新生儿缺血缺氧性脑病中TRPC3通道经SOC介导的钙内流调控内质网应激的研究

Neonatal hypoxic- ischemic encephalopathy (HIE) is an important cause of disability ，however its pathogenesis is not entirely clear．We first find that TRPC3 channel has a protective effect on neurons．Even though ，neurons intracellular calcium ion concentration increased significantly during HIE，however, after reduce the elevated calcium concentration by TRPC3 specific blocker , there is an increased death of neurons．What’s more，it seems to inconsistent with the classical theory of calcium overload．Unfortunately ，there is no related reported that if there is a new regulation mechanism．We believe that the decrease of SOC -mediated calcium influx and the endoplasmic reticulum stress induced by down-regulation of TRPC3 may be an important cause of neuronal death in HIE．The present study intends to use pharmacological blockade，over- expression of TRPC3 gene on the newborn rat HIE model and cultured cortical neurons OGD model．By this way，we want to delineates the effects of TRPC3 on HIE/OGD-induced injury in cortical neurons，depth exploration the relationships between the activity of TRPC3，membrane SOC，endoplasmic reticulum calcium homeostasis and endoplasmic reticulum stress．Through this study we expected to find new ways of neonatal hypoxic-ischemic injury in neuronal death, thus providing new molecular targets of neuroprotective treatment for HIE.

新生儿缺血缺氧性脑病（HIE）是新生儿致死、致残的重要原因，其发病机制尚不完全清楚。我们首先发现TRPC3通道对神经元有保护作用；OGD神经元细胞内钙明显增加，但特异性阻断TRPC3通道以降低升高的钙浓度时，神经元死亡反而增加，这看上去似乎与经典钙超载学说不完全一致，是否存在新的调控机制，未见报道。我们认为，TRPC3通道蛋白下调可能使膜上SOC介导的钙内流减少、内质网应激可能是缺血缺氧神经元死亡的重要原因。本课题拟采用新生鼠HIE模型和体外培养皮层神经元的OGD模型，运用药理学阻断、基因沉默和过表达等实验方法，首次研究HIE/OGD时TRPC3通道在神经元损伤中的作用，深入探讨缺血缺氧中神经元TRPC3通道活性与细胞膜SOC钙内流、内质网钙稳态和内质网应激的相互关系。通过本研究有望寻找到缺氧缺血损伤所致新生动物神经元死亡的新途径，从而为HIE后神经保护治疗措施提供新的分子靶点。

新生儿缺血缺氧性脑病（HIE）是新生儿致死、致残的重要原因，其发病机制尚不完全清楚。本课题采用新生鼠HI模型和体外培养皮层神经元的OGD模型，研究HIE/OGD时TRPC3通道在神经元损伤中的作用，深入探讨缺血缺氧中神经元TRPC3通道活性与细胞膜SOC钙内流、内质网钙稳态和内质网应激的相互关系。结果发现，缺血缺氧损伤可引起严重内质网应激，UPR发生，其原因可能与缺血缺氧因导致 SOC介导的钙内流减少，[Ca2+]i减少，破坏内质网钙稳态有关。进一步研究发现， TRPC3通道蛋白参与此重要过程。①TRPC3对缺血缺氧神经元具有保护效应，阻断TRPC3通道或者siRNA特异性抑制其表达可使神经元损伤死亡加重。②免疫共沉淀结果显示TRPC3蛋白和SOC通道蛋白成员STIM1/Orail具有相互作用，在OGD情况下，TRPC3与STIM1、orai1蛋白的结合减少，且当TRPC3被抑制表达后，二者的结合进一步降低，从而影响SOC钙内流，加重了内质网应激。③运用TRPC3通道阻断剂pry3或siRNA特异性抑制TRPC3表达，可明显上调内质网应激标志蛋白GRP78 及其下游信号通路的表达，加重内质网应激。⑤TRPC3过表达可减弱内质网应激，调控其下游信号通路，保护神经元。综上所述，我们的结果提示，在缺血缺氧损伤中，TRPC3通道可能通过调控SOC钙内流和内质网应激，保护神经元。TRPC3有望成为HIE后神经保护治疗措施新的分子靶点。