Effect of alveolar macrophage-derived exosome microRNA-380 on airway mucus hypersecretion in chronic obstructive pulmonary disease and its mechanism
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摘要:目的 探讨肺泡巨噬细胞来源外泌体中的miRNA-380（miR-380）对慢性阻塞性肺疾病（COPD）气道上皮细胞生物学功能的影响及其机制。方法 采用经鼻腔滴入脂多糖和熏香烟法建立大鼠COPD模型。将COPD大鼠肺泡巨噬细胞分为空白对照组、miR-380过表达（miR-380 mimic）组、miR-380低表达（miR-380 inhibitor）组及相应的载体对照组，提取并鉴定各组外泌体。将外泌体与COPD大鼠气道上皮细胞共培养，用蛋白质印迹法和/或免疫荧光法检测细胞黏蛋白（MUC）5AC、MUC5B、囊性纤维化跨膜传导调节因子（CFTR）、陷窝蛋白1（CAV-1）、磷酸化p38（p-p38）、磷酸化p65（p-p65）、磷酸化JNK（p-JNK）、磷酸化JAK（p-JAK）、磷酸化固醇调节元件结合蛋白（p-SREBP）表达，用ELISA法检测IL-1β、IL-6、TNF-α含量。对普通大鼠尾静脉注射过表达miR-380的外泌体（miR-380-OE-EXO），对COPD大鼠尾静脉注射miR-380拮抗剂（antagomiR-380），同时设置相应的对照组，取各组大鼠肺组织检测MUC5AC、MUC5B、p-p38、p-p65、p-JNK、p-JAK、p-SREBP表达，收集支气管肺泡灌洗液检测IL-1β、IL-6、TNF-α水平。结果 与对照组相比，miR-380 mimic组气道上皮细胞MUC5AC、MUC5B表达水平增高，CFTR表达水平降低，p65、p38、JNK蛋白磷酸化水平升高，炎症因子IL-1β、IL-6、TNF-α表达水平增高（均P＜0.05）；miR-380 inhibitor组上述蛋白和炎症因子变化趋势与miR-380 mimic组相反。经尾静脉注射miR-380-OE-EXO后的普通大鼠肺组织MUC5AC、MUC5B表达水平增高，肺组织p65、p38、JNK蛋白磷酸化水平以及支气管肺泡灌洗液中IL-1β、IL-6、TNF-α水平增高（均P＜0.05）。而经尾静脉注射antagomiR-380后的COPD大鼠肺组织MUC5AC、MUC5B表达下调，肺组织p65、p38、JNK蛋白磷酸化水平以及支气管肺泡灌洗液中IL-1β、IL-6、TNF-α水平也降低（均P＜0.05）。结论 COPD肺泡巨噬细胞通过旁分泌外泌体中miR-380促进气道上皮细胞黏液高分泌，其机制可能在于miR-380抑制上皮细胞CFTR功能，上调p65、p38及JNK磷酸化水平并促进IL-1β、TNF-α、IL-6等炎症因子分泌。Abstract:Objective To investigate the effect of alveolar macrophage-derived exosome microRNA-380 (miR-380) on the biological function of airway epithelial cells in chronic obstructive pulmonary disease (COPD) and its related mechanism.Methods A rat model of COPD was established by intranasal instillation of lipopolysaccharide combined with cigarette smoke exposure. Alveolar macrophages from COPD rats were assigned to blank control group, miR-380 overexpression (miR-380 mimic) group, miR-380 underexpression (miR-380 inhibitor) group, or corresponding vector control groups. Exosomes from each group were extracted and identified, then co-cultured with airway epithelial cells from COPD rats. The expression levels of mucin 5AC (MUC5AC), mucin 5B (MUC5B), cystic fibrosis transmembrane conductance regulator (CFTR), caveolin-1 (CAV-1), phosphorylated p38 (p-p38), phosphorylated p65 (p-p65), phosphorylated JNK (p-JNK), phosphorylated JAK (p-JAK), and phosphorylated sterol regulatory element-binding protein (p-SREBP) were detected by Western blotting and/or immunofluorescence. The levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay. Normal rats were injected with miR-380-overexpressing exosomes (miR-380-OE-EXO) via the tail vein, and COPD rats were injected with miR-380 antagonist (antagomiR-380) via the tail vein, with corresponding control groups set up simultaneously. Lung tissues were collected to detect the expression of MUC5AC, MUC5B, p-MAPK, p-NF-κB, p-JNK, p-JAK, and p-SREBP; and bronchoalveolar lavage fluid (BALF) was collected to detect the levels of IL-1β, IL-6, and TNF-α.Results Compared with the control group, the expression levels of MUC5AC and MUC5B in airway epithelial cells were significantly increased in the miR-380 mimic group, while the expression level of CFTR was significantly decreased. Additionally, the phosphorylation levels of p65, p38 and JNK and the expression levels of inflammatory cytokines IL-1β, IL-6 and TNF-α were significantly increased (all P<0.05). The levels of the above proteins and inflammatory factors in the miR-380 inhibitor group showed opposite trends to those in the miR-380 mimic group. In normal rats after miR-380-OE-EXO injection, the expression levels of MUC5AC and MUC5B in lung tissues were significantly increased, and the phosphorylation levels of p65, p38 and JNK in lung tissues and the levels of IL-1β, IL-6 and TNF-α in BALF were significantly increased (all P<0.05). In COPD rats after antagomiR-380 injection, the expression of MUC5AC and MUC5B in lung tissues was significantly decreased, and the phosphorylation levels of p65, p38 and JNK in lung tissues and the levels of IL-1β, IL-6 and TNF-α in BALF were also significantly decreased (all P<0.05).Conclusion Alveolar macrophages in COPD promote the hypersecretion of mucus in airway epithelial cells through paracrine exosomal miR-380. The underlying mechanism may be that miR-380 inhibits the CFTR function of epithelial cells, increases the phosphorylation levels of p65, p38 and JNK, and promotes the secretion of inflammatory mediators such as IL-1β, TNF-α, and IL-6.
