背景和目的

矽肺是我国最主要的致命性职业病之一，其特征是呼吸功能障碍，肺部广泛的纤维化型结节和全身炎症，通常因为长期接触可吸入的二氧化硅颗粒而导致。作为最主要也是危害性最大的一种尘肺病，矽肺不仅会导致肺间质纤维化，还会增加例如结核、肺癌以及其他各种自身免疫病的风险。尽管目前全球已经经历能源转型，但是与粉尘暴露的相关职业仍旧存在，矽肺的新发病例数也呈现增长趋势，主要存在于陶瓷、玻璃以及喷砂牛仔裤等工人人群中。在我国，每年报告新发的职业性尘肺病占到了全部职业病过半。虽然矽肺病是一个古老的疾病，但是发病机制仍未明确，目前较为确定的是肺泡巨噬细胞损伤在矽肺的发展过程中起到了重要作用，而肺泡巨噬细胞的氧化应激反应可能是其中重要的一环。与此同时，矽肺缺少有效的治疗药物和手段，因此继续深入研究矽肺的发病机制和发掘有效的治疗药物至关重要。

HDAC6是一种II类的组蛋白去乙酰化酶，其主要存在于细胞质之中。有研究表明抑制HDAC6可以减轻氧化应激水平。同时，HDAC6完全丧失也不会引起任何明显的表型变化，前期研究也表明靶向HDAC6可能对肺腺癌以及脂多糖所致的急性肺损伤存在一定的治疗作用，因此靶向HDAC6可能是治疗矽肺的一种较为有前途的策略。GHK是一种天然存在于人体血液、尿液和唾液的三肽，在血浆中GHK和铜离子结合形成复合物GHK-Cu，能够更充分的发挥生物学作用。在既往研究中已经明确GHK-Cu的抗炎抗氧化作用，前期研究也表明GHK-Cu可以减轻博来霉素诱导的小鼠肺纤维化，但目前尚无研究探究其在矽肺中的作用，因此我们猜想是否GHK-Cu在矽肺中也存在治疗作用。

矽肺是一种职业环境相关性间质性肺疾病，其确诊有时需要有肺活检病理结果，而冷冻肺活检已经广泛被推荐在间质性肺疾病的诊断中应用。作为一种侵入性操作，预置球囊是保证其安全性的主要措施之一，但是尚无相关研究对其作用进行验证。

因此本课题研究内容分为三个部分：第一部分：通过抑制肺泡巨噬细胞HDAC6靶向调控PRDX6减轻二氧化硅所致肺部炎症和纤维化；第二部分：GHK-Cu通过肺泡巨噬细胞靶向调控PRDX6减轻二氧化硅所致的肺部炎症和纤维化；第三部分：预置球囊在冷冻肺活检诊断间质性肺疾病患者中的安全性评价。

