紅景天苷對(duì)缺氧缺糖誘導(dǎo)的星形膠質(zhì)細(xì)胞線粒體自噬的影響

鄭麗云,黃慧芬,邱偉文

(1.溫州醫(yī)科大學(xué)附屬第五醫(yī)院 神經(jīng)內(nèi)科,浙江 麗水 323000;2.溫州醫(yī)科大學(xué)附屬第六醫(yī)院 神經(jīng)內(nèi)科,浙江 麗水 323000)

紅景天苷對(duì)缺氧缺糖誘導(dǎo)的星形膠質(zhì)細(xì)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(yī)科大學(xué)附屬第五醫(yī)院 神經(jīng)內(nèi)科,浙江 麗水 323000;2.溫州醫(yī)科大學(xué)附屬第六醫(yī)院 神經(jīng)內(nèi)科,浙江 麗水 323000)

目的:探討紅景天苷(Sal)對(duì)缺氧缺糖(OGD)誘導(dǎo)的星形膠質(zhì)細(xì)胞線粒體自噬的影響.方法:提取原代培養(yǎng)的新生鼠皮層星形膠質(zhì)細(xì)胞,進(jìn)行OGD干預(yù),建立OGD細(xì)胞模型.將實(shí)驗(yàn)分為對(duì)照組、OGD組和Sal+OGD組.用噻唑藍(lán)(MTT)法檢測(cè)細(xì)胞存活率,乳酸脫氫酶(LDH)法檢測(cè)LDH漏出率評(píng)估細(xì)胞損傷,Western blot法檢測(cè)自噬相關(guān)蛋白表達(dá)水平.結(jié)果:Sal終濃度為75、150、300 μmol/mL時(shí),細(xì)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(duì)照組(99.39%±2.17%),差異均有統(tǒng)計(jì)學(xué)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(duì)照組(16.68%±3.69%),差異均有統(tǒng)計(jì)學(xué)意義(P<0.05);Western blot結(jié)果顯示Sal+OGD組中LC3、Beclin1、PINK1和Parkin蛋白表達(dá)較OGD組明顯降低,但仍高于對(duì)照組(P<0.05).結(jié)論:Sal抑制OGD誘導(dǎo)線粒體自噬相關(guān)蛋白表達(dá),降低OGD誘導(dǎo)的星形膠質(zhì)細(xì)胞損傷.

紅景天苷;線粒體自噬;缺氧缺糖;星形膠質(zhì)細(xì)胞;細(xì)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(shí),Sal對(duì)細(xì)胞損傷具有重要保護(hù)作用[1-2].自噬是真核生物細(xì)胞進(jìn)行自我吞噬和自我保護(hù)的作用過(guò)程.但自噬又是一把"雙刃劍",自噬過(guò)度激活不僅可明顯誘導(dǎo)細(xì)胞損傷,亦可導(dǎo)致細(xì)胞器功能紊亂[3-4].線粒體自噬是指細(xì)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(guò)程[5-6],是研究心腦血管缺血缺氧病理機(jī)制的重要靶點(diǎn)[7-8].本研究以缺氧缺糖(oxygen-glucose deprivation,OGD)誘導(dǎo)的細(xì)胞損傷為模型分析Sal對(duì)OGD誘導(dǎo)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(lán)(MTT),乳酸脫氫酶(lactate dehydrogenase,LDH)檢測(cè)試劑盒購(gòu)買(mǎi)于美國(guó)Sigma公司;鼠源性抗體Actin、Beclin1、LC3、PINK1和Parkin單克隆抗體購(gòu)買(mǎi)于美國(guó)CST公司.SD大鼠(出生3 d內(nèi)),由溫州醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供,動(dòng)物生產(chǎn)許可證號(hào):SCXK(浙)2015-0009.

1.2 方法

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

1.2.2 建立細(xì)胞OGD模型及分組:待細(xì)胞在培養(yǎng)器皿中培養(yǎng)到一定密度時(shí),棄培養(yǎng)液,用PBS緩沖液洗滌細(xì)胞一遍,加入PBS緩沖液,將培養(yǎng)器皿放在密閉的OGD培養(yǎng)裝置中,向內(nèi)沖入95% N2/5% CO2的混合氣,持續(xù)5 min,達(dá)到無(wú)糖缺氧的狀態(tài),然后將OGD培養(yǎng)裝置放入37 ℃培養(yǎng)箱.實(shí)驗(yàn)分3組:對(duì)照組、OGD組和Sal+OGD組.