慢性气道黏液高分泌（chronic mucus hypersecretion，CMH）是慢性阻塞性肺疾病（chronic obstructive pulmonary disease，COPD）具有特殊病理生理改变的临床表型，合并该表型的患者往往症状重、急性发作频率高、预后差[1-2]。目前对CMH发生机制的研究以炎症反应为核心，多数学者认同包括炎症细胞和氧化应激在内的多种因素参与调节CMH形成的观点[3]。气道上皮细胞在炎症细胞调控下发生的表型和功能改变是CMH发生的重要机制[4]。气道上皮细胞是肺组织分泌黏液的主要细胞，参与合成和分泌黏蛋白（mucin，MUC），其中MUC5AC和MUC5B是气道黏液的主要大分子物质[5-6]。外泌体在体内扮演着“邮差信使”的角色，通过传递如miRNA等信号分子进行细胞间的信号调控，其生物学功能很大程度上取决于来源细胞的类型与状态[7-8]。肺泡巨噬细胞是肺组织中数量最多、功能最重要的免疫细胞[9]。研究表明肺泡巨噬细胞来源外泌体可被上皮细胞内吞，从而发挥生物学效应[10]。囊性纤维化跨膜传导调节子（cystic fibrosis transmembrane conductance regulator，CFTR）是一种位于气道上皮细胞膜上的离子通道，参与氯离子和碳酸氢盐的运输，通过影响气道表面液体（airway surface liquid，ASL）调控气道黏液黏稠度，参与CMH形成[11-13]。有研究发现外泌体参与调控CFTR，例如Dutta等[14]证实miRNA-145可抑制CFTR，阻断miRNA-145信号通路可上调CFTR，缓解CMH与COPD患者症状。
本课题组前期研究发现COPD大鼠巨噬细胞可增强气道上皮细胞的增殖、迁移和分泌能力，进一步检测发现肺泡巨噬细胞来源外泌体中miRNA-380（miR-380）明显上调，运用生物信息学方法对miR-380进行靶基因预测发现miR-380对CFTR基因具有潜在调控作用[15]。基于此，本研究从外泌体中miR-380对CFTR和MUC5AC、MUC5B表达调控角度探讨肺泡巨噬细胞与气道上皮细胞间的相互作用方式，并挖掘CMH病理生理过程中涉及到的相关分子信号通路。
1 材料和方法
1.1 实验动物和主要试剂
SD大鼠48只，8~10周龄，体重为300~350 g，购自上海斯莱克实验动物有限公司[实验动物生产许可证号：SCXK（沪）2022-0004]。DMEM、FBS购自美国Gibco公司；PBS购自南京拜睿生物技术有限公司；Triton X-100、脂多糖（lipopolysaccharide，LPS）、DAPI、胰酶、JNK抑制剂SP600125、MAPK抑制剂SB203580、DEPC购自美国Sigma公司；组织细胞裂解液、蛋白酶抑制剂、四甲基联苯胺（tetramethylbenzidine，TMB）显色剂、4%多聚甲醛、BCA蛋白浓度测定试剂盒、聚凝胺（polybrene）均购自上海碧云天生物技术有限公司；PVDF膜购自美国Millipore公司；山羊抗兔IgG、山羊抗鼠IgG、CD63抗体、CD81抗体、MUC5AC抗体、MUC5B抗体、CFTR抗体、β-肌动蛋白抗体、陷窝蛋白1（caveolin-1，CAV-1）抗体、磷酸化固醇调节元件结合蛋白（phosphorylated sterol regulatory element-binding protein，p-SREBP）抗体均购自英国Abcam公司，磷酸化JNK（phosphorylated JNK，p-JNK）抗体、磷酸化JAK（phosphorylated JAK，p-JAK）抗体、磷酸化p38（phosphorylated p38，p-p38）抗体、磷酸化p65（phosphorylated p65，p-p65）抗体均购自美国Cell Signaling公司；miR-380 mimic、miR-380 inhibitor购自上海吉凯基因医学科技股份有限公司，antagomiR-380购自广州市锐博生物科技有限公司；IL-1β、TNF-α、IL-6 ELISA试剂盒购自上海酶联生物科技有限公司；NF-κB抑制剂BAY11、JAK抑制剂ruxolitinib购自美国MCE公司。
1.2 COPD大鼠模型的构建
采用鼻腔滴入LPS和熏香烟法构建大鼠COPD模型。于第1、29天经鼻腔滴入LPS（30 μg/6 μL），第2~30天（除第29天）熏香烟（10支/次，30 min/次，5 d/周，共4周）。处死并解剖大鼠，收集大鼠肺组织。大鼠肺组织在4%多聚甲醛中固定24 h以上，梯度乙醇依次脱水，浸蜡后放于包埋机内进行包埋，－15 ℃冷冻凝固后切片，厚度为4~5 μm。通过H-E染色和过碘酸-希夫（periodic acid-Schiff，PAS）染色观察肺组织病理学变化，鉴定模型是否构建成功。
1.3 细胞的分离、提取和培养
取COPD模型大鼠3只，解剖大鼠游离出气管和肺，用100目钢丝网研磨肺组织，200×g离心10 min，加入低渗液静置5 min，再次200×g离心10 min，洗涤细胞后400×g离心20 min，可见一层乳白色细胞层，即为巨噬细胞富集层，收集细胞，采用锥虫蓝染色检测肺泡巨噬细胞的细胞数和活力，配成合适浓度。用DMEM冲洗气管，收集液体用红细胞裂解液处理后200×g离心7 min，重悬细胞后接种于培养皿中，待大多数成纤维细胞贴壁后，吸取上清液至新培养皿中培养，得到气道上皮细胞。
1.4 肺泡巨噬细胞来源外泌体的获取
采用慢病毒感染肺泡巨噬细胞的方式调控细胞内miR-380表达，包括miR-380过表达（感染负载miR-380 mimic的慢病毒载体）组和miR-380低表达（感染负载miR-380 inhibitor的慢病毒载体）组，另设空白对照组、mimic对照载体（NC mimic）组、inhibitor对照载体（NC inhibitor）组。相关慢病毒的设计及构建由上海吉凯基因医学科技股份有限公司完成。吸取各组肺泡巨噬细胞上清液，采用超速离心法（先后以300×g离心10 min、29 500×g离心20 min、120 000×g离心90 min）提取外泌体，通过纳米颗粒跟踪分析（nanoparticle tracking analysis，NTA）对外泌体进行分析鉴定，用于后续实验。