方法

第一部分：

通过在喉镜下气管插管滴注二氧化硅悬液构建矽肺小鼠模型，随后进行Western Blot检测HDAC6表达水平；采用免疫荧光对肺泡巨噬细胞和HDAC6进行共定位；
通过在喉镜下气管插管滴注二氧化硅悬液构建矽肺小鼠模型，采用HDAC6特异性抑制剂CAY-10603对矽肺小鼠进行腹腔注射治疗；随后通过肺泡灌洗液细胞分类计数、ELISA检测、肺部组织病理、Western Blot等实验明确抑制HDAC6可以减轻矽肺小鼠肺部炎症和纤维化；
构建HDAC6髓系特异性敲除小鼠，随后通过肺泡灌洗液细胞分类计数、ELISA检测、肺部组织病理、Western Blot等实验明确靶向敲除巨噬细胞HDAC6可以减轻二氧化硅所致的小鼠肺部炎症和纤维化；
通过野生型矽肺小鼠肺组织和HDAC6髓系特异性敲除矽肺小鼠的肺部组织进行蛋白质组学分析，筛选出HDAC6髓系特异性敲除矽肺小鼠的肺部氧化应激水平降低；
通过RAW264.7构建体外结晶型二氧化硅染尘模型；首先通过Western Blot明确HDAC6表达；随后采用HDAC6特异性抑制剂CAY-10603进行干预，通过活性氧检测、线粒体膜电位检测等实验方法明确抑制HDAC6对结晶型二氧化硅染尘巨噬细胞氧化应激水平的影响；
通过免疫沉淀后的蛋白质谱分析筛选出HDAC6结合蛋白PRDX6；随后通过免疫共沉淀，免疫荧光共定位以及分子对接明确HDAC6和PRDX6的结合；
通过敲减PRDX6，在RAW264.7体外结晶型二氧化硅染尘模型中通过活性氧检测、线粒体膜电位染色等实验方法验证抑制HDAC6所产生的抗氧化作用是通过PRDX6发挥的；
通过在RAW264.7中过表达PRDX6联合CAY-10603，随后采用免疫共沉淀明确抑制HDAC6对PRDX6乙酰化水平的影响；同时通过过表达PRDX6和HDAC6，随后采用免疫共沉淀进一步明确HDAC6对PRDX6乙酰化水平的影响；
构建巨噬细胞特异性腺相关病毒，靶向敲除小鼠肺部巨噬细胞HDAC6，进一步明确靶向肺部巨噬细胞抑制HDAC6对二氧化硅所致小鼠肺部炎症和纤维化的保护作用。

第二部分：

通过高效液相色谱法检测健康人群和矽肺患者血浆GHK水平，同时收集患者肺功能并检测患者血浆相关指标进行相关性分析；
通过在喉镜下气管插管滴注二氧化硅悬液构建矽肺小鼠模型，同时给予低剂量（2mg/kg）和高剂量（20mg/kg）的GHK-Cu进行腹腔注射治疗，观察GHK-Cu对肺部炎症和纤维化的影响；
利用RAW264.7细胞系构建结晶型二氧化硅染尘模型，检测GHK-Cu对染尘后的巨噬细胞的损伤以及氧化应激水平的影响；
用生物素标记GHK-Cu（Biotin-GHK-Cu），通过生物素化免疫沉淀后蛋白质谱分析，筛选与GHK-Cu结合并发挥作用的蛋白PRDX6；随后进行生物素化免疫共沉淀、表面等离子共振以及分子对接验证GHK-Cu和PRDX6的结合；
在RAW264.7二氧化硅染尘模型中敲除PRDX6，重复GHK-Cu对二氧化硅染尘RAW264.7氧化应激相关指标的影响，明确GHK-Cu通过PRDX6发挥抗氧化作用。

第三部分：

通过单中心前瞻性随机对照试验对预置球囊在冷冻肺活检诊断间质性肺疾病患者中的作用进行探究。将需要病理诊断进行确诊的疑似间质性肺疾病患者根据计算机生成的随机数字表进行随机分组（预置球囊组和非预置球囊组）。主要结局指标是两组的中度出血的发生率，次要结局指标是重度出血、气胸以及其他冷冻肺活检相关并发症的发生率。

结果

第一部分：

矽肺小鼠肺部高表达HDAC6，且巨噬细胞高表达HDAC6

通过在喉镜下气管插管滴注二氧化硅悬液成功构建矽肺小鼠模型，模型组小鼠肺部形成明显且均匀的矽肺结节；通过Western Blot对小鼠肺组织蛋白进行检测发现矽肺小鼠肺部HDAC6表达明显升高；采用F4/80和HDAC6抗体对巨噬细胞和HDAC6进行免疫荧光染色，结果显示巨噬细胞和HDAC6表达均增高且共定位。

HDAC6特异性抑制剂CAY-10603可以明显减轻矽肺小鼠肺部炎症和纤维化

通过在喉镜下气管插管滴注二氧化硅悬液成功构建矽肺小鼠模型；通过H&E染色、Masson染色、天狼星红染色、Western Blot检测、小鼠肺泡灌洗液炎症细胞分类计数以及小鼠肺泡灌洗液中各类炎症因子的ELISA检测，发现同矽肺模型小鼠，CAY-10603治疗矽肺模型小鼠肺部炎症和纤维化水平明显降低。

