紅景天苷對缺氧缺糖誘導的星形膠質細胞線粒體自噬的影響

鄭麗云,黃慧芬,邱偉文

(1.溫州醫科大學附屬第五醫院 神經內科,浙江 麗水 323000;2.溫州醫科大學附屬第六醫院 神經內科,浙江 麗水 323000)

紅景天苷對缺氧缺糖誘導的星形膠質細胞線粒體自噬的影響

鄭麗云1,黃慧芬1,邱偉文2

(1.溫州醫科大學附屬第五醫院 神經內科,浙江 麗水 323000;2.溫州醫科大學附屬第六醫院 神經內科,浙江 麗水 323000)

目的:探討紅景天苷(Sal)對缺氧缺糖(OGD)誘導的星形膠質細胞線粒體自噬的影響.方法:提取原代培養的新生鼠皮層星形膠質細胞,進行OGD干預,建立OGD細胞模型.將實驗分為對照組、OGD組和Sal+OGD組.用噻唑藍(MTT)法檢測細胞存活率,乳酸脫氫酶(LDH)法檢測LDH漏出率評估細胞損傷,Western blot法檢測自噬相關蛋白表達水平.結果:Sal終濃度為75、150、300 μmol/mL時,細胞存活率分別為83.15%±5.42%、86.75%±4.91%、91.41%±5.38%,明顯高于OGD組(71.22%±8.13%),但仍低于對照組(99.39%±2.17%),差異均有統計學意義(P<0.05);LDH漏出率為34.61%±5.19%、30.51%±8.15%、27.34±7.41%,明顯低于OGD組(40.51%±8.15%),但仍高于對照組(16.68%±3.69%),差異均有統計學意義(P<0.05);Western blot結果顯示Sal+OGD組中LC3、Beclin1、PINK1和Parkin蛋白表達較OGD組明顯降低,但仍高于對照組(P<0.05).結論:Sal抑制OGD誘導線粒體自噬相關蛋白表達,降低OGD誘導的星形膠質細胞損傷.

紅景天苷;線粒體自噬;缺氧缺糖;星形膠質細胞;細胞損傷

紅景天苷(salidroside,Sal)是紅景天科植物提取物中最為重要的活性成分之一,具有廣泛的抗氧化、抗炎等生物活性.研究證實,Sal對細胞損傷具有重要保護作用[1-2].自噬是真核生物細胞進行自我吞噬和自我保護的作用過程.但自噬又是一把"雙刃劍",自噬過度激活不僅可明顯誘導細胞損傷,亦可導致細胞器功能紊亂[3-4].線粒體自噬是指細胞在損傷刺激因素的作用下,將損傷的線粒體包裹至自噬體,并逐步降解的過程[5-6],是研究心腦血管缺血缺氧病理機制的重要靶點[7-8].本研究以缺氧缺糖(oxygen-glucose deprivation,OGD)誘導的細胞損傷為模型分析Sal對OGD誘導的線粒體自噬的影響.

1 材料和方法

1.1 材料 紅景天苷、噻唑藍(MTT),乳酸脫氫酶(lactate dehydrogenase,LDH)檢測試劑盒購買于美國Sigma公司;鼠源性抗體Actin、Beclin1、LC3、PINK1和Parkin單克隆抗體購買于美國CST公司.SD大鼠(出生3 d內),由溫州醫科大學實驗動物中心提供,動物生產許可證號:SCXK(浙)2015-0009.

1.2 方法

1.2.1 提取新生大鼠星形膠質細胞并純化培養:取出生3 d的SD大鼠的大腦皮質,剝除軟腦膜和血管,剪碎后經1.25 g/L胰蛋白酶37 ℃消化15 min,打散過濾后取沉淀,制成單細胞懸液(細胞存活率為90%).接種,孵育30 min,吸取細胞懸液,以1X105個/cm2密度散播在培養瓶中培養12 d,搖床18 h以去除少突膠質細胞和小膠質細胞;消化處理后孵育30 min,吸取細胞懸液進行純化傳代,以除去成纖維細胞;二次傳代后即可得純化的星形膠質細胞.

