基于线粒体MCU通道探究甲基莲心碱促血管再生抗脑缺血的作用机制

Exploring Mechanism of Neferine in Promoting Vascular Regeneration Against Cerebral Ischemia Based on Mitochondrial MCU Channel

目的:探究甲基莲心碱(Nef)通过调控线粒体钙单向转运体(MCU)离子通道促进血管再生,进而抗脑缺血的作用机制。方法:以微血管缺失斑马鱼肠下血管面积为指标,对Nef进行血管再生功效评价,并计算半数有效浓度(EC50)。将大鼠随机分为假手术组、模型组、阳性药组(丁苯酞,6 mg·kg^(-1))、Nef低、中、高剂量组(0.125、0.625、3.125μg·kg^(-1)),除假手术组外,其余各组建立大鼠大脑中动脉栓塞(MCAO)模型,造模完成后各组按相应剂量灌胃给药,假手术组、模型组灌胃给予等体积生理盐水,1次/d,连续7 d。对各组大鼠进行神经行为学评分,并通过2,3,5-氯化三苯基四氮唑(TTC)染色计算缺血侧脑组织梗死率;散斑血流成像系统测定各组大鼠局部脑血流量(rCBF);免疫荧光及蛋白免疫印迹法(Western blot)检测各组大鼠血管内皮生长因子(VEGF)、血小板内皮细胞黏附分子-1(CD31)、缺氧诱导因子-1α(HIF-1α)蛋白表达。将人脐静脉内皮细胞(HUVECs)分为正常组、模型组、阳性药(黄芪甲苷,10μmol·L^(-1))组、Nef组(32 nmol·L^(-1));在验证Nef线粒体保护作用及促血管再生机制时,增设精胺组(MCU激动剂)、Nef+精胺组,除正常组外,其余各组建立糖氧剥夺(OGD)的HUVECs模型,通过噻唑蓝(MTT)法检测细胞活力,划痕实验与管成实验评估细胞迁移能力,应用Rhod-2 AM、Fluo-3 AM、JC-1、Calcein AM荧光探针,以及细胞能量代谢分析仪分析Nef的线粒体保护作用。通过分子对接预测Nef与MCU、HIF-1α的结合能力,采用Western blot检测Nef对OGD模型HUVECs中MCU、B细胞淋巴瘤-2相关X蛋白(Bax)、胱天蛋白酶-3(Caspase-3)和HIF-1α蛋白表达的影响。结果:对微血管缺失斑马鱼的血管生成结果显示,与正常组比较,模型组的肠下血管面积显著下降(P<0.01);与模型组比较,不同浓度Nef组肠下血管面积显著提高(P<0.01),最大耐受浓度为10.24μmol·L^(-1),EC50为0.23μmol·L^(-1)。对MCAO大鼠的抗脑缺血结果显示,与假手术组比较,模型组rCBF显著下降,脑梗死率显著增加,CD31表达显著下降(P<0.01),VEGF、HIF-1α蛋白表达明显上升(P<0.05);与模型组比较,各给药组rCBF显著提高,梗死体积显著减少,CD31、VEGF、HIF-1α蛋白表达量显著增加(P<0.01)。细胞实验结果显示,与正常组比较,模型组细胞存活率降低,迁移能力下降,细胞质内Ca^(2+)和线粒体内Ca^(2+)水平上升,线粒体膜通透性转换孔(MPTP)开放程度降低,线粒体能量代谢能力下降,MCU、Bax、Caspase-3、HIF-1α的蛋白表达增加(P<0.05,P<0.01);与模型组比较,Nef组细胞存活率上升,迁移能力提高,细胞质内Ca^(2+)和线粒体内Ca^(2+)水平下降,MPTP开放上升,细胞线粒体能量代谢能力升高,MCU、Bax、Caspase-3的蛋白表达下降,HIF-1α蛋白表达增加(P<0.05,P<0.01)。结论:Nef能够稳定线粒体膜电位,抑制线粒体凋亡;并通过下调MCU的表达,抑制细胞内Bax、Caspase-3的激活,同时激活HIF-1α信号通路,增强VEGF、CD31的表达,进而促进血管再生来治疗缺血性脑损伤。