HDAC6髓系特异性敲除可以明显减轻矽肺小鼠肺部炎症和纤维化

成功构建HDAC6髓系特异性敲除小鼠；通过在喉镜下气管插管滴注二氧化硅悬液成功构建矽肺小鼠模型；通过H&E染色、Masson染色、天狼星红染色、Western Blot检测、小鼠肺泡灌洗液炎症细胞分类计数以及小鼠肺泡灌洗液中各类炎症因子的ELISA检测，发现同矽肺模型小鼠，CKO矽肺小鼠肺部炎症和纤维化水平明显降低。

蛋白质组学显示髓系特异性敲除HDAC6可以减轻肺部氧化应激水平

通过野生型矽肺小鼠肺组织和HDAC6髓系特异性敲除矽肺小鼠的肺部组织进行蛋白质组学分析，KEGG富集结果显示和氧化磷酸化以及线粒体自噬相关蛋白变化明显，其均一定程度反应氧化应激水平，表明髓系敲除HDAC6可以明显影响矽肺小鼠肺部组织氧化应激水平。

CAY-10603和敲减HDAC6均可以减轻结晶型二氧化硅染尘RAW264.7的氧化应激水平

通过RAW264.7构建体外结晶型二氧化硅染尘模型；Western Blot结果表明染尘巨噬细胞HDAC6表达明显增高；采用HDAC6特异性抑制剂CAY-10603进行干预，活性氧染色、线粒体膜电位检测结果显示，抑制HDAC6可以明显减轻染尘后巨噬细胞活性氧的产生，恢复线粒体膜电位的水平，表明抑制HDAC6可以明显减轻染尘后巨噬细胞的氧化应激水平；敲除HDAC6重复相关实验得出类似结果。

HDAC6和PRADX6能够结合

通过免疫沉淀后的蛋白质谱分析筛选出结合蛋白中排名靠前的PRDX6与氧化应激明显相关；随后通过免疫共沉淀进一步验证HDAC6和PRDX6结合；通过质粒过表达HDAC6和PRDX6，随后进一步通过免疫共沉淀和免疫荧光确定HDAC6和PRDX6共定位；通过分子对接模拟HDAC6和PRDX6的结合位点。

抑制HDAC6通过影响PRDX6发挥抗氧化应激作用

通过si-RNA在RAW264.7细胞系敲减PRDX6，随后在RAW264.7体外结晶型二氧化硅染尘模型中通过活性氧检测以及线粒体膜电位染色实验发现，加入HDAC6特异性抑制剂CAY-10603无法减少活性氧的产生并且无法恢复线粒膜电位水平，表明抑制HDAC6所产生的抗氧化作用是通过PRDX6发挥的。

HDAC6通过去乙酰化PRDX6在氧化应激中发挥作用

通过乙酰化免疫共沉淀结果显示，结晶型二氧化硅染尘的RAW264.7中PRDX6的乙酰化水平明显降低；通过在RAW264.7过表达PRDX6-Myc，同时用HDAC6特异性抑制剂CAY-10603进行处理，随后进行乙酰化免疫共沉淀，结果显示乙酰化PRDX6明显增高，并且PRDX6的乙酰化水平随CAY-10603剂量增高而升高，表明HDAC6剂量依赖性的对PRDX6产生去乙酰化作用。

靶向肺部巨噬细胞腺相关病毒敲减HDAC6（AAV6-F4/80-HDAC6）可以明显减轻矽肺小鼠肺部炎症和纤维化

成功构建靶向肺部巨噬细胞的腺相关病毒；感染3周后构建矽肺小鼠模型，28天后经H&E染色和Masson染色结果显示，靶向敲除肺部巨噬细胞HDAC6可以明显减轻肺部炎症和纤维化。

