阻断TRPC3通道加重新生大鼠缺氧缺血性脑损伤

经典瞬时受体电位3（transient receptor potential canonical 3，TRPC3)通道是胎儿期和围生期中枢神经系统中广泛表达的非特异性阳离子通道，参与体内众多生理和病理过程。有研究证明，TRPC3通道是细胞内钙稳态的重要调节者，调节包括细胞外信号调节激酶（extracellular signal-regulated kinase，ERK）通路在内的多条钙敏感胞内信号转导通路的活性，最终影响神经元的生存或死亡。但TRPC3通道在新生动物缺氧缺血性脑损伤（hypoxic- ischemic brain damage，HIBD）模型中的作用及其机制尚未见报道。本研究取新生7 d的SD大鼠，采用右侧颈总动脉结扎和缺氧（8% O2）2~5 h制备HIBD模型，观察腹腔注射选择性TRPC3阻断剂pyr3（5 mg/kg和20 mg/kg）对缺氧缺血处理后，急性期和长期神经行为学及脑组织损伤程度的影响。神经功能缺损评分和平衡木实验结果显示，用pyr3特异性阻断TRPC3可恶化缺氧缺血大鼠的神经行为学障碍；脑组织含水量检测、TTC染色和患/健侧脑重比等结果显示，pyr3可加重脑水肿，增加脑组织梗死区体积和加重脑萎缩程度。Western印迹实验显示，缺氧缺血可以导致患侧脑组织ERK1/2磷酸化水平一过性升高，阻断TRPC3可以显著抑制ERK1/2的磷酸化，并可上调促凋亡蛋白BAX和下调抗凋亡蛋白BCL-2的表达。上述结果证明，阻断TRPC3通道可以加重新生大鼠的缺氧缺血性脑损伤，其机制可能与其对ERK信号通路活性的调节作用有关，因此可能成为HIBD治疗的潜在作用靶点。

Aggravation of Hypoxic-ischemic Brain Damage of Neonatal Rats by Blocking TRPC3 Channel

The transient receptor potential canonical 3 (TRPC3) channel is a subtype of nonselective cation channels, which is widely distributed in the central nervous system of fetuses and neonates. It has been reported that TRPC3 channel exhibiting versatile functions is an important regulator of intracellular calcium homeostasis and cell fate. It is involved in the regulation of pivotal calcium-sensing signaling cascades including extracellular signal-regulated kinase (ERK) pathway, to modulate neuronal death or survival. However, much less is known about the potential effects and underlying mechanism of TRPC3 channel on neonatal hypoxic-ischemic brain damage (HIBD). In this study, HIBD model was induced by unilateral ligation of the right carotid artery followed by hypoxia (8% O2 for 2-5 h) in postnatal day 7 rat pups. Pyr3, a selective TRPC3 blocker, was injected (5 mg/kg or 100 mg/kg) intraperitoneally to investigate its acute and long-term effects on neurobehavior and brain injury after HI insult. Neurological deficit scores and balance beam test showed that specific blockade of TRPC3 activity by pyr3 aggravated the neurobehavioral deficits of HI animals. TTC staining, brain water content and ipsilateral/contralateral hemispheric weight ratio measurements revealed that pyr3 administration increased brain edema and infarct volume as well as exacerbated brain atrophy. Western blotting analysis demonstrated that p-ERK1/2 was transiently increased in the ipsilateral hemisphere after HI. Furthermore, blocking TRPC3 reduced p ERK1/2 expression, which accompanied a marked up-regulation of proapoptotic proteins, BAX, and a down-regulation of antiapoptotic proteins, BCL-2. Our data suggest that TRPC3 channel blockade aggravates hypoxic-ischemic brain damage by activating ERK signaling pathway in the neonatal rat brain and may be an effective target for HIBD.