甘草酸对博莱霉素诱导的实验性肺纤维化的干预作用

目的:探讨甘草酸(GA)对博莱霉素(BLM)诱导的小鼠肺纤维化的干预作用及其可能机制。方法:将160只雄性C57BL/6J小鼠随机分为生理盐水(NS)组、BLM组、BLM+NS组和BLM+GA组。通过口咽气管吸入法吸入博莱霉素(2.5 mg/kg)建立实验性肺纤维化模型,BLM+GA组及BLM+NS组每天给予40 mg/kg甘草酸或等体积的生理盐水灌胃,于术后第3、7、14、21天取材。采用HE染色和Masson染色观察肺组织病理学变化及纤维化程度,采用流式细胞术检测循环单核细胞和肺泡巨噬细胞的亚群比例变化,采用RT-qPCR检测肺组织中转化生长因子β1(TGF-β1)mRNA的表达水平,采用碱水解法检测肺组织中羟脯氨酸(HYP)含量。结果:与NS组相比,BLM组和BLM+NS组肺组织的炎症浸润及胶原含量明显增多,实验性肺纤维化模型制备成功。与BLM+NS组相比:BLM+GA组肺组织的炎症细胞浸润及胶原纤维沉积较少;第3、7天的Ly6C~(hi)单核细胞亚群比例和第7、14天肺泡巨噬细胞M2表型比例显著降低(P0.01);肺组织TGF-β1 mRNA表达量和HYP含量显著降低(P0.01)。结论:GA可以减轻博莱霉素诱导的小鼠肺组织的炎症反应和胶原纤维沉积,可能与GA对单核巨噬细胞的表型偏移和调控以及肺组织TGF-β1的表达下调有关。     

中华猕猴桃果仁非饱和脂肪酸通过激活Keap1/Nrf2信号通路增强肺纤维化大鼠抗氧化能力

目的观察中华猕猴桃果仁非饱和脂肪酸(果王素)对博莱霉素致大鼠肺纤维化的影响,探讨其是否通过Kelch样ECH相关蛋白1(Keap 1)/核因子E2相关因子2(Nrf 2)信号通路发挥作用。方法随机将60只SD大鼠分为对照组、模型组、(60、120、180)mg/kg中华猕猴桃果仁非饱和脂肪酸处理组和5 mg/kg醋酸泼尼松组,每组10只。对照组大鼠以生理盐水气管内注射,其余5组大鼠则以博莱霉素A5气管内注射建立肺纤维化模型。第2天起,中华猕猴桃果仁非饱和脂肪酸处理组大鼠分别予(60、120、180)mg/kg中华猕猴桃果仁非饱和脂肪酸灌胃,泼尼松组以5 mg/kg醋酸泼尼松灌胃,其余2组予生理盐水灌胃。所有大鼠第28天处死,留取肺组织,HE和Masson染色观察肺部病变,通过试剂盒测定肺组织匀浆中羟脯氨酸(HYP)、活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)含量,Western blot法检测肺组织中Keap 1、Nrf 2蛋白水平。结果与模型组比较,(60、120、180)mg/kg中华猕猴桃果仁非饱和脂肪酸处理组和泼尼松组肺泡炎症和肺纤维化程度明显减轻,肺组织HYP、ROS、MDA含量、胞质Keap 1蛋白水平降低,而SOD、CAT、GSH-Px含量、胞核Nrf 2蛋白水平升高。此外,与60 mg/kg中华猕猴桃果仁非饱和脂肪酸处理组相比,(120、180)mg/kg中华猕猴桃果仁非饱和脂肪酸处理组和泼尼松组上述指标明显改善,但后三者之间比较无显著性差异。结论中华猕猴桃果仁非饱和脂肪酸能抑制大鼠肺纤维化,与激活Keap 1/Nrf 2信号通路增加抗氧化物生成有一定关系。     

