研究背景：

特发性肺纤维化（idiopathic pulmonary fibrosis，IPF）是一种病因不详的、高致死性间质性肺疾病，其诊断后平均生存期仅2-3年。在遗传及环境等因素共同作用下，肺泡上皮细胞反复出现破坏，再上皮化修复异常，大量促纤维化介质释放，诱导成/肌成纤维细胞异常激活，促进细胞外基质（extracellular matrix，ECM）过度累积，最终引起肺组织纤维化。目前大量数据表明，氧化还原失衡（氧化应激增加和抗氧化能力下降）在IPF的发病过程中起重要作用。不仅在空气中可检测到氧化的蛋白质和脂质，而且在来源于IPF患者的样本（血清、支气管肺泡灌洗液和肺组织）中亦可检测到大量氧化及过氧化代谢物的产生及抗氧化介质含量的降低、活性的减弱等。研究发现，通过干预重塑小鼠肺组织内氧化还原平衡后，小鼠肺纤维化的严重程度可得到缓解。然而乙酰半胱氨酸等抗氧化治疗在进一步的临床试验中被认为无明显抗纤维化作用。抗氧化药物在肺纤维化治疗中的研究仍道阻且长。Liproxstatin-1（Lip-1）是一种强有效的细胞自氧化抑制剂，其通过直接抑制自由基链增殖反应发挥效用。已有研究发现，Lip-1可减轻急性辐射所致小鼠肺损伤，抑制香烟烟雾提取物引起的支气管上皮细胞死亡等。Lip-1是否可以缓解IPF目前尚无相关研究。

研究目的：

本研究旨在探讨Lip-1对博来霉素（bleomycin, BLM）诱导的小鼠肺纤维化及肺泡上皮细胞损伤是否具有缓解作用，并阐明其可能机制。

研究方法：

1. 通过给予8周龄的C57BL/6J雄性小鼠气道注射BLM构建肺纤维化模型。全部小鼠随机被分为4组：Con组（对照组），BLM组（博来霉素：3.5mg/kg处理组），Lip-1组（Lip-1：10mg/kg处理组），BLM+Lip-1组（博来霉素+ Lip-1处理组）。建模21天，称量小鼠体重及肺重，计算肺重/体重比；收获小鼠血清及肺泡灌洗液（bronchoalveolar lavage fluid，BALF），酶联免疫吸附测定（Enzyme linked immunosorbent assay，Elisa）法检测促炎因子水平；收获小鼠肺组织，石蜡包埋切片，进行苏木精-伊红染色（Hematoxylin-Eosin Staining，HE）、马松染色、天狼星红染色，观察小鼠肺组织结构改变、胶原产生及分布情况；通过Tunel染色，检测肺组织细胞凋亡程度；通过免疫荧光方法，评估肺泡上皮细胞分布范围; 借助透射电镜观察肺细胞超微形态；提取小鼠肺组织蛋白，评估氧化还原相关指标：活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）、还原型谷胱甘肽（glutathione，GSH）水平，及过氧化氢酶（catalase，CAT）、超氧化物歧化酶（superoxide dismutase，SOD）酶活性；通过Western blot及免疫荧光法，检测氧化应激相关通路（ROS/p53）。

2. 给予不同浓度的BLM（0、10、40、80、160、320、640、800ug/ml）刺激肺泡上皮细胞（A549细胞），采用MTT检测法评估肺泡上皮细胞的细胞活力变化情况; 给予不同刺激时间长，筛选BLM诱导肺泡上皮细胞损伤模型的适宜时间。

3. 体外构建BLM诱导的肺泡上皮细胞损伤模型，将A549细胞分为4组：Con组（对照组），BLM组（BLM：40ug/ml处理组），Lip-1组（Lip-1：2UM处理组），BLM+Lip-1组（Lip-1：2UM预给药30min，BLM：40ug/ml处理组）。采用MTT法检测A549细胞的细胞活力，乳酸脱氢酶（lactate dehydrogenase，LDH）法检测细胞毒性物质的释放；通过细胞荧光染色，评估ROS的产生情况；裂解细胞蛋白，检测氧化还原相关指标（MDA、GSH、CAT、T-SOD）的变化情况；通过Western blot法及免疫荧光法检测ROS/p53相关靶点Bcl-2相关X蛋白（BCL2-Associated X，BAX）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）及p21表达水平，探讨Lip-1的肺泡上皮细胞保护机制。

研究结果：

体内实验部分

1. 构建小鼠肺纤维化模型，HE染色发现BLM可诱导小鼠肺组织炎症浸润、肺泡结构破坏，Lip-1干预可减轻BLM诱导的小鼠肺组织损伤。

2. Lip-1干预还可减轻BLM诱导的小鼠BALF中炎症因子：转化生长因子β1（transforming growth factor β1，TGF-β1）、白介素6（Interleukin-6，IL-6）、白介素10（Interleukin-10，IL-10）、肿瘤坏死因子α（tumour necrosis factor-α，TNF-α）的释放（p<0.05）。

