研究背景及目的： 急性肺损伤(acute lung injury，ALI)是以急性进行性及顽固性低氧血症为主要表现的临床常见疾病，当病情发展至重度即氧合指数<200，则为急性呼吸窘迫综合征(acute respiratory distress syn&ome，ARDS)。目前ALI／ARDS治疗方式涉及所有危重患者的一般支持性治疗(如控制感染、以早期肠内营养为目标的营养支持、控制血糖、应激性溃疡及深静脉血栓形成的预防等)以及适当的保护性机械通气策略。这些治疗手段在一定程度上提高了ALI／ARDS患者的生存率，但总体来说，ALI／ARDS的治疗效果并未达到预期，其临床死亡率仍在36-44％之间。因此，迫切需要对急性肺损伤的分子机制展开进一步的研究，从而帮助开发新型治疗方案。目前认为，ALI／ARDS是由于直接或间接的肺损伤导致的过度及难以控制的全身炎症反应引起，其主要病理生理学特征是炎症细胞的迁徙、纤维增殖和细胞凋亡的激活。因此在ALI／ARDS的治疗中，采取干预措施控制肺部炎症反应的“瀑布”效应，抑制炎症因子生成，减少肺泡上皮细胞的凋亡是其治疗的有效措施。MicroRNAs(miRNAs)可参与一系列的生命进程，如参与调控细胞的增殖、分化、迁移和凋亡，调节机体炎症反应、免疫功能及肿瘤生成等。研究发现某些miRNAs在ALI／ARDS细胞凋亡及炎症反应等相关过程中显著上调或下调并发挥相应功能。NF-κB作为炎症反应重要的转录因子，在调节促炎因子如TNF-α、IL-1β和IL-6的产生中起着至关重要的作用，Wnt／β-catenin信号通路则与细胞增殖凋亡密切相关。在肿瘤等相关领域，已发现miRNA-1246可调控Wnt／β-catenin及NF-κB信号通路参与细胞凋亡及炎症反应过程。因此，我们推测miRNA-1246可能通过Wnt／β-catenin及NF-κB信号通路介导ALI／ARDS诱导的细胞凋亡及炎症反应。我们将通过本实验明确miRNA-1246对ALI／ARDS的潜在调节作用及相关机制，进而为ALI／ARDS的防治提供新的方向及思路。 研究方法： 1.选择SPF级C57BL／6小鼠(5-6周龄，18-20g)20只，经气道滴入脂多糖(LPS)构建小鼠急性肺损伤模型，造模1天后处死小鼠，HE染色检测肺损伤程度，同时使用RT-qPCR法检测肺组织中miRNA-1246的表达变化。 2.体外培养人肺泡上皮细胞株(A549细胞)，分别转染miRNA-1246， anti-miRNA-1246和negative mimic入A549细胞，然后给予LPS刺激模拟ALI／ARDS时的肺泡上皮细胞反应，RT-qPCR验证转染后各组miRNA-1246的表达情况。 3.应用MTT法检测各组细胞增殖情况，LDH活性试剂盒检测各组细胞LDH活性。 4.应用Annexin V-PI双染法及流式细胞术检测各组细胞凋亡。 5.应用比色法检测各组凋亡因子Caspase-3／9活性。 6.应用Western blot方法检测各组Wnt及β-catenin蛋白表达。 7.使用Wnt抑制剂，观察各组细胞增殖与凋亡、凋亡因子Caspase-3／9水平、 Wnt及β-catenin蛋白表达变化，探讨miRNA-1246在ALI细胞凋亡中的调控作用及机制。 8.应用酶联免疫吸附测定(ELISA)法检测各组炎症因子IL-1β、IL-6及趋化因子CCL2、CCL5水平。 9.应用Western blot方法检测各组p-NF-κB及p-IκBα蛋白表达。 10.使用NF-κB抑制剂，观察IL-1β、IL-6、CCL2、CCL5水平及p-NF-κB、 p-IκBα表达变化，探讨miRNA-1246在ALI炎症反应的调控作用及调控机制。 实验结果： 1.LPS滴入小鼠气道1天后造模成功，ALI组表现出严重的组织病理学改变变化，包括肺泡出血、肺组织间隔的增厚等，而对照组肺组织结构完整清晰，气道粘膜上皮完整，肺泡大小均匀，未见明显炎性细胞浸润。 2.RT-qPCR法检测肺组织miRNA-1246的表达发现与对照组相比，LPS诱导ALI组其肺组织miRNA-1246的表达显著上调。 3.在ALI细胞模型中，转染miRNA-1246 mimic后miRNA-1246的表达较对照组显著上调。上调miRNA-1246表达可有效抑制细胞增殖，增加细胞LDH活性，增加细胞凋亡，提高caspase-3及caspase-9的活性。 4.与对照组相比，转染anti-miRNA-1246 mimic后miRNA-1246的表达下调。下调miRNA-1246表达有效促进细胞增殖，抑制LDH活性，减少细胞凋亡，抑制caspase-3及caspase-9的活性。 5.与对照组相比，miRNA-1246组Wnt和β-catenin蛋白表达水平下调， anti-miRNA-1246组Wnt和β-catenin蛋白表达水平上调。 6.使用Wnt抑制剂后可以有效抑制Wnt及β-catenin蛋白表达；同时，与未使用Wnt抑制剂的miRNA-1246组相比，使用Wnt抑制剂后可以进一步抑制细胞增殖、增加LDH活性、促进细胞凋亡、促进easpase-3及caspase-9的活性。 7.与对照组相比，miRNA-1246组IL-1β、IL-6、CCL2、CCL5水平较对照组显著增加，anti-miRNA-1246组IL-1β、IL-6、CCL2、CCL5水平下降。 8.与对照组相比，miRNA-1246的上调诱导ALI细胞模型中p-NF-κB蛋白表达，抑制p-IκBα蛋白表达，miRNA-1246的下调则抑制p-NF-κB蛋白表达，诱导p-IκBα蛋白表达。 9.与未使用抑制剂的miRNA-1246组相比，使用NF-κB抑制剂后p-NF-κB蛋白表达下调，p-IκBα蛋白表达上调；同时抑制NF-κB后IL-1β、IL-6、CCL2、 CCL5水平均下降。 结论： 1.在LPS诱导的ALI模型中存在miRNA-1246的表达上调。 2.miRNA-1246通过抑制Wnt／β-catenin信号通路参与LPS诱导的ALI中的细胞凋亡过程。 3.miRNA-1246通过活化NF-κB信号通路参与LPS诱导的ALI中的炎症反应过程。 关键词：急性肺损伤；miRNA-1246；细胞凋亡；炎症反应

 Background and Objective: Acute lung injury (ALI) is a common clinical syndrome characterized by progressive and intractable hypoxemia. When the disease progresses to severe, and oxygenation index <200, it is called acute respiratory distress syndrome (ARDS). Treatment modalities for patients with ALI/ARDS involves general supportive care prescribed for all critically ill patients (e.g., infection control, nutritional support with an aim for early enteral feeding, good glycaemic control, stress ulceration prophylaxis, and deep venous thrombosis) combined with focused ventilator strategies. These