1.2.3 MTT法檢測(cè)細(xì)胞存活率:按照文獻(xiàn)方法[9],將增殖星形膠質(zhì)細(xì)胞取對(duì)數(shù)生長(zhǎng)期,調(diào)細(xì)胞密度為2X104/mL,接種于96孔培養(yǎng)板,每孔200 μL,常規(guī)培養(yǎng),待細(xì)胞貼壁之后長(zhǎng)至90%左右,予OGD處理0、2、4、8 h,Sal+OGD組用75、150、300 μmol/mL Sal預(yù)處理1 h,并給予OGD 4 h,設(shè)置調(diào)零孔,每組設(shè)置6個(gè)復(fù)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(yīng).以調(diào)零孔光密度值(OD)調(diào)零校準(zhǔn),用酶聯(lián)免疫檢測(cè)儀在492 nm波長(zhǎng)處測(cè)各孔的OD值.

1.2.4 LDH漏出率檢測(cè)細(xì)胞損傷:將星形膠質(zhì)細(xì)胞按1X106接種于12孔細(xì)胞培養(yǎng)板中,常規(guī)培養(yǎng)細(xì)胞24 h,待細(xì)胞貼壁之后長(zhǎng)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(yù)處理1 h,并給予OGD 4 h,設(shè)置調(diào)零孔.吸取細(xì)胞培養(yǎng)上清作為細(xì)胞培養(yǎng)液樣品,貼壁細(xì)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(xì)胞裂解液樣品.LDH活性按檢測(cè)試劑盒說(shuō)明書(shū)進(jìn)行,測(cè)得各樣品吸光度OD值,按公式LDH抽出率(%)=OD培養(yǎng)液/(OD培養(yǎng)液+OD細(xì)胞勻漿液)X100%,計(jì)算LDH漏出率.

1.2.5 Western blot檢測(cè)自噬相關(guān)蛋白的表達(dá):取上述處理后細(xì)胞,加入細(xì)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(jìn)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(zhuǎn)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(guò)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(guò)氧化物酶標(biāo)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(qiáng)化學(xué)發(fā)光試劑發(fā)光、顯影和定影.

1.3 統(tǒng)計(jì)學(xué)處理方法 采用SPSS22.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)處理.計(jì)量資料以表示,采用單因素方差分析.P<0.05為差異有統(tǒng)計(jì)學(xué)意義.

2 結(jié)果

2.1 Sal對(duì)OGD誘導(dǎo)細(xì)胞存活率的影響 75、150、300 μmol/mL Sal處理24 h后,細(xì)胞存活率分別為106.36%±5.96%、100.99%±6.64%和101.82%±10.69%,與對(duì)照組比差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05).OGD干預(yù)2、4、8 h后,細(xì)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(duì)照組的99.39%±2.17%,差異有統(tǒng)計(jì)學(xué)意義(P<0.05).Sal預(yù)處理星形膠質(zhì)細(xì)胞1 h,再進(jìn)行OGD干預(yù)4 h后,Sal終濃度為75、150、300 μmol/mL時(shí),細(xì)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(duì)照組(100%),差異均有統(tǒng)計(jì)學(xué)意義(P<0.05).

2.2 Sal對(duì)OGD誘導(dǎo)LDH漏出率的影響 LDH檢測(cè)結(jié)果顯示,OGD干預(yù)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(duì)照組(16.68±3.69%),差異具有統(tǒng)計(jì)學(xué)意義(P<0.05).Sal預(yù)處理星形膠質(zhì)細(xì)胞1 h,再進(jìn)行OGD干預(yù)4 h后,Sal終濃度為75、150、300 μmol/mL時(shí),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(duì)照組(16.68%±3.69%),差異有統(tǒng)計(jì)學(xué)意義(P<0.05).

2.3 Sal降低OGD誘導(dǎo)的線粒體自噬相關(guān)蛋白表達(dá)

Western blot結(jié)果顯示,與對(duì)照組比,OGD處理2、4、8 h后,星型膠質(zhì)細(xì)胞自噬蛋白LC3和Beclin1表達(dá)明顯增高,線粒體自噬相關(guān)蛋白PINK1和Parkin表達(dá)明顯增高,差異具有統(tǒng)計(jì)學(xué)意義(P<0.05);其中,以4 h時(shí)增高最為明顯(P<0.05),見(jiàn)圖1A.Sal終濃度為300 μmol/mL時(shí),OGD誘導(dǎo)的線粒體自噬蛋白PINK1、Parkin、LC3和Beclin1表達(dá)較OGD組明顯下降,但仍高于對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義(P<0.05),見(jiàn)圖1B.