1.5 气道上皮细胞相关实验
将纯化后的各组肺泡巨噬细胞外泌体50 μg加入细胞培养基中，与气道上皮细胞共培养24 h，检测相关蛋白和炎症因子的表达。在miR-380 mimic处理的基础上，加入NF-κB抑制剂BAY11、MAPK抑制剂SB203580、JNK抑制剂SP600125、JAK抑制剂ruxolitinib培养气道上皮细胞，检测相关蛋白和炎症因子的表达。
1.5.1 蛋白质印迹法检测MUC5AC、MUC5B、CFTR、CAV-1、p-p38、p-p65、p-JNK、p-JAK等蛋白的表达
待共培养至气道上皮细胞达90%左右融合度后将培养皿从培养箱中取出，用1 mL冷PBS洗涤细胞3次，向组织细胞裂解液中加入适量配制好的蛋白酶抑制剂和磷酸酶抑制剂，充分混匀后，缓慢加入细胞培养皿中，4 ℃裂解30 min，12 000×g离心15 min，收集上清液，使用BCA法测定蛋白浓度，经制胶、电泳、转膜、封闭、一抗孵育、洗膜、二抗孵育、洗膜后，滴加曝光液显影并采集图像，用ImageJ软件检测灰度值。
1.5.2 免疫荧光染色检测MUC5AC、MUC5B、CFTR、CAV-1蛋白的表达
收集细胞用EDTA-胰蛋白酶消化重悬并计数，爬片、浸洗、固定后，配制0.5% Triton X-100室温下通透20 min，再次浸洗及固定后依次加入一抗、二抗孵育，加入DAPI孵育5 min染核，用抗荧光淬灭封片剂封片，置于荧光显微镜下观察并采集图像。
1.5.3 qPCR法检测MUC5AC和MUC5B mRNA的表达
收集细胞提取总RNA，测定其纯度和浓度，使用反转录试剂盒进行第1链cDNA的合成，以β-肌动蛋白作为内参基因，进行qPCR检测。β-肌动蛋白正义链引物序列为5'-GGAGTACGATGAGTCCGGC-3'，反义链引物序列为5'-GTGTAAAACGCAGCTCAGTAACA-3'；MUC5AC正义链引物序列为5'-ACTCTACCACTCC-CTGCTTCTGC-3'，反义链引物序列为5'-TGAC-TAACCCTCTTGACCACCTG-3'；MUC5B正义链引物序列为5'-TTCTCTGTTGTGATTATGTGCCC-3'，反义链引物序列为5'-CTGTGATTCTGAGGATG-CTGTATG-3'。
1.5.4 ELISA法检测IL-1β、TNF-α、IL-6含量
收集细胞上清液，包被抗体后加入封闭液封闭，分别加入标准品与待测标本孵育2 h，洗板后加入检测抗体孵育1 h，洗板5次，每孔加入100 μL链霉亲和素，封板避光孵育20 min，洗板5次，每孔加入100 μL显色剂，封板避光孵育20 min，加入终止液。使用酶标仪测定450 nm波长处的光密度值，绘制标准曲线，计算待测炎症因子含量。
1.6 动物实验
将普通大鼠分为空白对照（control）组、尾静脉注射对照外泌体（control-EXO）组、尾静脉注射miR-380过表达外泌体（miR-380-OE-EXO）组，每组3只，予以相应的处理。将COPD模型大鼠分为模型对照（model）组、尾静脉注射PBS（model+PBS）组、尾静脉注射miR-380拮抗剂（model+antagomiR-380）组，每组3只，予以相应的处理。处死并解剖大鼠，收集大鼠肺组织和支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF），进行后续检测。
1.6.1 蛋白印迹法检测大鼠肺组织MUC5AC、MUC5B、p-p65、p-p38、p-JNK、p-JAK、p-SREBP等蛋白表达
取适量肺组织用蛋白酶抑制剂和RIPA液裂解（检测p-p65、p-p38、p-JAK、p-JNK和p-SREBP时需在裂解液中加入磷酸酶抑制剂），离心取上清液，制备总蛋白，使用BCA法测定蛋白浓度，经制胶、电泳、转膜、封闭、一抗孵育、洗膜、二抗孵育、洗膜后，滴加曝光液显影并采集图像，用ImageJ软件检测灰度值。
1.6.2 qPCR法检测大鼠肺组织MUC5AC和MUC5B mRNA表达
取肺组织样品1 520 mg，置于液氮预冷的研钵中磨碎，研磨过程中不断加入少量液氮，直到被磨为细粉状，后续总RNA提取、反转录、qPCR检测同1.5.3节。
1.6.3 ELISA法检测大鼠BALF中IL-1β、IL-6、TNF-α表达
取大鼠BALF进行ELISA检测，操作步骤同1.5.4节。
1.7 统计学处理
用SPSS 27.0软件进行统计学分析。所有实验均独立重复3次。计量资料以x±s表示，两组间比较采用独立样本t检验，多组间比较采用单因素方差分析（两两比较采用最小显著性差异法）。检验水准（α）为0.05。
2 结果
2.1 COPD大鼠模型的鉴定
H-E染色显示，与正常对照组相比，经LPS和持续烟熏处理的大鼠肺内气管、血管和肺泡周围出现大量炎症细胞浸润，管壁增厚，同时肺泡扩张甚至融合，提示COPD形成（图 1A）。PAS染色结果发现，与正常对照组相比，经LPS和持续烟熏处理的大鼠肺内气管、血管和肺泡周围出现白细胞浸润，肺泡壁增厚（图 1B）。
图 1 H-E和PAS染色检测大鼠肺组织病理学变化Fig. 1 Pathological changes in rat lung tissues detected by H-E and PAS stainingA: H-E staining of rat lung tissues; B: PAS staining of rat lung tissues. H-E: Hematoxylin-eosin; PAS: Periodic acid-Schiff; COPD: Chronic obstructive pulmonary disease.
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2.2 外泌体的鉴定
