STAT1 Antisense Oligonucleotides Attenuate the Proinflammatory Cytokine Release of Alveolar Macrophages in Bleomycin-Induced Fibrosis

To investigate the effect of signal transducers and activators of transcription 1 （STAT1） antisense oligonucleotides （ASON） on concentrations of TNF-α IL-8, NO secreted by alveolar macrophages （AMs） in bleomycin-induced rat pulmonary fibrosis, five adult female Wistar rats were intratracheally instilled with bleomycin. After 7 days, the rats were killed by right ventricle of heart exsanguinations under ketamine anaesthesia and bronchoalveolar lavage （BAL） was performed to obtain AMs. AMs were divided into four groups, treated with STAT1 ASON, STAT1 sense oligonucleotides （SON）, dexamethasone （DEX） and medium alone （control）, respectively. AMs and media were collected after culture for 36 h. The mRNA and protein expressions of STAT1 and ICAM-1 in AMs were detected by RT-PCR and ELISA, respectively. The concentrations of TNF-α IL-8, NO in cultured medium were detected. The STAT1 mRNA expression by AMs in the STAT1 ASON group was lower than those of AMs in the STAT1 SON group, the DEX group and the control group （p 〈 0.05）. Moreover, the STAT1 mRNA expression by AMs in the DEX group was also lower than those of AMs in the STAT1 SON group and the control group （p 〈 0.05）, but the STAT1 mRNA expression by AMs in the STAT1 SON group was not different from that of the control group （p 〉 0.05）. The protein expressions of STAT1 and ICAM-1 and the mRNA expression of ICAM-1 showed similar changes to the STAT1 mRNA expression by AMs. The concentrations of TNF-α IL-8, NO in cultured medium from STAT1 ASON group were lower than those from STAT1 SON, DEX and the control groups （p 〈 0.05）. Moreover, the concentrations of TNF-α IL-8, NO in cultured medium from DEX group were also lower than those from the control and STAT1 SON group （p 〈 0.05）, but no difference between STAT1 SON group and the control （p 〉 0.05）. The results suggest that STAT1 ASON could inhibit the secretion of TNF-α IL-8, NO in AMs, and STAT1 could become a target of treating pulmonary fibrosis. Cellular ＆ Molecular Immunology. 2005;2（3）：211-217.

Aerosolized STAT1 Antisense Oligodeoxynucleotides Decrease the Concentrations of Inflammatory Mediators in Bronchoalveolar Lavage Fluid in Bleomycin-Induced Rat Pulmonary Fibrosis

It has been demonstrated that alveolar macrophages （AMs） play a key role in the pathogenesis of pulmonary fibrosis by releasing a variety of cytokines and inflammatory mediators. In addition, abnormal signal transducer and activator of transcription-1 （STAT1） activation in AMs may play a pivotal role in the process of alveolitis and pulmonary fibrosis. In this study, we transfected STAT1 antisense oligodeoxynucleotide （ASON） into rats by aerosolization, and then investigated the effect of STAT1 ASON on inflammatory mediators such as TGF-β, PDGF and TNF-α in bronchoalveolar lavage fluid （BALF） from rats with bleomycln （BLM）-induced rat pulmonary fibrosis. Our results showed that STAT1 ASON by aerosolization could enter into lung tissues and AMs. STAT1 ASON could inhibit mRNA and protein expressions of STAT1 and ICAM-1 in AMs of rat with pulmonary fibrosis, and had no toxic side effect on liver and kidney. Aerosolized STAT1 ASON could ameliorate the alveolitis through inhibiting the secretion of inflammatory mediators in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis.