第二部分：

矽肺患者血浆GHK水平明显降低

矽肺患者的血浆GHK水平明显低于健康人群（35.67 ± 13.69 vs 105.50 ± 31.94 ng/ml, p<0.0001），血浆GHK水平不仅随着矽肺临床分期增加而减少，且与FEV1 %pred（p＜0.0001）、DLCO %pred（p=0.0002）以及SOD水平（p=0.0005）明显正相关，和血浆TNF-α（p＜0.0001）水平明显负相关。

外源性补充GHK-Cu可以明显减轻矽肺小鼠肺部炎症和纤维化

通过H&E染色、肺泡灌洗液炎症细胞分类计数以及肺泡灌洗液炎症因子检测结果显示，腹腔注射高剂量GHK-Cu可以明显减轻矽肺小鼠肺部炎症；通过Masson染色、肺脏指数、Western Blot以及免疫组化等结果显示，腹腔注射高剂量GHK-Cu可以明显减轻小鼠肺部纤维化。

GHK-Cu可以缓解结晶型二氧化硅诱导的巨噬细胞氧化应激

通过活性氧染色、线粒体膜电位染色以及线粒体自噬染色检测结果显示高剂量的GHK-Cu可以明显减轻结晶型二氧化硅诱导的活性氧的产生、恢复线粒体膜电位的水平并且减轻线粒体自噬的水平，减轻结晶型二氧化硅暴露后巨噬细胞的氧化应激水平。

GHK-Cu可以和巨噬细胞PRDX6结合

通过生物素化免疫沉淀后蛋白质谱分析筛选出GHK-Cu可以和PRDX6结合；通过生物素化免疫共沉淀、表面等离子共振以及分子对接模拟结果显示GHK-Cu可以和PRDX6直接结合。

GHK-Cu通过PRDX6发挥抗氧化应激作用

通过si-RNA敲减RAW264.7的PRDX6，重复活性氧检测以及线粒体膜电位检测，结果显示GHK-Cu缓解结晶型二氧化硅所致的氧化应激的水平的效果明显降低。

第三部分：

总共有250名疑诊为间质性肺疾病的患者最终纳入研究分析，非预置球囊组和预置球囊组各125人。非预置球囊组患者与预置球囊组患者的重度出血发生率无明显差异（1.6% vs 0.8%；adjusted p=0.520），但是非预置球囊组患者中度出血的发生率显著高于预置球囊组的患者（26.4% vs 6.4%，adjusted p=0.001）；非预置球囊组患者的止血药的使用率也显著高于预置球囊组的患者（28.0% vs 6.4%，adjusted p=0.001）。其中在非预置球囊组中有3名患者使用了后置的支气管球囊以进行大出血的止血。支气管镜医师在预置球囊组获取的样本显著多于非预置球囊组（3.8±0.9 vs 3.1±0.9，p＜0.001）。预置球囊组和非预置球囊组在多学科讨论诊断率没有明显的差异（89.6% vs 91.2%，adjusted p=0.182）。