NTA粒径分析结果显示，各组样本颗粒粒径数据均为单峰且曲线流畅，无多余杂峰，各组外泌体粒径峰值为119.6~165.9 nm（图 2A），有些许粒径较大的颗粒可能与颗粒之间的黏附有关，结果符合外泌体直径40~160 nm左右的范围定义；蛋白质印迹法检测各组外泌体表面特异性标志蛋白CD63、CD81的表达均呈阳性（图 2B）。以上结果说明从COPD大鼠肺泡巨噬细胞中提取到的外泌体符合定义，可用于后续实验。
图 2 不同组别外泌体粒径分析及标志蛋白检测Fig. 2 Particle size and marker protein detection of different groups of exosomesA: Exosome particle size of each group was detected by nanoparticle tracking analysis; B: Western blotting was used to detect the expression of exosome markers CD63 and CD81 in each group. NC: Negative control; miR-380: MicroRNA-380.下载:
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2.3 外泌体中miR-380对气道上皮细胞的作用及机制
2.3.1 外泌体中miR-380对气道上皮细胞MUC、CFTR和CAV-1表达的影响
蛋白质印迹法检测结果（图 3A）显示，气道上皮细胞在与不同外泌体共培养后，miR-380 mimic组气道上皮细胞MUC5AC和MUC5B表达水平较空白对照组及载体对照组增高（均P＜0.01），而miR-380 inhibitor组气道上皮细胞MUC5AC和MUC5B表达水平较空白对照组及载体对照组降低（均P＜0.01）。免疫荧光染色结果（图 3B）显示，miR-380 mimic组气道上皮细胞MUC5AC、MUC5B蛋白的荧光信号强度较对照组增强，而miR-380 inhibitor组气道上皮细胞MUC5AC、MUC5B蛋白的荧光信号强度较对照组减弱。
图 3 各组气道上皮细胞MUC5AC和MUC5B表达Fig. 3 Expression of MUC5AC and MUC5B in airway epithelial cells of each groupA: Expression of MUC was detected by Western blotting; B: Expression of MUC was detected by immunofluorescence staining. **P<0.01. n＝3, x±s. MUC: Mucin; NC: Negative control; miR-380: MicroRNA-380.下载:
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蛋白质印迹法检测结果（图 4A）显示，气道上皮细胞在与不同外泌体共培养后，miR-380 mimic组气道上皮细胞CFTR表达较空白对照组及载体对照组下降，而miR-380 inhibitor组的气道上皮细胞CFTR表达水平较空白对照组及载体对照组增高（与空白对照组相比均P＜0.01）；各组气道上皮细胞CAV-1的表达水平差异均无统计学意义（均P＞0.05）。免疫荧光染色结果（图 4B）显示，miR-380 mimic组气道上皮细胞CFTR蛋白荧光信号强度较对照组减弱，miR-380 inhibitor组气道上皮细胞CFTR蛋白荧光信号强度较对照组增强；各组气道上皮细胞的CAV-1蛋白荧光信号强度区别不明显。
图 4 各组气道上皮细胞CFTR和CAV-1表达Fig. 4 Expression of CFTR and CAV-1 in airway epithelial cells of each groupA: Expression of CFTR and CAV-1 was detected by Western blotting; B: Expression of CFTR and CAV-1 was detected by immunofluorescence staining. **P<0.01. n＝3, x±s. CFTR: Cystic fibrosis transmembrane conductance regulator; CAV-1: Caveolin-1; NC: Negative control; miR-380: MicroRNA-380.下载:
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2.3.2 miR-380下游信号通路机制
蛋白质印迹法检测结果（图 5）显示，miR-380 mimic组气道上皮细胞NF-κB通路的p65以及MAPK通路的p38和JNK蛋白磷酸化水平上调（均P＜0.01），而miR-380 inhibitor组气道上皮细胞p38、p65、JNK的蛋白磷酸化水平下调（均P＜0.01）。各组气道上皮细胞的JAK和SREBP蛋白磷酸化水平差异无统计学意义（均P＞0.05）。在miR-380 mimic处理的基础上加入NF-κB抑制剂BAY11、MAPK抑制剂SB203580、JNK抑制剂SP600125、JAK抑制剂ruxolitinib培养气道上皮细胞，qPCR和蛋白质印迹法检测结果（图 6A、6B）显示miR-380 mimic组气道上皮细胞MUC5AC、MUC5B表达较空白对照组和载体对照组增强（均P＜0.05），而在miR-380 mimic组中加入NF-κB抑制剂、MAPK抑制剂或JNK抑制剂可下调MUC5AC、MUC5B的表达，但JAK抑制剂不具有该效果。此外，ELISA检测结果（图 6C）显示，相较于空白对照组和载体对照组，miR-380 mimic组气道上皮细胞炎症因子IL-1β、TNF-α、IL-6的分泌水平增高（均P＜0.01），而在加入NF-κB、MAPK、JNK或JAK通路抑制剂后炎症因子的分泌水平有所下降（均P＜0.05）。
