Paraquat에 의한 백서의 폐섬유화증에서 비선택적 Endothelin-1 receptor blocker($Bosentan^{(R)}$)의 치료효과

The Effect of Nonspecific Endothelin-1 Receptor Blocker ($Bosentan^{(R)}$) on Paraquat Induced Pulmonary Fibrosis in Rat

연구배경 : IPF에 의한 유병률과 사망률은 점차 증가하는 추세이나 좋은 치료는 없는 상태이다. 폐 섬유화 과정에서 TGF-${\beta}_1$, TNF-$\alpha$, ET-1, IFN-$\gamma$등의 사이토카인이 중요한 역할을 함이 알려져 있다. 본 실험은 파라콰트를 기관지 내로 주입하여 섬유화가 유발되는 과정의 백서의 폐 조직 내에서 ET-1과 TGF-${\beta}_1$의 발현을 살펴보고, 또한 비선택적 ET-1 receptor blocker인 Bosentan이 폐 섬유화의 치료에 효과가 있는지를 보고자 하였다. 방 법 : 웅성 7-8 주령의 백서 120 마리를 세 그룹으로 나누고 제 1그룹은 대조군으로 하여 기관지 내로 생리 식염수를 투여하였고, 제2그룹은 파라콰트를 투여하였으며, 제3그룹은 첫날 파라콰트를 투여한 후 매일 gastric gavage 방법으로 보센탄을 투여하였다. 파라콰트 혹은 생리식염수를 투여한 지 1, 3, 5, 7, 10, 14일째 각각 세 그룹의 일정 수를 희생하여 폐의 병리조직을 보고 면역세포화학염색으로 ET-1과 TGF-${\beta}_1$의 발현 율을 조사하여 분석하였다. 폐 섬유화의 정도는 H&E 염색과 Masson trichrome 염색을 하여 컴퓨터 영상분석을 시행하였고, 면역세포화학염색은 염색정도에 따라 반정량화하여 분석하였다. 결 과 : 파라콰트를 투여한 군이 대조군에 비해 콜라겐의 침착이 실험 3일째부터 현저히 증가하였고, ET-1과 TGF-${\beta}_1$의 발현이 주로 실험 초기에 증가하였다. 그러나 보센탄을 투여한 경우 콜라겐이 침착된 양에는 유의한 변화가 없었고 파라콰트군과 비교해서 ET-1과 TGF-${\beta}_1$의 발현에 뚜렷한 변화는 없었다. 결 론: 파라콰트를 투여한 경우 폐 섬유화가 증가하였다. 그리고 ET-1과 TGF-${\beta}_1$의 발현이 증가하였다. 그러나 ET-1 에 대한 receptor blocker인 보센탄이 폐 섬유화를 막지는 못하였다. 파라콰트에 의한 폐 섬유화에 ET-1이 연관성이 있으나 그 역할에 대해서는 추후 더 연구가 필요할 것으로 사료된다.

