延髓腹外侧头端炎症反应调控PER基因参与高血压时血压稳态失衡的机制研究

A disrupted homeostasis of blood pressure (BP) caused by circadian misalignment contributes to a significantly increased target organ damage in patients with hypertension. Therefore, it is particularly necessary to investigate the molecular mechanism that leads to the abnormal homeostasis of BP and explore the effectively therapeutic strategies. With this in mind, 24-hour ambulatory BP and H2S levels in hypertensive patients, 24-hour BP, renal sympathetic nerve activity, and H2S levels in SHR rats are determined to investigate the sympathetic over-activation in hypertension association with circadian BP rhythm and H2S levels. In order to clarify that sympathetic over-activation and the abnormal homeostasis of BP is due to the change of circadian clock gene in RVLM caused by central inflammation, the electrophysiological activity of neuron in RVLM was detected by patch clamp, the circadian clock gene expression and inflammatory factor levels were assessed by molecular biology techniques, such as Western blotting, RT-PCR. Silenced or over expressed the circadian gene expression using adenovirus transfection to elucidate the circadian gene expression are responsible for the sympathetic over-activation. Some techniques including immunohistochemistry and cell culture is used to confirm that the increased MPO activity of microglia is involved in the inflammatory response in the RVLM. In vitro, H2S and MPO protein are incubated together and various spectroscopy techniques (infrared spectroscopy, circular dichroism, UV-visible spectroscopy, Raman spectroscopy and X-ray absorption fine structure spectroscopy) are used to clarify the molecular mechanism that H2S inhibit the catalytic activity of MPO. This will shed light on the action targets of H2S regulates the circadian clock gene in RVLM by reducing inflammation, thereby inhibiting excessive sympathetic activation, maintaining the homeostasis of blood pressure, and mitigating the pathogenesis and development of hypertension. It will provide theoretical and experimental evidence for the effective treatment of hypertension and damage of target organ.

血压昼夜节律紊乱所致血压稳态失衡显著增加高血压患者靶器官损伤。探讨血压稳态失衡的发生机制，寻找有效的治疗手段尤为必要。本项目通过检测高血压患者24小时动态血压和H2S水平，SHR大鼠的24小时血压、肾神经放电和H2S水平，明确高血压时交感神经过度激活与血压节律紊乱、H2S水平改变有关。应用脑片膜片钳检测RVLM区神经元放电，western blot、RT-PCR检测RVLM区生物钟基因表达和炎症因子水平，并采用腺病毒调控相关基因的表达，明确中枢炎症导致的RVLM生物钟基因改变引起了交感过度激活，血压稳态失衡。应用免疫组化、细胞培养等技术，明确小胶质细胞MPO活性增高参与了RVLM区炎症反应。应用多种光谱学方法明确H2S可抑制MPO活性，进而减低炎症，调控RVLM区生物钟基因，抑制交感神经过度激活，从而维持血压的稳态，缓解高血压的发生发展，为有效治疗高血压、缓解靶器官损伤提供理论及实验依据。

血压昼夜节律紊乱所致血压稳态失衡显著增加高血压患者靶器官损伤。探讨血压稳态失衡的发生机制，寻找有效的治疗手段尤为必要。本项目拟在整体、组织、细胞、蛋白和分子水平探索RVLM区炎症介导的交感神经过度激活在血压稳态调控中的分子机制，观察H2S是否抑制小胶质细胞MPO活性，调节RVLM区炎症因子水平，恢复RVLM区生物钟基因表达的昼夜节律，抑制交感神经系统的过度激活，从而维持血压的稳态，缓解高血压的发生发展。我们通过检测高血压患者24小时动态血压和H2S水平，SHR大鼠的24小时血压、肾神经放电和H2S水平，发现高血压患者和SHR大鼠血浆H2S水平明显下降，交感紧张性活动明显增强，血压昼夜节律紊乱，血压稳态失衡，两者之间存在显著的相关性。随后SHR和WKY大鼠分别每4小时取RVLM区组织进行PCR-array实验，并通过WB实验进行验证，证实RVLM生物钟基因Per昼夜节律表达明显紊乱引起了交感神经过度激活，血压稳态失衡。应用免疫组化、细胞培养等技术，明确小胶质细胞MPO活性增高导致了RVLM区炎症反应，从而引起了RVLM区生物钟基因表达的改变。我们将不同浓度的H2S与MPO纯化共同孵育发现H2S可以在一定浓度范围内抑制MPO活性，在动物和细胞实验发现H2S可抑制RVLM区小胶质细胞M1型极化，减少小胶质细胞炎症因子的分泌，减轻中枢炎症，恢复生物钟基因的表达，降低交感紧张性，维持血压稳态，为有效治疗高血压、缓解靶器官损伤提供理论及实验依据。

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