纤溶酶原激活物抑制剂1 siRNA对大鼠肺纤维化的治疗作用

Inhibition of plasminogen activator inhibitor-1 by small interfering RNA attenuates pulmonary fibrosis in rats

目的:观察纤溶酶原激活物抑制剂1(PAI-1)siRNA对博来霉素(BLM)诱导的大鼠肺纤维化的治疗作用,并初步探讨其机制。方法:72只Wister大鼠分为4组,即对照组(control组)、BLM组、BLM+非特异siR-NA组(BLM+N组)和BLM+PAI-1 siRNA组(BLM+P组)。BLM 5 mg/kg气管内滴入制作肺纤维化模型,control组注入等体积的生理盐水。造模后,于局麻下BLM+P组每周2次气管内注入PAI-1 siRNA 7.5 nmol(0.2 mL);BLM+N组注入相同剂量的非特异siRNA;control组和BLM组注入等体积的生理盐水,28 d共给药8次。分别于第7 d、14 d和28 d每组处死6只,左肺行肺泡灌洗测定PAI-1活性,右中叶肺组织RT-PCR法检测Ⅲ型胶原、α-平滑肌肌动蛋白(α-SMA)和金属蛋白酶组织抑制物1(TIMP-1)的mRNA表达。结果:造模后,7 d、14 d和28 d肺泡灌洗液中PAI-1活性持续升高,每周2次气管内注入PAI-1 siRNA可以使肺泡灌洗液中PAI-1的活性降低,与同一时点BLM组比较有明显差异(均P<0.05);模型组7 d、14 d和28 dⅢ型胶原、α-SMA和TIMP-1的mRNA表达明显增高,BLM+P组3个时点Ⅲ型胶原、α-SMA和TIMP-1的mRNA表达明显减少,分别与同一时点BLM组比较有明显差异(均P<0.05)。结论:每周2次气管内给予PAI-1 siRNA可以持续减少PAI-1表达。PAI-1 siRNA不但直接抑制肺纤维化进展,而且可以打破基质金属蛋白酶及组织抑制因子之间的平衡。

AIM ： To examine the effects of silencing of plasminogen activator inhibitor-1 （PAI-1） expression by small interfering RNA （siRNA） on bleomycin （ BLM）-induced rat pulmonary fibrosis. METHODS ： Total 72 Wistar rats were divided into 4 groups： control, BLM, BLM ＋ non-specific siRNA （BLM ＋ N）, and BLM ＋ PAI-1 siRNA （BLM ＋ P）. Pulmonary fibrosis was induced by intratracheal injection of BLM （5 mg/kg）, whereas equal volume of normal sa- line was used in control group. After the administration of BLM or normal saline, the rats were treated with tracheal injec- tion of PAI-I-siRNA （7.5 nmol/0.2 mL per rat） in BLM ＋ P group, non-specific siRNA （7.5 nmol/0.2 mL per rat） in BLM ＋ N group, and 0.2 mL normal saline in BLM group and control group, twice a week, 8 times in 28 d. On day 7, 14, and 28, the rats （n =6 at each time point） were sacrificed. The bronchoalveolar lavage fluid （BALF） from the left lung was harvested to examine the activity of PAI-1. The mRNA expression of collagen typeⅢ, a-smooth muscle actin （ a- SMA） and tissue inhibitor of metalloproteinase-1 （TIMP-1） in the middle lobe of the right lung was detected by RT-PCR. RESULTS ： PAI-1 activity and the expression of collagen type Ⅲ, a-SMA and TIMP-1 were increased in BLM group on day 7, 14 and 28. Intratracheal injection of PAl-1 siRNA twice a week continuously reduced PAI-1 activity in the BALF （ P 〈0. 05） ,and decreased the expression of collagen type Ⅲ, a-SMA and TIMP-1 in the fibrotic lung tissues on day 7, 14 and 28. Statistical differences in the expression of collagen type Ⅲ, a-SMA and TIMP-1 between BLM ＋ P group and BLM group at the same time point were observed. CONCLUSION： Intratracheal injection of PAI-1 siRNA twice a week contin- uously inhibits the expression of PAI-1. PAl-1 siRNA ameliorates BLM-induced pulmonary fibrosis by down-regulation of TIMP-1 expression.