3. 马松染色、天狼星染色及羟脯氨酸检测（hydroxyproline，HYP）检测发现Lip-1干预可减轻BLM诱导的小鼠肺组织胶原分泌。

4. 氧化应激相关检测发现，在BLM刺激下小鼠肺组织氧化产物ROS、MDA水平升高，抗还原相关介质GSH、CAT、T-SOD的酶活性降低，Lip-1干预可重塑小鼠肺组织氧化还原的平衡。

5. Lip-1干预可缓解BLM诱导的小鼠肺组织细胞凋亡、肺泡上皮细胞减少以及线粒体破坏。

6. BLM可激活小鼠肺组织氧化还原相关通路ROS/p53，Lip-1干预可降低上述通路激活水平，减轻肺纤维化。

体外实验部分

1. BLM可引起A549细胞生存力减低，且以浓度依赖的方式发挥作用，浓度越高细胞生存力越低。

2. BLM可诱导A549细胞发生上皮样向间质样细胞的改变，且以时间依赖的方式发挥作用，刺激24h细胞形态改变明显。

3. Lip-1可改善BLM引起的A549细胞活力减低，并减少LDH的产生。

4. Lip-1可减轻BLM造成的 A549细胞氧化损伤，降低ROS及MDA的产生。

5. Lip-1可缓解BLM对A549细胞还原能力的破坏，提升GSH水平，但对CAT及T-SOD的酶活力影响不大。

6. BLM可激活A549细胞氧化还原相关通路ROS/p53，Lip-1干预可减弱ROS/p53下游BAX（凋亡相关）及α-SMA（纤维化相关）的表达，但不影响p21（衰老相关）的表达。

研究结论：

1. BLM诱导的小鼠肺纤维化模型中，肺组织炎症浸润广泛、氧化还原紊乱、胶原分泌增加、细胞凋亡增加、肺泡上皮细胞破坏并减少。

2. Lip-1可以改善BLM诱导的小鼠肺纤维化进程中炎症反应，重塑氧化还原平衡，抑制胶原分泌，保护肺泡上皮细胞及线粒体，减轻肺纤维化程度。

3. Lip-1可以减弱BLM诱导的A549细胞破坏，减轻氧化损伤，提高抗氧化能力。

4. Lip-1缓解BLM诱导的小鼠肺纤维化及肺泡上皮细胞损伤的机制可能与其对氧化还原相关通路ROS/p53的调控有关。

Background:

Idiopathic pulmonary fibrosis (IPF), a terminal disease with undetermined etiology and limited treatment option is extremely lethal, with an average life expectancy of only 2-3 years after diagnosis. Both genetic susceptibility and environmental factors or damage contribute to the recurrent injury and abnormal repair of epithelial cells, cause chronic inflammatory infiltration, induce the abnormal activation of fibroblast/myofibroblast, promote superabundant extracellular matrix (ECM) protein deposition, finally result in progressive and irreversible fibrosis. Previous studies found that redox imbalance (increased oxidative stress and compromised antioxidant ability) played an important role in the development of IPF. Not only the IPF samples (serum, bronchus alveolar lavage fluid, and lung tissue) but also the the atmosphere were demonstrated to fill with oxidized proteins and lipids. In view of the crucial role of redox imbalance in IPF development, the maintainence of redox homeostasis was deemed to be a potential therapeutic target to IPF for years. Variety studies found that the maintainence of antioxidant function of mouse can alleviate the progression of fibrosis in experimental animal model. However, several antioxidant including N-acetylcysteine (NAC) were prove to have no antfibrotic funtion in clinical trials. Thus, the way of applying antioxidants to treat pulmonary fibrosis still long and full of thorns. As a radical-trapping antioxidant, Liproxstatin-1 (Lip-1) is a potent lipid autoxidation inhibitor which can directly repress the radical chain propagation. Lip-1 was proved to alleviate acute radiation induced mice lung injury and inhibit cigarette smoke extract caused bronchial epithelial cells death. Based on the protective function of Lip-1 in previous researches, it hypothesizes that Lip-1 might suppress pulmonary fibrosis by reshaping redox equilibrium.

Objective:

This study aim to investigate whether and how Lip-1 regulates bleomycin (BLM) induced pulmonary fibrosis both in vivo and in vitro.

