BCL6抑制肺微血管内皮细胞焦亡对支气管肺发育不良肺血管新生的作用

Impaired vasculogenesis caused by chronic inflammation is the key cause of bronchopulmonary dysplasia (BPD). Inhibition of NLRP3 inflammasome activation can attenuate bronchopulmonary dysplasia. We have found that BCL6 inhibited NLRP3 transcription. In the following study, we showed the increased levels of NLRP3, caspase-1, Gasdermin D (key molecular of pyroptosis) and pro-inflammatory cytokines (IL-1β and IL-6), and the decreased expression of BCL6 in lung tissues of hyperoxia-induced newborn mice. Therefore, we hypothesis that BCL6 inhibits hyperoxia-induced pyroptosis in pulmonary microvascular endothelial cells (PMECs) and promotes angiogenesis through inhibiting NLRP3 transcription. In this project, wild-type neonatal mice and PMECs will be applied for hyperoxia stimuli and interventing BCL6 expression with BCL6 inhibitor and BCL6 overexpression plasmid in vivo and in vitro to determine the effects of BCL6 on hyperoxia-induced pyroptosis and angiogenesis. Secondly, hyperoxia-induced NLRP3 knockout mice will also be used to clarify the role of NLRP3-mediated pyroptosis in angiogenesis. Moreover, we will use the BCL6 inhibitor 79-6 to determine the role of BCL6/NLRP3 pathway in hyperoxia-induced pyroptosis and angiogenesis in NLRP3 knockdown mice. Finally, dual luciferase reporter assay will be performed to clarify the specific BCL6 domains that functions in inhibiting NLRP3 transcription. With the completion of this project, we will offer new strategy to inhibit lung inflammation and therefore treat patients suffered from BPD.

慢性炎症引发的肺血管发育异常是支气管肺发育不良(BPD)的关键诱因。抑制NLRP3炎性小体激活可减轻BPD。本人前期发现BCL6抑制NLRP3转录；预实验发现高氧诱导的BPD小鼠肺组织NLRP3炎性小体激活、焦亡关键蛋白和炎性因子增多及BCL6减少。据此，本人提出假说：BCL6通过抑制NLRP3转录、减轻BPD肺微血管内皮细胞(PMECs)焦亡促进肺血管新生。本研究拟分别利用BCL6抑制剂和BCL6过表达质粒干预高氧刺激的新生小鼠和PMECs中BCL6的表达，观察其对BPD焦亡及血管新生的作用；NLRP3基因敲除小鼠建立BPD模型观察焦亡及血管新生情况；对NLRP3基因敲除小鼠给予BCL6抑制剂，建立BPD模型后初探BCL6/NLRP3/焦亡在BPD中的作用；双荧光素酶报告基因验证高氧状态下BCL6抑制NLRP3转录的关键结构域。预期研究结果将为抑制肺部炎症改善BPD提供新方法。

慢性炎症引发的肺血管发育异常是引发支气管肺发育不良（BPD）的关键诱因。因此，抑制炎症促进肺微血管发育是治疗BPD的重要措施。本项目主要分成两大部分，分别探讨了（1）B细胞淋巴瘤6（BCL6）对高氧诱导的新生小鼠BPD的作用及机制；（2）短链脂肪酸（丙酸）对脂多糖（LPS）诱导的新生小鼠BPD的作用及机制。.一、BCL6通过抑制NLRP3介导的炎症反应减轻高氧诱导的新生小鼠BPD.我们利用高氧（95% O2）和腹腔注射LPS两种方法构建BPD小鼠模型。发现高氧和LPS均可抑制肺组织BCL6的mRNA和蛋白表达。过表达BCL6显著抑制了高氧引起的NLRP3炎性小体的激活和炎症反应，减轻了BPD小鼠的肺泡简化和肺血管生成障碍。同时，BCL6过表达促进了高氧诱导的HPMECs的增殖和血管生成，抑制了细胞凋亡和炎症反应。此外，BCL6抑制剂FX1抑制了BPD小鼠的生长发育。腹腔注射FX1的BPD小鼠表现出肺泡病理恶化和肺血管通透性的增加，促炎细胞因子和促纤维化因子的mRNA水平的上调。总之，我们发现BCL6抑制NLRP3介导的炎症反应，减轻高氧诱导的BPD小鼠的肺泡简化和肺血管发育障碍，我们认为BCL6可作为治疗BPD和新生儿相关疾病的靶点。.二、丙酸钠通过激活Nrf2介导的抗炎和抗氧化作用改善LPS诱导的新生小鼠BPD.我们使用WT、Nrf2-/-小鼠和肺微血管内皮细胞（HPMECs）分别在体内和体外研究丙酸钠（SP）对LPS诱导的新生小鼠BPD的作用及机制。在LPS诱导的新生小鼠BPD模型中，SP能显著增加Nrf2的表达，并且降低Keap-1的表达。SP减轻了LPS诱导的野生型小鼠肺部炎症和氧化应激，改善肺泡简化和肺血管新生异常，然而在Nrf2-/-新生小鼠中却并未观察到此现象。在LPS诱导的HPMECs中，我们观察到SP促进Nrf2入核，并且促进内皮细胞血管生成。除此之外，利用ML385（一种Nrf2特异性抑制剂）预处理，可抵消SP对炎症、氧化应激和血管生成的有益作用。该研究首次揭示了SP可通过激活Nrf2介导的抗炎和抗氧化通路改善LPS诱导的BPD小鼠肺泡的简化和异常血管新生。这些结果为利用SP预防BPD提供了理论依据。