Objective:To investigate the mechanism of neferine(Nef)in promoting vascular regeneration against cerebral ischemia through modulation of mitochondrial calcium uniporter(MCU)ion channel.Method:Taking the area of subintestinal vessels in microvascular deficiency zebrafish as an index,the vascular regenerative efficacy of Nef was evaluated,and the median effective concentration(EC50)was calculated.Rats were randomly divided into a sham operation group,a model group,a positive drug group(butylphthalide,6 mg·kg^(-1)),and Nef low,medium,and high dose groups(0.125,0.625,3.125μg·kg^(-1)).Except for the sham operation group,the middle cerebral artery occlusion(MCAO)model was established in other groups.After modeling,the groups were administered the corresponding dose of drugs by gavage,while the sham operation and model groups received equal volumes of saline,once a day for 7 consecutive days.Neurobehavioral scores were assessed for each group of rats,and the infarct rate of ischemic brain tissue was calculated by 2,3,5-triphenyltetrazolium chloride(TTC)staining.The regional cerebral blood flow(rCBF)of each group was measured using a speckle contrast imaging.Immunofluorescence and Western blot were conducted to detect the expression of vascular endothelial growth factor(VEGF),platelet endothelial cell adhesion molecule-1(CD31),and hypoxia-inducible factor-1α(HIF-1α)proteins in each group.Human umbilical vein endothelial cells(HUVECs)were divided into the normal group,model group,positive drug group(astragalosideⅣ,10μmol·L^(-1)),and Nef group(32 nmol·L^(-1)).In the verification of mitochondrial protection of Nef and its mechanism in promoting vascular regeneration,the spermine(MCU agonist)and Nef+spermine group were added.HUVECs model of oxygen-glucose deprivation(OGD)was established in all groups except the normal group,the cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,and cell migration ability was evaluated through scratch and tube formation assays.Fluorescent probes(Rhod-2 AM,Fluo-3 AM,JC-1,Calcein AM)and a cellular energy metabolism analyzer were used to analyze the mitochondrial protective effects of Nef.Molecular docking was performed to predict the binding ability of Nef with MCU and HIF-1α,and Western blot was used to detect the effects of Nef on the protein expressions of MCU,B-cell lymphoma-2 associated X protein(Bax),Caspase-3 and HIF-1αin the OGD model HUVECs.Result:The results of vascular regeneration in microvascular deficiency zebrafish showed that compared to the normal group,the area of subintestinal vessels in the model group significantly decreased(P<0.01).Compared to the model group,different concentrations of Nef could significantly increase the area of subintestinal vessels(P<0.01),with the maximum tolerated concentration of 10.24μmol·L^(-1) and the EC50 of 0.23μmol·L^(-1).Anti-cerebral ischemia results on MCAO rats showed that compared to the sham operation group,the model group had a significant decrease in rCBF and a significant increase in infarct rate,while CD31expression significantly decreased(P<0.01),and VEGF and HIF-1αprotein expressions significantly increased(P<0.05).Compared to the model group,the treated groups showed significant increases in rCBF,significant reductions in infarct volume,and significant increases in CD31,VEGF,and HIF-1αprotein expression(P<0.01).Cell experiment results showed that compared to the normal group,the model group had decreased cell viability and migration ability,increased intracellular Ca^(2+)and mitochondrial Ca^(2+)levels,reduced mitochondrial permeability transition pore(MPTP)opening,and decreased mitochondrial energy metabolism capability,with increased expressions of MCU,Bax,Caspase-3 and HIF-1αproteins(P<0.05,P<0.01).Compared to the model group,the Nef group showed increased cell viability and migration ability,decreased intracellular Ca^(2+)and mitochondrial Ca^(2+)levels,increased MPTP opening,enhanced mitochondrial energy metabolism capability,decreased expressions of MCU,Bax and Caspase-3 proteins,and increased HIF-1αprotein expression(P<0.05,P<0.01).Conclusion:Nef can stabilize mitochondrial membrane potential and inhibit mitochondrial apoptosis.By down-regulating the expression of MCU,it suppresses the activation of intracellular Bax and Caspase-3 while activating the HIF-1αsignaling pathway,enhancing the expression of VEGF and CD31,thereby promoting vascular regeneration to treat ischemic brain injury.