脂氧素A4调控let-7c干预支气管肺发育不良的机制研究

Bronchopulmonary dysplasia (BPD) is one of the most serious complication and vexing challenges in premature infant around the world. A 'silver bullet' for BPD treatment probably does not exist at present. The transforming growth factor-β (TGF-β) superfamily has been shown to be essential for proper lung development and chronic lung diseases. Prolonged alterations of TGF-β1 signaling have been verifed to result in BPD. Lipoxin A4 (LXA4) is a lipid mediator generated during inflammation, promotes resolution of inflammation, and acts as an endogenous "braking signals" in inflammation process. In a pilot experiment, LXA4 was verified to ameliorate the structural changes in neonatal mice lung induced by hyperoxia and increase the expression of microRNA let-7c which has been shown to involved in both homeostasis and pathophysiology of lung development. Furthermore, our investigation suggested LXA4 inhibited the mRNA and protein expressions of TGF-β1 in MLE-12 cells. We therefore surveyed a computational algorithm (TargetScan) to identify distinct seed regions of let-7c in the 3'UTR of the TGF-β1-receptor type I (TGF-βR1) gene. Based on computational algorithms, 3'UTR of TGF-βR1 has been shown to be a direct target of let-7c and regulated by let-7c. Taken together, we speculate that the pathophysiologic intervention on BPD of let-7c induced by LXA4 was related to the reduced production of TGF-β1. However, the mechanisms by which LXA4 induced protective role on pulmonary hyperoxia injury remains unclear. Therefore, the object of this study was to investigate the effects of LXA4 on the regulation of let-7c/TGF-β1 signal pathway during hyperoxia lesion. Consequently, we will investigate mechanisms that lipoxin A4 attenuates hyperoxia injury via regulation of let-7c/TGF-β1 pathway by using overexpression, luciferase analysis, real-time reverse transcription polymerase chain reaction and western blot, etc. in an experimental model of BPD induced by prolonged exposure to hyperoxia, which strongly resembles BPD in premature infants. Our research provides the first evidence that LXA4 therapy might provide useful information in the future BPD prevention. Our findings also afford evidences and important theoretical basis for prevention and treatment by LXA4 analogs on BPD in premature.

支气管肺发育不良(BPD)是新生儿常见的危重疾病，治疗棘手。脂氧素A4 (LXA4)是机体最重要的抗炎、促消退介质，为炎症反应的刹车信号。我们的预试验发现：LXA4能够改善高氧诱导的肺发育异常，上调肺发育、肺损伤密切相关micro RNA let-7c的表达；LXA4还可抑制BPD发病关键信号分子转化生长因子β1 (TGF-β1)的表达；TGF-β1受体基因3'UTR区存在let-7c的保守结合位点。推测LXA4通过影响let-7c参与TGF-β1调控，从而干预BPD。本研究拟从分子、细胞、组织、器官和整体水平研究LXA4在BPD中的保护作用以及诱导let-7c/TGF-β1的信号机制。本课题具有源头创新性，为BPD治疗提供新的突破口，为应用LXA4类似物治疗新生儿BPD提供实验依据，具有重要的理论意义和临床应用前景。

【背景】. 支气管肺发育不良（BPD）是早产儿常见的慢性肺疾病，迄今缺乏有效治疗方法。脂氧素A4 (LXA4)是机体最重要的抗炎、促消退介质，能够改变肺发育、肺损伤密切信号通路TGF-β的表达。.【研究内容】.①LXA4抗炎、促进炎症消散作用，保护BPD所致肺损伤；.②LXA4诱导let-7c的过表达，抑制TGF-β1表达，干预BPD；.【结果】. LXA4提高高氧暴露MLE-12细胞的存活率、细胞活力和SOD活性；LXA4上调高氧暴露MLE-12细胞的抗氧化应激关键酶HO-1表达；LXA4抑制的高氧损伤炎性因子MCP-1和IL-6表达。LXA4处理组和TGF-β中和抗体组的MLE-12和NIH/3T3细胞高氧生存状况明显改善。与BPD模型组相比，LXA4处理组和TGF-β中和抗体组活细胞数目增多，凋亡细胞减少。LXA4组let-7c mRNA表达上调。LXA4干预的α-SMA、collagen I、fibronectin、tenascin-C以及TGF-βR1、TGF-βR2、Smad2、Smad3 mRNA表达降低，MMP-1升高，TIMP-1降低；TGF-β中和抗体干预的α-SMA、collagen I、fibronectin、tenascin-C以及TGF-βR1、TGF-βR2、Smad3 mRNA表达均明显降低，MMP-1升高。1D11、1D11+LXA4组、let-7c过表达组细胞的TGFβR1、Smad2、Smad3、Smad4、p-Smad2、p-Smad3蛋白的表达明显降低。体内实验证实，高氧环境新生小鼠生长状态不良，肺组织结构紊乱。LXA4与1D11干预的小鼠肺形态结构趋于正常。 LXA4组和1D11组fibronectin、α-SMA、elastin、tenascin-C、collagen I表达量降低。LXA4可恢复BPD组MMP-1/TIMP-1表达平衡；LXA4干预下调TGF-βR1和TGF-βR2的表达。.【意义】.研究阐述LXA4能够保护MLE-12细胞对抗高氧损伤，其机制可能涉及上调HO-1的表达，并抑制炎性因子MCP-1 和IL-6的表达。LXA4通过上调let-7c的表达，抑制TGF-β1信号通路下调肺纤维化相关因子实现高氧肺损伤的保护作用。本研究为新型抗炎药物LXA4保护与高氧肺损伤提供了实验依据，为BPD治疗提供新思路。