核因子E2相关因子2对人永生化角质形成细胞氧化损伤的保护作用

目的 探讨核因子E2相关因子2 (Nrf2)对过氧化氢(H2O2)诱导的人永生化角质形成细胞(HaCaT)氧化损伤的保护作用。 方法 用慢病毒转染方式使HaCaT过表达Nrf2(Nrf2/HaCaT),并用MTT法和抗Ki67免疫荧光染色法检测其增殖活性。实验分为Nrf2/HaCaT组和HaCaT组,每组5个样本。应用200 μmol/L H2O2处理两组细胞24 h以诱导氧化损伤,并用MTT法检测细胞活性,乳酸脱氢酶(LDH)法检测细胞损伤情况,TUNEL染色检测细胞凋亡,ELISA法和比色法检测氧化应激相关指标Nrf2、谷胱甘肽（GSH）、超氧化物歧化酶（SOD）、丙二醛（MDA）和活性氧（ROS）,以及炎症相关因子白细胞介素（IL)-6、肿瘤坏死因子（TNF）-α、核因子κB（NF-κB）P65的含量。 结果 Nrf2/HaCaT细胞增殖活性显著高于HaCaT(P<0.05)。经H2O2诱导后,Nrf2/HaCaT活性较HaCaT高(P<0.05),LDH释放及凋亡率较HaCaT低(P<0.05),其上清液中的抗氧化物Nrf2、GSH和SOD水平较HaCaT高(P<0.05),而氧化产物ROS、MDA以及炎症因子IL-6、TNF-α和NF-κB P65水平较HaCaT低(P<0.05)。 结论 Nrf2过表达可促进HaCaT增殖及减轻H2O2诱导的细胞氧化和炎性损伤。     

苦参碱通过调节单核吞噬细胞表型偏移改善博莱霉素诱导的肺纤维化

目的:研究苦参碱(matrine,MA)对博菜霉素(bleomycin,BLM)诱导的循环单核细胞和肺泡巨噬细胞表型偏移的调节作用。方法:160只C57BL/6雄性小鼠随机分为生理盐水(NS)组、BLM组、苦参碱干预组(BLM+MA组)及溶剂对照组(BLM+NS组)经口咽吸入法给予BLM(2.5 mg/kg)建立实验性肺纤维化模型,对照组给予等体积NS,BLM+MA组和BLM+NS组分别在术后每天灌胃给予MA(15 mg·kg~(-1)·d~(-1))或等量NS。术后第3、7、14和21天处死小鼠,采用HE染色和Masson染色观察肺组织病理学变化及纤维化程度,采用碱水解法测定肺组织羟脯氨酸的含量,用流式细胞术分别检测循环单核细胞亚群和支气管肺泡灌洗液细胞表型的变化。结果:与对照组相比,MA干预可以明显减轻BLM诱导的小鼠肺组织炎症反应及纤维化程度(P0.05);与NS组相比,BLM组的Ly6C~(hi)单核细胞比例升高,肺泡巨噬细胞表型由M1型向M2型偏移且与炎症反应和纤维化程度呈现一定的相关性;MA干预后可以部分逆转BLM诱导的循环单核细胞和肺泡巨噬细胞的表型偏移。结论:苦参碱可以减轻BLM诱导的急性肺泡炎症和肺纤维化程度,可能是部分通过逆转循环单核细胞和肺泡巨噬细胞表型的偏移而实现的。     