结论

1. 抑制HDAC6可以减轻矽肺小鼠肺部炎症和纤维，同时可以减轻结晶型二氧化硅暴露后巨噬细胞的损伤和氧化应激。

2.HDAC6通过结合PRDX6并去乙酰化PRDX6增强结晶型二氧化硅暴露后的巨噬细胞氧化应激。

3. 外源性补充GHK-Cu可以减轻矽肺小鼠肺部炎症和纤维，同时可以减轻结晶型二氧化硅暴露后巨噬细胞的损伤和氧化应。

4. GHK-Cu通过结合PRDX6减轻结晶型二氧化硅暴露后的巨噬细胞氧化应激。

5. 预置球囊可以有效降低冷冻肺活检诊断间质性肺疾病中的中等量出血发生率并且提高支气管镜医师的取材信心。

Background and Objective

Silicosis, usually resulting from prolonged exposure to respirable silica particles, is one of the leading fatal occupational diseases in our country, characterized by respiratory dysfunction, extensive fibrotic-type nodules in lungs, and systemic inflammation. As the primary and most damaging form of pneumoconiosis, silicosis not only causes interstitial fibrosis, but also increases the risk of diseases such as tuberculosis, lung cancer, and various other autoimmune diseases. Despite the current global energy transition, dust-related occupations exposure still exist, what’s more the number of new cases of silicosis is showing an upward trend, mainly among workers in ceramics, glass and sandblasted jeans. In China, new cases of occupational pneumoconiosis account for more than half of all occupational diseases reported each year. Although silicosis is an ancient disease, the pathogenesis is still largely unclear. It is certain that alveolar macrophage damage plays an important role in the development of silicosis, with oxidative stress in these macrophages potentially being a significant factor. Furthermore, there is a lack of effective therapeutic drugs and treatments for silicosis, so it is crucial to continue the in-depth investigation of the pathogenesis of silicosis and development of effective treatment measures.

HDAC6 is a class II histone deacetylase, which is mainly found in the cytoplasm. Inhibition of HDAC6 has been shown to reduce the level of oxidative stress, and the complete loss of HDAC6 does not cause any significant phenotypes. Previous studies also suggested that targeting HDAC6 might have a therapeutic effect on lung adenocarcinoma and lipopolysaccharide-induced acute lung injury. Thus, targeting HDAC6 may be a promising treatment strategy for silicosis. GHK is a tripeptide naturally found in human blood, urine, and saliva. In plasma, GHK binds to copper ions to form a complex (GHK-Cu), which can more effectively fulfill its biological role. The anti-inflammatory and antioxidant effects of GHK-Cu have been clearly demonstrated in previous studies. Additionally, previous studies have also shown that GHK-Cu attenuates bleomycin-induced pulmonary fibrosis in mice. Therefore, we conjecture that GHK-Cu may have a therapeutic effect on silicosis.

Silicosis, as an occupation / environment-related interstitial lung disease, often requires pathologic confirmation for its diagnosis, and transbronchial lung cryobiopsy has been widely recommended for use in the diagnosis of interstitial lung diseases. As an invasive procedure, pre-placing of the bronchial balloon is one of the main measures to ensure the safety, but there were no studies exploring the role of pre-placing the bronchial balloon in the diagnosis of interstitial lung diseases using transbronchial lung cyrobiopsy.

The present study was divided into two parts: Part I: GHK-Cu attenuates silica-induced pulmonary inflammation and fibrosis by targeting alveolar macrophage PRDX6. Part II: Inhibition of alveolar macrophage HDAC6 attenuates silica-induced pulmonary inflammation and fibrosis by targeting PRDX6. Part III: Safety evaluation of preplaced balloons in patients with interstitial lung disease diagnosed by transbronchial lung crybiopsy.

Methods

Part I:

1. Silicosis mice model was constructed by giving crystalline silica suspension through the non-exposed airway. The expression level of HDAC6 was detected by Western Blot. The alveolar macrophages and HDAC6 were co-localized by immunofluorescence.

2. The silicosis mice was treated with the HDAC6-specific inhibitor CAY-10603. It was clarified that the inhibition of HDAC6 could reduce the inflammation and fibrosis in the lungs of the silicosis mice by the experiments of alveolar lavage cell counting, ELISA, lung tissue staining, and Western Blot.

3. Myeloid-specific HDAC6 knockout mice was constructed to clarify that targeted knockout of macrophage HDAC6 could attenuate silica-induced lung inflammation and fibrosis in silicosis mice.

4. Proteomic analysis of lung tissues from wild-type silicosis mice and lung tissues from myeloid-specific HDAC6 knockout silicosis mice was conducted to screen the possible mechanisms.

5. An in vitro silica-exposed model by RAW264.7 was conducted, and the expression of HDAC6 was clarified by Western Blot. The HDAC6-specific inhibitor CAY-10603 was subsequently used, and the level of oxidative stress in silica-exposed macrophages was measured by reactive oxygen species assay, mitochondrial membrane potential assay, and other experimental methods.

