红景天苷对缺氧缺糖诱导的星形胶质细胞线粒体自噬的影响

郑丽云,黄慧芬,邱伟文

(1.温州医科大学附属第五医院 神经内科,浙江 丽水 323000;2.温州医科大学附属第六医院 神经内科,浙江 丽水 323000)

红景天苷对缺氧缺糖诱导的星形胶质细胞线粒体自噬的影响

郑丽云1,黄慧芬1,邱伟文2

(1.温州医科大学附属第五医院 神经内科,浙江 丽水 323000;2.温州医科大学附属第六医院 神经内科,浙江 丽水 323000)

目的:探讨红景天苷(Sal)对缺氧缺糖(OGD)诱导的星形胶质细胞线粒体自噬的影响.方法:提取原代培养的新生鼠皮层星形胶质细胞,进行OGD干预,建立OGD细胞模型.将实验分为对照组、OGD组和Sal+OGD组.用噻唑蓝(MTT)法检测细胞存活率,乳酸脱氢酶(LDH)法检测LDH漏出率评估细胞损伤,Western blot法检测自噬相关蛋白表达水平.结果:Sal终浓度为75、150、300 μmol/mL时,细胞存活率分别为83.15%±5.42%、86.75%±4.91%、91.41%±5.38%,明显高于OGD组(71.22%±8.13%),但仍低于对照组(99.39%±2.17%),差异均有统计学意义(P<0.05);LDH漏出率为34.61%±5.19%、30.51%±8.15%、27.34±7.41%,明显低于OGD组(40.51%±8.15%),但仍高于对照组(16.68%±3.69%),差异均有统计学意义(P<0.05);Western blot结果显示Sal+OGD组中LC3、Beclin1、PINK1和Parkin蛋白表达较OGD组明显降低,但仍高于对照组(P<0.05).结论:Sal抑制OGD诱导线粒体自噬相关蛋白表达,降低OGD诱导的星形胶质细胞损伤.

红景天苷;线粒体自噬;缺氧缺糖;星形胶质细胞;细胞损伤

红景天苷(salidroside,Sal)是红景天科植物提取物中最为重要的活性成分之一,具有广泛的抗氧化、抗炎等生物活性.研究证实,Sal对细胞损伤具有重要保护作用[1-2].自噬是真核生物细胞进行自我吞噬和自我保护的作用过程.但自噬又是一把"双刃剑",自噬过度激活不仅可明显诱导细胞损伤,亦可导致细胞器功能紊乱[3-4].线粒体自噬是指细胞在损伤刺激因素的作用下,将损伤的线粒体包裹至自噬体,并逐步降解的过程[5-6],是研究心脑血管缺血缺氧病理机制的重要靶点[7-8].本研究以缺氧缺糖(oxygen-glucose deprivation,OGD)诱导的细胞损伤为模型分析Sal对OGD诱导的线粒体自噬的影响.

1 材料和方法

1.1 材料 红景天苷、噻唑蓝(MTT),乳酸脱氢酶(lactate dehydrogenase,LDH)检测试剂盒购买于美国Sigma公司;鼠源性抗体Actin、Beclin1、LC3、PINK1和Parkin单克隆抗体购买于美国CST公司.SD大鼠(出生3 d内),由温州医科大学实验动物中心提供,动物生产许可证号:SCXK(浙)2015-0009.

1.2 方法

1.2.1 提取新生大鼠星形胶质细胞并纯化培养:取出生3 d的SD大鼠的大脑皮质,剥除软脑膜和血管,剪碎后经1.25 g/L胰蛋白酶37 ℃消化15 min,打散过滤后取沉淀,制成单细胞悬液(细胞存活率为90%).接种,孵育30 min,吸取细胞悬液,以1X105个/cm2密度散播在培养瓶中培养12 d,摇床18 h以去除少突胶质细胞和小胶质细胞;消化处理后孵育30 min,吸取细胞悬液进行纯化传代,以除去成纤维细胞;二次传代后即可得纯化的星形胶质细胞.

1.2.2 建立细胞OGD模型及分组:待细胞在培养器皿中培养到一定密度时,弃培养液,用PBS缓冲液洗涤细胞一遍,加入PBS缓冲液,将培养器皿放在密闭的OGD培养装置中,向内冲入95% N2/5% CO2的混合气,持续5 min,达到无糖缺氧的状态,然后将OGD培养装置放入37 ℃培养箱.实验分3组:对照组、OGD组和Sal+OGD组.

1.2.3 MTT法检测细胞存活率:按照文献方法[9],将增殖星形胶质细胞取对数生长期,调细胞密度为2X104/mL,接种于96孔培养板,每孔200 μL,常规培养,待细胞贴壁之后长至90%左右,予OGD处理0、2、4、8 h,Sal+OGD组用75、150、300 μmol/mL Sal预处理1 h,并给予OGD 4 h,设置调零孔,每组设置6个复孔,每孔加入20 μL MTT,37 ℃恒温箱孵育4 h后,弃去上清液,加入DMSO 120 μL终止反应.以调零孔光密度值(OD)调零校准,用酶联免疫检测仪在492 nm波长处测各孔的OD值.