Rho激酶-1在肺纤维化小鼠肺组织中的表达变化与上皮-间充质转化

目的 观察Rho激酶-1(Rock-1) 在博莱霉素（BLM）致小鼠肺纤维化中的表达变化, 探讨其在肺纤维化中与上皮间充质细胞转化(EMT) 的关系。 方法 将60只小鼠随机分成正常对照组（Cont组）, BLM致肺纤维化模型组, 地塞米松（DXM）治疗组。BLM组给予0.04ml BLM (5mg/kg)一次性气管内灌注,治疗组在BLM一次性气管内灌注后每天给予DXM 5mg/kg腹腔注射。各组分别于第3、7、14和28天各处死5只小鼠, 取肺组织, 做病理切片,HE染色观察小鼠肺炎症和纤维化的程度, 以样本碱水解法检测肺组织中羟脯氨酸(HYP)含量, 免疫组织化学检测不同实验组小鼠间充质细胞标记物α-平滑肌肌动蛋白(α-SMA) 和上皮细胞标记物上皮钙黏素( E-Cad) 的表达,应用Western blotting法测定不同实验组小鼠Rock-1的表达水平。 结果 BLM组肺组织病理切片按时间顺序呈现由肺泡炎至纤维化的动态改变,肺组织胶原含量在第7天明显增加, 第28天达高峰, HYP含量测定动态增高。与Cont组比较、BLM组、DXM组E-Cad的蛋白表达降低,α-SMA蛋白表达增高。Western blotting结果显示,BLM组Rock-1的表达与正常对照组比较显著增高( P<0.05),第14天时达到高峰。DXM组Rock-1的表达逐步下降。 结论 Rock-1在小鼠肺纤维化中表达增高, 表明其可能是肺纤维化形成的促进因子, 且随着Rock-1的增高,α-SMA逐步增高 E-Cad逐步降低, 推测Rock-1在肺纤维化形成中的机制可能与促进EMT有关。     

中和IL－17对博莱霉素诱导的肺纤维化小鼠肺组织Bax／Bcl－2表达的影响

目的：研究中和IL－17 A对博莱霉素（ bleomycin, BLM）诱导的肺纤维化小鼠肺组织Bax／Bcl－2表达的影响。方法将小鼠分别分成生理盐水组、 BLM组、 IL－17 A抗体组和IL－17 A抗体对照组,各30只,利用HE染色、 Masson三色胶原纤维染色检测小鼠肺组织纤维化程度,免疫组化方法检测小鼠肺组织Bax／Bcl－2的表达。结果从生理盐水组、到IL－17 A抗体组、再到BLM组和IL－17 A抗体对照组,小鼠肺组织肺泡炎症程度、肺泡壁的增厚情况以及肺泡壁的纤维化程度逐渐加重,差异有显著性意义（P＜0．01）; BLM组和IL－17 A抗体对照组间无明显差异（P＞0．05）。4组间Bax蛋白、 Bcl－2蛋白的表达也逐渐加重,差异均有统计学意义（ P＜0．01和P＜0．05）。 BLM组与抗体对照组间Bax、 Bcl－2的表达均无显著差异（P＞0．05）。结论 BLM可通过IL－17 A来诱导肺纤维化的形成,肺纤维化时肺实质细胞凋亡增多,而中和IL－17 A可通过调节Bax／Bcl－2的表达来抑制肺组织气道上皮的凋亡、达到抑制肺纤维化的目的。     

MyD88基因缺失型与野生型小鼠在博莱霉素诱导肺纤维化过程中的病理学比较

目的探讨小鼠体内My D88在博莱霉素(bleomycin,BLM)诱导肺纤维化后的表达变化,从而进一步探索肺纤维化的发病机制。方法利用BLM分别诱导My D88基因缺失型与野生型小鼠肺纤维化,HE、Masson染色检测肺部纤维化程度,Western blot检测野生型小鼠模型My D88蛋白的表达变化,并测定肺组织中羟脯氨酸含量。结果基因缺失型小鼠模型肺纤维化程度均轻于野生型,野生型造模组小鼠My D88表达量及羟脯氨酸含量均明显高于对照组(P0.05),My D88基因缺失型小鼠造模组羟脯氨酸含量与对照组相比差异无统计学意义。结论 My D88在博莱霉素诱导肺纤维化进程中有一定的促进作用。     

