下丘脑室旁核TSPYL2激活GluN2A/B转录参与高血压发病的机制

Hypertension is one of the leading chronic diseases in China. With its rapid increase in young population, the society presents a soaring demand for an effective long-term control method for patients’ blood pressure. Yet there hasn’t been a satisfying answer. Recent researches have shown that injection of GluN2 (subunit of NMDA receptor) antagonist into the hypothalamic paraventricular nucleus (PVN) can reduce the blood pressure of the hypertensive rats. Since the transcription factor TSPYL2 has been shown to be critical for the expression of GluN2 subtypes, GluN2A/B, we hypothesize that interfering the transcriptional activation GluN2A/B by TSPYL2 can block GluN2A/B expression, reduce the number of NMDA receptors on the neurons in PVN and thus counteract the high blood pressure. In this project, effect of up-regulated GluN2A/B will be first tested by injecting lentivirus carrying GluN2A/B-overexpression vector into the PVN of normotensive rats; then similar procedure will be performed to test the effect of TSPYL2 overexpression on GluN2A/B transcription; also, shRNA designed to knock-down TSPYL2 will be inserted into a lentivirus vector and the virus will be injected into the PVN of the Spontaneous Hypertensive Rats (SHR) to determine if blocking TSPYL2 can reduce the transcription of GluN2A/B genes and lower the blood pressure; meanwhile, ChIP-qPCR, reporter gene assay and point-mutation assay will be performed with cultured hypothalamic cells, seeking the trans-acting-element(s) near GluN2A/B genes and the binding site(s) of TSPYL2 on these trans-acting-element(s) that enable TSPYL2 to activate GluN2A/B transcription. Successful completion of this project will provide ideas for the long-term treatment for hypertensive and new targets for gene therapy.

研究表明转录因子TSPYL2可能参与高血压发病时下丘脑室旁核神经元上NMDA受体亚基亚型GluN2A/B基因的转录激活，使NMDA受体数量增加、交感神经活动增强、血压升高，但其分子机制尚不清楚。本项目首先利用慢病毒介导的基因过表达上调正常大鼠室旁核GluN2A/B，观察对室旁核神经元功能、交感神经活动和血压的影响；其次分别通过基因过表达上调正常大鼠室旁核TSPYL2或通过干扰RNA下调高血压大鼠室旁核TSPYL2，观察对室旁核中GluN2A/B基因转录、交感神经活动和血压的影响；最后培养原代高血压大鼠下丘脑神经元，通过染色质免疫共沉淀、qPCR、报告基因检测、点突变检测等方法探寻高血压时GluN2A/B基因转录激活所需的顺式作用元件，以及TSPYL2在该顺式作用元件上的结合位点。本项目有望阐明高血压时室旁核中TSPYL2激活GluN2A/B基因转录的分子机制，为高血压治疗提供新的分子靶点。

高血压是严重危害我国国民健康的疾病之一。本项目中针对高血压发生发展的中枢机制，探讨了下丘脑室旁核中转录因子Tspyl2对N-甲基-D-天冬氨酸受体(NMDAR)的重要调控作用及该调控过程参与高血压发生发展的机制。首先在高血压大鼠中使用拮抗剂分别检验NMDAR的GluN2亚基两种亚型GluN2A/B在高血压发展过程中各自的作用，发现拮抗GluN2A可降低血压。其次在自发性高血压大鼠(SHR)的室旁核中分别敲减或过表达Tspyl2，发现其对GluN2A/B均有调控作用；敲减Tspyl2后血压下降、交感神经活动减弱，室旁核活性氧簇、炎性因子水平降低，GluN2A/B表达水平下降；而过表达Tspyl2后各指标变化不明显。再次在原代SHR下丘脑细胞中敲减Tspyl2或分别给予NMDAR、GluN1A/B阻断剂D-AP5、PEAQX、IFPD，四种处理后均发现神经元细胞钙离子内流减少、活性氧簇及炎性因子水平降低；而在正常血压的对照WKY大鼠下丘脑细胞中过表达Tspyl2后神经元细胞钙离子内流增加，炎性反应和氧化应激水平无显著变化；在WKY大鼠下丘脑细胞中过表达Tspyl2的同时给予D-AP5或PEAQX、IFPD后，神经元细胞钙离子内流情况、炎性反应和氧化应激水平均无显著变化，说明Tspyl2在维持高血压时神经元钙离子通道高水平开放中起重要作用，其主要通过调控NMDAR调节钙离子内流。最后，通过在SHR模型鼠室旁核给予PEAQX或IFPD、或在WKY大鼠室旁核中过表达Tspyl2的同时给予PEAQX或IFPD对细胞实验的内容进行了验证，证明Tspyl2通过调控NMDAR参与高血压发生发展。本项目明确了转录因子Tspyl2在高血压发生发展过程中具有的重要作用，并验证了其通过调控NMDAR影响血压的机制，为寻找能够以其为靶点调节心血管功能、治疗心血管疾病的新药物提供了新的思路及研究基础。