图 5 各组气道上皮细胞下游信号通路蛋白磷酸化水平Fig. 5 Phosphorylation levels of downstream signaling pathway proteins in airway epithelial cells in each groupThe phosphorylation levels of signaling pathway proteins were detected by Western blotting. **P<0.01. n＝3, x±s. NC: Negative control; miR-380: MicroRNA-380; p-: Phosphorylated; JNK: c-Jun N-terminal kinase; JAK: Janus kinase; SREBP: Sterol-regulatory element binding protein.下载:
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图 6 信号通路抑制剂对各组气道上皮细胞MUC表达和炎症因子分泌的影响Fig. 6 Effects of signaling pathway inhibitors on MUC expression and inflammatory cytokine secretion in airway epithelial cells of each groupA: MUC mRNA expression was detected by qPCR; B: MUC protein expression was detected by Western blotting; C: Inflammatory cytokines were detected by ELISA. *P<0.05, **P<0.01. n＝3, x±s. BAY11: NF-κB inhibitor; SB203580: MAPK inhibitor; SP600125: JNK inhibitor; Ruxolitinib: JAK inhibitor. MUC: Mucin; qPCR: Quantitative polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assay; MAPK: Mitogen activated protein kinase; JNK: c-Jun N-terminal kinase; JAK: Janus kinase; NC: Negative control; miR-380: MicroRNA-380; NF-κB: Nuclear factor κB; IL: Interleukin; TNF-α: Tumor necrosis factor α.下载:
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2.4 外泌体中miR-380对普通大鼠肺组织的作用及机制
2.4.1 外泌体中miR-380对普通大鼠肺组织MUC表达的影响
qPCR和蛋白质印迹法检测结果（图 7）显示，普通大鼠尾静脉注射miR-380-OE-EXO后肺组织中MUC5AC和MUC5B转录及表达水平较control组和control-EXO组升高（均P＜0.01）。
图 7 各组普通大鼠肺组织MUC5AC、MUC5B的表达Fig. 7 Expression of MUC5AC and MUC5B in lung tissues of normal rats in each groupA: MUC mRNA expression in rat lung tissues was detected by qPCR; B: MUC protein expression in rat lung tissues was detected by Western blotting. **P<0.01. n＝3, x±s. MUC: Mucin; qPCR: Quantitative polymerase chain reaction; EXO: Exosome; miR-380: MicroRNA-380; OE: Overexpression.下载:
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2.4.2 外泌体中miR-380对普通大鼠肺组织下游信号通路及BALF中炎症因子的影响
蛋白质印迹法检测结果（图 8A）显示，相较于control组和control-EXO组大鼠，尾静脉注射miR-380-OE-EXO后大鼠肺组织p38、p65、JNK蛋白磷酸化水平增高（均P＜0.01），而JAK和SREBP蛋白磷酸化水平各组间差异无统计学意义（均P＞0.05），该结果与细胞学实验结果一致。此外，ELISA检测结果（图 8B）显示，普通大鼠经尾静脉注射miR-380-OE-EXO后，BALF中炎症因子IL-1β、TNF-α、IL-6水平升高（均P＜0.05）。
图 8 各组普通大鼠肺组织中信号通路蛋白磷酸化水平和BALF中炎症因子水平Fig. 8 Phosphorylation levels of signaling pathway proteins in lung tissues and levels of inflammatory factors in BALF of normal rats in each groupA: The phosphorylation levels of signaling pathway proteins in rat lung tissues were detected by Western blotting; B: The levels of inflammatory factors in rat BALF were detected by ELISA. *P<0.05, **P<0.01. n＝3, x±s. BALF: Bronchoalveolar lavage fluid; ELISA: Enzyme-linked immunosorbent assay; EXO: Exosome; miR-380: MicroRNA-380; OE: Overexpression; p-: Phosphorylated; JNK: c-Jun N-terminal kinase; JAK: Janus kinase; SREBP: Sterol-regulatory element binding protein; IL: Interleukin; TNF-α: Tumor necrosis factor α.下载:
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2.5 antagomiR-380对COPD大鼠肺组织的作用及机制
2.5.1 antagomiR-380对COPD大鼠肺组织MUC表达的影响
qPCR和蛋白质印迹法检测结果（图 9）显示，经尾静脉注射antagomiR-380后，COPD大鼠肺组织中MUC5AC和MUC5B转录及表达水平较2个对照组降低（均P＜0.01）。
图 9 各组COPD模型大鼠肺组织MUC5AC、MUC5B的表达Fig. 9 Expression of MUC5AC and MUC5B in airway epithelial cells of COPD model rats in each groupA: MUC expression was detected by qPCR; B: MUC expression was detected by Western blotting. **P<0.01. n＝3, x±s. COPD: Chronic obstructive pulmonary disease; MUC: Mucin; PBS: Phosphate-buffered saline; qPCR: Quantitative polymerase chain reaction; antagomiR-380: Antagonist of microRNA-380.下载:
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2.5.2 antagomiR-380对COPD大鼠肺组织下游信号通路及BALF中炎症因子的影响
蛋白质印迹法检测结果（图 10A）显示，尾静脉注射antagomiR-380后，COPD大鼠肺组织p38、p65、JNK蛋白磷酸化水平较2个对照组降低（均P＜0.01），JAK和SREBP蛋白磷酸化水平在各组间差异无统计学意义（均P＞0.05）。此外，ELISA检测结果（图 10B）显示，尾静脉注射antagomiR-380后，COPD大鼠BALF中炎症因子IL-1β、TNF-α、IL-6的水平较2个对照组降低（均P＜0.05）。
图 10 各组COPD大鼠肺组织中信号通路蛋白磷酸化水平和BALF中炎症因子水平Fig. 10 Phosphorylation levels of signaling pathway proteins in lung tissues and levels of inflammatory factors in BALF of COPD rats in each groupA: The phosphorylation levels of signaling pathway proteins in rat lung tissues were detected by Western blotting; B: The levels of inflammatory factors in rat BALF were detected by ELISA. *P<0.05, **P<0.01. n＝3, x±s. COPD: Chronic obstructive pulmonary disease; BALF: Bronchoalveolar lavage fluid; ELISA: Enzyme-linked immunosorbent assay; PBS: Phosphate-buffered saline; antagomiR-380: Antagonist of microRNA-380; p-: Phosphorylated; JNK: c-Jun N-terminal kinase; JAK: Janus kinase; SREBP: Sterol-regulatory element binding protein; IL: Interleukin; TNF-α: Tumor necrosis factor α.下载:
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3 讨论
CMH是COPD常见的病理生理改变之一，给患者带来巨大的痛苦和负担。研究显示合并CMH的COPD患者急性加重频率及死亡风险更高，MUC可作为预测COPD进展及预后的潜在指标[16-17]。揭示CMH发生机制及改善CMH是未来COPD个体化治疗的重要方向。肺泡巨噬细胞和气道上皮细胞之间的相互作用与CMH的关键特征（如炎症反应、气流受限及黏液分泌等）密切相关[18]。越来越多的证据表明，外泌体中富集的某些miRNA在COPD进展过程中发挥重要作用。有研究发现与吸烟者相比，COPD患者外周血中miRNA-24-3p、miRNA-93-5p、miRNA-320a、miRNA-320b、miRNA-191-5p、miRNA-342-3p和miRNA-92a-3p的表达上调，而miRNA-3613-3p、miRNA-1273g-3p和miRNA-4668-5p的表达下调[19]。Tasena等[20]通过支气管活检的方法分析miRNA表达谱，确定了10个与CMH相关的miRNA，其中miRNA-31-5p和miRNA-708-5p的表达率较高，而miRNA-134-5p、miRNA-146a-5p、miRNA-193-5p、miRNA-500a-3p和miRNA-1207-5p的表达率较低。