6. Protein profiling after immunoprecipitation was conducted to screen the binding protein of HDAC6 (PRDX6). Immunoprecipitation, immunofluorescence co-localization and molecular docking were subsequently conducted to clarify the binding of HDAC6 and PRDX6.

7. After knocking down PRDX6, reactive oxygen species detection, mitochondrial membrane potential staining and other experimental methods were subsequently conducted in the silica-exposed RAW264.7 model to clarify that HDAC6 acts by regulating PRDX6.

8. Immunoprecipitation was conducted to determinate the deacetylation of PRDX6 by HDAC6 after overexpression of PRDX6 combined with CAY-10603, and overexpression of both PRDX6 and HDAC6 in RAW264.7.

9. Macrophage-specific adeno-associated virus which was targeted knockdown of HDAC6 in mouse lung macrophages was constructed to further define the protective effect of inhibition of HDAC6 on silica-induced inflammation and fibrosis in mice.

Part II:

1. Plasma GHK levels were measured by high-performance liquid chromatography in healthy people and silicosis patients. Data of lung function and related plasma indexes of patients were collected and then used for correlation analysis.

2. Silicosis mice model was successfully constructed by drip infusion of silica suspension under laryngoscope-guided tracheal intubation, while low-dose (2 mg/kg) and high-dose (20 mg/kg) GHK-Cu were given through intraperitoneal injection for treatment, and the effects of GHK-Cu on inflammation and fibrosis of lung tissue were observed.

3. Using RAW264.7 cell line to construct a silica-exposed model to detect the effect of GHK-Cu on macrophages injury and oxidative stress in dust-stained macrophages.

4. Biotin was used to label GHK-Cu (Biotin-GHK-Cu) and protein profiling after biotinylated immunoprecipitation was conducted to screen the binding protein of GHK-Cu (PRDX6). Biotinylated immunoprecipitation, surface plasmon resonance, and molecular docking were conducted to verify the binding of GHK-Cu and PRDX6

5. After knockdown of PRDX6 in the silica-exposed RAW264.7 model which was also treated with GHK-Cu, oxidative stress-related indexes were detected to clarify whether GHK-Cu exerts antioxidant effect via PRDX6.

Part III:

A single-center prospective randomized controlled trial was conducted to investigate the effect of pre-placing bronchial balloons in the diagnosis of patients with interstitial lung disease by transbronchial lung cryobiopsy. Patients with suspected interstitial lung disease requiring pathological diagnosis for confirmation were randomized into two groups (pre-placed balloon and non-preplaced balloon groups) according to a computer-generated table of random numbers. The primary outcome was the incidence of moderate bleeding in both groups, and the secondary outcome were the incidence of severe bleeding, pneumothorax, and other transbronchial lung cryobiopsy-related complications.

Results

Part I:

1. High expression of HDAC6 in lungs and alveolar macrophages of silicosis mice.

A mouse model of silicosis was successfully constructed by administering crystalline silica suspension through the non-exposed airways, and uniform silicosis nodules were formed in the lungs. Western Blot showed that the expression of HDAC6 in the lungs of the mice with silicosis was significantly elevated. The results of immunofluorescence staining of macrophages and HDAC6 using F4/80 and HDAC6 antibodies demonstrated that the number of macrophages increased and the expression of HDAC6 was high and co-located with macrophages.

2. The HDAC6-specific inhibitor CAY-10603 significantly reduced lung inflammation and fibrosis in silicosis mice.

A mouse silicosis model was constructed by administering crystalline silica suspension to non-exposed airways. The results of H&E staining, Masson staining, Sirius red staining, Western Blot, BALF cell counting and BALF inflammatory factor detected by ELISA showed that compared with the wild-type model group, the lung inflammation and fibrosis levels were significantly reduced in the silicosis mice treated with CAY-10603.