The Effects of Aerosolized STAT1 Antisense Oligodeoxynucleotides on Rat Pulmonary Fibrosis

Previous study showed that aerosolized signal transducer and activator of transcription-1 （STAT1） antisense oligodeoxynucleotide （ASON） inhibited the expression of STAT1 and ICAM-1 mRNA and protein in alveolar macrophages （AMs） and decreased the concentrations of TGF-β, PDGF and TNF-α in bronchioalveolar lavage fluid （BALF） in bleomycin （BLM）-induced rat pulmonary fibrosis. Administration of STAT1 ASON ameliorated alveolitis in rat pulmonary fibrosis. However, further investigations are needed to determine whether there is an effect from administration of STAT1 ASON on fibrosis. This study investigated the effect of aerosolized STAT1 ASON on the expressions of inflammatory mediators, hydroxyproline and type Ⅰ and type Ⅲcollagen mRNA in BLM-induced rat pulmonary fibrosis. The results showed that STAT1 ASON applied by aerosolization could ameliorate alveolitis and fibrosis, inhibit the expressions of inflammatory mediators, decrease the content of hydroxyproline, and suppress the expressions of type Ⅰand type Ⅲcollagen mRNA in lung tissue in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis. Cellular ＆ Molecular Immunology.

肺间质纤维化大鼠肺泡巨噬细胞STAT_1的活化及其依赖性免疫应答基因ICAM-1的表达

目的:探讨肺泡巨噬细胞(AM)中STAT1(signal transducersand activators of transcription 1)的活化在大鼠肺间质纤维化中的作用。方法:50只健康雌性Wistar大鼠随机分成博莱霉素(BLM)组和生理盐水(NS)组各25只,气管内分别灌注BLM和NS。用Western-blot法测定AM中STAT1活化的动态变化;用免疫组化染色法测定AM的ICAM-1(intercellular adhesionmolecule-1)的表达。结果:NS组中的AM内只有少许STAT1活化,气管内灌注BLM后1d,AM中STAT1的活化即开始显著增高,于第7天达高峰,以后逐渐下降,但28 d后仍高于NS组(P<0.05)。NS与BLM组的AM中表达ICAM-1的细胞数增高的模式,与AM中STAT1活化的模式相同。AM中STAT1活化的程度与AM中ICAM-1的表达呈正相关(r=0.913,P<0.01),而后者又与肺组织炎症的积分呈正相关(r=0.947,P<0.01)。结论:实验性肺间质纤维化大鼠AM中存在STAT1的异常活化,并参与了肺泡炎和肺纤维化的形成。

小鼠肺纤维化模型中基质金属蛋白酶-9及其抑制剂表达水平的变化

目的 :研究小鼠博来霉素在肺纤维化模型中肺泡巨噬细胞(AM)分泌的基质金属蛋白酶 9(MMP 9)和基质金属蛋白酶抑制剂 1(TIMP 1)的表达随着时间的变化 ,观察肺纤维化中MMP 9和TIMP 1的表达是否存在失衡。方法 :以博来霉素诱导建立小鼠肺纤维化模型 ,采用HE染色观察肺部胶原沉积状况。选用抗CD6 8mAb ,采用微小免疫磁珠法分离纯化肺泡灌洗液中的AM ,用Hoechst 332 5 8染色AM ,在下光镜高倍视野计数法评估AM的纯度和活性 ,用EIA法检测AM上清液中MMP 9和TIMP 1的表达。结果 :肺组织切片HE染色显示小鼠肺部胶原沉积在 1、3、7、14、2 8d呈逐渐加重趋势。粗分离的AM纯度为 (82 .5± 2 .5 ) %。采用微小免疫磁珠法分离肺泡灌洗液中的AM纯度可达到 (99.3± 0 .7) % (P <0 .0 5 )。纯化后的细胞存活率为 (92 .5± 1.8) % ,与纯化前的 (92 .7± 2 .0 ) % ,相差不大 (P >0 .0 5 )。随着小鼠肺间质纤维化的进展 ,MMP 9的分泌量逐渐减少 ,而TIMP 1的表达量逐渐增加。结论 :在小鼠肺纤维化中AM分泌的MMP 9和TIMP 1之间存在失衡 。

