siRNA against plasminogen activator inhibitor-1 ameliorates bleomycin-induced lung fibrosis in rats.
OBJECTIVE
Plasminogen activator inhibitor-1 (PAI-1) is involved in the progression of pulmonary fibrosis. The present study was undertaken to examine the effects on pulmonary fibrosis of silencing PAI-1 expression with small interfering RNA (siRNA) and to assess the possible underlying mechanisms.


METHODS
Male Wistar rats were subjected to intratracheal injection of bleomycin (BLM, 5 mg/kg, 0.2 mL) to induce pulmonary fibrosis. Histopathological changes of lung tissue were examined with HE or Masson's trichrome staining. The expression levels of α-smooth muscle actin (α-SMA), collagen type-I and type-III, caspase-3, as well as p-ERK1/2 and PI3K/Akt in the lung tissue were evaluated using imunohistochemistry and Western blot analyses. The fibroblasts isolated from BLM-induced fibrotic lung tissue were cultured and transfected with pcDNA-PAI-1 or PAI-1siRNA. The expression level of PAI-1 in the fibroblasts was measured using real time RT-PCR and Western blot analysis. The fibroblast proliferation was evaluated using MTT assay.


RESULTS
Intratracheal injection of PAI-1-siRNA (7.5 nmoL/0.2 mL) significantly alleviated alveolitis and collagen deposition, reduced the expression of PAI-1, α-SMA, collagen type-I and collagen type-III, and increased the expression of caspase-3 in BLM-induced fibrotic lung tissue. In consistence with the in vivo results, the proliferation of the cultured fibroblasts from BLM-induced fibrotic lung tissue was inhibited by transfection with PAI-1-siRNA, and accelerated by overexpression of PAI-1 by transfection with pcDNA-PAI-1. The expression of caspase-3 was increased as a result of PAI-1 siRNA transfection, and decreased after transfection with pcDNA-PAI-1. In addition, the levels of p-ERK1/2 and PI3K/Akt in the fibrogenic lung tissue were reduced after treatment with PAI-1siRNA.


CONCLUSIONS
The data demonstrate that PAI-1 siRNA inhibits alveolitis and pulmonary fibrosis in BLM-treated rats via inhibiting the proliferation and promoting the apoptosis of fibroblasts. Suppression ERK and AKT signalling pathways might have at least partly contributed to this process. Targeting PAI-1 is a promising therapeutic strategy for pulmonary fibrosis.
Department of Pathophysiology, Hebei Medical University, Shijiazhuang, China.
Yan-ping
YP
Zhang
Wen-bin
WB
Li
Wei-li
WL
Wang
Jian
J
Liu
Shu-xia
SX
Song
Lin-lin
LL
Bai
Yu-yan
YY
Hu
Ya-dong
YD
Yuan
Min
M
Zhang
D000199:Actins / Q000032:analysis / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001761:Bleomycin; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D006909:Hydroxyproline / Q000032:analysis / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000473:pathology; D020935:MAP Kinase Signaling System; D008297:Male; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000627:therapeutic use*; D051381:Rats; D017208:Rats, Wistar
D000199:Actins; D001761:Bleomycin; D053148:Caspase 3; D003094:Collagen; D006909:Hydroxyproline; D058539:Phosphatidylinositol 3-Kinase; D017395:Plasminogen Activator Inhibitor 1; D034741:RNA, Small Interfering; C498862:Acta2 protein, rat