摘要
目的探讨唑尼沙胺(zonisamide,ZNS)对创伤性脑损伤(traumatic brain injury,TBI)糖氧剥夺(oxygen-glucose deprivation,OGD)细胞模型的保护作用及其机制。方法体外培养人神经母细胞瘤(human neuroblastoma cells,SH-SY5Y)细胞,按随机数字表法分为对照组(Control组)、糖氧剥夺组(OGD组)和给药组(OGD+ZNS组)。采用糖氧剥夺法建立创伤性脑损伤细胞模型。造模后,检测细胞活性;检测细胞乳酸脱氢酶(LDH)释放情况;β-半乳糖苷酶试剂盒对细胞染色,观察细胞衰老情况;线粒体红色荧光探针(Mito Tracker Red)、JC-1线粒体膜电位试剂盒对线粒体染色,观察线粒体形态及膜电位变化;检测细胞ATP浓度;从SH-SY5Y细胞中提取蛋白,用蛋白质印迹法(Western blot)法检测内质网应激相关指标:葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78),增强子结合蛋白同源蛋白(C/EBP-homologous protein antibody,CHOP)、蛋白二硫键异构酶(protein disulfide isomerase,PDI)以及内参β-肌动蛋白(β-actin)的表达变化。结果OGD组细胞存活率较Control组明显减少(P<0.01),OGD+ZNS组细胞存活率较OGD组明显增加(P<0.01);OGD组LDH释放率较Control组明显增加(P<0.01),OGD+ZNS组LDH释放率较OGD组明显减少(P<0.01)。细胞染色结果显示,与Control组和OGD+ZNS组相比,OGD组细胞明显受损衰老严重,染色更深。线粒体染色结果显示,与Control组和OGD+ZNS组相比,OGD组线粒体线性连接明显减少,线粒体活性明显下降。与Control组和OGD+ZNS组相比,OGD组细胞线粒体染色红色荧光明显减弱,绿色荧光明显增强,线粒体膜电位明显下降;OGD组ATP浓度较Control组明显下降(P<0.01),OGD+ZNS组ATP浓度较OGD组明显上升(P<0.01)。Western blot结果显示OGD组GRP78、CHOP、PDI表达较Control组明显增加(P均<0.05),OGD+ZNS组GRP78、CHOP、PDI较OGD组明显下降(P均<0.05)。结论唑尼沙胺可以通过保护线粒体活性以及抑制内质网应激保护创伤性脑损伤糖氧剥夺细胞模型。
Objective To explore the protective effect of zonisamide(ZNS)on oxygenglucose deprivation(OGD)cell model of traumatic brain injury(TBI),and its underlying mechanism.Methods Human neuroblastoma cells(SH-SY5Y)were cultured in vitro and divided into the control group,OGD group,and drug administration group(OGD+ZNS group)according to the random number table method.The OGD method was used to establish a TBI cell model.After modeling,the cell activity,the release of lactate dehydrogenase(LDH),and β-galactosidase staining were detected to evaluate cell function and senescence.Additionally,mitochondrial morphology and potential membrane changes were observed using Mito Tracker Red and JC-1 mitochondrial membrane potential staining.ATP concentration was measured,and protein was extracted from SH-SY5Y cells and then subjected to Western blot analysis to detect endoplasmic reticulum stress-related markers,including glucose-regulated protein 78(GRP78),C/EBP homologous protein(CHOP),protein disulfide isomerase(PDI),and β-actin.Results The OGD group had a significantly lower cell survival rate compared to the control group(P<0.01),while the OGD+ZNS group had a significant higher cell survival rate than the OGD group(P<0.01).The LDH release rate was significantly higher in the OGD group than in the control group(P<0.01),while the OGD+ZNS group had a significant lower LDH release rate compared to the OGD group(P<0.01).Moreover,the cell staining results indicated that compared to the control and OGD+ZNS groups,the cells in the OGD group exhibited significant damage and senescence with darker staining while the mitochondrial staining results demonstrated a significant reduction in mitochondrial linear junctions and decreased mitochondrial activity in the OGD group compared to the control and OGD+ZNS groups.Compared to the control and OGD+ZNS groups,the OGD group exhibited a significant reduction in mitochondrial staining red fluorescence,a significant increase in green fluorescence,and a significant decrease in mitochondrial membrane potential.The OGD group demonstrated a significant decrease in ATP concentration compared to the control group(P<0.01),whereas the OGD+ZNS group exhibited a significant higher ATP concentration compared to the OGD group(P<0.01).Western blot analysis revealed significant upregulation of GRP78,CHOP,and PDI in the OGD group compared to the control group(all P<0.05),while in the OGD+ZNS group,the expression levels of these proteins were significantly downregulated compared to the OGD group(all P<0.05).Conclusions Zonisamide can protect OGD TBI cell model by preserving mitochondrial activity and inhibiting endoplasmic reticulum stress.
作者
滕陈怀
吴芳芳
张开睿
张仁侃
徐绩
卢磊磊
陈大庆
Teng Chenhuai;Wu Fangfang;Zhang Kairui;Zhang Renkan;Xu Ji;Lu Leilei;Chen Daqing(Department of Emergency Medicine,Second Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000,China)
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2023年第7期912-918,共7页
Chinese Journal of Emergency Medicine
基金
浙江省自然科学青年基金(LQ22H150004)
温州市重大科技创新攻关项目(ZY2021024)。
关键词
唑尼沙胺
创伤性脑损伤
糖氧剥夺
人神经母细胞瘤细胞
线粒体
内质网应激
Zonisamide
Traumatic brain injury
Oxygen-glucose deprivation
Human neuroblastoma cells
Mitochondria
Endoplasmic reticulum stress