Methods:

1. Eight-week-old male C57BL/6 mice weighed about 23g were induced by BLM intratracheally to performe the pulmonary fibrosis model. All the mice were randomly divided into four groups: control group (Con), bleomycin treated group (BLM: 3.5mg/kg), Liproxstatin-1 treated group (Lip-1: 10mg/kg) and bleomycin plus Liproxstatin-1 co-treated group (BLM+Lip-1). After 21 days, the weight of mice and lungs, as well as the macroscopic structure of lungs were recorded; the serum and bronchoalveolar lavage fluid (BALF) were prepared to calculate the level of proinflammatory cytokine; the 4% paraformaldehyde-fixed, paraffin-embedded lung tissues were sliced to investigate the pathologic change and  production of collagen by performing hematoxylin-eosin solution (HE) staining, Masson’s Trichrome staining and Sirius Red staining; Tunel staining was carried out to evaluate cell apoptosis in the fibrosis lung; Immunofluorescence staining was implemented to show the amount and distribution of alveolar epithelial cell; transmission electron microscope (TEM) was applied to observe the ultra microstructure of cells; the level of oxidative products including reactive oxygen species (ROS) and malondialdehyde (MDA), the activity of various antioxidant glutathione (GSH), catalase (CAT), and total superoxide dismutase (T-SOD) were tested using protein lysate; oxidative stress related profibrotic pathways (ROS/p53) were analyzed by Western blot and Immunofluorescence staining.

2. The viability of A549 cells treated with different concentration of BLM (0, 10, 40, 80, 160, 320, 640 and 800ug/ml) for 24 h were performed by MTT assay; the optimum time of BLM in inducing A549 cells injury and fibrosis was evaluated by Western blot.

3. In the AECs injury model induced by BLM, the A549 cells were divided into four groups: control group (Con), BLM treated group (BLM: 40ug/ml), Lip-1 treated group (Lip-1: 2UM) and BLM plus Lip-1 co-treated group (BLM+Lip-1). MTT assay was performed to assess the cell viability; lactate dehydrogenase (LDH) assay was carried out to measure the cellular damage; a DCFH-DA probe was used to estimate the level of ROS; the amount of MDA and the activity of various antioxidant (GSH, CAT, T-SOD) were tested using protein lysate; Western blot and Immunofluorescence staining were adopted to calculate the expression of p53 and its possible targets including BCL2-Associated X (BAX), α-smooth muscle actin (α-SMA), and p21, to evaluate the function of Lip-1 in alleviating pulmonary fibrosis.

Result:

In vivo:

1. In mice model of pulmonary fibrosis, HE staining confirmed that BLM could induce inflammation and destroy the structure in pulmonary, while the addition of Lip-1 alleviated such inflammatory infiltration and structure damage.

2. Lip-1 can also mitigate the expression of transforming growth factor β1， interleukin-6 (IL-6), interleukin-10 (IL-10), and (TGF-β1), tumour necrosis factor α (TNF-α) in BALF induced by BLM.

3. Masson’s trichrome staining, Sirius red staining, and hydroxyproline (HYP) test found that Lip-1 could reduce the generation of collagen induced by BLM in mice.

4. The tests to evaluate oxidative stress identified that BLM promoted the produce of ROS and MDA, inhibited the activity of GSH, CAT, and T-SOD, while the addition of Lip-1 reversed these alterations in mice model.

5. Lip-1 can relieve the cell apoptosis, alveolar epithelial cells (AECs) reduction and mitochondria damage induced by BLM in mice lung.

6. Lip-1 can attenuate ROS/p53 pathway related to redox which was activated by BLM in mice.

In vitro:

1. The cell viability in A549 cells was reduced with increasing BLM concentration in a concentration-dependent manner.

2. The addition of BLM resulted in A549 cells mesenchymal like changes in a time-dependent manner.

3. Lip-1 can recover the cell viability and decreased LDH relaese, all which were disordered by BLM in A549 cells.

4. Lip-1 can alleviate the oxidative damage (ROS and MDA) in A549 cells induced by BLM.

5. Lip-1 can restore the reductive ability in A549 cell damaged by BLM through enhancing GSH expression, with little effect on CAT and T-SOD activity.

6. Lip-1 can attenuate ROS/p53 related pathways including BAX (apoptosis associated) and α-SMA (fibrosis associated) pathways, but did not affect the p21 (senescence associated) pathway, all which are activated by BLM.

Conclusion:

1. The inflammatory infiltration was extensive, redox balance was disturbed, collagen secretion was increased, apoptosis was increased, and AECs were destroyed and decreased in BLM induced mice pulmonary fibrosis model.

2. Lip-1 can improve the inflammatory response, inhibit collagen secretion, reshape redox balance, and protect AECs in BLM induced mice pulmonary fibrosis model.

3. Lip-1 can improve BLM induced A549 cells destruction, reduce oxidative damage and enhance antioxidant capacity.

4. Lip-1 alleviates bleomycin-induced pulmonary fibrosis in mice and A549 cells damage, which may relate to the regulation of ROS/p53 pathway (apoptosis and fibrosis associated, other than senescence associated).