1.2.4 LDH漏出率检测细胞损伤:将星形胶质细胞按1X106接种于12孔细胞培养板中,常规培养细胞24 h,待细胞贴壁之后长至90%左右,予OGD处理0、2、4、8 h处理,或者用0、75、150、300 μmol/mL Sal预处理1 h,并给予OGD 4 h,设置调零孔.吸取细胞培养上清作为细胞培养液样品,贴壁细胞用1%Triton X-100于37 ℃裂解30 min,吹打吸取裂解液后离心(3 000 r/min,5 min),取上清作为细胞裂解液样品.LDH活性按检测试剂盒说明书进行,测得各样品吸光度OD值,按公式LDH抽出率(%)=OD培养液/(OD培养液+OD细胞匀浆液)X100%,计算LDH漏出率.

1.2.5 Western blot检测自噬相关蛋白的表达:取上述处理后细胞,加入细胞裂解液冰上裂解15 min,12 000 r/min离心15 min,吸取上清.采用BCA法进行蛋白定量,取20 μg总蛋白4%~12%聚丙烯酰胺梯度凝胶电泳分离,电转至聚偏二氟乙烯膜上,3%脱脂牛奶室温封闭1 h.加入一抗4 ℃孵育过夜,随后洗涤缓冲液洗聚偏二氟乙烯膜3次,每次8 min,加入辣根过氧化物酶标记的第二抗体室温孵育1~2 h,再用洗涤缓冲液洗聚偏二氟乙烯膜3次,每次8 min,增强化学发光试剂发光、显影和定影.

1.3 统计学处理方法 采用SPSS22.0统计软件进行统计学处理.计量资料以表示,采用单因素方差分析.P<0.05为差异有统计学意义.

2 结果

2.1 Sal对OGD诱导细胞存活率的影响 75、150、300 μmol/mL Sal处理24 h后,细胞存活率分别为106.36%±5.96%、100.99%±6.64%和101.82%±10.69%,与对照组比差异无统计学意义(P>0.05).OGD干预2、4、8 h后,细胞存活率分别为78.32%±6.91%、71.22%±8.13%和67.36%±9.47%,均明显低于对照组的99.39%±2.17%,差异有统计学意义(P<0.05).Sal预处理星形胶质细胞1 h,再进行OGD干预4 h后,Sal终浓度为75、150、300 μmol/mL时,细胞存活率分别为83.15%±5.42%、86.75%±4.91%和91.41%±5.38%,明显高于OGD组(71.22%±8.13%),但仍低于对照组(100%),差异均有统计学意义(P<0.05).

2.2 Sal对OGD诱导LDH漏出率的影响 LDH检测结果显示,OGD干预2、4、8 h后,LDH漏出率为:38.23%±2.75%、40.51%±8.15%、46.34%±7.41%,均明显高于对照组(16.68±3.69%),差异具有统计学意义(P<0.05).Sal预处理星形胶质细胞1 h,再进行OGD干预4 h后,Sal终浓度为75、150、300 μmol/mL时,LDH漏出率为34.61%±5.19%、30.51%±8.15%、27.34%±7.41%,明显低于OGD组(40.51%±8.15%),但仍高于对照组(16.68%±3.69%),差异有统计学意义(P<0.05).

2.3 Sal降低OGD诱导的线粒体自噬相关蛋白表达

Western blot结果显示,与对照组比,OGD处理2、4、8 h后,星型胶质细胞自噬蛋白LC3和Beclin1表达明显增高,线粒体自噬相关蛋白PINK1和Parkin表达明显增高,差异具有统计学意义(P<0.05);其中,以4 h时增高最为明显(P<0.05),见图1A.Sal终浓度为300 μmol/mL时,OGD诱导的线粒体自噬蛋白PINK1、Parkin、LC3和Beclin1表达较OGD组明显下降,但仍高于对照组,差异具有统计学意义(P<0.05),见图1B.

