肺间质病变是系统性硬皮病重要并发症，易导致呼衰和死亡，临床尚无有效治疗方法。其发病机制未完全阐明，但炎症、免疫失控，巨噬细胞极化是其重要机制之一。我们的研究发现硬皮病肺组织中钙蛋白酶（calpain） 活性升高而 PI3K/AKT 信号通路下调，选择性抑制钙蛋白酶能上调PI3K活性、而敲除髓系细胞calpain能抑制巨噬细胞向M1型极化和减轻肺间质病变。已证实AKT是巨噬细胞极化的关键调控因子，本研究提出钙蛋白酶负调控PI3K/AKT信号从而诱导巨噬细胞向M1型极化，导致下游促炎症因子TNF-α等释放而促进肺间质病变的假说，将开展应用髓系细胞特异性calpain敲除小鼠明确钙蛋白酶对巨噬细胞极化的调控机制及在硬皮病肺间质病变中的作用地位。为阐明其发病机制，最终为临床早期防治提供新的靶点。

Interstitial lung disease is an important complication in patients with systemic scleroderma(systemic sclerosis,SSc), usually resulting in respiratory failure and death, however, there is no effective clinical treatment. The exact mechanism is still unclear, but macrophage polarization is one of the important mechanisms with uncontrolled inflammation and immune. Our preliminary research observed an increase of calpain activity and down-regulation of PI3K/AKT signaling in scleroderma-induced pulmonary lesion tissues. Furthermore, selective inhibition of calpain can prevent down-regulation the activity of PI3K in pulmonary lesion tissues, and knock-out of calpain in myeloid cells can inhibit the modulation of macrophage(Mφ) polarization to M1 type and reduce interstitial lung disease. It's known that AKT is a key regulator of macrophage polarization. In this grant, we hypothesize that calpain negatively regulates PI3K/AKT signal to induce macrophage polarization to M1 type, resulting in the release of pro-inflammatory cytokines of TNF-α and leading to scleroderma-associated pulmonary interstitial inflammation and fibrosis. To address this hypothesis, we put forth the following specific aims: using mice with myeloid cell-specific calpain deletion, we will investigate the exact mechanism of calpain-regulated macrophage polarization, and confirm the role of calpain in the scleroderma-associated interstitial lung inflammation and fibrosis in vivo. To clarify the pathogenesis and ultimately provide new potential targets for early clinical prevention and treatment of scleroderma associated interstitial lung disease.

肺间质病变（Interstitial lung disease，ILD）是导致系统性硬皮病（systemic scleroderma,SSc)）死亡的重要并发症，防治方法有限。发病机制不明，但炎症、免疫失控，巨噬细胞极化是重要机制之一。目的：探讨calpain在调控巨噬细胞极化及其在系统性硬皮病肺间质病变（SSc-ILD）发生中的作用。方法：本课题分别应用博来霉素诱导的SSc-ILD小鼠和RAW264.7巨噬细胞为模型，用组织染色确认肺组织和皮肤组织的炎症与纤维化程度，羟脯氨酸检测肺和皮肤组织的胶原蛋白含量，qRT-PCR检测mRNA，Western Blot和免疫组化检测蛋白质表达，流式细胞技术分析巨噬细胞极化类型，ELISA检测calpain活性，Transwell方法检测巨噬细胞移行情况。结果：第一部分：成功构建髓系细胞Capn4特异性基因敲除小鼠和SSc-ILD小鼠模型；calpain活性在SSc模型小鼠肺组织中比正常小鼠有显著增高(P<0.05）；与野生型对照组相比，SSc-ILD野生型组，纤维化相关的胶原面积率和相关羟脯氨酸显著增加(P<0.05）；PD150606对SSc-ILD小鼠干预后，与对照小鼠相比，BLM诱导的炎症程度显著减轻(P<0.05）；BLM诱导的SSc-ILD小鼠模型中巨噬细胞向M1极化，F4/80+MHCⅡ+细胞明显增加(P<0.05）；pAKT1和PI3K蛋白水平在Capn4-/-SSc-ILD与野生型SSc-ILD相比明显下降(P<0.05）。第二部分：不同浓度LPS和IL-4分别刺激巨噬细胞向M1和M2极化；M1、M2型巨噬细胞内calpain-mRNA表达明显不同，与PBS比较有差异(P<0.05）；在M1型巨噬细胞内，calpain2 蛋白表达明显增加(P<0.05）；而在M2型巨噬细胞内，calpain 2蛋白表达却明显减少（P<0.05）；Calpain2可能促进巨噬细胞向M1型极化；Calpain2可能通过促进NF-κB，抑制STAT3促进巨噬细胞向M1型极化；Calpain干预后巨噬细胞的迁移能力下降(P<0.05）。结论：Calpain能够调控巨噬细胞的极化并且对其干预抑制后肺部组织的炎症和纤维化均减轻；同样，Calpain2 可能通过 NF-κB/STAT3 信号通路促使巨噬细胞向 M1 极化，并调控巨噬细胞