Neuroscience Research Center, Suleyman Demirel University, 32260, Isparta, Turkey.
Department of Biophysics, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey.
Mol Cell Biochem. 2019 Mar;453(1-2):143-155. doi: 10.1007/s11010-018-3439-0. Epub 2018 Aug 29.
The expression level of TRPV1 is high in hippocampus which is a main epileptic area in the brain. In addition to the actions of capsaicin (CAP) and reactive oxygen species (ROS), the TRPV1 channel is activated in neurons by endogenous cannabinoid, anandamide (AEA). In the current study, we investigated the role of inhibitors of TRPV1 (capsazepine, CPZ), AEA transport (AM404), and FAAH (URB597) on the modulation of Ca entry, apoptosis, and oxidative stress in in vitro seizure-induced rat hippocampus and human glioblastoma (DBTRG) cell line. The seizure was induced in the hippocampal and DBTRG neurons using in vitro 4-aminopyridine (4-AP) to trigger a seizure-like activity model. CPZ and AM404 were fully effective in reversing 4-AP-induced intracellular free Ca concentration of the hippocampus and TRPV1 current density of DBTRG. However, AEA and CAP did not activate TRPV1 in the URB597-treated neurons. Hence, we observed TRPV1 blocker effects of URB597 in the DBTRG neurons. In addition, the AM404 and CPZ treatments decreased intracellular ROS production, mitochondrial membrane depolarization, apoptosis, caspases 3 and 9 values in the hippocampus. In conclusion, the results indicate that inhibition of AEA transport, FAAH synthesis, and TRPV1 activity can result in remarkable neuroprotective effects in the epileptic neurons. Possible molecular pathways of involvement of capsazepine (CPZ) and AM4040 in anandamide and capsaicin (CAP)-induced apoptosis, oxidative stress, and Ca accumulation through TRPV1 channel in the seizure-induced rat hippocampus and human glioblastoma neurons. The TRPV1 channel is activated by different stimuli including reactive oxygen species (ROS), anandamide (AEA), and CAP and it is blocked by capsazepine (CPZ). Cannabinoid receptor type 1 (CB1) is also activated by AEA. The AEA levels in cytosol are decreased by fatty acid amide hydrolase (FAAH) enzyme. Inhibition of FAAH through URB597 induces stimulation of CB1 receptor through accumulation AEA. URB597 acts antiepileptic effects through inhibition of TRPV1. Overloaded Ca concentration of mitochondria can induce an apoptotic program by stimulating the release of apoptosis-promoting factors such as caspases 3 and caspase 9 by generating ROS due to respiratory chain damage. AM404 and CPZ reduce TRPV1 channel activation and Ca entry in the in vitro 4-AP seizure model-induced hippocampal and glioblastoma neurons.
TRPV1 蛋白在大脑的主要癫痫发作区——海马体中表达水平较高。除了辣椒素（CAP）和活性氧（ROS）的作用外，内源性大麻素，即花生四烯酸乙醇胺（AEA）也能激活神经元中的 TRPV1 通道。在本研究中，我们研究了 TRPV1 抑制剂（辣椒素、CPZ）、AEA 转运抑制剂（AM404）和 FAAH 抑制剂（URB597）对体外致痫大鼠海马体和人神经胶质瘤（DBTRG）细胞系中 Ca 内流、细胞凋亡和氧化应激的调节作用。使用 4-氨基吡啶（4-AP）在海马体和 DBTRG 神经元中诱导癫痫发作，以触发类似癫痫发作的活动模型。CPZ 和 AM404 可完全逆转 4-AP 诱导的海马体细胞内游离 Ca 浓度和 DBTRG 中的 TRPV1 电流密度。然而，URB597 处理的神经元中，AEA 和 CAP 不能激活 TRPV1。因此，我们观察到 URB597 在 DBTRG 神经元中的 TRPV1 阻断作用。此外，AM404 和 CPZ 处理降低了海马体中细胞内 ROS 的产生、线粒体膜去极化、细胞凋亡以及半胱天冬酶 3 和 9 的值。总之，这些结果表明，抑制 AEA 转运、FAAH 合成和 TRPV1 活性可导致癫痫神经元产生显著的神经保护作用。在致痫大鼠海马体和人神经胶质瘤神经元中，CPZ 和 AM4040 通过 TRPV1 通道参与辣椒素和 CAP 诱导的细胞凋亡、氧化应激和 Ca 积累的可能分子途径。TRPV1 通道可被不同的刺激物激活，包括活性氧（ROS）、花生四烯酸乙醇胺（AEA）和 CAP，可被辣椒素（CPZ）阻断。大麻素受体 1（CB1）也可被 AEA 激活。细胞溶质中的 AEA 水平通过脂肪酸酰胺水解酶（FAAH）酶降低。URB597 通过抑制 FAAH 诱导 AEA 积累来刺激 CB1 受体，从而发挥抗癫痫作用。URB597 通过抑制 TRPV1 发挥抗癫痫作用。由于呼吸链损伤导致 ROS 产生，线粒体中 Ca 浓度过载会通过刺激凋亡促进因子（如半胱天冬酶 3 和 9）的释放，从而引发凋亡程序。AM404 和 CPZ 减少了体外 4-AP 癫痫发作模型诱导的海马体和神经胶质瘤神经元中 TRPV1 通道的激活和 Ca 内流。
