抗β2-肾上腺素受体自身抗体通过调控MMP-9表达参与COPD相关肺气肿的发生发展

COPD patients with severe emphysema have lower survival rate and there is no effective treatment yet. Protease-antiprotease imbalance is the main pathogenesis of COPD related emphysema, MMP-9 is one of the key proteases involved in this regulation. We previously showed that anti beta-2 adrenergic receptor autoantibodies (β2-AAbs) in the serum of patients with COPD increased and was positively correlated with RV% predicted. Rats serum β2-AAbs levels, lung MMP-9 expression were increased and showed emphysema-like changes in the lung after repeatedly immunized with polypeptide of rat beta 2-adrenergic receptor extracellular second loop. Stimulation of β2-AAbs in rat alveolar macrophage cell line caused increased MMP-9 expression through β2-AR. Passive smoking, active and passive immunization rat models will be applied in this research to determine the function of β2-AAbs in the occurrence and development of chronic obstructive related emphysema. Alveolar macrophages will be applied to observe the MMP-9 expression changes and explore the underlying molecular mechanism. The different signaling pathways will be compared between the agonist and β2-AAbs stimulation. The following chemical molecules, gene knockout intervention and small sample clinical study will be carried out to further clarify the relationship between β2-AAbs and emphysema, thus providing new clues for inhibiting the occurrence and development of pulmonary emphysema.

严重肺气肿导致COPD患者生存率下降，目前尚无有效治疗方法。蛋白酶-抗蛋白酶失衡是COPD相关肺气肿的主要发病机制，MMP-9是参与此调节的关键蛋白酶之一。我们前期发现COPD患者血清中抗β2-肾上腺素受体自身抗体(β2-AAbs)水平升高，与残气量占预计值百分比呈正相关；预实验使用β2AR胞外第二环肽段主动免疫大鼠，血清β2-AAbs水平升高、肺部MMP-9增加，出现肺气肿。用β2-AAbs刺激大鼠肺泡巨噬细胞系，可通过β2AR介导MMP-9表达。本研究拟制备烟雾暴露、主动、被动免疫大鼠模型，确定β2-AAbs在COPD肺气肿发生发展中的作用，观察肺泡巨噬细胞中MMP-9的表达变化规律，探讨β2-AAbs调节MMP-9表达的分子机制及与β2AR激动剂经典信号通路的差别。继采用化学分子、基因敲除等干预；进行小样本临床研究，进一步明确β2-AAbs与肺气肿的关系，为抑制肺气肿病程提供新依据。

背景：慢性阻塞性肺疾病（慢阻肺）是以不可逆的气流受限为主要特征的疾病，肺气肿是其重要的病理表现之一，肺气肿患者体内出现大量循环自身抗体并与肺气肿的严重程度相关，β2-肾上腺素受体自身抗体（β2-adrenergic receptor autoantibody，β2-AAbs）是否与肺气肿相关尚无报道，本研究通过对β2-AAbs在肺气肿中的作用进行深入研究，寻找改善肺气肿预后的方法。方法：1.慢性香烟烟雾暴露（CS）诱导大鼠肺气肿，观察血清β2-AAbs水平变化和与肺气肿的关系；观察β2-AR胞外第二环多肽主动免疫和主动免疫联合CS是否诱导或加重大鼠肺气肿，检测大鼠肺部基质金属蛋白酶-9 (MMP-9)表达，观察地塞米松和普萘洛尔干预是否降低血清β2-AAbs水平及改善肺气肿；2.检测吸烟者血浆β2-AAbs水平，分析其与肺气肿严重程度的关系；3.纯化β2-AAbs刺激肺泡巨噬细胞（Alveolar Macrophage, AM），检测下游经典和非经典通路的激活情况及MMP-9表达，干预β2-AR下游经典/非经典信号转导分子探讨β2-AAbs促进AM细胞MMP-9表达的机制。结果：1.CS大鼠血清β2-AAbs水平升高且与MLI正相关（r=0.370, P=0.036）；β2-AR胞外第二环多肽主动免疫大鼠血清β2-AAbs水平明显升高，MLI增大（77.05±2.65vs94.10±10.86μm），RV增大（0.99±0.29vs1.90±0.28ml），且肺组织MMP-9表达升高，主动免疫联合CS加重肺气肿且不能被地塞米松和普萘洛尔抑制；2.吸烟者血浆β2-AAbs水平与RV%pred及RV/TLC正相关（r=0.454, P＜0.001；r=0.455, P＜0.001），高血浆β2-AAbs水平组肺气肿评分更高；3.纯化的β2-AAbs促进AM细胞NF-κB磷酸化和MMP-9表达且不能被β-AR拮抗剂普萘洛尔抑制，siRNA敲减β-arrestin1/2可明显抑制β2-AAbs刺激后AM细胞NF-κB磷酸化及MMP-9表达。结论：1.吸烟者以及CS大鼠中，循环β2-AAbs水平与肺气肿相关；2.β2-AAbs主动免疫加重大鼠肺气肿，促进肺组织MMP-9表达；3.β2-AAbs通过β-arrestin2依赖方式促进AM细胞MMP9表达。