Background : Idiopathic pulmonary fibrosis(IPF) is a devastating illness for which there is little effective treatment. The key cytokines currently implicated in the fibrotic process are the transforming growth factor-${\beta}_1$(TGF-${\beta}_1$), tumor necrosis factor-$\alpha$(TNF-$\alpha$), endothelin-1(ET-1) and interferon-$\gamma$(IFN-$\gamma$). The rat model for paraquat-induced pulmonary fibrosis was chosen to investigate the role of ET-1 in this disease. Both ET-1 and TGF-${\beta}_1$ expression in lung lesions were examined using immunohistochemical staining. After $Bosentan^{(R)}$ administration, an orally active ET-$l_A$ and ET-$1_B$ receptor antagonist, the degree of pulmonary fibrosis and ET-1 and TGF-${\beta}_1$ expression were analyzed. Method : Sprague-Dawley rats were divided into three groups, the control group, the fibrosis group, and the fibrosis-$Bosentan^{(R)}$-treated group. The animals were sacrificed periodically at 1, 3, 5, 7, 10, 14 days after administering saline or paraquat. The effects between groups were compared with the results of light microscopy and immunohistochemical staining for ET-1 and TGF-${\beta}_1$. The degree of fibrosis was evaluated by H&E and Masson's trichrome staining, which were graded by a computerized image analyzer. The degree of immunohistochemical staining was categorized by a semi-quantitative analysis method. Results : The lung collagen content had increased in the paraquat instillated animals by day 3, and continued to increase up to day 14. A daily treatment by gavage with $Bosentan^{(R)}$ (100mg/kg) did not prevent the increase in collagen deposition on the lung that was induced by paraquat instillation. There were increased immunohistochemical stains of ET-1 on the exudate, macrophages, vascular endothelial cells and pneumocytes in the paraquat instillated group. Furthermore, TGF-${\beta}_1$ expression was higher on the exudate, macrophages, some inflammatory cells, pneumocytes( type I, and II), vascular endothelium and the respiratory epithelial cells around the fibrotic area. After Bosentan treatment, there were no definite changes in ET-1 and TGF-${\beta}_1$ expression. Conclusion : Fibrosis of the Paraquat instillated group was more advanced when compared with the control group. In addition, there was increased ET-1 and TGF-${\beta}_1$ expression around the fibrotic area. ET-1 is associated with lung fibrosis but there was little effect of the ET-1 receptor blocker($Bosentan^{(R)}$) on antifibrosis.Background : Idiopathic pulmonary fibrosis(IPF) is a devastating illness for which there is little effective treatment. The key cytokines currently implicated in the fibrotic process are the transforming growth factor-${\beta}_1$(TGF-${\beta}_1$), tumor necrosis factor-$\alpha$(TNF-$\alpha$), endothelin-1(ET-1) and interferon-$\gamma$(IFN-$\gamma$). The rat model for paraquat-induced pulmonary fibrosis was chosen to investigate the role of ET-1 in this disease. Both ET-1 and TGF-${\beta}_1$ expression in lung lesions were examined using immunohistochemical staining. After $Bosentan^{(R)}$ administration, an orally active ET-$1_A$ and ET-$1_B$ receptor antagonist, the degree of pulmonary fibrosis and ET-1 and TGF-${\beta}_1$ expression were analyzed. Method : Sprague-Dawley rats were divided into three groups, the control group, the fibrosis group, and the fibrosis-$Bosentan^{(R)}$-treated group. The animals were sacrificed periodically at 1, 3, 5, 7, 10, 14 days after administering saline or paraquat. The effects between groups were compared with the results of light microscopy and immunohistochemical staining for ET-1 and TGF-${\beta}_1$. The degree of fibrosis was evaluated by H&E and Masson's trichrome staining, which were graded by a computerized image analyzer. The degree of immunohistochemical staining was categorized by a semi-quantitative analysis method. Results : The lung collagen content had increased in the paraquat instillated animals by day 3, and continued to increase up to day 14. A daily treatment by gavage with $Bosentan^{(R)}$ (100mg/kg) did not prevent the increase in collagen deposition on the lung that was induced by paraquat instillation. There were increased immunohistochemical stains of ET-1 on the exudate, macrophages, vascular endothelial cells and pneumocytes in the paraquat instillated group. Furthermore, TGF-${\beta}_1$ expression was higher on the exudate, macrophages, some inflammatory cells, pneumocytes( type I, and II), vascular endothelium and the respiratory epithelial cells around the fibrotic area. After Bosentan treatment, there were no definite changes in ET-1 and TGF-${\beta}_1$ expression. Conclusion : Fibrosis of the Paraquat instillated group was more advanced when compared with the control group. In addition, there was increased ET-1 and TGF-${\beta}_1$ expression around the fibrotic area. ET-1 is associated with lung fibrosis but there was little effect of the ET-1 receptor blocker($Bosentan^{(R)}$) on antifibrosis.