LncRNA LXLOC_003839/miR-377/SIRT1通路调控巨噬细胞极化介导糖尿病合并脓毒症肠损伤的机制研究

Intestinal injury has become a major course of multiple organ dysfunction syndrome (MODS) and increased mortality in patients with diabetes and concomitant sepsis whose morbidity is rapidly increasing. Our preliminary study demonstrated that in mice with diabetes and concomitant sepsis the severities of intestinal injury and the symptoms of sepsis were significantly higher than those in non-diabetic mice, accompanied with higher mortality. We firstly identified that the level of a long noncoding RNA, LXLOC_003839 which we named as LncRNA-DSI was significantly overexpressed in the intestinal tissue of mice with diabetes and concomitant sepsis as compared to non-diabetic mice, which was accompanied by significant downregulation of miR-377 and SIRT1 and significant increase of the biomarkers of M1 type macrophages. In in vitro cell culture studies, LncRNA-DSI silence resulted in increases of miR-377 and SIRT1 expression which was accompanied by the increased transforming of M1 to M2 macrophages and alleviation of intestinal cell injury when co-cultured with the endotoxin and macrophages. We, thus, hypothesized that via miR-377-SIRT1 signaling regulates macrophage polarization-mediated intestinal injury in mice with diabetes and sepsis. We will establish sepsis models in non-diabetic and in db/db diabetic mice, and also in CD11c-DRT transgenic mice following the establishment of diabetes and the ensuing removal of M1 macrophages. The present study was designed to test the hypothesis in vivo and in vitro experiments in an effort to identify effective therapies for the treatment and prevention of intestinal injury in subjects with diabetes and sepsis.

糖尿病合并脓毒症患者日益增多；肠损伤是脓毒症患者发生多器官功能不全和死亡的重要原因。预实验发现糖尿病合并脓毒症小鼠肠损伤较非糖尿病严重，脓毒症状更重，死亡率更高；肠组织LncRNA LXLOC_003839(我们命名为LncRNA-DSI)显著上调，伴随miR-377及SIRT1显著下调及促炎M1巨噬细胞的标志物增加。体外细胞实验沉默LncRNA-DSI后，miR-377和SIRT1上调，M1巨噬细胞向抗炎的M2型转化，与内毒素及巨噬细胞共培养的肠粘膜上皮损伤减轻。据此，提出“LncRNA-DSI/miR-377/SIRT1通路调控巨噬细胞极化介导糖尿病合并脓毒症肠损伤”的科学假设。本研究拟建立正常与db/db糖尿病小鼠脓毒症模型及CD11c-DRT转基因小鼠建立糖尿病模型剔除M1巨噬细胞后的脓毒症模型，结合体外细胞学研究，验证假设，为糖尿病合并脓毒症肠损伤的防治提供有效的靶标和实验依据。

糖尿病合并脓毒症患者日益增多，肠损伤是脓毒症患者发生多器官功能障碍和死亡的重要原因。然其损伤机制未完全阐明,缺乏有效防治方法。巨噬细胞在组织损伤中的作用已得到确认，同时我们前期研究发现糖尿病合并脓毒症小鼠肠组织中巨噬细胞浸润明显上调，与肠损伤严重程度一致。在此基础上，我们提出：“糖尿病合并脓毒症肠损伤病程进展过程，由于LncRNA Gm16023表达上调，导致miR-377 和Sirt1表达降低，巨噬细胞大量向M1表型转化，导致肠损伤的发生、发展”的科学假设。为证实这个假设，本课题通过三个部分予以论证:1）观察糖尿病合并脓毒症小鼠不同时期肠损伤特点、巨噬细胞极化特征、炎症和氧化应激的变化；结果发现糖尿病小鼠发生脓毒症时肠损伤严重，同时M1表型的巨噬细胞增加，伴随炎症介质释放增加；肠损伤的严重程度与M1巨噬细胞呈正相关；2）应用野生型及CD11b-DTR转基因小鼠的在体实验、细胞学研究，结果发现巨噬细胞特异性剔除后可缓解糖尿病合并脓毒症肠损伤；3）通过细胞学研究，探讨LncRNA Gm16023/miR-377/Sirt1信号轴对巨噬细胞极化的调控作用，结果发现lncRNA Gm16023作为miR-377的miRNA海绵，促进Sirt1的表达从而抑制巨噬细胞向M1型极化。本研究从表观遗传学角度将LncRNA Gm16023/miR-377/Sirt1信号轴和巨噬细胞极化联系起来并进入深入研究，极大丰富了巨噬细胞极化的调控机制。这一新的学术思路，为糖尿病合并脓毒症肠损伤的防治提供新的理论依据和实验证据，为糖尿病合并脓毒症肠损伤的防治提供潜在的靶点。具有较大的理论意义与潜在的临床应用价值。