心肌肥厚预适应通过调节成纤维细胞早衰上调Nrf2信号通路抑制心肌纤维化

Recently we first provided experimental evidence that preconditioning by prohypertrophic factors increases the resistance of the heart to subsequent hypertrophic stress and delays progression from hypertrophy and fibrosis to heart failure, indicting the existence of hypertrophic preconditioning phenomenon, implying that induction of hypertrophic preconditioning has the potential to become a new approach to protect cardioprotection for patients with pressure overload.,In this study, we focus on the possible mechanisms that myocardial hypertrophy preconditioning preconditioning exert effects of anti-fibrosis. Fibroblasts senescence triggered by Oxidative stress and other pathologic stimulation plays an important role in tissue fibrosis.In general, transient induction of senescence followed by tissue remodelling is beneficial, because it contributes to the elimination of damaged cells. Conversely, persistent senescence or the inability to eliminate senescent cells is detrimental. Nrf2 is known as a master transcription factor in cellular antioxidant and a negative regulator of cardiac pathological remodeling and dysfunction in diverse pathological settings.Previous report have demonstrated that, Nrf2 deficiency alters redox balance,which contributes to the acquisition of an apoptosis-resistant fibroblast phenotype. we hypothesize that Myocardial hypertrophy preconditioning inhibit myocardial fibrosis through regulation of premature senescence of myofibroblasts after transient short-term hypertrophic stimulation, as well as induce the expression of Nrf-2 render the heart resistant to subsequent hypertrophic stress, and slow the progression of myocardial fibrosis.We plan to perform cardiovascular physiologic and molecular biological experiments in mice subjected to hypertrophy preconditioning and subsequent pressure overload. This project contains 2 research parts: (1) To clarify that myocardial fibroblasts premature senescence as a regulatory mechanism of myocardial hypertrophy preconditioning; (2) If so, what are the changes on Nrf2-regulated signal pathway and the relative mechanism. We believe such studies will be of clinical significance to optimize hypertrophic preconditioning for clinical application

我们的前期实验首次证实了心肌肥厚预适应现象，即短暂的病理性肥厚刺激可通过诱导肥厚消退因子抵抗后续的持续肥厚刺激，发挥抗肥厚及心肌纤维化的作用。本实验延续前期研究着眼于肥厚预适应改善心肌纤维化的可能机制，氧化应激等病理刺激下成纤维细胞发生早衰在组织纤维化中起着重要作用，某些短暂的病理刺激因素下，细胞衰老有利于损伤细胞的清除，相反，持续存在的病理刺激促进细胞衰老但失去清除衰老的细胞能力则是有害的，已知Nrf2是调节细胞抗氧化应激的重要转录因子, 研究认为Nrf2可通过调节衰老细胞分泌表型在纤维化中发挥作用。因此我们假设肥厚预适应短暂间断的促肥厚刺激方式可以通过调节心肌成纤维细胞的早衰改善心肌纤维化，并通过上调Nrf2抑制后续持久的病理性肥厚刺激引起的心肌纤维化。本项目拟包括两个研究部分：（1）验证心肌肥厚预适应后心肌成纤维细胞早衰情况；（2）验证成纤维细胞早衰后Nrf2的表达及相关机制。

本实验中我们假设肥厚预适应短暂间断的促肥厚刺激方式可以通过调节心肌成纤维细胞的早衰改善心肌纤维化，并通过上调Nrf2抑制后续持久的病理性肥厚刺激引起的心肌纤维化。本项目拟包括两个研究部分：（1）验证运动肥厚预适应后心肌成纤维细胞早衰情况；（2）验证成纤维细胞早衰后Nrf2的表达及相关机制。因运动可以引起生理性心肌肥厚，我们以前曾报道过，病理性心肌肥厚的消退后仍会产生抗肥厚作用。因此，我们假设生理性肥厚消退后也存在抗肥厚性记忆，它对随后的病理性肥厚应激具有抵抗力。故我们在试验中改用运动作为预适应模型。方法和结果：采用成年小鼠进行游泳运动诱导生理性心肌肥厚、小鼠压力负荷模型、分离大鼠乳鼠以及小鼠乳鼠成纤维细胞进行了实验，检测组织中纤维细胞标志分子 α-SMA，衰老相关指标：SA-β-Gal，P16，P53 的表达情况，心肌组织中 Nrf2 的表达情况以及组织中 Nrf2 下游相关抗氧化基因的表达水平。直接心肌内注射重组腺病毒，敲低NRF2的表达水平，观察NRF2与心肌纤维化、细胞衰老指标的表达，及成纤维细胞增殖、凋亡情况。1、C57BL/6小鼠接受21天的游泳训练以引起生理性肥厚，在运动终止后1周消退。然后运动训练后小鼠与对照组分别接受横行主动脉缩窄（TAC）或假手术。TAC 1或4周后与对照组相比，明显改善了心肌纤维化，肺充血程度更低，左室（LV）内径和舒张期末压力较小，LV射血分数更大；2、运动组的心肌成纤维细胞衰老相关指标表达增加，EDU检测证实运动预适应组的心肌成纤维细胞相比对照组增殖增加，凋亡减少；同时心肌中Nrf2 及下游抗氧化基因表达升高，Nrf2的沉默减弱了运动预适应对接受TAC的小鼠和经血管紧张素II处理的成纤维细胞中的抗心肌纤维化作用。结论：运动预适应产生的短暂间断的促肥厚刺激方式可以通过调节心肌成纤维细胞的早衰改善心肌纤维化，并通过上调Nrf2抑制后续持久的病理性肥厚刺激引起的心肌纤维化。