依那西普抑制博来霉素诱导的小鼠肺纤维化

 目的：观察肿瘤坏死因子 α(TNF-α)拮抗剂依那西普对博来霉素诱导的肺纤维化小鼠的抑制纤维化作用,并探讨依那西普治疗肺纤维化的可能机制。方法：将45只SPF级雌性昆明小鼠随机分为3组：对照组（气管内雾化生理盐水）、纤维化组（气管内博来霉素3 mg/kg溶于100 μL生理盐水内雾化）和依那西普干预组（气管内雾化博来霉素后,4 mg/kg依那西普溶于100 μL生理盐水内腹腔注射,每3 d注射1次）。处理后第28 d收集样本,小鼠左肺置于10%中性甲醛固定,石蜡包埋切片后行HE与Masson染色;右肺碱水解法检测组织羟脯氨酸(HYP)的含量;酶联免疫法检测血清TNF-α和转化生长因子 β（TGF-β）的含量;提取肺组织总蛋白,Western blotting 检测磷酸化ERK1/2、JNK和p38的表达。结果：依那西普干预组肺组织病理损伤及气道上皮下胶原沉积较纤维化组减轻,肺叶炎症损伤评分和纤维化评分明显下降（均P<0.01）,肺组织HYP含量显著降低（P<0.05）,血清TNF-α 和TGF-β的浓度明显减少（均P<0.01）,肺组织ERK1/2、JNK和p38蛋白的磷酸化水平也显著下降（P<0.01,P<0.05,P<0.01）。结论：依那西普能显著下调TNF-α 和TGF-β的水平,从而抑制ERK1/2、JNK和p38的活化,缓解博来霉素诱导的小鼠肺纤维化病变。     

小鼠间质性肺病循环纤维细胞的水平与肺部炎症和纤维化有关

目的研究博来霉素(BLM)诱导的间质性肺病(ILD)和胶原蛋白诱导关节炎联合博来霉素诱导的间质性肺病(CIA-ILD)小鼠模型肺部病变的发展、病理特点及其与循环纤维细胞(CF)消长的相关性。方法 90只C57BL/6小鼠随机分为3组:对照组(S)组、BLM诱导(B)组和CIA联合BLM诱导(CB)组。分别于BLM处理后第2、7、14、21、28天,HE染色观察肺组织急性炎症,天狼星红染色观察肺组织纤维化情况,免疫组织化学染色观察α-平滑肌肌动蛋白的表达,流式细胞术检测外周血CF(CD45+Col1+细胞)并与病变的程度进行相关性分析。结果 2组小鼠肺组织病理变化均呈现为肺泡炎至纤维化的动态改变,B组在BLM刺激后第7~14天炎性病变最明显,第21、28天病变趋于好转;CB组第14天开始出现肺泡结构明显破坏伴有胶原沉积,病变逐渐加重至第28天达最高峰;与B组相比,CB组肺组织羟脯氨酸含量在第14天后明显增加(P0.05);2组外周血CF均在给药后出现先增高后下降的趋势,且CB组在第14、21、28天明显高于同一时间点B组的CF数量(P0.05);免疫组化结果显示,CB组亦在第14天至第28天有大量肌纤维母细胞聚集;外周血CF数量与肺部炎症、纤维化评分及肺组织羟脯氨酸含量呈正相关(r=0.847、0.826、0.735,P0.01)。结论 CIA-ILD小鼠模型更为接近RA-ILD病理过程,循环纤维细胞可能参与了肺部炎症和纤维化病理进程。     

