基于线粒体生物发生的雌激素减轻脓毒症相关急性肺损伤作用机制研究

In sepsis, mitochondrial dysfunction triggered lung epithelial cell apoptosis pathway, which contributed to the development of ALI. Enhanced mitochondrial function by mitochondrial biogenesis decreased cell death, and therefore alleviated ALI. But the mechanism for regulating mitochondrial biogenesis in sepsis induced-ALI remains unknown. Estrogen was proved to increase cerebral vascular mitochondrial biogenesis by regulating PGC1β/NRF1/TFAM signaling pathway. Our pre-experiment revealed that estrogen might enhance the expression of lung PGC1β and play an important role in the attenuation of ALI. Thus, we hypothesized that estrogen might improve lung mitochondrial biogenesis by regulating PGC1β/NRF1/TFAM signaling pathway, which contributed to the underlying mechanism for estrogen-mediated attenuation of ALI. In this study, mitochondrial biogenesis, epithelial cell apoptosis and activation of PGC1β/NRF1/TFAM signaling pathway will be investigated in lung tissue from the rat model of LPS induced-ALI with/without ovariectomy. PGC1β siRNA will be used to evaluate the role of PGC1β in estrogen-mediated attenuation of pulmonary alveolar epithelial cells injury. Our study will explore the mechanism for regulating mitochondrial biogenesis in sepsis induced-ALI.

线粒体损伤诱导肺泡上皮细胞凋亡是脓毒症相关急性肺损伤（ALI）发病的重要机制。线粒体生物发生可以抑制线粒体损伤进而减轻ALI，然而其上游调控机制尚未明确。研究发现雌激素可以通过PGC1β/NRF1/TFAM信号通路增强脑组织的线粒体生物发生。我们的预实验提示雌激素能够增加肺组织PGC1β的表达，并能减轻脓毒症相关ALI。为此，我们推测雌激素可能通过PGC1β/NRF1/TFAM信号通路增强肺组织的线粒体生物发生，进而抑制肺泡上皮细胞凋亡，减轻脓毒症相关ALI。本课题将通过脂多糖诱导大鼠ALI模型，采用卵巢去势从动物水平观察雌激素对PGC1β/NRF1/TFAM信号通路、线粒体和ALI的影响；采用RNA干扰抑制人肺泡Ⅱ型上皮细胞系A549的PGC1β表达，深入探讨PGC1β在雌激素减轻脓毒症相关ALI中的作用机制，即线粒体生物发生调控机制。本研究的完成将为雌激素治疗ALI提供新的理论依据。

本课题假设PGC1β/NRF1/TFAM调控的线粒体生物发生是雌激素减轻脂多糖（LPS）诱导的脓毒症大鼠急性肺损伤（ALI）的重要机制。利用HE染色、肺干湿重和MPO检测研究雌激素的肺保护作用，发现雌性大鼠去势加重LPS诱导的脓毒症大鼠ALI，而给予雌激素减轻ALI。利用流式细胞术和TUNEL染色研究LPS诱导的肺泡Ⅱ型上皮细胞（ATⅡ）凋亡，发现雌激素减轻LPS诱导的ATⅡ细胞凋亡。利用电镜、线粒体荧光染色、mtDNA和ATP检测研究雌激素对线粒体数量和功能的调控，发现雌性大鼠去势后，LPS对大鼠肺组织线粒体数量和功能的抑制更严重，而给予雌激素减轻该作用。利用Realtime PCR和Western Blot研究雌激素对线粒体生物发生相关PGC1β/NRF1/TFAM信号通路的影响，发现雌性大鼠去势对肺组织PGC1β和NRF1 mRNA的表达没有影响，可显著降低NRF2和TFAM mRNA的表达；而给予雌激素可显著增强脓毒症大鼠肺组织PGC1β、NRF2和TFAM mRNA 和蛋白的表达。利用慢病毒介导的RNAi抑制ATⅡ细胞NRF2的表达，研究NRF2/TFAM信号通路对雌激素调节线粒体数量功能以及ATⅡ细胞凋亡的影响，发现雌激素通过NRF2/TFAM调控线粒体数量和功能，进而抑制LPS诱导的ATⅡ细胞凋亡。雌激素通过NRF2/TFAM调节ATⅡ细胞线粒体数量和功能，进而抑制ATⅡ细胞凋亡是雌激素减轻LPS诱导的脓毒症大鼠ALI的重要机制。