TRPC6对内毒素血症致心肌功能障碍的重要调控作用及其机制

Endotoxemia (ETM) is a leading cause of death in hospital intensive care units. Mortality rate can reach is 70-90% in ETM with cardiac dysfunction. Our previous study found that, lipopolysaccharide (LPS)-caused ETM mice showed cardiac dysfunction, following a high mortality rate. However, high mortality rate rarely happens, when TRPC6 knockout (Trpc6-/-) mice were challenged by LPS. These findings strongly indicated that TRPC6 may be a key molecular in myocardial dysfunction induced by LPS. Previous research found that inflammatory reaction, cell apoptosis and oxidative stress signaling pathways were importantly involved in ETM-caused cardiac dysfunction. This study will be performed by dual wavelength ion imaging technique, confocal laser scanning microscopy, patch clamp and molecular biology techniques. Also, pharmacological intervention with a combination of genetic strategy (such as RNAi technique, gene knockout) will be used to confirm the functional roles of TRPC6 in ETM-caused cardiac dysfunction. Further, the efforts will be put to explore and elucidate the novel regulatory mechanism in ETM-caused pathological cardiac dysfunction, which is mediated by TRPC6 through inflammatory reaction (MAPKs pathway), cell apoptosis (death receptor and mitochondrial pathways) and oxidative stress (NADPH-ROS pathway) signaling. The current study will provide the experimental basis to find the new target for prevention and treatment of ETM.

内毒素血症（ETM）是医院ICU患者死亡的首要原因，伴心功能障碍是ETM高死亡率的主因，死亡率可达70～90％。我们前期研究发现，脂多糖诱导的ETM小鼠出现心功能障碍并呈现出高死亡率，而在TRPC6基因敲除小鼠死亡却很少发生。提示：TRPC6可能是ETM致心肌功能障碍的关键分子。以往研究发现，炎症反应、细胞凋亡和氧化应激等信号途径在ETM致心肌功能障碍的发生发展过程中具有重要作用。本研究拟采用药理学和基因干预（基因沉默和敲除）相结合的手段，应用双波长离子影像和激光共聚焦等细胞影像技术、膜片钳及分子生物学技术，从分子、细胞和整体水平确证TRPC6在ETM致心肌功能障碍中的作用；探讨和阐明TRPC6通过炎症反应（MAPKs通路）、细胞凋亡（死亡受体或线粒体通路）以及氧化应激（NADPH-ROS通路）等相关途径对ETM时心肌功能障碍可能的调控新机制；以期为发现有效防治ETM的新靶点提供实验依据。

内毒素血症（Endotoximia, ETM）是现代临床危重病医学面临的棘手问题，本课题应用基因敲除策略以及免疫荧光染色，小动物超声影像，生存分析，WB、ELLSA、Co-IP、TUNEL法和基因组测序等实验技术方法，从分子、细胞、组织和整体水平探讨了TRPC6在ETM致心肌功能障碍中的作用，明确了TRPC6对其调控的机制：（1）LPS刺激后，WT小鼠呈现典型的ETM特征，心肌TRPC1/6蛋白表达呈现先升高后降低的变化趋势，3-4 h最显著；敲除TRPC6基因可显著提高小鼠生存率、延长生存时间、减轻心肌炎性病理损伤、改善心功能；（2）TRPC1/6经由炎症反应通路TLR4-MyD88-TRAF6，NF-kappaB和MAPKs（ERK1/2, p38, JNKs和ERK5）等，对ETM所致心肌功能障碍具有重要调控作用；（3）TRPC1/6可调控CaM的表达，进而与TLR4，MyD88和TRAF6蛋白等相互作用，调控心肌炎症反应；（4）TRPC1/6通过Mito凋亡通路（相关的Bax，Bcl-2和Caspase-9表达水平显著变化）和DR凋亡通路（相关的p53和Caspase-8及Caspase-3表达水平显著变化），对ETM所致心肌功能障碍具有重要的调控作用；（5）TRPC6通过调控NADPH酶核心亚基NOX-2，NOX-4的表达和ROS的产生，重要性地参与了ETM心肌功能障碍的进展。（6）TRPC抑制剂SKF96365可剂量依赖性地减轻ETM小鼠心肌组织炎性病理损伤、改善ETM小鼠心功能、提高其生存率。本课题在研究TRPC6的同时，对TRPC1在心肌功能障碍中的作用也进行了深入探讨。综合结果提示TRPC1/6是ETM心功能障碍治疗的新靶点；TRPC抑制剂是潜在的治疗药物，且尚无临床应用报道。本课题结果拓展了ETM致心肌功能障碍的分子机制，发掘了ETM有效治疗新靶点，为新药研发奠定了坚实的实验基础。