3-羥基丁酸衍生物對(duì)PC12細(xì)胞凋亡的保護(hù)作用*

鄒湘輝,劉博聰,2,莊東紅,查廣才,吳云影

(1.韓山師范學(xué)院生物系,潮州 521041;2.汕頭大學(xué)生物系,汕頭 515063)

3-羥基丁酸衍生物對(duì)PC12細(xì)胞凋亡的保護(hù)作用*

鄒湘輝1,劉博聰1,2,莊東紅1,查廣才1,吳云影1

(1.韓山師范學(xué)院生物系,潮州 521041;2.汕頭大學(xué)生物系,汕頭 515063)

目的 觀察3-羥基丁酸甲酯和3-羥基丁酸乙酯對(duì)PC12缺血清誘導(dǎo)凋亡細(xì)胞的保護(hù)作用。方法化學(xué)法降解聚羥基丁酸酯制備3-羥基丁酸甲酯和3-羥基丁酸乙酯。培養(yǎng)PC12細(xì)胞,以含血清培養(yǎng)組細(xì)胞為陰性對(duì)照組,缺血清培養(yǎng)組細(xì)胞為陽性對(duì)照組,以在缺血清條件下添加不同濃度3-羥基丁酸甲酯或3-羥基丁酸乙酯細(xì)胞為樣品組,通過形態(tài)學(xué)觀察法、噻唑藍(lán)(MTT)法檢測(cè)各組細(xì)胞形態(tài)及活性變化。結(jié)果與陰性對(duì)照組比較,陽性對(duì)照組細(xì)胞出現(xiàn)大量凋亡,形態(tài)變小變細(xì),活性降低。樣品處理組細(xì)胞活性不同程度提高,低濃度(0.01和0.001 mg·mL-1)3-羥基丁酸甲酯和3-羥基丁酸乙酯對(duì)凋亡細(xì)胞有保護(hù)作用,濃度為1 mg·mL-1時(shí)效果最好。結(jié)論通過化學(xué)法能夠制備高純度3-羥基丁酸甲酯和3-羥基丁酸乙酯,上述兩種樣品對(duì)缺血清誘導(dǎo)凋亡的PC12細(xì)胞有保護(hù)作用。

3-羥基丁酸;3-羥基丁酸甲酯;3-羥基丁酸乙酯;PC12細(xì)胞;細(xì)胞凋亡

3-羥基丁酸甲酯和3-羥基丁酸乙酯是3-羥基丁酸衍生物,3-羥基丁酸是人體脂肪酸代謝后產(chǎn)生的3種酮體之一,通常人體血液和組織中濃度約0.1 mmol·L-1,在糖饑餓情況下可以作為腦部的一種應(yīng)急性能源使用[1]。已有研究表明,3-羥基丁酸與糖尿病、體內(nèi)能量代謝紊亂等有著密切聯(lián)系[2]。在以往的研究中發(fā)現(xiàn),聚羥基脂肪酸酯(polyhydroxyalkanoates,PHA)在組織工程和材料學(xué)方面具有十分突出的應(yīng)用潛力,目前PHA的主要應(yīng)用是作為一種環(huán)境友好型生物可降解塑料。3-羥基丁酸作為高分子PHA的一種最普遍的降解產(chǎn)物,受到越來越多的關(guān)注[3],3-羥基丁酸和通過對(duì)聚羥基丁酸酯(polyhydroxybutyrate,PHB)進(jìn)行醇解衍生化生成3-羥基丁酸甲酯和-3羥基丁酸乙酯能夠促進(jìn)神經(jīng)膠質(zhì)細(xì)胞、成骨細(xì)胞等的生長[3]。筆者前期的工作發(fā)現(xiàn),3-羥基丁酸及其衍生物具有增強(qiáng)大鼠腦部神經(jīng)遞質(zhì)表達(dá)和提高大鼠認(rèn)知功能的作用[4]。筆者在本實(shí)驗(yàn)中采用缺血清誘導(dǎo)PC12細(xì)胞凋亡,研究3-羥基丁酸甲酯和3-羥基丁酸乙酯對(duì)凋亡細(xì)胞的保護(hù)作用。

1 材料與方法

1.1 試劑及設(shè)備 PHB(清華大學(xué)生命科學(xué)學(xué)院微生物實(shí)驗(yàn)室提供,該材料為其實(shí)驗(yàn)室微生物發(fā)酵產(chǎn)物,純度:97.5%);達(dá)爾伯克必需基本培養(yǎng)液(Dulbecco's Modified Eagle Medium,DMEM)高糖培養(yǎng)液(批號(hào): 1154412)購自美國Gibco公司;噻唑藍(lán)(thiazole blue, MTT,批號(hào):C18H16N5SBr)購自美國Sigma-Aldrich公司;胎牛血清購自四季青公司(批號(hào):130620);二甲亞砜(dimethyl sulfoxide,DMSO)購自天津希恩思公司; Shimadzu氣質(zhì)聯(lián)用(GasChromatograph-Mass Spectrometer,GC-MS)儀(GC-MSQP5050A);OLYMPUS 1X7/1X51倒置顯微鏡。