techniques have improved the prognosis of ALI/ARDS to a certain extent. But overall, the effect is not satisfactory, and the ALI/ARDS mortality rate is still above 40%. Therefore, there is an urgent need for further research on the molecular mechanisms of acute lung injury to help develop new treatment options. It is currently believed that ALI/ARDS is caused by excessive and uncontrolled systemic inflammatory response caused by direct or indirect lung injury, and its main pathophysiological features are migration of inflammatory cells, activation of fibroproliferation and apoptosis. Therefore, controlling the "cascade" reaction of inflammation in the lung, preventing the expansion of the inflammatory cascade, and reducing the apoptosis of alveolar epithelial cells are effective measures for the treatment of ALI/ARDS. MicroRNAs (miRNAs) can participate in a series of life processes, such as participating in the regulation of cell proliferation, differentiation, migration and apoptosis, regulating the body's inflammatory response, immune function, tumor formation and other responses. Studies have found that certain miRNAs are significantly up-regulated or down-regulated in ALI/ARDS cell apoptosis and inflammatory responses. NF-κB plays an important role in the regulation of the production of pro-inflammatory factors such as TNF-α, IL-1β and IL-6, and Wnt/β-catenin signaling pathway is closely related to cell proliferation and apoptosis. In tumors and other related fields, it has been found that miRNA-1246 regulates Wnt/β-catenin and NF-κB signaling pathways involved in apoptosis and inflammatory processes. Therefore, we hypothesized that miRNA-1246 may mediate ALI/ARDS-induced apoptosis and inflammatory responses via Wnt/β-catenin and NF-κB signaling pathways. Through this experiment, the potential regulation of miRNA-1246 on ALI/ARDS and related mechanisms were clarified, which provided a new direction and ideas for the prevention and treatment of ALI/ARDS. Methods: 1.TwentySPF grade C57BL/6 mice (5-6 weeks old, 18-20 g) were selected. The acute lung injury model was established by intratracheal instillation of lipopolysaccharide (LPS). After 1 day of modeling, we sacrificed mice and detected the degree of lung injury by HE staining, and the expression of miRNA-1246 in lung tissue by RT-qPCR. 2.Humanalveolar epithelial cell lines (A549 cells) were cultured in vitro and transfected with miRNA-1246, anti- miRNA-1246 and negative mimic into A549 cells. Then, LPS stimulation was given to simulate the alveolar epithelial cell response in ALI/ARDS. RT-qPCR was performed to verify the expression of miRNA-1246 in each group after transfection. 3.Theproliferation of each group was detected by MTT assay, and the LDH activity of each group was detected by LDH activity kit. 4.Apoptosiswas detected by Annexin V-PI double staining and flow cytometry. 5.Colorimetricassay was used to detect the activity of apoptotic factor caspase-3/9 in each group. 6.Western blot was used to detect the expression of Wnt and β-catenin protein in each group 7.Wntinhibitor were used to observe the cell proliferation and apoptosis, the level of apoptosis factor caspase-3/9, the expression changes of Wnt and beta-catenin protein in each group, and to explore the regulatory role and mechanism of miRNA-1246 in the apoptosis of ALI. 8.Thelevels of inflammatory factors IL-1β, IL-6 and chemokines CCL2 and CCL5 in each group were detected by enzyme-linked immunosorbent assay (ELISA). 