摘要
目的观察“电针血清”对新生乳鼠皮质神经细胞糖氧剥夺/复氧(oxygen-glucose deprivation and reoxygenation,OGD/R)损伤的作用。方法将成年SD大鼠随机分为对照组、模型组、电针组,采用电凝法复制大脑中动脉闭塞模型;7 d后对各组大鼠进行腹主动脉取血,离心,抽取上清,分离新生24 h内SD大鼠乳鼠皮质神经细胞,随机分为对照组、模型组、“电针血清”组。对照组以含正常大鼠血清的培养基培养,模型组进行OGD 2 h后复糖复氧24 h处理,“电针血清”组先以含“电针血清”的培养基培养24 h,其余操作同模型组。MTT法检测各组神经细胞的存活率;TUNEL法检测各组神经细胞凋亡情况;乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验检测各组神经细胞培养液中LDH漏出水平;检测各组神经细胞内超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)水平。Western blot法检测各组神经细胞Wnt蛋白1(Wnt-1)、糖原合成酶激酶3β(glycogen synthase kinase-3β,GSK-3β)、β-连环蛋白(β-catenin)、神经细胞核抗原(neuronal nuclear protein,NeuN)蛋白的表达情况。结果与对照组比较,模型组神经细胞存活率显著降低(P<0.05),神经细胞凋亡率显著增多(P<0.05);与模型组比较,“电针血清”组神经细胞存活率显著升高(P<0.05),神经细胞凋亡率显著减少(P<0.05)。与对照组比较,模型组SOD活性显著降低(P<0.05),MDA含量、LDH漏出量显著增多(P<0.05);与模型组比较,“电针血清”组SOD活性显著增加(P<0.05),MDA含量、LDH漏出量显著减少(P<0.05)。与对照组比较,模型组Wnt-1、GSK-3β、β-catenin、NeuN蛋白表达水平显著降低(P<0.05);与模型组比较,“电针血清”组Wnt-1、β-catenin、NeuN蛋白表达水平显著升高(P<0.05),GSK-3β表达水平显著降低(P<0.05)。结论“电针血清”可以减轻缺糖低氧诱导的皮质神经细胞损伤,减少神经细胞的凋亡,其机制可能与激活Wnt/β-catenin信号通路相关。
Objective To investigate the effect of electroacupuncture serum on oxygen-glucose deprivation/reoxygenation(OGD/R)injury of cortical neurons from neonatal mice.Methods Adult Sprague-Dawley(SD)rats were randomly divided into control group,model group,and electroacupuncture group.A rat model of middle cerebral artery occlusion model(MCAO)was established by electrocoagulation;at 7 days after modeling,blood was collected from the abdominal aorta and supernatant was extracted by centrifugation.Cortical neurons were isolated from neonatal SD rats within 24 hours after birth and were then randomly divided into control group,model group,and electroacupuncture serum group.The neurons in the control group were cultured in the medium of normal rat serum,those in the model group were treated with oxygen-glucose deprivation for 2 hours followed by glucose and oxygenation for 24 hours,and those in the electroacupuncture serum group were cultured in the medium containing electroacupuncture serum for 24 hours,followed by the same treatment as the model group.MTT assay was used to measure the viability of neurons;TUNEL method was used to measure neuronal apoptosis;the lactate dehydrogenase(LDH)release assay was used to measure the level of LDH leakage in neuronal culture medium;the kit was used to measure the levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in neurons;Western blot was used to measure the protein expression levels of Wnt-1,glycogen synthase kinase-3β(GSK-3β),β-catenin,neuronal nuclear protein(NeuN)in neurons.Results Compared with the control group,the model group had a significant reduction in the viability of neurons(P<0.05)and a significant increase in the apoptosis rate of neurons(P<0.05);compared with the model group,the electroacupuncture serum group had a significant increase in the viability of neurons(P<0.05)and a significant reduction in the apoptosis rate of neurons(P<0.05).Compared with the control group,the model group had a significant reduction in SOD activity(P<0.05)and significant increases in MDA content and LDH leakage(P<0.05);compared with the model group,the electroacupuncture serum group had a significant increase in SOD activity(P<0.05)and significant reductions in MDA content and LDH leakage(P<0.05).Compared with the control group,the model group had significant reductions in the protein expression levels of Wnt-1,GSK-3β,β-catenin,and NeuN(P<0.05);compared with the model group,the electroacupuncture serum group had significant increases in the protein expression levels of Wnt-1,β-catenin,and NeuN(P<0.05)and a significant reduction in the protein expression level of GSK-3β(P<0.05).Conclusion Electroacupuncture serum can alleviate the injury of cortical neurons induced by hypoxia and glucose deprivation and reduce the apoptosis of neurons,possibly by activating the Wnt/β-catenin signaling pathway.
作者
李笑笑
李姝洁
董健健
韩永升
LI Xiao-xiao;LI Shu-jie;DONG Jian-jian;HAN Yong-sheng(Graduate School of Anhui University of Chinese Medicine, Anhui Hefei 230012, China;Institute of Neurology of Anhui University of Chinese Medicine, Anhui Hefei 230038, China;Anhui Hospital of Integrated Traditional Chinese and Western Medicine, Anhui Hefei 230031, China)
出处
《安徽中医药大学学报》
CAS
2022年第1期43-49,共7页
Journal of Anhui University of Chinese Medicine
基金
国家自然科学基金项目(81774425)。
作者简介
李笑笑(1995-),女,硕士研究生;通信作者:韩永升(1976-),男,主任医师,教授,hyssp@126.com。
