脓毒性休克过程中生物钟基因Per2的作用机制

Recent studies have revealed that core mammalian clock genes regulate immune functions. Previously, we reported mPer2-/- mice displayed an altered and down-regulated circadian immune response to lipopolysaccharide (LPS). .Corticosterone (CORT), a hormone whose serum levels oscillate, plays a crucial role in immune suppression.When LPS was injected at two time points (ZT3 and ZT8) into mPer2-/- and wild type (WT) mice, respectively. In WT mice, CORT was significantly increased at 1 or 6 hours post LPS injection at ZT3 or ZT8. However, in mPer2-/- mice, CORT was significantly increased at 0.5 hour post LPS injection at ZT3 or ZT8. Similar earlier induction of mStAR (a rate-limiting regulatory gene for CORT production) expression was observed in mPer2-/- mice as compared to that in WT mice post LPS injection at ZT3 or ZT8. Expression of mStAR, mBmal1 and mClock in mPer2-/- mice are pronounced and long lasting compared to the ones in WT mice post LPS injection at ZT3 or ZT8, suggesting loss of mPer2 function rendered the expression of mStAR, mBmal1 and mClock more susceptible to LPS.In this study we will investigate whether CORT contributes to the circadian immune defect in mPer2-/- mice at more timepoint. Chromatin immunoprecipitation (ChIP) and luciferase assay will be used to examine whether CLOCK/BMAL1 heterodimer directly regulates mStAR expression via binding to two E-box elements in the mStAR's promoter region, or Per2 knockout enhanced the expression of mStAR through PPRE element in the mStAR's promoter region by the binding of PPAR-gamma protein. Our results will demonstrate mPer2's protective role in modulating induction of CORT production through clock-controlled mStAR's expression following LPS administration,and provide some insights into the endotoxemia resistance in mPer2-/- mice. The proposed studies will yield important information about the temporal and spatial relationship between circadian clock genes and immune regulatory genes, therefore, laying the ground work for deciphering how clock genes control immune functioning in host defenses.

脓毒性休克是机体炎症反应过度激活的后果，是世界范围内重症病人的最大杀手。生物钟在机体的病理感染过程中起重要作用，为感染条件下机体的防御提供了新的途径。我们前期研究发现Per2敲除小鼠对脓毒性休克反应具有更高的抵抗力，并且发现无论野生型还是Per2敲除小鼠，其死亡率和皮质酮水平升高呈现一定的相关性。在此基础上，本项目拟综合运用分子生物学、免疫学、表观遗传学等多学科的研究手段和技术，从正常小鼠到基因敲除小鼠到手术肾上腺切除小鼠，从生物钟调控环路和非生物钟调控环路角度，从动物行为、生理生化、分子水平系统研究生物钟基因Per2在脂多糖诱导的内毒素休克中作用的分子机制，尤其是从表观遗传修饰的角度研究生物钟基因在脂多糖诱导的脓毒性休克过程中的保护作用。本研究将为全面系统了解生物钟在先天免疫和宿主防御中的作用提供崭新的视角，特别是为内毒素诱导的脓毒性休克的治疗提供关键数据。

Per2敲除小鼠对脓毒性休克反应具有更高的抵抗力，但在Per2和免疫功能之间行使功能的具体分子及Per2对其的调控机制仍不清楚。通过项目执行，我们证明了皮质酮在LPS诱导的急性炎症反应中对Per2功能缺失小鼠的存活起保护作用，发现Per2功能缺失小鼠皮质酮合成的限速蛋白Star基因及生物钟基因(Clock/Bmal1)的mRNA表达显著升高，通过染色质免疫共沉淀技术证明了在Per2功能缺失小鼠中，生物钟调控环路的关键蛋白CLOCK:BMAL1异二聚体在LPS刺激的条件下对Star基因启动子区的转录调控元件的结合活性增强。在LPS刺激下，Clock功能缺失致使Clock功能缺失小鼠血清中皮质酮的产生和Star基因的表达在LPS刺激后没有得到显著升高，Clock功能缺失小鼠也相应地表现出了高死亡率，但是当我们在Clock功能缺失小鼠中注射皮质酮，能够显著提高Clock功能缺失小鼠的存活率。本研究证明了在LPS刺激下，Per2能通过生物钟调控环路调控Star基因的表达，介导了皮质酮的产生，表现出了对脓毒性休克反应具有更高的抵抗力，为全面系统了解生物钟在先天免疫和宿主防御中的作用提供崭新的视角。我们还发现在持续黑暗条件下，野生型小鼠肝脏中Star基因的表达呈现节律性，而Per2功能缺失小鼠肝脏中Star基因与核心节律基因的表达紊乱，生物钟调控环路的关键蛋白CLOCK:BMAL1异二聚体对Star基因启动子区转录调控元件的结合活性具有节律性，此结合的节律性与该调控元件24小时组蛋白H3K9乙酰化水平的模式呈现一致性，通过萤光素酶报告基因系统分析发现hHAT缺失的Clock蛋白对Star基因的转录调控活性变弱，我们认为Clock蛋白是通过hHAT结构域驱动了Star基因的24小时节律表达模式，并可能参与了一些胆固醇代谢产物的生理节律。此外，为了研究皮质酮合成的限速蛋白Star基因在不同物种中的功能，我们分析了硬骨鱼急性类固醇调节相关的脂转运基因（START）家族的功能分化。发现了硬骨鱼急性类固醇调节相关的脂转运基因（START）家族成员在不同组间、组内的非对称进化，并且发现通过硬骨鱼特异的基因组复制而来的35个复制对中有25个复制对的蛋白序列发生了不对称的快速进化，同一组内的START基因表达出现了分化，相互之间可能会补偿彼此的功能。