Ambroxol mitigates cyclophosphamide-induced liver injury by suppressing TLR-4/NF-κB signaling and oxidative stress and upregulating cytoglobin, TXNRD1 and HMGB1

Figure 1 Ambroxol ameliorated liver function markers in cyclophosphamide-administered rats. A: Serum alanine aminotransferase; B: Aspartate aminotransferase; C: Alkaline phosphatase; D: Albumin. Data are expressed as mean ± SD (n = 6). ^aP < 0.05 and ^cP < 0.001 vs control group. ^fP < 0.001 vs cyclophosphamide. ABX: Ambroxol; CP: Cyclophosphamide; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase.

Figure 2 Ambroxol prevented liver tissue injury in cyclophosphamide-administered rats. A-D: Photomicrographs of hematoxylin & eosin-stained sections from the control (A and B) and ABX-supplemented rats (C and D) showing normal liver architecture, including hepatocytes (arrowheads), hepatic lamina (L), central veins (arrows), and sinusoids (S); E-H: Cyclophosphamide-treated rats showing congested vessels (arrows), hepatocyte vacuolation (black arrowheads), enlarged hepatocytes with enlarged nuclei (white arrowheads), inflammatory cells (IC) infiltration, dilated sinusoids (S), and disorganized hepatic laminae (L); and I and J: ABX treatment prevented tissue injury and hepatic tissues relatively appear as control group showing normal non-congested vessels (arrows), hepatocytes (arrowheads), hepatic laminae (L), and sinusoids (S). A, C, E, and I: 100 × and scale bar = 200 µm; B, D, F, G, H and J: 400 × and scale bar = 50 µm. ABX: Ambroxol; CP: Cyclophosphamide; S: Sinusoids; L: Laminae; IC: Inflammatory cells.

Figure 3 Ambroxol prevented cyclophosphamide-induced collagen, mucopolysaccharides, and iron deposition in rat liver. Photomicrographs of Sirius red-, periodic acid-Schiff (PAS)- and Prussian blue-stained liver sections. Sirius red staining shows few collagen fibers (arrows) in hepatic parenchyma around the central vein in control and ambroxol (ABX)-treated rats, increased collagen fibers (arrows) in cyclophosphamide (CP)-administered rats and normal collagen fiber (arrows) content in CP-administered rats treated with ABX. The liver of control and ABX-treated rats shows normal PAS stain intensity and distribution (arrows), whereas CP-administered rats show an increase in the PAS stain intensity (arrows). ABX treatment decreased PAS staining in CP-administered rats. Control and ABX-supplemented rats show negative Prussian blue staining affinity, CP-administered rats show hemosiderin deposits (arrows), and CP-administered rats treated with ABX show few hemosiderin deposits (arrows). (400 × and scale bar = 50 µm). ABX: Ambroxol; CP: Cyclophosphamide; PAS: Periodic acid-Schiff.

Figure 4 Ambroxol attenuated cyclophosphamide-induced oxidative stress. A-D: Ambroxol decreased hepatic malondialdehyde (A), and increased reduced glutathione (B), superoxide dismutase (C), and catalase (D) in cyclophosphamide (CP)-administered rats. Data are expressed as mean ± SD (n = 6). ^aP < 0.05 and ^cP < 0.001 vs control group, ^fP < 0.001 vs CP. MDA: Malondialdehyde; SOD: Superoxide dismutase; GSH: Reduced glutathione; ABX: Ambroxol; CP: Cyclophosphamide.

Figure 5 Ambroxol suppressed inflammation in cyclophosphamide-treated rats. A-C: Ambroxol decreased liver toll-like receptor-4 (A and B), nuclear factor-kappaB (NF-κB) p65 (A and C); D: Tumor necrosis factor-α; E: Interleukin-1β. Data are expressed as mean ± SD (n = 6). ^bP < 0.01 and ^cP < 0.001 vs control group, ^eP < 0.01 and ^fP < 0.001 vs cyclophosphamide. ABX: Ambroxol; CP: Cyclophosphamide; TLR: Toll-like receptor; NF-κB: Nuclear factor-kappa B; TNF: Tumor necrosis factor; IL: Interleukin.

Figure 6 Ambroxol upregulated cytoglobin in the liver of cyclophosphamide-administered rats. A and B: Ambroxol (ABX) enhanced the expression of cyctoglobin in the liver of rats that received cyclophosphamide (CP). Data are expressed as mean ± SD (n = 6). ^cP < 0.001 vs control group and ^fP < 0.001 vs CP; C: Molecular docking showing the interaction between ABX and cytoglobin. ABX: Ambroxol; CP: Cyclophosphamide.

Figure 7 Ambroxol increased thioredoxin reductase 1 in the liver of cyclophosphamide-administered rats. A and B: Ambroxol (ABX) upregulated the expression of thioredoxin reductase 1 (TXNRD1) in the liver of rats that received cyclophosphamide (CP). Data are expressed as mean ± SD (n = 6). ^aP < 0.05 and ^cP < 0.001 vs control group, ^fP < 0.001 vs CP; C: Molecular docking showing the interaction between ABX and TXNRD1. ABX: Ambroxol; CP: Cyclophosphamide; TXNRD1: Thioredoxin reductase 1.

Figure 8 Ambroxol upregulated high-mobility group box 1 in the liver of cyclophosphamide-administered rats. A and B: Ambroxol (ABX) increased the expression of high-mobility group box 1 (HMGB1) in the liver of rats that received cyclophosphamide (CP). Data are expressed as mean ± SD (n = 6). ^bP < 0.01 and ^cP < 0.001 vs control group, ^fP < 0.001 vs CP; C: Molecular docking showing the interaction between ABX and HMGB1. ABX: Ambroxol; CP: Cyclophosphamide; HMGB1: High-mobility group box 1.