吡非尼酮和尼达尼布抑制博来霉素诱导的小鼠肺纤维化药效比较

目的:比较不同时期给予吡非尼酮和尼达尼布对博来霉素诱导的小鼠肺纤维化的作用。方法:根据给药时间和给药时长,建立5类小鼠模型:炎症时期给药模型、纤维化早期预防给药模型、纤维化早期治疗模型、纤维化晚期治疗模型和全程给药模型,分别检测炎症指标和纤维化指标。结果:(1)抗炎抗氧化评估:吡非尼酮和尼达尼布均能降低炎症细胞数目,抑制炎症因子分泌。吡非尼酮对白细胞介素1β(IL-1β)和IL-4的抑制效果较好(P<0.01),尼达尼布对IL-6、IFN-γ的抑制作用较好(P<0.05)。吡非尼酮可显著提高超氧化物歧化酶(SOD)活性(P<0.01),而尼达尼布可显著降低丙二醛(MDA)和髓过氧化物酶(MPO)含量(P<0.01)。(2)肺组织胶原含量检测:在纤维化早期治疗模型、纤维化晚期治疗模型、全程给药模型中,尼达尼布对羟脯氨酸的抑制作用均优于吡非尼酮(P<0.05)。而吡非尼酮在纤维化早期预防给药模型中对羟脯氨酸的抑制作用较好(P<0.01)。(3)肺组织病理学评价:吡非尼酮和尼达尼布均可减少肺组织炎症浸润和纤维化面积,抑制效果对比结果同胶原检测结果一致。结论:在博来霉素诱导的小鼠肺纤维化模型中,吡非尼酮和尼达尼布均具有抗炎、抗氧化及抗肺纤维化作用,其中吡非尼酮在预防给药模型中作用效果更优,尼达尼布在早期、晚期和全程治疗模型中作用效果更优。     

Therapeutic effects of alpha-lipoic acid on bleomycin-induced pulmonary fibrosis in rats

Pulmonary fibrosis (PF) is a major side effect of radiotherapy and chemotherapy. Recent clinical trials, unfortunately, have failed to identify any therapeutic agent which has the potential to reduce the consequences of this devastating condition. Reactive oxygen species and tissue remodeling regulators, such as metalloproteinases (MMPs) and their inhibitors (TIMPs), are thought to be involved in the development of PF. We investigated these factors to determine the protective effects of antioxidant alpha-lipoic acid (LA) against antineoplastic agent bleomycin (BLM)-induced oxidant lung toxicity in Sprague-Dawley rats. At different time intervals after BLM administration, pathological changes of the lung were analyzed with the measurement of total protein in bronchoalveolar lavage fluid (BALF), hydroxyproline (HYP) content and the level of three oxidative stress markers, i.e. malondialdehyde (MDA), the GSH/GSSG ratio, and total antioxidative capability (T-AOC). Also, the expression changes of MMP-1 and TIMP-1 were measured. At day 14 or 28 after BLM administration, protein content in BALF, and HYP, MDA and T-AOC contents of the lung increased significantly with a decreased GSH/GSSG ratio, implicating an increased efflux of GSSG from the lung and consumption of GSH. In contrast, treatment with LA protected BLM-induced pulmonary injury by suppressing oxidative stress with the reduction of MDA, and the enhancement of the GSH/GSSG ratio and T-AOC. The BLM-stimulated symptoms of PF were relieved with significant reduction of HYP and total proteins in LA-treated rats. LA also ameliorated the MMP-1/TIMP-1 ratio. These results suggest that LA inhibits BLM-induced lung toxicity associated with oxidative damage. Therefore, antioxidant LA has a potential therapeutic effect in the prevention and alleviation of PF.     

Oxymatrine attenuates bleomycin-induced pulmonary fibrosis in mice via the inhibition of inducible nitric oxide synthase expression and the TGF-β/Smad signaling pathway