图1 Western blot检测自噬相关蛋白表达

3 讨论

研究证实Sal对各种氧化应激诱导的细胞及器官损害具有重要的保护作用,例如:ZHENG等[10]研究发现Sal通过Akt-Nrf2信号通路抑制OGD诱导H9c2心肌细胞坏死.ZHONG等[11]研究证实Sal通过调节线粒体凋亡通路降低OGD诱导的心肌细胞LDH漏出率和细胞凋亡率,但其具体机制并未完全明确.本研究以提取的原代星形胶质细胞作为研究对象,构建OGD模型,分析Sal对OGD诱导的星形胶质细胞损伤的作用,重点分析Sal对OGD诱导的星形胶质细胞线粒体相关蛋白表达的影响.结果显示,成功提取星形胶质细胞后,予75、150、300 μmol/mL Sal处理24 h,细胞存活率无明显降低.予OGD干预2 h后,细胞存活率开始降低,LDH漏出率增高,干预8 h后,细胞存活率降至67.36%±9.47%,LDH漏出率则增高至46.34%±7.41%.说明OGD诱导星形胶质细胞损伤,但Sal对星形胶质细胞无明显毒性作用.取4 h作为时间点,用75、150、300 μmol/mL预处理1 h后,再予OGD干预,细胞存活率较OGD组明显增加,而LDH漏出率则明显降低,以Sal终浓度为150、300 μmol/mL时,差异具有统计学意义,说明Sal对OGD诱导的星形胶质细胞损伤具有保护作用.

研究发现线粒体自噬在缺氧再灌注损伤过程中起重要调控作用[8],其中,PINK1/Parkin信号通路是线粒体自噬的关键通路[5-6].正常情况下,位于线粒体外膜的PINK1,经转位酶的调节,转移至线粒体内膜,但当线粒体受损后,PINK1膜内转移受阻,线粒体外膜的PINK1累积增加,导致自身磷酸化而被激活[5].Parkin作为PINK1的下游分子,在接受PINK1刺激活化后,募集至线粒体基质与微管相关蛋白1轻链3(LC3)结合,促进线粒体包裹进入自噬体,从而导致线粒体自噬的发生[6].Beclin1蛋白和LC3是Beclin1发生和成熟的两种关键蛋白,可反映细胞内自噬水平的高低.O'DONNELL等[12]研究发现OGD可诱导海马星形胶质细胞线粒体自噬的发生.DI等[8]通过体内外研究发现美蓝可抑制缺血缺氧诱导的线粒体自噬.另外,Sal亦可降低皮层神经元LC3II/I,上调自噬水平,抑制神经毒性[13].本研究也证实OGD干预不同时间后,自噬标志蛋白Beclin1、LC3和线粒体自噬特异蛋白PINK1、Parkin表达均明显增高,以OGD干预4 h时最为明显.Sal终浓度为300 μmol/mL时可明显降低PINK1、Parkin、LC3和Beclin1表达,说明Sal抑制OGD诱导的线粒体自噬的发生.

综上所述,Sal可明显降低OGD诱导的线粒体自噬相关蛋白PINK1、Parkin、LC3和Beclin1表达,增加细胞存活率,起到保护作用.

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(本文编辑:赵翠翠)

The effect of salidroside on mitophagy induced by oxygen-glucose deprivation in astrocytes

ZHENG Liyun1,HUANG Huifen1, QIU Weiwen2.
1.Department of Neurology, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui, 323000; 2.Department of Neurology, the Sixth Affiliated Hospital of Wenzhou Medical University, Lishui, 323000

Objective:To investigate the effect of salidroside (Sal) on mitophagy induced by oxygen-glucose deprivation (OGD) in astrocytes.Methods:The primary astrocytes were isolated from cerebral cortex of neonatal rats and subjected to OGD for imitating hypoxia and glucose deprivation model in vitro. Cells were divided into sham group, OGD model group and Sal pretreatment+OGD group (Sal+OGD). Cell survival rate was detected with MTT and LDH leakage was analyzed with lactate dehydrogenase activity assay kit. Western blot was used to detect the expression of autophagy related protein.ResultsThe survival rate of cells with final concentration of 75, 150, 300 mol/mL Sal pretreatment and OGD were 83.15%±5.42%, 86.75%±4.91%, 91.41%±5.38%,which were significantly higher than that of the model group (71.22%±8.13%) (P<0.05), but still lower than that of the control group (99.39%±2.17%), the difference was statistically significant (P<0.05). Meanwhile, the LDH leakage rate of cell with final concentration of 75, 150, 300 mol/mL Sal pretreatment were 34.61%±5.19%,30.51%±8.15%, 27.34%±7.41%, which was significantly lower than that of model group (40.51%±8.15%),but still higher than that of the control group (16.68%±3.69%), the difference was statistically significant (P<0.05). Western blot showed the expression of LC3, Beclin1, PINK1 and Parkin protein in the Sal+OGD group significantly lower than that in model group, but still higher than that in the control group (P<0.05).Conclusion:Sal decreases mitophagy related protein and restored the cell proliferation inhibition induced by OGD in astrocytes.

salidroside; mitophagy; oxygen-glucose deprivation; astrocyte; cell injury

R285;R743.1

A

10.3969/j.issn.2095-9400.2017.10.009

2017-03-07

郑丽云(1987-),女,浙江丽水人,住院医师,硕士.

邱伟文,主任医师,Email:weiwenq@Hotmail.com.