蛋白激酶Akt2调控巨噬细胞介导特发性肺纤维化的机制研究与新型药物靶标鉴定

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by a progressive lung fibrosis that is induced by non-special chronic interstitial pneumonia, which is currently lack of effective treatments. Therefore, it is of importance to determine the molecular and pathological mechanisms of IPF and identify new potential drug targets. Although protein kinase Akt2 is widely expressed in inflammatory cells, its role in IPF hasn’t been defined. Our supporting data not only reveal that the phosphorylation of Akt2 was significantly increased in alveolar macrophages of both pulmonary fibrosis patients and bleomycin-induced mouse pulmonary fibrosis, but also find that Akt2 deficiency ameliorated bleomycin-induced pulmonary fibrosis. Therefore, we will comprehensively determine the mechanisms of IPF through studying Akt2-mediated inflammatory microenvironment by the following specific aims: (1) To determine the role of Akt2 in bleomycin-induced pulmonary fibrosis and discover whether Akt2 regulates IPF through modulating macrophage functions; (2) To determine how Akt2-FoxO3a signal modulates IL-33-induced the production of IL-13 and TGF-beta; (3) To determine whether interference of Akt2 or IL-33 can inhibit bleomycin-induced pulmonary fibrosis. Collectively, our studies will not only reveal the novel molecular and pathological mechanisms that Akt2-FoxO3a signal regulates IPF via facilitating macrophages to produce IL-13 and TGF-beta, but also provide several novel drug targets for therapeutic intervention of IPF.

特发性肺纤维化（IPF）是由慢性间质性肺炎引起的进行性肺纤维化，最终导致病人死亡的严重呼吸系统疾病，尚无有效治疗药物，因此，阐明IPF病理机制和鉴定新型药物靶标具有重大意义。蛋白激酶Akt2虽广泛表达于炎症细胞，但是否参与调控IPF尚未见报道。依据前期研究结果，肺纤维化病人和肺纤维化小鼠模型的肺泡巨噬细胞Akt2磷酸化水平显著上调，博来霉素诱导肺纤维化损伤在Akt2敲除小鼠中明显减弱，本课题拟从Akt2介导的炎症微环境角度研究IPF病理进程，阐明Akt2调控IPF的病理机制；探明Akt2通过转录因子FoxO3a调节IL-33诱导IL-13和TGF-beta表达的分子机制；评价阻断Akt2或IL-33能否缓解IPF病理损伤。最终揭示信号通路Akt2-FoxO3a诱导巨噬细胞释放IL-13和TGF-beta等炎症因子，介导IPF损伤的分子病理机制，为IPF的治疗提供数个潜在新型药物作用靶标。

特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）属于发病原因不明的慢性进行性，不可逆转的间质性肺病，最终导致病人死亡。发病机理和分子机制复杂，目前临床上尚没有有效的治疗药物。因此，探索特发性肺纤维化的发病机制，开发有效治疗肺纤维化的药物靶点意义重大。大量研究证明早期肺损伤引起的炎症反应是特发性肺纤维化发病的重要原因之一。我们的研究主要通过AKT2, AKT1基因敲除小鼠，探究其在调控特异性肺纤维化中的作用及药物靶标鉴定。前期我们在临床肺纤维化病人肺组织标本和博来霉素诱导的小鼠纤维化肺组织内发现磷酸化AKT2，即pAKT2 (Ser474)的表达升高，且主要表达在巨噬细胞上。进一步我们通过利用博来霉素诱导小鼠肺纤维化模型，发现与野生型小鼠相比，博来霉素诱导的Akt2-/-小鼠，AKT1-/-小鼠肺组织内成纤维细胞聚集、细胞外基质均明显减少、胶原含量明显降低和羟脯氨酸含量明显减少，同时显示SMA和fibronectin的mRNA表达水平降低。生存率分析证实AKT2基因敲除后博来霉素诱导小鼠生存率显著增加。我们还发现AKT2， AKT1敲除后炎症反应明显降低，肺泡灌洗液蛋白浓度、细胞计数减少，肺组织MPO降低，以上结果表明AKT2，AKT1基因缺失能够减轻博来霉素诱导的肺纤维化及炎症反应。进一步的，体内实验检测发现AKT2缺失降低了博来霉素诱导的TGF-β1和IL-13的表达。使用IL-33或IL-4刺激巨噬细胞，比较野生型和AKT2基因缺失的骨髓来源巨噬细胞（BMDMs），发现AKT2缺失BMDMs内TGF-β1和IL-13产生减少，FoxO3a的丝氨酸318位点被明显磷酸化，而AKT2基因缺失骨髓巨噬细胞的FoxO3a磷酸化明显降低。抑制小鼠骨髓巨噬细胞内FoxO3a表达后，IL-33诱导产生的细胞因子IL-13明显增加。而AKT1缺失能抑制IL-13的表达。综上，我们研究结果表明AKT2，AKT1可能通过调控巨噬细胞产生与肺纤维化密切相关的细胞因子IL-13含量，最终调控特发性肺纤维化。我们的研究不仅阐明了巨噬细胞在肺纤维化中的作用，还为肺纤维的治疗提供了新的药物作用靶标。