MircroRNA-146a在吸入性损伤中的保护效应及机制研究

The mortality of smoke inhalation injury was high and easily lead to pulmonary fibrosis; Its mechanism has not been elucidated and lack of targeted treatment measures. The study showed NF-κB over-expression through the synthesis of inducible nitric oxide synthase and pro-inflammatory cytokine to mediate inflammatory response. TGF-β/Smad4 regulated alveolar type Ⅱ epithelial cells epithelial mesenchymal transformation (loss of steady-state), involved in pulmonary fibrosis. Our cell research showed: MiR-146a has the ability to negative regulate the activity NF-κB and TGF-β/Smad4. Therefore, we hypothesized that: exogenous MiR-146a administering can specifically inhibit smoke inhalation associated inflammation response and fibrosis. In our present study, with animal and cell model, using small animal CT, fluorescent tracer, protein double standard technology, to show whether MiR-146a has a protective effect on smoke inhalation injury. Furthermore, using the Northern blot, bioinformatics, target gene reporter vector validation experiments to state which target gene regulate the alveolar type Ⅱ epithelial cells and its underlying mechanism. In conclusion, our research provides new ideas for small molecule drug discovery in smoke inhalation injury and also lays the foundation for the clinical use of MiR-146a in the future.

吸入性损伤死亡率高，易导致肺纤维化；其发生机制尚未阐明，缺乏针对性治疗措施。研究发现NF-κB通过诱导一氧化氮合酶的合成和促炎因子的过表达，介导吸入性损伤的早期炎症反应；TGF-β/Smad4通过调控肺Ⅱ型上皮细胞上皮间质的转化（失稳态），参与肺纤维化。我们的细胞实验发现：MiR-146a可负性调控NF-κB及TGF-β/Smad4的活化。因此，我们推测：外源性MiR-146a可特异性抑制吸入性损伤的炎症反应及纤维化发生。本研究拟采用整体动物与离体细胞模型结合的方式，利用小动物CT、荧光示踪及蛋白双标等技术，明确MiR-146a对吸入性损伤是否具有保护效应；通过Northern blot、生物信息学预测、靶基因报告载体验证实验等方法研究MiR-146a通过何种靶基因调控肺Ⅱ型上皮细胞的稳态并阐明其作用机制；为吸入性损伤的小分子药物研发提供新思路，也为MiR-146a的临床转化奠定理论依据。

烟雾吸入性肺损伤的病死率高，发病机制复杂，主要是肺巨噬细胞NF-κB信号通路激活，诱导大量细胞因子释放，促使炎症反应失控，导致ALI/ARDS的发生；MiRNA作为新发现的一类内源性调控因子，广泛参与调控机体的各种应答。基于课题组前期研究结果，我们开展了烟雾吸入性肺损伤相关miR-146a和miR-155的研究。1初步筛选出miR-146a在巨噬细胞中的潜在作用靶点FAF2、Notch2、TMEM33，构建靶基因的荧光素酶报告质粒；结果显示miR-146a能与靶基因的3′-UTRs结合，从而抑制报告基因的表达。2建立香烟烟雾抽提物(CSE)刺激巨噬细胞模型：CSE感染能够明显诱导 RAW细胞分泌促炎细胞因子 IL-8、MCP-1、GRO-α (P<0.01)。3.当RAW细胞分别转染 miR-146a mimics或miR-Control，检测CSE感染后诱导的炎性因子IL-8、MCP-1、GRO-α的mRNA水平，结果表明miR-146a mimics组显著低于miR-Control (P<0.05)。4.建立烟雾吸入性肺损伤大鼠模型：结果显示烟雾致伤组明显的炎细胞浸润、充血及肺泡改变；肺湿干比及COX2的灰度值均大于正常组（P<0.01）。5大鼠miRNA芯片和RTA芯片联合分析显示烟雾致伤组miR-155对NFAT5具有靶向调控趋势；构建靶基因-荧光素酶报告质粒，结果显示miR-155与NFAT5 mRNA 3’-UTR互补结合。6烟雾致伤组NFAT5明显高于正常组（P<0.01。NFAT5基因敲除小鼠和正常组烟雾致伤后24小后，病理检测较正常组损伤明显减轻；肺组织NFAT5、P-P65和COX2表达明显低于正常组（P<0.01）。7病理检测结果显示烟雾致伤组+miRNA-155 mimics大鼠肺组织的炎症充血、炎症细胞的浸润、肺泡结构破环程度、NFAT5、P-P65、P65、COX2四种蛋白表达均较烟雾致伤组+miRNA-155 control和烟雾致伤组+miRNA-155 inhibitors明显弱，仅次于正常组；过表达miR-155可以减弱烟雾导致急性肺损伤相关蛋白COX2和炎症因子(TNF-α、MCP-1、IL- 1β、IL-8)的表达(P<0.05)。