Q) Bcl-2 percentage expression in the hepatic sections of different treatment groups; data are presented as mean ± SEM (n = 10). a, b, and c p < 0.05 significantly different compared to normal, TAA, and ivabradine 10 + TAA, respectively, using one-way ANOVA followed by the Tukey–Kramer multiple comparisons post hoc test.