1.2 3-羥基丁酸甲酯及3-羥基丁酸乙酯的制備及鑒定 稱取PHB 5 g,溶于50 mL三氯甲烷,轉(zhuǎn)移至圓底燒瓶。安裝上水浴冷凝管,并放入恒溫水浴鍋,緩慢加熱攪拌溶解。另量取甲醇100 mL放入150 mL錐形瓶,同時(shí)加入濃硫酸2 mL常溫?cái)嚢杌旌?將上述乙醇-濃硫酸溶液加入PHB三氯甲烷溶液圓底燒瓶,混合。溫度控制在約90℃。反應(yīng)時(shí)間48 h。反應(yīng)結(jié)束后,抽濾除雜。將濾液轉(zhuǎn)移至250 mL分液漏斗,加入純化水25 mL,靜置分層,收集下層有機(jī)相,加入5%碳酸氫鈉(NaHCO3)25 mL中和1次,純化水25 mL洗滌1次,分液漏斗分離,有機(jī)相用無水硫酸鈉干燥,過濾出硫酸鈉。有機(jī)相用旋轉(zhuǎn)蒸發(fā)儀蒸發(fā)三氯甲烷并干燥,減壓蒸餾得到產(chǎn)物。通過以上過程制備的產(chǎn)物用GC-MS分析其組成和比例。

3-羥基丁酸乙酯制備及鑒定與3-羥基丁酸甲酯制備方法相同,將其中的甲醇更換成乙醇。通過以上過程制備的產(chǎn)物用GC-MS分析其組成和比例。

1.3 PC12細(xì)胞的培養(yǎng)及損傷模型的建立 PC12細(xì)胞用含10%胎牛血清的DMEM高糖培養(yǎng)液于37℃、5%二氧化碳(CO2)細(xì)胞培養(yǎng)箱中培養(yǎng),當(dāng)細(xì)胞覆蓋瓶底面積約80%時(shí),0.25%胰蛋白酶消化離心,完全培養(yǎng)液重懸后進(jìn)行繼代培養(yǎng)。取對(duì)數(shù)期生長期細(xì)胞用于分組實(shí)驗(yàn)。

陰性對(duì)照組更換新鮮含5%胎牛血清的高糖DMEM培養(yǎng)液,陽性對(duì)照組更換新鮮不含血清的DMEM高糖培養(yǎng)液,3-羥基丁酸甲酯保護(hù)組更換新鮮不含胎牛血清但3-羥基丁酸甲酯終濃度為1,0.1, 0.01,0.001 mg·mL-1DMEM高糖培養(yǎng)液,3-羥基丁酸乙酯保護(hù)組更換新鮮不含胎牛血清但3-羥基丁酸乙酯終濃度為1,0.1,0.01,0.001 mg·mL-1DMEM高糖培養(yǎng)液。繼續(xù)培養(yǎng)24 h。

1.4 細(xì)胞形態(tài)學(xué)觀察 倒置顯微鏡下觀察各組細(xì)胞形態(tài)并攝像。

1.5 MTT法檢測(cè)細(xì)胞活性 各組細(xì)胞每孔加入5 mg·mL-1MTT 20 μL,孵育4 h后去上清液,每孔加入DMSO 150 μL,水平搖床震蕩10 min,酶標(biāo)儀[芬蘭/雷勃(美國熱電),型號(hào):Multiskan MK3]492 nm波長下測(cè)定吸光度(A)值,調(diào)零孔加入DMSO 150 μL為空白對(duì)照。

2 結(jié)果

2.1 PHB醇解產(chǎn)物的鑒定分析 通過醇解PHB制得3-羥基丁酸甲酯和3-羥基丁酸乙酯,經(jīng)過GC-MS檢測(cè)后各產(chǎn)物鑒定分析結(jié)果見圖1,2和表1,2所示。

1.3-羥基丁酸乙酯;2.3-羥基丁酸乙酯圖1 PHB乙醇解產(chǎn)物GC-MS分析1.ethyl-3-hydroxybutyrate;2.ethyl-3-hydroxybutyrateFig.1 GC-MS analysis on ethanolysis product of PHB

表1 PHB乙醇解產(chǎn)物成分Tab.1 Component of ethanolysis product of PHB

1.3-羥基丁酸甲酯;2.3-羥基丁酸甲酯圖2 PHB甲醇解產(chǎn)物GC-MS分析1.methyl-3-hydroxybutyrate;2.methyl-3-hydroxybutyrateFig.2 GC-MS analysis on methanolysis product of PHB

表2 PHB甲醇解產(chǎn)物成分Tab.2 Component of methanolysis product of PHB

由表1,2可以看出,通過醇解方法對(duì)PHB進(jìn)行降解可以制備高純度3-羥基丁酸甲酯和3-羥基丁酸乙酯,產(chǎn)物在GC-MS分析下分別有兩個(gè)不同的出峰時(shí)間。查閱數(shù)據(jù)庫后分析可知,是制備過程中同種產(chǎn)物出現(xiàn)左旋和右旋兩種不同構(gòu)型形成的。