9.Western blot was used to detect the expression of p-NF-κB and p-IκBα in each group. 10.NF-B inhibitor were used to observe levels of IL-1β, IL-6, CCL2, CCL5 and the expression of p-NF-κB and p-IκBα, and to investigate the regulation effect and mechanism of miRNA-1246 in inflammatory response of ALI. Results: 1.Lung tissues were collected at 1 day after LPS treatment in mice, and subjected to H&E staining. LPS-induced ALI group showed severe histopathologic changes, including alveolar wall thickness, pulmonary congestion and pulmonary congestion. 2.miRNA-1246 expression was effectively up-regulated in LPS-induced ALI group, compared with the control group. 3.In ALI cell model, the expression of miRNA-1246 was effectively increased using miRNA-1246 mimic, compared with the control group. Over-expression of miRNA-1246 effectively inhibited cell proliferation and increased LDH activity in comparison with the control group. However, up-regulation of miRNA-1246 effectively increased cell apoptosis and promoted caspase-3 and caspase-9 activity . 4.miRNA-1246 expression was reduced using anti-miRNA-1246 mimic, compared with the control group. Down-regulation of miRNA-1246 effectively increased cell proliferation, inhibited LDH activity ,decreased cell apoptosis and inhibited caspase-3 and caspase-9 activity in ALI cell model. 5.Over-expression of miRNA-1246 effectively suppressed Wnt and β-catenin protein expression, and the down-regulation of miRNA-1246 effectively induced Wnt and β-catenin protein expression in ALI cell model, compared with the control group. 6.Wnt and β-catenin protein expression was effectively inhibited by Wnt inhibitor. At the same time, Wnt inhibitor further inhibited cell proliferation, increased LDH activity, promoted cell apoptosis and increased the activity of caspase-3 and caspase-9 compared with miRNA-1246 group without Wnt inhibitor. 7.Over-expression of miRNA-1246 increased IL-1β, IL-6, CCL2 and CCL5 levels and down-regulation of miRNA-1246 decreased IL-1β, IL-6, CCL2 and CCL5 levels in ALI cell model, compared with the control group. 8.Up-regulationof miRNA-1246 induced p-NF-κB and suppressed p-IκBα protein expression. In contrast, down-regulation of miRNA-1246 suppressed p-NF-κB and induced p-IκBα protein expression in ALI cell model, compared with the control group. 9.Comparedwith miRNA-1246 group without inhibitor, the expression of P-NF-κB protein was down-regulated and the expression of p-IκBα protein was up-regulated after the use of NF-κB inhibitor. At the same time, the levels of IL-1β, IL-6, CCL2 and CCL5 decreased after the inhibition of NF-κB . Conclusions: 1.The expression of miRNA-1246 is up-regulated in the LPS-induced ALI model. 2.miRNA-1246may participate in the apoptosis process of LPS-induced ALI by inhibiting Wnt/β-catenin signaling pathway. 3.miRNA-1246may participate in the inflammatory process of LPS-induced ALI through activating NF-κB signaling pathway. Keywords: Acute lung injury; miRNA-1246; Apoptosis; Inflammation.