Brg1调控的信号通路在脓毒症心肌自适应中的作用及机制研究

The presence of cardiovascular dysfunction in sepsis is associated with much higher mortality rate compared with septic patients without cardiovascular dysfunction. Some clinical observations revealed that patients who could survive severe sepsis with cardiac dysfunction undergo acute left ventricle (LV) remodeling in respond to aggressive fluid resuscitation, the LV of survivors dilates and temporary reduces its ejection fraction in respond to aggressive fluid resuscitation, while those from non-survivors had an higher ejection fractions but smaller diastolic ventricular diameters. Adaptive LV dilation thus has been proposed to be important to sepsis patients with cardiac dysfunction. However, its molecular mechanism has not yet to be elucidated. Our preliminary data shows that the myocardial Brg1 expression is significantly reduced in LPS treated rats that was coincident with impaired LV diastolic dysfunction, and antioxdiant treatment mediated improvement of LV diastolic funciton is associated with enhancement of cardiac Brg1 expression. Further, we find that antioxidant mediated attenucation of LPS-induced cardiomyocyte toxicity and enhnacement of HO-1 expression can be cancelled by Brg-1 silencing with siRNA. Of note, Brg1 has been newly confirmed to play an indispensable role in heart development and remodeling, and can interact with Nrf2 to markedly enhance HO-1 induction in response to oxidative stress, and cardiac HO-1 induction has been shown to improve cardiovascular performance and survival in sepsis. We therefore hypothesize that Brg1 plays a critical role in inducing HO-1 expression in hearts that undergo remodeling in sepsis. To investigate this hypothesis, we will use the doxycyclin inducible cardiac specific Brg1 knockout mice to make in vivo septic animal model and treat with fluids. Also in vitro adult mouse cardiomyocytes will be treated with LPS(10μg/ml) incorporating Brg1 gene transfection ,Nrf2 or HO-1 activator and/or interference to identify the role of Brg1 and its interaction with the Nrf2/HO-1 signaling in mediating myocardial remodeling response to sepsis. The results of this research may provide a new perspective and experimental base for treatment of sepsis.

研究发现脓毒症患者若在液体治疗后左心室能适应性扩大的则生存率显著增高，其分子机制目前尚无报道。申请者前期研究发现左心室未适应性扩大的脓毒症大鼠心肌Brg1表达显著低于左心室扩大者，而抗氧化治疗能增加心肌Brg1表达，并显著改善脓毒症大鼠的心功能。鉴于Brg1是心肌重塑及维持心脏功能不可缺少的调节基因，并在氧化应激时是Nrf2诱导HO-1大量表达所必需的，而HO-1的抗氧化应激作用能减轻脓毒症心肌损伤，显著提高生存率，我们推测Brg1缺失引起的Nrf2/HO-1失活是容量治疗后左心室未扩大的脓毒症患者死亡率高的关键原因。本课题拟用特异性心肌Brg1敲除小鼠为研究对象，腹腔注射LPS制备脓毒症模型并模拟临床液体治疗及LPS诱导的离体心肌细胞脓毒症模型，并结合腺病毒转染及 siRNA技术，探讨Brg1在脓毒症心肌自适应中的作用及信号转导机制，为临床脓毒症心肌损伤的防治提供新的视角及实验依据。

研究发现脓毒症患者若在液体治疗后左室能适应性扩大的则生存率显著增高，其分子机制迄今国内外尚无报道。鉴于Brg1是心脏发育及重塑不可缺少的调节基因，并在氧化应激时是Nrf2诱导HO-1大量表达所必需的，而HO-1的抗氧化应激作用能减轻脓毒症心肌损伤，明显提高生存率，我们推测Brg1缺失引起的Nrf2/HO-1失活是容量治疗后左室未扩大的脓毒症小鼠死亡率高的关键原因。我们在正常小鼠CLP模型上观察脓毒症心肌损伤小鼠心肌Brg1表达的变化，以及与左室扩张、心脏功能、小鼠存活率的相关性。检测Nrf2、HO-1在脓毒症小鼠心肌组织中蛋白表达变化。同时检测心肌或血清氧化应激相关指标及炎症因子表达的变化。在LPS诱导离体培养成熟小鼠心肌细胞制备的脓毒症模型上探讨Brg1调控Nrf2/HO-1通路在脓毒症心肌细胞保护中的作用及分子机制。我们的研究结果发现CLP小鼠容量治疗后30小时，31.25%小鼠出现心室扩张。扩张小鼠72h生存率明显高于非扩张组（P<0.05,70% vs54.5%）。在30小时，与对照组比较，非扩张组血清中MDA 水平显著增高，抗氧化酶SOD活性明显降低，并伴随着心肌炎性因子IL-6 和TNF-alpha的水平显著升高（P<0.05），且非扩张小组心肌Brg1及 HO-1表达均较对照组降低。与非扩张组比较，扩张组血清中抗氧化酶SOD活性明显升高，MDA水平显著下降，同时心肌炎性因子IL-6 和TNF-alpha的水平显著降低（P<0.05），且心肌Brg1及 HO-1表达均较非扩张组明显增高（P<0.05），虽然扩张组HO-1表达仍低于对照组（P<0.05）。在LPS诱导离体培养成熟小鼠心肌细胞制备的脓毒症模型上，研究发现LPS在6小时引起Brg1和HO-1表达显著增高,在12h和24h 逐渐下降至低于基础水平（P<0.05），NAC可以显著升高Brg1和HO-1的表达并在24h内维持在一个较高水平（P<0.05）。然而Brg1基因沉默后逆转了NAC 的抗炎抗氧化的作用。以上研究结果表明Brg1调控的Nrf2/HO-1信号通路在脓毒症心肌损伤中起着重要的保护作用。此研究结果为探索脓毒症心肌损伤的发病机制提供了新的视角和实验依据。