3. HDAC6 myeloid-specific knockdown significantly reduced lung inflammation and fibrosis in mice with silicosis.

We successfully constructed HDAC6 myeloid-specific knockout mice, and a mouse silicosis model was constructed by administering crystalline silica suspension through the non-exposed airways. The results of H&E staining, Masson staining, Sirius red staining, Western Blot, BALF cell counting, and BALF inflammatory factor detected by ELISA showed that the levels of lung inflammation and fibrosis were significantly lower in the CKO model mice compared with those in the wild-type model group.

4. Proteomics showed that myeloid-specific knockdown of HDAC6 attenuated oxidative stress in the lungs.

KEGG enrichment of proteomic analysis of lung tissues from wild-type silicosis mice and HDAC6 myeloid-specific knockout silicosis mice showed significant changes in oxidative phosphorylation as well as mitochondrial autophagy-related proteins, which all responded to the level of oxidative stress to a certain extent, suggesting that the myeloid-specific knockout of HDAC6 can significantly affect the level of oxidative stress in lung tissues of mice with silicosis.

5. Both CAY-10603 and knockdown of HDAC6 could alleviate the level of oxidative stress in dust-stained RAW264.7.

An in vitro model of dust staining was constructed by RAW264.7, and Western Blot results showed that HDAC6 expression in dust-stained macrophages was significantly increased. Subsequently, the HDAC6-specific inhibitor CAY-10603 was used for the intervention, and the results of  the staining of reactive oxygen species, as well as mitochondrial membrane potential, demonstrated that the inhibition of HDAC6 could significantly attenuate the generation of reactive oxygen species in macrophages after dust staining, and recover the level of mitochondrial membrane potential, indicating that inhibition of HDAC6 could significantly reduce the level of oxidative stress in macrophages after dust staining. Knockdown of HDAC6 achieved similar experimental results as inhibition of HDAC6.

6. HDAC6 can bind PRADX6.

Protein profiling after immunoprecipitation screened that PRDX6, the top-ranked protein among the binding proteins, was significantly associated with oxidative stress. Subsequently, the binding of HDAC6 and PRDX6 was further verified by immunoprecipitation. HDAC6 and PRDX6 were overexpressed by plasmid, and co-localization of HDAC6 and PRDX6 was determined by immunofluorescence thereafter. Molecular docking was used to simulate the binding sites of HDAC6 and PRDX6.

7. Inhibition of HDAC6 can present anti-oxidative stress effect of via PRDX6.

Knockdown of PRDX6 by si-RNA in RAW264.7 cell line, followed by the staining of reactive oxygen species, as well as the staining of mitochondrial membrane potential staining in the RAW264.7 dust-stained model, revealed that the addition of the HDAC6 specific inhibitor, CAY-10603, was unable to attenuate the reactive oxygen species production the recover level of mitochondrial membrane potential.

8. HDAC6 acts in oxidative stress by deacetylating PRDX6

After overexpression of PRDX6-Myc in RAW264.7 and concomitant treatment with the HDAC6-specific inhibitor CAY-10603, immunoprecipitation was conducted with acetylation antibody, and we found that the acetylated PRDX6 was significantly increased, while the level of PRDX6 acetylation increased with the increase of CAY-10603 dose.

9. Targeted pulmonary macrophage adeno-associated virus knockdown of HDAC6 (AAV5-F4/80-HDAC6) significantly attenuated lung inflammation and fibrosis in silicosis mice.

Lung-targeted pulmonary macrophage adeno-associated virus was successfully constructed, and the infection was clarified by immunofluorescence after 3 weeks of infection. The silicosis mouse model was constructed after 3 weeks of AAV infection, and the results of H&E staining and Masson staining after 28 days showed that targeted knockdown of pulmonary macrophage HDAC6 significantly attenuated lung inflammation and fibrosis.