Oxymatrine (OM) is an alkaloid extracted from the Chinese herb Sophora flavescens Ait. with a variety of pharmacological activities. The aim of this study was to investigate the preventive effects of OM on bleomycin (BLM)-induced pulmonary fibrosis (PF) and to further explore the underlying mechanisms. C57BL/6 mice were randomly assigned to five groups: the saline sham group; the BLM group, in which mice were endotracheally instilled with BLM (3.0?mg/kg); and the BLM plus OM groups, in which OM was given to mice daily (10, 20 or 40?mg/kg) one day after BLM instillation for 21?days. The bronchoalveolar lavage fluid (BALF) and lung tissues were collected at 15 and 22?days post BLM administration, respectively. Lung tissues were stained with hematoxylin and eosin (H&E) for histological evaluation. Levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and nitric oxide (NO) in mouse BALF were measured, as well as myeloperoxidase (MPO) activity and malondialdehyde (MDA) content in lung homogenates. The inducible nitric oxide synthase (iNOS) expression in the lung tissues was determined by immunohistochemical staining, quantitative real-time PCR and western blot analysis. Moreover, the expression of transforming growth factor (TGF)-β1, Smad2, Smad3, p-Smad2 and p-Smad3 were also detected. We found that OM improved BLM-induced lung pathological changes, inhibited MPO activity and reduced MDA levels in a dose-dependent manner. OM also dose-dependently inhibited the release of TNF-α and IL-6, and decreased the expression of iNOS in lung tissues and thus prevented NO release in response to BLM challenge. In addition, OM decreased the expression of TGF-β1, p-Smad2 and p-Smad3, which are all important members of the TGF-β/Smad signaling pathway. Our study provides evidence that OM significantly ameliorated BLM-induced PF in mice via the inhibition of iNOS expression and the TGF-β/Smad pathway.     

Human placental mesenchymal stem cells of fetal origins-alleviated inflammation and fibrosis by attenuating MyD88 signaling in bleomycin-induced pulmonary fibrosis mice

Pulmonary fibrosis is a progressive lung disease that its pathogenic mechanism currently is incompletely understood. Toll-like receptor (TLR) signaling has recently been identified as a regulator of inflammation and pulmonary fibrosis. In addition, mesenchymal stem cells (MSCs) of different origins offer a great promise in treatment of idiopathic pulmonary fibrosis (IPF). However mechanisms of pathogenic roles of TLR signaling and therapeutic effects of MSCs in the IPF remain elusive. In present study, the involvement of TLR signaling and the therapeutic role of MSCs were interrogated in MyD88-deficient mice using human placental MSCs of fetal origins (hfPMSCs). The results showed an alleviated pulmonary inflammation and fibrosis in myeloid differentiation primary response gene 88 (MyD88)-deficient mice treated with bleomycin (BLM), accompanied with a reduced TGF-β signaling and production of pro-fibrotic cytokines, including TNF-α, IL-1β. An exposure of HLF1 lung fibroblasts, A549 epithelial cells and RAW264.7 macrophages to BLM led an increased expression of key components of MyD88 and TGF-β signaling cascades. Of interest, enforced expression and inhibition of MyD88 protein resulted in an enhanced and a reduced TGF-β signaling in above cells in the presence of BLM, respectively. However, the addition of TGF-β1 showed a marginally inhibitory effect on MyD88 signaling in these cells in the absence of BLM. Importantly, the administration of hfPMSCs could significantly attenuate BLM-induced pulmonary fibrosis in mice, along with a reduced hydroxyproline (HYP) deposition, MyD88 and TGF-β signaling activation, and production of pro-fibrotic cytokines. These results may suggest an importance of MyD88/TGF-β signaling axis in the tissue homeostasis and functional integrity of lung in response to injury, which may offer a novel target for treatment of pulmonary fibrosis.     