本课题组前期研究发现在COPD大鼠模型中，肺泡巨噬细胞外泌体中的miR-380表达上调，提示其可能作为信号物质参与调控气道上皮细胞功能[15]。本研究发现miR-380过表达的肺泡巨噬细胞来源外泌体可促进气道上皮细胞MUC5AC、MUC5B的表达，体外细胞学免疫荧光染色同样发现过表达miR-380组气道上皮细胞MUC5AC、MUC5B的表达增强，说明外泌体中的miR-380可促进气道上皮细胞黏液分泌，与CMH形成密切相关。在动物实验中，本研究同样发现外泌体中的miR-380在大鼠体内可促进肺组织MUC5AC、MUC5B的转录和表达。此外，本研究观察到在COPD大鼠模型中注射miR-380拮抗剂antagomiR-380后，大鼠肺组织中MUC5AC、MUC5B的转录和表达水平下调。这些研究结果表明外泌体中的miR-380参与调控COPD的CMH发病进程，而抑制外泌体中的miR-380表达或许可成为治疗COPD和缓解CMH的潜在方式。
生物信息数据库预测外泌体中的miR-380对CFTR基因具有潜在调控作用[15]。CFTR是一种广泛存在于上皮细胞表面的氯离子通道，参与对ASL的调控，CFTR功能障碍可促使高浓度和高黏弹性黏液的形成，致使黏液堵塞、不易咳出、诱导慢性细菌感染等[21]。先天性CFTR突变是囊性纤维化的常见致病基因，然而越来越多的研究发现在COPD患者中也可观察到获得性CFTR的功能障碍[13]。CFTR多与CAV-1共同存在于细胞膜脂筏结构上，构成内化平台，负责对感染等外界刺激产生免疫应答[22]。本研究证实外泌体中的miR-380可下调气道上皮细胞CFTR表达并促进MUC分泌，表明CFTR功能障碍与CMH病理过程密切相关，CFTR蛋白是CMH进展的负性调节器。但在本研究中，与miR-380过表达或低表达外泌体共培养后的气道上皮细胞中CAV-1的表达无明显差异，提示CAV-1在CMH发病机制中的功能仍不明确。
过度的肺部炎症反应被认为是COPD及CMH形成的关键因素，具体表现为免疫细胞的激活以及细胞因子和炎症介质的大量释放[3]。NF-κB信号通路在炎症反应中发挥重要作用，激活后的NF-κB通路可调节体内数十种炎症和其他生物学过程，因此，NF-κB表达的变化也被认为是机体炎症反应的标志物[23]。此外，MAPK、JNK、JAK等细胞内常见信号通路也广泛参与肺部炎症反应进程[24-26]。本研究发现外泌体中的miR-380能够促进气道上皮细胞IL-1β、IL-6、TNF-α等炎症因子的分泌，同时上调了NF-κB、MAPK及JNK信号通路蛋白的磷酸化水平；抑制外泌体中的miR-380可阻断气道上皮细胞NF-κB、MAPK及JNK信号通路的激活并下调炎症因子的表达，但不管是过表达或是抑制外泌体中的miR-380后对JAK和SREBP信号通路的影响都不明显。由此可见，在外泌体中的miR-380通过CFTR-MUC5AC/MUC5B轴调控CMH的分子通路中，伴随着NF-κB、MAPK及JNK通路的激活以及IL-1β、IL-6、TNF-α等炎症介质的释放，提示外泌体中的miR-380通过调控信号通路蛋白及炎症级联反应共同导致COPD和CMH的进展。
本研究的局限性在于未对外泌体进行透射电子显微镜形态学观察、动物实验中未对大鼠肺组织CFTR表达情况进行检测；研究观察到的各项平行指标变化不足以确立因果机制，关于miR-380-CFTR-MUC5AC/MUC5B以及下游炎症通路的激活等机制，仍需通过双萤光素酶报告基因实验以及免疫共沉淀等方法进一步证实；另外，在细胞及动物实验中未直接检测外泌体中的miR-380表达水平，未来研究将对提取的外泌体进行直接的miRNA测序或qPCR分析，以精确量化其内容物的变化。
综上所述，本研究通过实验证明了CMH的可能机制：肺泡巨噬细胞通过分泌富含miR-380的外泌体将miR-380转运到气道上皮细胞中，下调气道上皮细胞CFTR表达，并促进气道上皮细胞分泌MUC，此过程中伴随着NF-κB、MAPK、JNK信号通路的激活以及炎症因子IL-1β、IL-6、TNF-α的表达上调，从而诱发气道慢性炎症、加重CMH。此外，本研究发现miR-380拮抗剂在大鼠体内可降低肺组织MUC和炎症因子表达，具有潜在的治疗及缓解COPD和CMH的作用，该结果也将为研发新型COPD治疗药物提供一定的参考。
图 1 H-E和PAS染色检测大鼠肺组织病理学变化
Fig. 1 Pathological changes in rat lung tissues detected by H-E and PAS staining
A: H-E staining of rat lung tissues; B: PAS staining of rat lung tissues. H-E: Hematoxylin-eosin; PAS: Periodic acid-Schiff; COPD: Chronic obstructive pulmonary disease.
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图 2 不同组别外泌体粒径分析及标志蛋白检测
Fig. 2 Particle size and marker protein detection of different groups of exosomes
A: Exosome particle size of each group was detected by nanoparticle tracking analysis; B: Western blotting was used to detect the expression of exosome markers CD63 and CD81 in each group. NC: Negative control; miR-380: MicroRNA-380.