Part II:

1. Plasma GHK levels were significantly reduced in silicosis patients.

Plasma GHK levels were significantly lower in silicosis patients than in the healthy population (35.67 ± 13.69 vs 105.50 ± 31.94 ng/ml, p<0.0001), and were decreased with increasing clinical stage of silicosis. Plasma GHK levels were positively correlated with FEV1 %pred (p<0.0001), DLCO %pred (p=0.0002), and SOD levels (p=0.0005), while were negatively correlated with plasma TNF-α (p<0.0001) levels in silicosis patients;

2. Exogenous supplementation of GHK-Cu significantly attenuated lung inflammation and fibrosis in silicosis mice

The results of H&E staining, BALF inflammatory cell classification count, and BALF inflammatory factor assay showed that intraperitoneal injection of high-dose GHK-Cu could significantly reduce lung inflammation in mice with silicosis. The results of Masson staining, lung index, Western Blot, and immunohistochemistry showed that intraperitoneal injection of high-dose GHK-Cu could significantly reduce lung fibrosis in mice.

3. GHK-Cu alleviates silica dust-induced oxidative stress in macrophages.

The results of the staining of reactive oxygen species, the staining of mitochondrial membrane potential and the staining of the mitochondrial autophagy staining showed that a high dose of GHK-Cu could significantly alleviate the production of reactive oxygen species induced by silica, restore the level of mitochondrial membrane potential and reduce the level of mitochondrial autophagy, which alleviate the level of oxidative stress in the macrophage cells after silica exposure.

4. GHK-Cu binds to macrophage PRDX6.

Biotinylated immunoprecipitation followed by protein profiling showed that GHK-Cu could bind to PRDX6. Furthermore, biotinylated immunoprecipitation, surface plasmon resonance and molecular docking simulation showed that GHK-Cu could directly bind to PRDX6.

5. GHK-Cu exerts anti-oxidative stress effects by binding to PRDX6.

After knockdown of PRDX6 in RAW264.7 by si-RNA, the staining of reactive oxygen species, as well as the staining of mitochondrial membrane potential were conducted and the results showed that GHK-Cu alleviated the level of silica-induced oxidative stress significantly.

Part III：

A total of 250 patients with suspected interstitial lung disease were finally included in the study and analyzed, 125 in each of the non-preplaced balloon group and pre-placed balloon group. There was no significant difference in the incidence of severe bleeding between patients in the non-placed balloon group and patients in the pre-placed balloon group (1.6% vs 0.8%; adjusted p=0.520), but the incidence of moderate bleeding was significantly higher in patients in the non-preplaced balloon group than in patients in the preplaced balloon group (26.4% vs 6.4%, adjusted p=0.001). The use of hemostatic drug was also significantly higher in patients in the non-preplaced balloon group than in patients in the preplaced balloon group (28.0% vs 6.4%, adjusted p=0.001). Posteriorly placed bronchial balloons were used due to severe bleeding in 3 patients in the non-pre-placed balloon group. Bronchoscopists obtained significantly more samples in the preplaced balloon group than in the non-preplaced balloon group (3.8±0.9 vs 3.1±0.9, p<0.001). There was no significant difference in the rate of multidisciplinary discussion of the diagnosis between the preplaced balloon group and the non-preplaced balloon group (89.6% vs 91.2%, adjusted p=0.182).

Conclusion

1. Inhibition of HDAC6 attenuates lung inflammation and fibrosis in silicosis mice, as well as attenuates macrophage damage and oxidative stress after crystalline silica exposure.

2. HDAC6 enhances macrophage oxidative stress after crystalline silica exposure by binding to and deacetylating PRDX6.

3. Exogenous supplementation of GHK-Cu attenuates not only lung inflammation and fibrosis in silicosis mice, but also macrophage damage and oxidative stress after crystalline silica exposure.

4. GHK-Cu attenuates macrophage oxidative stress after crystalline silica exposure by binding to PRDX6.

5. A pre-placed bronchial balloon can reduce the incidence of moderate bleeding and increase the confidence of the bronchoscopists.