Korean J Physiol Pharmacol.  2021 Sep;25(5):385-393. 10.4196/kjpp.2021.25.5.385.

Loss of RAR-α and RXR-α and enhanced caspase-3-dependent apoptosis in N-acetyl-p-aminophenol-induced liver injury in mice is tissue factor dependent

• Abdel-Bakky MS
• Helal GK
• El-Sayed ESM
• Amin E
• Alqasoumi A
• Alhowail A
• Said Abdelmoti ES
• Saad AS

• KMID: 2519401
• DOI: http://doi.org/10.4196/kjpp.2021.25.5.385

Abstract

Tissue factor (TF) activates the coagulation system and has an important role in the pathogenesis of various diseases. Our previous study stated that retinoid receptors (RAR-α and RXR-α) are released as a lipid droplet in monocrotaline/ lipopolysaccharide-induced idiosyncratic liver toxicity in mice. Herein, the interdependence between the release of retinoid receptors RAR-α and RXR-α and TF in Nacetyl-p-aminophenol (APAP)-induced mice liver toxicity, is investigated. Serum alanine transaminase (ALT) level, platelet and white blood cells (WBCs) counts, protein expression of fibrin, TF, cyclin D1 and cleaved caspase-3 in liver tissues are analyzed. In addition, histopathological evaluation and survival study are also performed. The results indicate that using of TF-antisense (TF-AS) deoxyoligonucleotide (ODN) injection (6 mg/kg), to block TF protein synthesis, significantly restores the elevated level of ALT and WBCs and corrects thrombocytopenia in mice injected with APAP. TF-AS prevents the peri-central overexpression of liver TF, fibrin, cyclin D1 and cleaved caspase-3. The release of RXR-α and RAR-α droplets, in APAP treated sections, is inhibited upon treatment with TF-AS. In conclusion, the above findings designate that the released RXR-α and RAR-α in APAP liver toxicity is TF dependent. Additionally, the enhancement of cyclin D1 to caspase-3-dependent apoptosis can be prevented by blocking of TF protein synthesis.

Keyword

Acetaminophen; Retinoid A receptor alpha; Retinoid X receptor alpha; Thromboplastin; Tissue factor antisense

Figure

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  Fig. 1 Serum alanine transaminase (ALT) and hepatic tissue factor (TF) expression in N-acetyl-p-aminophenol (APAP)-treated mice in the presence or absence of TF deoxyoligonucleotides (ODNs). (A) Increased serum ALT in APAP treated group (800 mg/kg, single i.p. dose) significantly compared to control vehicle-treated mice (0.2 ml per mice, i.p.). APAP + TF-SC group (3.5 h prior to APAP injection in a dose of 6 mg/kg in 100 µl saline, i.v.) has no significant difference compared to APAP-treated mice. APAP + TF-AS (similar dose as TF-SC) reduced significantly serum ALT level compared to APAP treated mice. Data of the figure are represented as means ± SD. (B) TF protein expression is increased in hepatocytes (arrows) and hepatic stellate cells in the pericentral zone in APAP injected animals. No difference has been detected in APAP + TF-SC compared to APAP treated animals. Low level of expression is seen in APAP + TF-AS sections compared to APAP-treated group. CV, central vein (magnification 200X, scale bar 100 µm and 100X, scale bar 250 µm). (C) TF fluorescence intensity is blotted in bar chart. (D) Quantitative histogram of TF in green fluorescence measuring the intensity of six fields for at least six mice per group. (E) Western blot analysis of liver tissue samples from normal and APAP with or without TF ODNs using TF and β-actin Abs as an internal control (n = 3). Data in the figures are represented as mean ± SD. aSignificantly different from vehicle treated group. bSignificantly different from APAP-treated group using one-way ANOVA followed by Tukey-Kramer test as multiple comparisons (p < 0.05).

