TRPV1通道介导MAPK信号通路调控缺血后脑损伤及解毒通络中药复方干预机制研究

Calcium overload is reported to be the key player in the biochemical cascade after cerebral ischemia, and reduce the Ca2+ influx could be very important in the treatment of cerebral ischemia. According to recent studies, Transient receptor potential vanilloid-1 (TRPV1) channel, regarded as thermo-TRP, can transport Ca2+ across the membrane, regulate the intracellular calcium concentration when activated, and mediate the inflammation-related MAPK signaling pathway to regulate cerebral ischemic injufy, and is considered to be the potential target in treating cerebral ischemia. In this study, focal cerebral ischemic/reperfusion injury is established using middle cerebral artery occlusion (MCAO) model in rat, the expression of TRPV1 and the TRPV1 current will be measured, the phosphorylated and nonphosphorylated protein expression of p38 MAPK, ERK1/2 MAPK and JNK MAPK signaling pathway will be detected, and the inflammatory biomarkers in the brain tissue, the cerebral temperature, the thermal hyperalgesia threshold, the cerebral blood flow, the blood-brain barrier permeability and neurological function deficit will be observed to demonstrate the mechanism of TRPV1 activation as well as TRPV1-mediated MAPK signaling pathway on regulation of cerebral ischemic injury; the interventional effects by toxin-relieving and collateral-unblocking TCM Compound Formula that has the role of inhibiting post-ischemia inflammatory reaction on TRPV1 and MAPK signal pathway will be observed to demonstrate the cellular and molecular mechanism on improving brain circulation and neuroprotection of the TCM Compound Formula.

细胞内钙超载是脑缺血后生化级联过程中的关键环节，减少Ca2+流入细胞是治疗脑缺血的重要手段。热敏感的瞬时受体电位通道TRPV1活化时可引起钙内流，直接调控细胞内钙离子浓度，激活炎症相关的MAPK信号通路，参与诱导缺血后脑损伤，因而成为治疗脑缺血的潜在靶标。我们拟建立大鼠脑缺血再灌注模型，通过检测TRPV1通道蛋白表达、通道电流和细胞内游离钙离子浓度，以及磷酸化与非磷酸化p38 MAPK、ERK1/2 MAPK及JNK MAPK信号通路蛋白表达，并观察脑内炎症反应相关指标、动物脑温、热痛敏阈值、脑血流量及血脑屏障通透性的变化以及神经功能缺损情况，阐明脑缺血后TRPV1通道活化及介导MAPK信号通路参与调控缺血后脑损伤的机制；同时通过观察具有抑制缺血性脑损伤炎性反应作用的解毒通络中药复方对TRPV1通道及MAPK信号转导通路的干预作用，探讨该中药复方发挥改善微循环及神经保护作用的细胞分子机制。

细胞内钙超载是脑缺血后生化级联过程中的关键环节，减少钙离子流入细胞是治疗脑缺血的重要手段。热敏感的瞬时受体电位通道TRPV1活化时可引起钙内流，直接调控细胞内钙离子浓度，激活炎症相关的MAPK信号通路，参与诱导缺血后脑损伤，因而成为治疗脑缺血的潜在靶标。本项目研究结果表明，脑缺血再灌注引起TRPV1通道蛋白表达及mRNA表达增加，通过调控MAPK信号通路参与炎症级联反应过程，进而影响体温及伤害性热感受刺激的阈值；通络清脑注射液能够作用于TRPV1通道，抑制炎症级联反应过程，从而起到抗缺血性脑损伤作用，发挥其清热解毒活血通络的功效。本项目明确了TRPV1通道在缺血性脑损伤中的作用，探讨了TRPV1通过影响MAPK信号通路参与调控缺血后脑损伤的机制，并初步揭示了TRPV1与NLRP3炎症小体信号通路的关系；同时，本项目还观察了解毒活血中药复方通络清脑方对TRPV1通道及相关信号通路的影响。本研究从离子通道功能异常这一病理生理学角度对脑缺血损伤的机制进行了积极探索，这对于阐明中风病热毒血瘀病机的实质，完善中风病热毒理论具有重要意义，同时本研究从离子通道角度阐释具有清热解毒功效的复方中药抗脑缺血的作用及其机制，这为寻找治疗缺血性脑血管病的新药提供了方向，具有一定的意义和应用前景。