圖1 Western blot檢測(cè)自噬相關(guān)蛋白表達(dá)

3 討論

研究證實(shí)Sal對(duì)各種氧化應(yīng)激誘導(dǎo)的細(xì)胞及器官損害具有重要的保護(hù)作用,例如:ZHENG等[10]研究發(fā)現(xiàn)Sal通過(guò)Akt-Nrf2信號(hào)通路抑制OGD誘導(dǎo)H9c2心肌細(xì)胞壞死.ZHONG等[11]研究證實(shí)Sal通過(guò)調(diào)節(jié)線粒體凋亡通路降低OGD誘導(dǎo)的心肌細(xì)胞LDH漏出率和細(xì)胞凋亡率,但其具體機(jī)制并未完全明確.本研究以提取的原代星形膠質(zhì)細(xì)胞作為研究對(duì)象,構(gòu)建OGD模型,分析Sal對(duì)OGD誘導(dǎo)的星形膠質(zhì)細(xì)胞損傷的作用,重點(diǎn)分析Sal對(duì)OGD誘導(dǎo)的星形膠質(zhì)細(xì)胞線粒體相關(guān)蛋白表達(dá)的影響.結(jié)果顯示,成功提取星形膠質(zhì)細(xì)胞后,予75、150、300 μmol/mL Sal處理24 h,細(xì)胞存活率無(wú)明顯降低.予OGD干預(yù)2 h后,細(xì)胞存活率開(kāi)始降低,LDH漏出率增高,干預(yù)8 h后,細(xì)胞存活率降至67.36%±9.47%,LDH漏出率則增高至46.34%±7.41%.說(shuō)明OGD誘導(dǎo)星形膠質(zhì)細(xì)胞損傷,但Sal對(duì)星形膠質(zhì)細(xì)胞無(wú)明顯毒性作用.取4 h作為時(shí)間點(diǎn),用75、150、300 μmol/mL預(yù)處理1 h后,再予OGD干預(yù),細(xì)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(shí),差異具有統(tǒng)計(jì)學(xué)意義,說(shuō)明Sal對(duì)OGD誘導(dǎo)的星形膠質(zhì)細(xì)胞損傷具有保護(hù)作用.

研究發(fā)現(xiàn)線粒體自噬在缺氧再灌注損傷過(guò)程中起重要調(diào)控作用[8],其中,PINK1/Parkin信號(hào)通路是線粒體自噬的關(guān)鍵通路[5-6].正常情況下,位于線粒體外膜的PINK1,經(jīng)轉(zhuǎn)位酶的調(diào)節(jié),轉(zhuǎn)移至線粒體內(nèi)膜,但當(dāng)線粒體受損后,PINK1膜內(nèi)轉(zhuǎn)移受阻,線粒體外膜的PINK1累積增加,導(dǎo)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(zhì)與微管相關(guān)蛋白1輕鏈3(LC3)結(jié)合,促進(jìn)線粒體包裹進(jìn)入自噬體,從而導(dǎo)致線粒體自噬的發(fā)生[6].Beclin1蛋白和LC3是Beclin1發(fā)生和成熟的兩種關(guān)鍵蛋白,可反映細(xì)胞內(nèi)自噬水平的高低.O'DONNELL等[12]研究發(fā)現(xiàn)OGD可誘導(dǎo)海馬星形膠質(zhì)細(xì)胞線粒體自噬的發(fā)生.DI等[8]通過(guò)體內(nèi)外研究發(fā)現(xiàn)美藍(lán)可抑制缺血缺氧誘導(dǎo)的線粒體自噬.另外,Sal亦可降低皮層神經(jīng)元LC3II/I,上調(diào)自噬水平,抑制神經(jīng)毒性[13].本研究也證實(shí)OGD干預(yù)不同時(shí)間后,自噬標(biāo)志蛋白Beclin1、LC3和線粒體自噬特異蛋白PINK1、Parkin表達(dá)均明顯增高,以O(shè)GD干預(yù)4 h時(shí)最為明顯.Sal終濃度為300 μmol/mL時(shí)可明顯降低PINK1、Parkin、LC3和Beclin1表達(dá),說(shuō)明Sal抑制OGD誘導(dǎo)的線粒體自噬的發(fā)生.

綜上所述,Sal可明顯降低OGD誘導(dǎo)的線粒體自噬相關(guān)蛋白PINK1、Parkin、LC3和Beclin1表達(dá),增加細(xì)胞存活率,起到保護(hù)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(yī)師,碩士.

邱偉文,主任醫(yī)師,Email:weiwenq@Hotmail.com.