• 

  Fig. 2 Effect of N-acetyl-p-aminophenol (APAP) treatment with or without tissue factor (TF) deoxyoligonucleotides (ODNs) on liver tissue expression of fibrin protein. (A) Microscopic appearance of fibrin protein expression in APAP treated mice in the presence or absence of TF ODNs using immunofluorescence technique. Fibrin protein is increased in the liver of mice treated with APAP and APAP + TF-SC. However, APAP + TF-AS treated mice section showed lower fibrin protein expression compared to APAP treatment alone (arrows). CV, central vein (magnification 200×). (B) Histogram of green fluorescence intensity. (C) Figure of TF representing the quantitative fluorescence intensity of six fields per section for at least six mice per group. Data are represented as means ± SD. aSignificantly different from vehicle treated mice. bSignificantly different from APAP-treated mice using one-way ANOVA followed by Tukey-Kramer test as multiple comparison at p < 0.05 (magnification 200×, scale bar 100 µm).

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  Fig. 3 Protein expression of RXR-α and RAR-α in mice livers tissues treated with N-acetyl-p-aminophenol (APAP) with or without tissue factor (TF) deoxyoligonucleotides (ODNs). Control group showed basal level of RXR-α expression (arrows), APAP and APAP + TF-SC treated mice displaying an intense speckled RXR-α expression (arrows) around CV in liver. Mice injected with APAP + TF-AS showed normal RXR-α expression around CV (A). Quantitative fluorescence intensity of RXR-α using ImageJ software of six fields per section for at least six mice per group (B). Constitutive expression of RAR-α around central vein in control sections (arrows), APAP treated group and APAP + TF-SC treated mice showing an intense diffuse speckled expression of RAR-α (arrows) around CV. Normal RAR-α expression in tissues of animals injected with APAP + TF-AS (C). Fluorescence intensity of RAR-α using ImageJ software (D). aSignificantly different from vehicle treated mice, bSignificantly different from APAP-treated mice, cSignificantly different from APAP + TF-SC treated mice using one-way ANOVA followed by Tukey-Kramer test as multiple comparison at p < 0.05. CV, central vein (magnification 400×, scale bar 50 µm).

• 

  Fig. 4 Effect of N-acetyl-p-aminophenol (APAP) treatment on cyclin D1 and cleaved caspase-3 (c Caspase-3) protein expression in the presence or absence of TF-SC and TF-AS. Elevated level of cyclin D1 (arrows) (A) and cleaved caspase-3 (arrows) (C) protein expressions are mostly in the pericentral area in APAP treated animals compared to vehicle treated animals after 24 h. Normal, low, expression of cyclin D1 and cleaved caspase-3 in vehicle treated mice sections. Strong expression of both proteins is detected in hepatocytes around central vein in APAP and APAP + TF-SC-treated treated mice. Low expression of cyclin D1 and cleaved caspase-3 is seen in APAP + TF-AS injected mice. CV, central vein (magnification 200×, scale bar 100 µm). Fluorescence intensity of cyclin D1 (B) and cleaved caspase-3 (D) using ImageJ software (D) of six fields per section for at least six mice per group. aSignificantly different from vehicle treated mice, bSignificantly different from APAP-treated mice, cSignificantly different from APAP + TF-SC treated mice using one-way ANOVA followed by Tukey-Kramer test as multiple comparison at p < 0.05.

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  Fig. 5 Histological analysis of hepatic tissue sections from vehicle and N-acetyl-p-aminophenol (APAP)-treated mice in the presence or absence of tissue factor (TF) deoxyoligonucleotides (ODNs). Normal histologic features are seen in vehicle treated animals. However, APAP-injected and APAP + TF-SC mice sections displayed alteration in the general architectures in the form of ischemia, necrosis (black arrowheads), hemorrhage (long arrows), loss of sinusoidal architecture and fatty degeneration (yellow arrowheads). Whereas APAP + TF-AS treated mice showed normal tissue architectures (H&E, magnification 200×, scale bar 100 µm).

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  Fig. 6 Effect of N-acetyl-p-aminophenol (APAP) in the presence or absence of tissue factor (TF) deoxyoligonucleotides (ODNs) injection on the survival rate of mice. Animal survivability was analyzed in mice treated with APAP with or without TF ODNs for four days. Mice treated with APAP alone exhibit 6 out of 10 were survived at the end of the experiment. Similarly, 60% of the animals were survived after 4 days of treatment in APAP + TF-SC. On the other hand, mice treated with APAP in the presence of TF-AS showed 100% survivability (p < 0.05). MCT/LPS, monocrotaline/lipopolysaccharide.

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