莱鲍迪甙B通过线粒体凋亡途径对实验性自身免疫性脑脊髓炎小鼠的保护作用  

The protective effect of rebaudioside B for experimental autoimmune encephalomyelitis through inhibiting mitochondrial apoptosis pathway

作　　者：柳宁 孙梦娇 周丹 张雯静 王满侠[1] LIU Ning;SUN Mengjiao;ZHOU Dan;ZHANG Wenjing;WANG Manxia(不详;Department of Neurology,Qinghai Provincial People's Hospital,Qinghai Xining 810000,China;Department of Neurology,The Second Hospital of Lanzhou University,Gansu Lanzhou 730000,China)

机构地区：[1]兰州大学第二医院神经内科,730000 [2]青海省人民医院神经内科,810000

出　　处：《中国神经免疫学和神经病学杂志》2024年第3期159-166,173,共9页Chinese Journal of Neuroimmunology and Neurology

基　　金：甘肃省重点人才项目(编号:2022-77-6);青海省人民医院科学技术研究专项项目(编号:ZX-63000001-2024-008)。

摘　　要：目的探讨莱鲍迪甙B在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)模型中的保护作用及相关机制。方法培养BV2细胞,将细胞按完全随机法分为正常对照组、脂多糖(lipopolysaccharide,LPS)炎症细胞模型组、莱鲍迪甙B干预组。给予BV2细胞1μg/mL LPS培养24 h制备LPS炎症细胞模型。莱鲍迪甙B干预组于造模前给予10μmol/L莱鲍迪甙B干预4 h。利用流式细胞术检测细胞周期和细胞凋亡,采用免疫荧光法观察细胞形态学变化,采用Western blot检测细胞抗活化半胱氨酸蛋白酶蛋白-3抗体(Caspase-3)、抗Bcl-2相关X蛋白抗体(Bax)和抗B细胞淋巴瘤因子-2抗体(Bcl-2)表达情况。选取6~8周龄雌性C57BL/6小鼠30只,随机分为正常对照组、EAE模型组、莱鲍迪甙B干预组,每组各10只。利用MOG_(35-55)多肽皮下注射法构建EAE动物模型。莱鲍迪甙B干预组小鼠于造模后第8天给予腹腔注射莱鲍迪甙B(按体重1 mg/kg),持续15 d。采用HE染色观察小鼠脊髓组织病理学变化,采用Western blot检测脊髓组织Caspase-3、Bax和Bcl-2表达情况,采用透射电镜观察小鼠脑组组织内凋亡小体。结果(1)细胞实验结果:与正常对照组相比,LPS炎症细胞模型组的细胞S期比例下降,细胞核染色质固缩明显,细胞凋亡率上升,Caspase-3表达水平增加,Bax/Bcl-2比值升高(均P<0.01)。与LPS炎症细胞模型组相比,莱鲍迪甙B干预组细胞S期比例上调,细胞核染色质固缩现象改善,细胞凋亡率下降,Caspase-3表达水平降低及Bax/Bcl-2比值降低(均P<0.01)。(2)动物实验结果:与正常对照组相比,EAE模型组小鼠脊髓组织炎症细胞浸润增多,Caspase-3表达水平增加及Bax/Bcl-2比值升高(均P<0.01),小鼠脑组织的细胞形态异常,出现凋亡小体。与EAE模型组小鼠相比,莱鲍迪甙B干预组小鼠脊髓组织Caspase-3表达水平下降及Bax/Bcl-2比值下降(均P<0.01),小鼠脑组织细胞形态基本正常。结论莱鲍迪�Objective To study the protective effect and related mechanisms of rebaudioside B in the experimental autoimmune encephalomyelitis(EAE)model.Methods BV2 cells were cultured and randomly divided into normal control group,lipopolysaccharide(LPS)inflammatory cell model group,and rebaudioside B intervention group.The LPS inflammatory cell model was prepared by incubating BV2 cells with 1μg/mL LPS for 24 hours.The rebaudioside B intervention group was given 10μmol/L rebaudioside B for 4 hours before modeling.Flow cytometry was used to detect cell cycle and apoptosis,immunofluorescence was used to observe cell morphology,and western blot was used to detect the expression of Caspase-3,Bax,and Bcl-2 in cells.Thirty female C57BL/6 mice of 6-8 weeks of age were selected and randomly divided into a normal control group,EAE model group,and rebaudioside B intervention group,with 10 mice in each group.The EAE animal model was constructed by subcutaneous injection of MOG_(35-55)peptide.On the 8th day after the modeling,mice in the rebaudioside B intervention group were injected intraperitoneally with rebaudioside B(1 mg/kg)for 15 d.HE staining was used to observe the histopathological changes in the spinal cord of mice,western blot was used to detect the expression of Caspase-3,Bax,and Bcl-2 in the spinal cord,and the apoptotic bodies in the brain of mice were observed by transmission electron microscopy.Results(1)The results of cellular experiments:compared with the normal control group,the LPS inflammatory cell model group showed a decreased proportion of cells in the S phase,significant nuclear chromatin condensation,increased apoptosis rate,increased expression level of Caspase-3,and elevated Bax/Bcl-2 ratio(all P<0.01).Compared with the LPS inflammatory cell model group,the proportion of cells in the S phase was up-regulated in the rebaudioside B intervention group,and chromatin condensation in the nucleus was improved.The apoptosis rate,the expression level of Caspase-3,and the Bax/Bcl-2 ratio was decreased(all P<0.01).

关 键 词：多发性硬化 脑脊髓炎 自身免疫性 实验性 莱鲍迪甙B 小胶质细胞 细胞凋亡 

分 类 号：R744.51[医药卫生—神经病学与精神病学]