Angiotensin converting enzyme 2 abrogates bleomycin-induced lung injury

Despite substantial progress, mortality and morbidity of the acute respiratory distress syndrome (ARDS), a severe form of acute lung injury (ALI), remain unacceptably high. There is no effective treatment for ARDS/ALI. The renin-angiotensin system (RAS) through Angiotensin-converting enzyme (ACE)-generated Angiotensin II contributes to lung injury. ACE2, a recently discovered ACE homologue, acts as a negative regulator of the RAS and counterbalances the function of ACE. We hypothesized that ACE2 prevents Bleomycin (BLM)-induced lung injury. Fourteen to 16-week-old ACE2 knockout mice-male (ACE2(-/y)) and female (ACE2(-/-))-and age-matched wild-type (WT) male mice received intratracheal BLM (1.5U/kg). Male ACE2(-/y) BLM injured mice exhibited poorer exercise capacity, worse lung function and exacerbated lung fibrosis and collagen deposition compared with WT. These changes were associated with increased expression of the profibrotic genes α-smooth muscle actin (α-SMA) and Transforming Growth Factor ?1. Compared with ACE2(-/y) exposed to BLM, ACE2(-/-) exhibited better lung function and architecture and decreased collagen deposition. Treatment with intraperitoneal recombinant human (rh) ACE2 (2?mg/kg) for 21?days improved survival, exercise capacity, and lung function and decreased lung inflammation and fibrosis in male BLM-WT mice. Female BLM WT mice had mild fibrosis and displayed a possible compensatory upregulation of the AT2 receptor. We conclude that ACE2 gene deletion worsens BLM-induced lung injury and more so in males than females. Conversely, ACE2 protects against BLM-induced fibrosis. rhACE2 may have therapeutic potential to attenuate respiratory morbidity in ALI/ARDS.     

Metformin Reduces Bleomycin-induced Pulmonary Fibrosis in Mice

Metformin has anti-inflammatory and anti-fibrotic effects. We investigated whether metformin has an inhibitory effect on bleomycin (BLM)-induced pulmonary fibrosis in a murine model. A total of 62 mice were divided into 5 groups: control, metformin (100 mg/kg), BLM, and BLM with metformin (50 mg/kg or 100 mg/kg). Metformin was administered to the mice orally once a day from day 1. We sacrificed half of the mice on day 10 and collected the bronchoalveolar lavage fluid (BALF) from their left lungs. The remaining mice were sacrificed and analyzed on day 21. The right lungs were harvested for histological analyses. The messenger RNA (mRNA) levels of epithelial-mesenchymal transition markers were determined via analysis of the harvested lungs on day 21. The mice treated with BLM and metformin (50 mg/kg or 100 mg/kg) showed significantly lower levels of inflammatory cells in the BALF compared with the BLM-only mice on days 10 and 21. The histological examination revealed that the metformin treatment led to a greater reduction in inflammation than the treatment with BLM alone. The mRNA levels of collagen, collagen-1, procollagen, fibronectin, and transforming growth factor-β in the metformin-treated mice were lower than those in the BLM-only mice on day 21, although statistical significance was observed only in the case of procollagen due to the small number of live mice in the BLM-only group. Additionally, treatment with metformin reduced fibrosis to a greater extent than treatment with BLM alone. Metformin suppresses the inflammatory and fibrotic processes of BLM-induced pulmonary fibrosis in a murine model.     

Inhibition of activin receptor-like kinase 5 attenuates bleomycin-induced pulmonary fibrosis

Activin receptor-like kinase 5 (ALK5) is a type I receptor of transforming growth factor (TGF)-beta. ALK5 inhibition has been reported to attenuate the tissue fibrosis including pulmonary fibrosis, renal fibrosis and liver fibrosis. To elucidate the inhibitory mechanism of ALK5 inhibitor on pulmonary fibrosis in vivo, we performed the histopathological assessment, gene expression analysis of extracellular matrix (ECM) genes and immunohistochemistry including receptor-activated Smads (R-Smads; Smad2/3), CTGF, myofibroblast marker (alpha-smooth muscle actin; aSMA) and type I collagen deposition in the lung using Bleomycin (BLM)-induced pulmonary fibrosis model. ALK5 inhibitor, SB-525334 (10 mg/kg or 30 mg/kg) was orally administered at twice a day. Lungs were isolated 5, 7, 9 and 14 days after BLM treatment. BLM treatment led to significant pulmonary fibrotic changes accompanied by significant upregulation of ECM mRNA expressions, Smad2/3 nuclear translocation, CTGF expression, myofibroblast proliferation and type I collagen deposition. SB-525334 treatment attenuated the histopathological alterations in the lung, and significantly decreased the type I and III procollagen and fibronectin mRNA expression. Immunohistochemistry revealed that SB-525334 treatment showed significant attenuation in Smad2/3 nuclear translocation, decrease in CTGF-expressing cells, myofibroblast proliferation and type I collagen deposition. These results suggest that ALK5 inhibition attenuates R-Smads activation thereby attenuates pulmonary fibrosis.     