1.2.2 建立細胞OGD模型及分組:待細胞在培養器皿中培養到一定密度時,棄培養液,用PBS緩沖液洗滌細胞一遍,加入PBS緩沖液,將培養器皿放在密閉的OGD培養裝置中,向內沖入95% N2/5% CO2的混合氣,持續5 min,達到無糖缺氧的狀態,然后將OGD培養裝置放入37 ℃培養箱.實驗分3組:對照組、OGD組和Sal+OGD組.

1.2.3 MTT法檢測細胞存活率:按照文獻方法[9],將增殖星形膠質細胞取對數生長期,調細胞密度為2X104/mL,接種于96孔培養板,每孔200 μL,常規培養,待細胞貼壁之后長至90%左右,予OGD處理0、2、4、8 h,Sal+OGD組用75、150、300 μmol/mL Sal預處理1 h,并給予OGD 4 h,設置調零孔,每組設置6個復孔,每孔加入20 μL MTT,37 ℃恒溫箱孵育4 h后,棄去上清液,加入DMSO 120 μL終止反應.以調零孔光密度值(OD)調零校準,用酶聯免疫檢測儀在492 nm波長處測各孔的OD值.

1.2.4 LDH漏出率檢測細胞損傷:將星形膠質細胞按1X106接種于12孔細胞培養板中,常規培養細胞24 h,待細胞貼壁之后長至90%左右,予OGD處理0、2、4、8 h處理,或者用0、75、150、300 μmol/mL Sal預處理1 h,并給予OGD 4 h,設置調零孔.吸取細胞培養上清作為細胞培養液樣品,貼壁細胞用1%Triton X-100于37 ℃裂解30 min,吹打吸取裂解液后離心(3 000 r/min,5 min),取上清作為細胞裂解液樣品.LDH活性按檢測試劑盒說明書進行,測得各樣品吸光度OD值,按公式LDH抽出率(%)=OD培養液/(OD培養液+OD細胞勻漿液)X100%,計算LDH漏出率.

1.2.5 Western blot檢測自噬相關蛋白的表達:取上述處理后細胞,加入細胞裂解液冰上裂解15 min,12 000 r/min離心15 min,吸取上清.采用BCA法進行蛋白定量,取20 μg總蛋白4%~12%聚丙烯酰胺梯度凝膠電泳分離,電轉至聚偏二氟乙烯膜上,3%脫脂牛奶室溫封閉1 h.加入一抗4 ℃孵育過夜,隨后洗滌緩沖液洗聚偏二氟乙烯膜3次,每次8 min,加入辣根過氧化物酶標記的第二抗體室溫孵育1~2 h,再用洗滌緩沖液洗聚偏二氟乙烯膜3次,每次8 min,增強化學發光試劑發光、顯影和定影.

1.3 統計學處理方法 采用SPSS22.0統計軟件進行統計學處理.計量資料以表示,采用單因素方差分析.P<0.05為差異有統計學意義.

2 結果

2.1 Sal對OGD誘導細胞存活率的影響 75、150、300 μmol/mL Sal處理24 h后,細胞存活率分別為106.36%±5.96%、100.99%±6.64%和101.82%±10.69%,與對照組比差異無統計學意義(P>0.05).OGD干預2、4、8 h后,細胞存活率分別為78.32%±6.91%、71.22%±8.13%和67.36%±9.47%,均明顯低于對照組的99.39%±2.17%,差異有統計學意義(P<0.05).Sal預處理星形膠質細胞1 h,再進行OGD干預4 h后,Sal終濃度為75、150、300 μmol/mL時,細胞存活率分別為83.15%±5.42%、86.75%±4.91%和91.41%±5.38%,明顯高于OGD組(71.22%±8.13%),但仍低于對照組(100%),差異均有統計學意義(P<0.05).

2.2 Sal對OGD誘導LDH漏出率的影響 LDH檢測結果顯示,OGD干預2、4、8 h后,LDH漏出率為:38.23%±2.75%、40.51%±8.15%、46.34%±7.41%,均明顯高于對照組(16.68±3.69%),差異具有統計學意義(P<0.05).Sal預處理星形膠質細胞1 h,再進行OGD干預4 h后,Sal終濃度為75、150、300 μmol/mL時,LDH漏出率為34.61%±5.19%、30.51%±8.15%、27.34%±7.41%,明顯低于OGD組(40.51%±8.15%),但仍高于對照組(16.68%±3.69%),差異有統計學意義(P<0.05).

