不同时间点雾化吸入STAT1反义寡核苷酸对肺纤维化大鼠肺组织Ⅰ、Ⅲ型胶原表达的影响

EFFECTS OF AEROSOLIZED STAT1 ANTISENSE OLIGONUCLEOTIDES INHALATION ON EXPRESSION OF COLLAGEN TYPEⅠ AND Ⅲ IN BLEOMYCIN-INDUCED RAT PULMONARY FIBROSIS AT DIFFERENT TIME POINT

目的:探讨雾化吸入信号转导子与转录活化子1(STAT1)反义寡核苷酸(ASON)干预肺纤维化大鼠的最佳时机。方法:清洁级健康雌性Wistar大鼠25只,随机分为生理盐水(NS)组、博来霉素(BLM)组和ASON 0 d组、ASON 7 d组、ASON14d组各5只。BLM组、ASON 0 d组、ASON 7 d组和ASON 14 d组大鼠气管内灌注BLM,NS组大鼠气管内灌注NS。大鼠气管内灌注BLM或NS后第0、2、4、6 d,NS组和BLM组大鼠雾化吸入NS,ASON 0 d组大鼠雾化吸入STAT1 ASON,ASON 7天组大鼠于气管内灌注BLM后第7、9、11、13d雾化吸入STAT1 ASON,ASON 14 d组于灌注BLM后第14、16、18、20d雾化吸入STAT1 ASON,各组均于气管内灌注BLM或NS后28d处死大鼠。左肺组织HE染色观察肺泡炎程度,Masson染色观察肺纤维化程度,免疫组织化学SP法测定肺组织中PAI-1、TGF-β1的表达。右肺测定肺组织Ⅰ、Ⅲ型胶原mRNA的表达及羟脯氨酸含量。结果:①STAT1 ASON雾化吸入后,ASON 0 d组与BLM组、ASON 7 d组、ASON 14d组比较,肺泡炎和肺纤维化程度均明显减轻(P<0.05)。②ASON 0d组与BLM组比较,肺组织转移生长因子β1(TGF-β1)表达减少(P<0.05)。ASON 14d组与ASON 0d组比较,肺组织TGF-β1表达增多(P<0.05)。③与BLM组比较,ASON 0d组和ASON 7d组肺组织PAI-1表达降低(P<0.05);与ASON 0d组和ASON 7d组比较,ASON 14d组肺组织PAI-1表达增加(P<0.05)。④与BLM组比较,ASON 0d组肺组织Ⅰ、Ⅲ型胶原mRNA表达水平降低,ASON 7d组肺组织Ⅲ型胶原mRNA表达水平降低(P<0.05);与ASON 0d比较,A-SON 7d组和ASON 14d组肺组织Ⅰ、Ⅲ型胶原mRNA表达水平增高(P<0.05)。⑤与BLM组比较,ASON 0d组、ASON 7d组羟脯氨酸含量均减少(P<0.05);与ASON 0d组比较,ASON 7d组和ASON 14d组羟脯氨酸含量增高(P<0.05);与ASON 7d组比较,ASON 14d组羟脯氨酸含量增高(P<0.05)。结论:不同时间点雾化吸入STAT1 ASON对肺纤维化的干预作用是不同的,0d、7d可以减轻肺纤维化,其中0d雾化吸入是治疗的最佳时间。

Objective： To investigate the best treatment time of aerosolized STAT1 antisense oligonucleotides inhalation in bleomycin-induced rat pulmonary fibrosis.Methods： Twenty-five adult female Wistar rats were randomly divided into normal saline（NS） group,BLM group,ASON 0 d group,ASON 7 d group,and ASON 14 d group.BLM group and ASON groups were intratracheally instilled with BLM,while NS group was instilled with NS.NS group and BLM group were inhaled with aerosolized NS,while ASON 0 d group was inhaled with aerosolized STAT1 ASON on day 0,2,4,6,ASON 7 d group on day 7,9,11,13,and ASON 14 d group on day 14,16,18,20.Then the rats were killed on day 28.The lung tissues were removed.HE and Masson staining were performed on lung tissue sections to observe the degree of alveolitis and fibrosis.TGF-β1 and PAI-1 proteins in tissue of left lung were assessed by immunohistochemistry.The mRNA levels of collagen type Ⅰand Ⅲ of right lung were measured by Reverse Transcription-Polymerase Chain Reaction（RT-PCR）.The content of hydroxyproline was also examined.Results： ①Compared with BLM group,ASON 7 d and 14 d group,the scores of alveolitis and fibrosis in ASON 0 d group remarkably decreased.②Compared with BLM group and ASON 14 d group,the expression of TGF-β1 protein in lung tissue in ASON 0 d group was reduced.All the difference had statistical significance.③The expression of PAI-1 protein in lung tissue in ASON 0 d and 7 d group decreased compared with BLM group.The expression of PAI-1 protein in lung tissue in ASON 14 d group increased compared with ASON 0 d and 7 d group.④mRNA levels of collagen typeⅠand Ⅲ in ASON 0 d group were reduced compared with BLM group.All the difference had statistical significance.Compared with ASON 0 d group,the mRNA levels of collagen typeⅠand Ⅲ in ASON 7 d and 14 d group markably increased.⑤Compared with BLM group,the content of hydroxyproline in lung tissue in ASON 0 d and 7 d group significantly decreased.Compared with ASON 0 d group,the content of hydroxyproline in lung tissue in ASON 7 d and 14 d group significantly increased.Compared with ASON 7 d group,the content of hydroxyproline in lung tissue in ASON 14 d group significantly increased.All the difference had statistical significance.Conclusion： The effects of aerosolized STAT1 antisense oligonucleotides on expression of collagen in bleomycin-induced rat pulmonary fibrosis at different time point were different.Aerosolized STAT1 ASON on day 0 and 7 could abate the alveolitis and fibrosis in BLM-induced rat pulmonary fibrosis.The best treatment time should be on day 0.