In the central nervous system, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential to maintain normal neuronal function. Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury, but the mechanisms remain unclear. Autophagy is activated in cerebral ischemia, but its role in cell death/survival remains controversial. In this study, our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) in hippocampal HT22 neurons. Furthermore, in the OGD/R group, p-mTOR, p-ULK1 (Ser⁷⁵⁷), and p62 were significantly decreased, while p-ULK1 (Ser³¹⁷), atg5, and beclin1 were remarkably increased. ZD7288 did not change the expression of p-ULK1 (Ser⁷⁵⁷), ULK1 (Ser³¹⁷), p62, Beclin1, and atg5, which are involved in regulating autophagosome formation. Besides, we found that OGD/R induced a significant increase in Cathepsin D expression, but not LAMP-1. Treatment with ZD7288 at 10 μmol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1. However, chloroquine (CQ), which decreases autophagosome-lysosome fusion, eliminated the correction of excessive autophagy and neuroprotection by ZD7288. Besides, shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-II and increased neuron survival in the OGD/R and transient global cerebral ischemia (TGCI) models, and CQ also eliminated the effects of HCN2-shRNA. Furthermore, we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNA-transfected HT22 neurons exposed to OGD/R or CQ. In HCN2-shRNA-transfected HT22 neurons, the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R; however, the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons. The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.

中文翻译：

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阻断 HCN2 通道可通过加速海马神经元的自噬降解来提供针对缺血性损伤的神经保护。


在中枢神经系统中，超极化激活的环核苷酸门控（HCN）通道对于维持正常的神经元功能至关重要。近年来研究表明HCN通道可能参与缺血性脑损伤的病理过程，但其机制尚不清楚。自噬在脑缺血时被激活，但其在细胞死亡/存活中的作用仍存在争议。在本研究中，我们的结果表明，HCN 通道阻滞剂 ZD7288 显着降低了海马 HT22 神经元凋亡神经元的百分比，并纠正了氧糖剥夺再灌注 (OGD/R) 诱导的过度自噬。此外，在OGD/R组中，p-mTOR、p-ULK1（Ser ⁷⁵⁷ ）和p62显着减少，而p-ULK1（Ser ³¹⁷ ）、atg5和beclin1显着增加。 ZD7288 不会改变参与调节自噬体形成的 p-ULK1 (Ser ⁷⁵⁷ )、ULK1 (Ser ³¹⁷ )、p62、Beclin1 和 atg5 的表达。此外，我们发现 OGD/R 诱导组织蛋白酶 D 表达显着增加，但不诱导 LAMP-1 表达。 OGD/R组用10 μmol/L ZD7288处理后，组织蛋白酶D和LAMP-1的表达没有变化。然而，减少自噬体-溶酶体融合的氯喹 (CQ) 消除了 ZD7288 对过度自噬和神经保护的纠正。此外，HCN2通道的shRNA敲低显着减少了OGD/R和短暂性全脑缺血（TGCI）模型中LC3-II的积累并增加了神经元存活，并且CQ还消除了HCN2-shRNA的影响。 此外，我们发现在接触 OGD/R 或 CQ 的 Con-shRNA 转染的 HT22 神经元中，与 LAMP-1 阳性溶酶体共定位的 LC3 阳性斑点的百分比降低。在 HCN2-shRNA 转染的 HT22 神经元中，与 LAMP-1 阳性溶酶体共定位的 LC3 阳性斑点的百分比在 OGD/R 下增加；然而，向 HCN2-shRNA 转染的 HT22 神经元中添加 CQ 后，该百分比显着降低。目前的结果表明，HCN2 通道的阻断通过加速自噬降解（归因于自噬体和溶酶体融合的促进）提供针对 OGD/R 和 TGCI 的神经保护。