FTS reduces bleomycin-induced cytokine and chemokine production and inhibits pulmonary fibrosis in mice

Bleomycin (BLM), an antitumour drug, is known to cause interstitial pneumonia followed by pulmonary fibrosis, and has often been used to produce an animal model of pulmonary fibrosis. In the present study, we examined the effect of a nonapeptide thymic hormone, facteur thymique serique (FTS), on the murine lung fibrosis induced by intratracheal instillation of BLM. Treatment with FTS ameliorated BLM-induced fibrotic changes in a dose-dependent manner, as indicated by the reduced accumulation of hydroxyproline (HP). In addition, FTS suppressed BLM-induced cellular inflammatory response in the lungs, as evidenced by inhibition of increased lung weight, reduced accumulation of inflammatory leucocytes, including lymphocytes and neutrophils, but not macrophages, and less pronounced histopathological changes. Finally, BLM challenge increased the local synthesis of proinflammatory cytokines, TNF-alpha and IL-1beta and chemokines, MCP-1, MIP-1alpha RANTES, MIP-2 and KC, while administration of FTS suppressed the production of these cytokines, except for MCP-1. These effects of FTS were observed only when mice received intratracheal instillation with BLM. Considered collectively, our results indicated that FTS treatment ameliorated the cellular inflammatory responses and fibrotic changes in the lungs caused by BLM and such inhibition was well correlated with reduced synthesis of several fibrosis-related cytokines, and suggested that FTS may be potentially useful for the treatment of pulmonary fibrosis.     

Role of interleukin-6 in bleomycin-induced lung inflammatory changes in mice

Interleukin-6 (IL-6) is known to be involved in the pathogenesis of various inflammatory diseases, but its role in bleomycin (BLM)-induced lung injury and subsequent fibrotic changes remains to be determined. We evaluated the role of IL-6 in the lung inflammatory changes induced by BLM using wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice. The mice were treated intratracheally with 1 mg/kg BLM and killed 2, 7, or 21 days later. Lung Inflammation in the acute phase (Days 2 and 7) was assessed by differential cell counts in bronchoalveolar lavage (BAL) fluid and cytokine levels in the lung. Lung fibrotic changes were evaluated on Day 21 by histopathology and collagen assay. On Day 2, BLM administration induced significant increases in the numbers of total cells, macrophages, and neutrophils in BAL fluid, which were attenuated in IL-6(-/-) mice (P < 0.05). Lung pathology also showed inflammatory cell accumulation, which was attenuated in the IL-6(-/-) mice compared with WT mice. In WT mice, elevated levels of TGF-beta(1) and CCL3 were observed 2 and 7 days after BLM challenge, respectively. On Day 7, BLM-induced inflammatory cell accumulation did not differ between the genotypes. Lung pathology 21 days after BLM challenge revealed significant fibrotic changes with increased collagen content, which was attenuated in IL-6(-/-) mice. Although the TGF-beta(1) level in the lung did not differ between the genotypes on Day 21, CCL3 was significantly lower in IL-6(-/-) mice. These results indicate that IL-6 may play an important role in the pathogenesis of BLM-induced lung injury and subsequent fibrotic changes.