2.3 Sal降低OGD誘導的線粒體自噬相關蛋白表達

Western blot結果顯示,與對照組比,OGD處理2、4、8 h后,星型膠質細胞自噬蛋白LC3和Beclin1表達明顯增高,線粒體自噬相關蛋白PINK1和Parkin表達明顯增高,差異具有統計學意義(P<0.05);其中,以4 h時增高最為明顯(P<0.05),見圖1A.Sal終濃度為300 μmol/mL時,OGD誘導的線粒體自噬蛋白PINK1、Parkin、LC3和Beclin1表達較OGD組明顯下降,但仍高于對照組,差異具有統計學意義(P<0.05),見圖1B.

圖1 Western blot檢測自噬相關蛋白表達

3 討論

研究證實Sal對各種氧化應激誘導的細胞及器官損害具有重要的保護作用,例如:ZHENG等[10]研究發現Sal通過Akt-Nrf2信號通路抑制OGD誘導H9c2心肌細胞壞死.ZHONG等[11]研究證實Sal通過調節線粒體凋亡通路降低OGD誘導的心肌細胞LDH漏出率和細胞凋亡率,但其具體機制并未完全明確.本研究以提取的原代星形膠質細胞作為研究對象,構建OGD模型,分析Sal對OGD誘導的星形膠質細胞損傷的作用,重點分析Sal對OGD誘導的星形膠質細胞線粒體相關蛋白表達的影響.結果顯示,成功提取星形膠質細胞后,予75、150、300 μmol/mL Sal處理24 h,細胞存活率無明顯降低.予OGD干預2 h后,細胞存活率開始降低,LDH漏出率增高,干預8 h后,細胞存活率降至67.36%±9.47%,LDH漏出率則增高至46.34%±7.41%.說明OGD誘導星形膠質細胞損傷,但Sal對星形膠質細胞無明顯毒性作用.取4 h作為時間點,用75、150、300 μmol/mL預處理1 h后,再予OGD干預,細胞存活率較OGD組明顯增加,而LDH漏出率則明顯降低,以Sal終濃度為150、300 μmol/mL時,差異具有統計學意義,說明Sal對OGD誘導的星形膠質細胞損傷具有保護作用.

研究發現線粒體自噬在缺氧再灌注損傷過程中起重要調控作用[8],其中,PINK1/Parkin信號通路是線粒體自噬的關鍵通路[5-6].正常情況下,位于線粒體外膜的PINK1,經轉位酶的調節,轉移至線粒體內膜,但當線粒體受損后,PINK1膜內轉移受阻,線粒體外膜的PINK1累積增加,導致自身磷酸化而被激活[5].Parkin作為PINK1的下游分子,在接受PINK1刺激活化后,募集至線粒體基質與微管相關蛋白1輕鏈3(LC3)結合,促進線粒體包裹進入自噬體,從而導致線粒體自噬的發生[6].Beclin1蛋白和LC3是Beclin1發生和成熟的兩種關鍵蛋白,可反映細胞內自噬水平的高低.O'DONNELL等[12]研究發現OGD可誘導海馬星形膠質細胞線粒體自噬的發生.DI等[8]通過體內外研究發現美藍可抑制缺血缺氧誘導的線粒體自噬.另外,Sal亦可降低皮層神經元LC3II/I,上調自噬水平,抑制神經毒性[13].本研究也證實OGD干預不同時間后,自噬標志蛋白Beclin1、LC3和線粒體自噬特異蛋白PINK1、Parkin表達均明顯增高,以OGD干預4 h時最為明顯.Sal終濃度為300 μmol/mL時可明顯降低PINK1、Parkin、LC3和Beclin1表達,說明Sal抑制OGD誘導的線粒體自噬的發生.

綜上所述,Sal可明顯降低OGD誘導的線粒體自噬相關蛋白PINK1、Parkin、LC3和Beclin1表達,增加細胞存活率,起到保護作用.

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(本文編輯:趙翠翠)

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

鄭麗云(1987-),女,浙江麗水人,住院醫師,碩士.

邱偉文,主任醫師,Email:weiwenq@Hotmail.com.