STAT_1对实验性肺纤维化大鼠肺组织炎症的调控作用

目的 探讨肺泡巨噬细胞 (AM)内转录活化蛋白 1(STAT1 )活化在大鼠肺纤维化中的作用。方法 取 5 0只健康雌性 Wistar大鼠随机分成博莱霉素组 (BL M)和生理盐水组 (NS) ,每组各 2 5只。用 Western- blot法测定 AM内 STAT1 活化的动态变化 ,同时用免疫组化方法测定 AM的细胞间粘附分子 - 1(ICAM- 1)表达。结果 NS组大鼠的 AM内有少许 STAT1 活化 ,气管内灌注博莱霉素 1d后 AM内 STAT1 活化就开始显著增高 ,第 7d达高峰 ,以后逐渐下降 ,第 2 8d仍高于 NS组 (P<0 .0 5 ) ;NS组的 AM中有少许细胞 ICAM- 1表达呈阳性 ,气管内灌注博莱霉素 1d后 AM的 ICAM- 1表达阳性细胞数就显著增高 ,第 7d达高峰 ,以后逐渐下降 ,第 2 8d仍高于 NS组(P<0 .0 5 ) ;AM内 STAT1 活化程度与 AM的 ICAM- 1表达呈正相关 (r=0 .913,P<0 .0 1) ,而后者又与肺组织炎症积分呈正相关 (r=0 .94 7,P<0 .0 1)。结论 实验性肺纤维化大鼠 AM内存在 STAT1 的异常活化 ,异常活化的STAT1 参与肺泡炎和肺纤维化的形成。

白细胞介素-1受体拮抗剂对博莱霉素致大鼠肺纤维化模型的影响

目的：探讨白细胞介素１受体拮抗剂（ｉｎｔｅｒｌｅｕｋｉｎ１ｒｅｃｅｐｔｏｒａｎｔａｇｏｎｉｓｔ，ＩＬ１ｒａ）治疗博莱霉素（ｂｌｅｏｍｙｃｉｎ，ＢＬＭ）致大鼠肺纤维化模型的作用机制。方法：利用ＭＴＴ法测定经ＩＬ１ｒａ作用前、后１周时该模型大鼠肺泡巨噬细胞产生肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒα，ＴＮＦα），及其培养上清促肺成纤维细胞增殖能力的变化；利用Ｎｏｒｔｈｅｒｎ杂交测定肺泡巨噬细胞ＴＮＦα、血小板衍化生长因子（ｐｌａｔｅｌｅｔｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ，ＰＤＧＦ）ｍＲＮＡ的表达。结果：ＢＬＭ致肺纤维化模型１周时，肺泡巨噬细胞ＴＮＦα的产生及该组细胞上清的促成纤维细胞增殖活性与正常对照组相比均明显增强，而ＩＬ１ｒａ（１０ｍｇ·Ｌ－１）对肺泡巨噬细胞ＴＮＦα的产生及该组细胞上清的促成纤维细胞增殖活性有明显的抑制作用。ＢＬＭ致肺纤维化模型１周时肺泡巨噬细胞ＴＮＦα、ＰＤＧＦｍＲＮＡ的表达较正常对照组增高，而ＩＬ１ｒａ对其表达无明显影响。结论：ＩＬ１ｒａ通过对肺泡巨噬细胞的ＴＮＦα产生及其对成纤维细胞增殖活性的抑制，可能对该模型的肺纤维化起到抑制作用?

