Korean Circ J.  2014 Mar;44(2):97-104. 10.4070/kcj.2014.44.2.97.

Apoptosis and Inflammation Associated Gene Expressions in Monocrotaline-Induced Pulmonary Hypertensive Rats after Bosentan Treatment

• Hong YM
• Kwon JH
• Choi S
• Kim KC

• KMID: 2223898
• DOI: http://doi.org/10.4070/kcj.2014.44.2.97

Abstract

BACKGROUND AND OBJECTIVES
Vascular wall remodeling in pulmonary hypertension can be caused by an aberration in the normal balance between proliferation and apoptosis of endothelial cell in the pulmonary artery. The objective of this study was to evaluate the effect of bosentan on apoptosis in monocrotaline (MCT)-induced pulmonary hypertension.
MATERIALS AND METHODS
Sprague-Dawley rats were divided into three groups: control (C) group, M group (MCT 60 mg/kg) and B group (MCT 60 mg/kg plus bosentan 20 mg/day orally). Gene expressions of Bcl (B cell leukemia/lymphoma)-2, caspase-3, complement component (C)-6, vascular endothelial growth factor (VEGF), interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-alpha) were analyzed by real time polymerase chain reaction and western blot analysis.
RESULTS
The messenger ribonucleic acid (mRNA) expressions of caspase-3 and VEGF were significantly increased in the M group compared with the C group, and significantly decreased in the B group compared with the M group in week 4. mRNA expression of IL-6 was significantly decreased in weeks 1, 2, and 4 in the B group compared with the M group. mRNA expression of TNF-alpha was significantly decreased on day 5 and in weeks 1 and 2 in the B group compared with the M group.
CONCLUSION
Bosentan may have potential for preventing apoptosis and inflammation.

Keyword

Hypertension, pulmonary; Monocrotaline; Apoptosis; Gene expression; Bosentan

MeSH Terms

Animals
Apoptosis*
Blotting, Western
Caspase 3
Complement System Proteins
Endothelial Cells
Gene Expression*
Hypertension, Pulmonary
Inflammation*
Interleukin-6
Interleukins
Monocrotaline
Pulmonary Artery
Rats*
Rats, Sprague-Dawley
Real-Time Polymerase Chain Reaction
RNA
RNA, Messenger
Tumor Necrosis Factor-alpha
Vascular Endothelial Growth Factor A
Caspase 3
Complement System Proteins
Interleukin-6
Interleukins
Monocrotaline
RNA
RNA, Messenger
Tumor Necrosis Factor-alpha
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Representative RT-PCR product of each gene. The RT-PCR products from the transcripts of Bcl-2, caspase-3, C-6, VEGF, IL-6, and TNF-α were 177 bp, 100 bp, 145 bp, 97 bp, 161 bp, and 113 bp. RT-PCR: reverse transscription-polymerase chain reaction, Bcl: B cell leukemia/lymphoma, C: complement component, VEGF: vascular endothelial growth factor, IL: interleukin, TNF: tumor necrosis factor.

• 

  Fig. 2 Gene expressions of Bcl-2 (A), caspase-3 (B) in rat lung tissues after bosentan treatment. The gene expression of Bcl-2 mRNA was not significantly different after bosentan treatment (A). The expression of caspase-3 mRNA was significantly decreased after bosentan treatment in week 4 (B). *p<0.05 significantly different from C group, †p<0.05 significantly different from M group. Bcl: B cell leukemia/lymphoma, C: control group, M: monocrotaline group, B: bosentan group.

• 

  Fig. 3 Gene expressions of C-6 (A) and VEGF (B) in rat lung tissues after bosentan treatment. Expressions of C-6 (A) and VEGF mRNA (B) were significantly decreased after bosentan treatment in week 4. *p<0.05 significantly different from C group, †p<0.05 significantly different from M group. C-6: complement component, VEGF: vascular endothelial growth factor, C: control group, M: monocrotaline group, B: bosentan group.

• 

  Fig. 4 Gene expressions of IL-6 and TNF-α in rat lung tissues after bosentan treatment. mRNA expression of IL-6 was significantly decreased in weeks 1, 2, and 4 after bosentan treatment (A). mRNA expressions of TNF-α had significantly decreased on day 5, in weeks 1 and 2 after bosentan treatment (B). *p<0.05 significantly different from C group, †p<0.05 significantly different from M group. mRNA: messenger ribonucleic acid, IL: interleukin, TNF: tumor necrosis factor, C: control group, M: monocrotaline group, B: bosentan group.

• 

  Fig. 5 Protein contents of C-6 (A) and VEGF (B) in rat lung tissues after bosentan treatment. C-6 and VEGF protein contents significantly decreased in the B group in week 4. *p<0.05 significantly different from C group, †p<0.05 significantly different from M group. C: complement component, VEGF: vascular endothelial growth factor, C: control group, M: monocrotaline group, B: bosentan group.

• 

  Fig. 6 Protein contents of IL-6 (A) and TNF-α (B) in rat lung tissues after bosentan treatment. IL-6 and TNF-α protein contents were significantly decreased in the B group in week 4. *p<0.05 significantly different from C group, †p<0.05 significantly different from M group. IL: interleukin, TNF: tumor necrosis factor, C: control group, M: MCT group, B: bosentan group.

• 

  Fig. 7 TUNEL assay results in lung tissues. There were no TUNEL positive cells (green) in the C group. TUNEL positivity was progressively increased by MCT treatment. Copious cell death was evident in the M group compared with the C group. In contrast, apoptotic cell death in the B group was ameliorated than the M group. TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling, C: control group, M: monocrotaline group, B: bosentan group.

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