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图 3 各组气道上皮细胞MUC5AC和MUC5B表达
Fig. 3 Expression of MUC5AC and MUC5B in airway epithelial cells of each group
A: Expression of MUC was detected by Western blotting; B: Expression of MUC was detected by immunofluorescence staining. **P<0.01. n＝3, x±s. MUC: Mucin; NC: Negative control; miR-380: MicroRNA-380.
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图 4 各组气道上皮细胞CFTR和CAV-1表达
Fig. 4 Expression of CFTR and CAV-1 in airway epithelial cells of each group
A: Expression of CFTR and CAV-1 was detected by Western blotting; B: Expression of CFTR and CAV-1 was detected by immunofluorescence staining. **P<0.01. n＝3, x±s. CFTR: Cystic fibrosis transmembrane conductance regulator; CAV-1: Caveolin-1; NC: Negative control; miR-380: MicroRNA-380.
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图 5 各组气道上皮细胞下游信号通路蛋白磷酸化水平
Fig. 5 Phosphorylation levels of downstream signaling pathway proteins in airway epithelial cells in each group
The phosphorylation levels of signaling pathway proteins were detected by Western blotting. **P<0.01. n＝3, x±s. NC: Negative control; miR-380: MicroRNA-380; p-: Phosphorylated; JNK: c-Jun N-terminal kinase; JAK: Janus kinase; SREBP: Sterol-regulatory element binding protein.
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图 6 信号通路抑制剂对各组气道上皮细胞MUC表达和炎症因子分泌的影响
Fig. 6 Effects of signaling pathway inhibitors on MUC expression and inflammatory cytokine secretion in airway epithelial cells of each group
A: MUC mRNA expression was detected by qPCR; B: MUC protein expression was detected by Western blotting; C: Inflammatory cytokines were detected by ELISA. *P<0.05, **P<0.01. n＝3, x±s. BAY11: NF-κB inhibitor; SB203580: MAPK inhibitor; SP600125: JNK inhibitor; Ruxolitinib: JAK inhibitor. MUC: Mucin; qPCR: Quantitative polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assay; MAPK: Mitogen activated protein kinase; JNK: c-Jun N-terminal kinase; JAK: Janus kinase; NC: Negative control; miR-380: MicroRNA-380; NF-κB: Nuclear factor κB; IL: Interleukin; TNF-α: Tumor necrosis factor α.
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图 7 各组普通大鼠肺组织MUC5AC、MUC5B的表达
Fig. 7 Expression of MUC5AC and MUC5B in lung tissues of normal rats in each group
A: MUC mRNA expression in rat lung tissues was detected by qPCR; B: MUC protein expression in rat lung tissues was detected by Western blotting. **P<0.01. n＝3, x±s. MUC: Mucin; qPCR: Quantitative polymerase chain reaction; EXO: Exosome; miR-380: MicroRNA-380; OE: Overexpression.
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图 8 各组普通大鼠肺组织中信号通路蛋白磷酸化水平和BALF中炎症因子水平
Fig. 8 Phosphorylation levels of signaling pathway proteins in lung tissues and levels of inflammatory factors in BALF of normal rats in each group
A: The phosphorylation levels of signaling pathway proteins in rat lung tissues were detected by Western blotting; B: The levels of inflammatory factors in rat BALF were detected by ELISA. *P<0.05, **P<0.01. n＝3, x±s. BALF: Bronchoalveolar lavage fluid; ELISA: Enzyme-linked immunosorbent assay; EXO: Exosome; miR-380: MicroRNA-380; OE: Overexpression; p-: Phosphorylated; JNK: c-Jun N-terminal kinase; JAK: Janus kinase; SREBP: Sterol-regulatory element binding protein; IL: Interleukin; TNF-α: Tumor necrosis factor α.
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图 9 各组COPD模型大鼠肺组织MUC5AC、MUC5B的表达
Fig. 9 Expression of MUC5AC and MUC5B in airway epithelial cells of COPD model rats in each group
A: MUC expression was detected by qPCR; B: MUC expression was detected by Western blotting. **P<0.01. n＝3, x±s. COPD: Chronic obstructive pulmonary disease; MUC: Mucin; PBS: Phosphate-buffered saline; qPCR: Quantitative polymerase chain reaction; antagomiR-380: Antagonist of microRNA-380.
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图 10 各组COPD大鼠肺组织中信号通路蛋白磷酸化水平和BALF中炎症因子水平
Fig. 10 Phosphorylation levels of signaling pathway proteins in lung tissues and levels of inflammatory factors in BALF of COPD rats in each group
A: The phosphorylation levels of signaling pathway proteins in rat lung tissues were detected by Western blotting; B: The levels of inflammatory factors in rat BALF were detected by ELISA. *P<0.05, **P<0.01. n＝3, x±s. COPD: Chronic obstructive pulmonary disease; BALF: Bronchoalveolar lavage fluid; ELISA: Enzyme-linked immunosorbent assay; PBS: Phosphate-buffered saline; antagomiR-380: Antagonist of microRNA-380; p-: Phosphorylated; JNK: c-Jun N-terminal kinase; JAK: Janus kinase; SREBP: Sterol-regulatory element binding protein; IL: Interleukin; TNF-α: Tumor necrosis factor α.
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