茶黄素调节CaMKK2/AMPK信号通路对脑出血大鼠神经元凋亡和血脑屏障的影响

Influences of theaflavins on neuronal apoptosis and blood-brain barrier in rats with cerebral hemorrhage by regulating CaMKK2/AMPK signaling pathway

目的:探讨茶黄素(TFs)通过调节钙调蛋白激酶激酶2(CaMKK2)/单磷酸腺苷活化蛋白激酶(AMPK)信号通路对脑出血大鼠神经元凋亡和血脑屏障(BBB)的影响。方法:选取90只大鼠随机分为假手术组、模型组、TFs低剂量组(20 mg/kg TFs)、TFs高剂量组(40 mg/kg TFs)、TFs高剂量+STO-609组(40 mg/kg TFs+10μL CaMKK2抑制剂-STO-609)、阳性对照组(2 mg/kg尼莫地平注射液),每组15只。采用VII型胶原酶诱导脑出血大鼠模型。对大鼠行为学以及脑组织含水量进行检测;分离大鼠血清,检验炎症因子-血管黏附因子-1(VCAM-1)、肿瘤坏死因子-α(TNF-α)、细胞间黏附因子-1(ICAM-1)水平;取血肿周围脑组织,检测神经元凋亡、BBB通透性参数-伊文思蓝(EB)水平以及p-CaMKK2/CaMKK2、p-AMPK/AMPK和凋亡相关蛋白Bax表达情况。结果:模型组大鼠神经行为学评分(mNSS评分)、ICAM-1、TNF-α、VCAM-1、脑组织含水量、凋亡率、EB水平以及Bax蛋白表达较假手术组均增加,p-CaMKK2/CaMKK2、p-AMPK/AMPK均降低(P<0.05);TFs低、高剂量组、阳性对照组大鼠mNSS评分、ICAM-1、TNF-α、VCAM-1、脑组织含水量、凋亡率、EB水平以及Bax表达较模型组均降低,p-CaMKK2/CaMKK2、p-AMPK/AMPK均增加(P<0.05);与TFs高剂量相比,TFs高剂量+STO-609组大鼠mNSS评分、ICAM-1、TNF-α、VCAM-1、脑组织含水量、凋亡率、EB水平以及Bax表达均增加,p-CaMKK2/CaMKK2、p-AMPK/AMPK降低(P<0.05)。结论:TFs可降低神经元凋亡、炎症反应、BBB通透性,对脑出血损伤大鼠发挥保护作用,其作用机制可能与激活CaMKK2/AMPK信号通路有关。

AIM:To investigate the impacts of theaflavins(TFs)on neuronal apoptosis and blood-brain barrier(BBB)by regulating the calcium/calmodulin-dependent protein kinase kinase 2(CaMKK2)/5'-adenosine monophosphate-activated protein kinase(AMPK)signaling pathway.METHODS:Ninety rats were randomly separated into sham operation group,model group,low-dose TFs group(20 mg/kg TFs),high-dose TFs group(40 mg/kg TFs),and high-dose TFs+STO-609 group(40 mg/kg TFs+10μL CaMKK2 inhibitor-STO-609),positive control group(2 mg/kg nimodipine injection),with 15 rats in each group.A rat model of intracerebral hemorrhage was induced by collagenase type VII.The behavior of rats and the water content of brain tissue were detected;the serum of rats was isolated,and the levels of inflammatory factors-vascular cell adhesion molecule-1(VCAM-1),tumor necrosis factor-α(TNF-α),and intercellular adhesion molecule-1(ICAM-1)were detected;brain tissue around the hematoma was collected to detect neuronal apoptosis,BBB permeability parameter-EB level,and expressions of p-CaMKK2/CaMKK2,p-AMPK/AMPK and apoptosis-related protein Bax.RESULTS:Compared with the sham operation group,the mNSS score,ICAM-1,TNF-α,VCAM-1,brain tissue water content,apoptosis rate,EB level and Bax protein expression in the model group were all increased,both p-CaMKK2/CaMKK2 and p-AMPK/AMPK were decreased(P<0.05);compared with the model group,the mNSS score,ICAM-1,TNF-α,VCAM-1,brain water content,apoptosis rate,EB level and Bax expression in the low-and high-dose TFs groups and the positive control group were all lower than those in the model group,both p-CaMKK2/CaMKK2 and p-AMPK/AMPK were increased(P<0.05);compared with the high-dose TFs group,the mNSS score,ICAM-1,TNF-α,VCAM-1,brain tissue water content,apoptosis rate,EB level and Bax expression were all increased in the high dose TFs+STO-609 group,both p-CaMKK2/CaMKK2 and p-AMPK/AMPK were decreased(P<0.05).CONCLUSION:TFs can reduce neuronal apoptosis,inflammatory response,BBB permeability,and play a protective role in rats with cerebral hemorrhage injury.Its mechanism is related to the activation of CaMKK2/AMPK signaling pathway.