用RT-PCR方法动态分析博莱霉素致肺纤维化大鼠TGF-β_1的基因表达

目的 :探讨转化生长因子 β1(TGF β1)在肺纤维化中的作用。方法 :采用博莱霉素 (BLM)致肺纤维化大鼠模型 ,分离纯化肺泡巨噬细胞 (alveolarmacrophage ,AM) ,用RT PCR及图像分析方法 ,对不同时期AM以及肺组织释放的TGF β1进行动态观察。结果 :BLM组AM和肺组织中TGF β1mRNA第 3天即明显增加 (P <0 .0 5和P>0 .0 5 ) ,第 7天达到高峰 (P<0 .0 1) ,第 142 8天逐渐下降 ,但仍高于对照组 (P>0 .0 5 )。结论 :TGF β1mRNA在正常大鼠及肺纤维化大鼠中均有表达 ,后者表达明显高于前者 ;肺纤维化大鼠肺内TGF β1mRNA可能主要来源于AM。

白细胞介素Ⅰ在矽肺纤维化过程中的作用

二氧化硅(SiO_2)所引起的肺纤维化可能与一系列细胞间的互相作用有关,其中包括肺泡巨噬细胞(AM)和肺成纤维细胞(PFB)。PFB的增殖是肺组织对SiO_2刺激的早期反应。以往许多研究曾经证明,用SiO_2染尘的AM释放的白细胞介素I(IL-I)明显高于未染尘者。当大鼠AM与致纤维化的SiO_2培养时,培液中IL-1活力明显升高。本研究用经SiO_2刺激的肺泡巨噬细胞(AM)上清液与肺成纤维细胞(PFB)共培养,在不同时间作观察,发现PFB明显增殖。提示在矽肺纤维化过程中AM、白细胞介素-I(IL-I)和PFB起了关键性作用。

肌醇需求激酶1抑制剂对染矽尘大鼠肺纤维化干预作用研究

目的研究肌醇需求激酶1(IRE1)抑制剂对染矽尘大鼠肺纤维化内质网应激(ERS)凋亡途径的影响,评价IRE1抑制剂对大鼠矽肺纤维化的干预作用。方法无特定病原体(SPF)级健康成年雄性SD大鼠24只随机分为对照组、矽肺组和IRE1组,每组8只。矽肺组和IRE1组大鼠均采用非气管暴露法一次性注入质量浓度为50.0 g/L的二氧化硅混悬液1.0mL造模,对照组注入等体积灭菌生理氯化钠溶液。造模28 d后,IRE1组大鼠腹腔注射剂量为1.0 mg/kg体质量的IRE1抑制剂,其余2组予等体积灭菌生理氯化钠溶液,每7天注射1次,共4次。造模后第56天处死大鼠,收集左侧肺泡灌洗液,分离、纯化和培养肺泡巨噬细胞(AM)。观察肺组织病理变化情况,实时荧光定量聚合酶链式反应测定肺组织Ⅰ型和Ⅲ型前胶原mRNA相对表达水平,流式细胞术检测AM活性氧(ROS)阳性率和线粒体去极化率,免疫印迹法测定肺组织内IRE1α、天冬氨酸特异性半胱氨酸蛋白酶(Caspase)-12、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)和Bcl-2相关X蛋白(Bax)的相对表达水平。结果肺组织病理学检查结果显示,矽肺组肺组织可见炎细胞浸润和典型矽结节形成;IRE1组可见普遍炎细胞浸润和肺泡间隔变大,有弥漫性纤维化改变,但未见矽结节形成。IRE1组、矽肺组大鼠肺组织Ⅰ型、Ⅲ型前胶原mRNA相对表达水平和AM的ROS阳性率、线粒体去极化率均高于对照组(P<0.01),IRE1组上述4个指标均低于矽肺组(P<0.01)。IRE1组、矽肺组大鼠肺组织IRE1α、Caspase-12和Bax蛋白的相对表达水平均高于对照组(P<0.01),IRE1组大鼠上述3个指标均低于矽肺组(P<0.01);但IRE1组和矽肺组大鼠肺组织Bcl-2相对表达水平均低于对照组(P<0.01),IRE1组大鼠Bcl-2相对表达水平高于矽肺组(P<0.01)。结论 IRE1参与的ERS凋亡通路可能在矽肺肺纤维化的发生和发展中发挥着重要作用;IRE1抑制剂可以通过阻断IRE1通路拮抗ERS诱导的AM凋亡,从而拮抗肺纤维化。