2.2 細(xì)胞形態(tài) 見圖3,4。陰性對(duì)照組細(xì)胞長勢(shì)良好,單個(gè)細(xì)胞呈現(xiàn)粗壯多變性,有3或4個(gè)突起,折光性高,細(xì)胞生長均勻且密度較大。陽性對(duì)照組細(xì)胞呈細(xì)絲線形或圓形欲浮起狀,兩端無法看見類似陽性對(duì)照組細(xì)胞的突起,折光性低,細(xì)胞生長不均勻且密度較小。在兩種樣品實(shí)驗(yàn)組中可以看出在各個(gè)劑量下的細(xì)胞形態(tài)與陽性對(duì)照比較較粗壯,折光性恢復(fù),無明顯細(xì)胞突起,但細(xì)胞數(shù)目明顯較模型對(duì)照組多。在3-羥基丁酸甲酯保護(hù)組中,0.01 mg·mL-13-羥基丁酸甲酯組細(xì)胞形態(tài)較陽性對(duì)照組有較大變化,細(xì)胞寬度明顯加大,少數(shù)細(xì)胞還能觀察到細(xì)胞突起存在,折光性較好。1 mg·mL-13-羥基丁酸乙酯保護(hù)組細(xì)胞寬度加大,折光性好,細(xì)胞突觸數(shù)量多,細(xì)胞數(shù)目也較陽性對(duì)照組多。

2.3 細(xì)胞活性檢測(cè) 經(jīng)過缺血清誘導(dǎo)處理后,陽性對(duì)照組細(xì)胞活性較陰性對(duì)照組差,而添加了不同劑量3-羥基丁酸甲酯和3-羥基丁酸乙酯的細(xì)胞活性均有不同程度提高,3-羥基丁酸甲酯和3-羥基丁酸乙酯在1 mg·mL-1時(shí)均呈現(xiàn)出最好的保護(hù)作用,劑量為0.1 mg·mL-1時(shí)保護(hù)作用不明顯,劑量為0.01和0.001 mg·mL-1時(shí)均具有一定保護(hù)作用(表3)。

3 討論

3-羥基丁酸是最普遍的PHA降解產(chǎn)物,近年來對(duì)于3-羥基丁酸的生理作用的研究逐漸增多,包括為大腦提供能量[3],作為腦的谷氨酸抑制性神經(jīng)遞質(zhì)(γ-氨基丁酸)的代謝前體物[4],和作為細(xì)胞凋亡的抑制劑作用等[5]。目前利用生物方法來進(jìn)行3-羥基丁酸及其衍生物的合成很困難,且有關(guān)3-羥基丁酸甲酯和3-羥基丁酸乙酯在PC12細(xì)胞凋亡保護(hù)方面的研究還未見報(bào)道。筆者通過化學(xué)降解法成功制備得到3-羥基丁酸甲酯和3-羥基丁酸乙酯,具有很高的得率,再利用GC-MS對(duì)產(chǎn)物進(jìn)行檢測(cè),發(fā)現(xiàn)產(chǎn)物的純度也比較高,化學(xué)法降解PHA制備3-羥基丁酸衍生物作為一種簡單的制備手性藥物分子的途徑具有推廣利用的價(jià)值。

A.陰性對(duì)照組;B.陽性對(duì)照組;C.1 mg·mL-13-羥基丁酸乙酯組;D.0.1 mg·mL-13-羥基丁酸乙酯組;E.0.01 mg·mL-13-羥基丁酸乙酯組;F.0.001 mg·mL-13-羥基丁酸乙酯組圖4 3-羥基丁酸乙酯對(duì)缺血清誘導(dǎo)細(xì)胞凋亡保護(hù)作用A.negative control group;B.positive control group;C.1 mg·mL-1Ethyl-3-hydroxybutyrate group;D.0.1 mg·mL-1Ethyl-3-hydroxybutyrate group;E.0.01 mg·mL-1Ethyl-3-hydroxybutyrate group;F.0.001 mg·mL-1Ethyl-3-hydroxybutyrate groupFig.4 Protection of ethyl-3-hydroxybutyrate on the apoptosis of PC12 induced by FBS deficiency

表3 3-羥基丁酸衍生物對(duì)PC12細(xì)胞活性影響分析Tab.3 Effect of derivate of 3-hydroxybutyrate on the activity of PC12 cells ±s

表3 3-羥基丁酸衍生物對(duì)PC12細(xì)胞活性影響分析Tab.3 Effect of derivate of 3-hydroxybutyrate on the activity of PC12 cells ±s

與陽性對(duì)照組比較,*1P＜0.05Compared with positive control group,*1P＜0.05

組別吸光度值(A492nm)組別吸光度值(A492nm)陰性對(duì)照組0.176±0.008*1,χ2=4.017陰性對(duì)照組0.127±0.003*1,χ2=3.115陽性對(duì)照組0.112±0.002陽性對(duì)照組0.087±0.001 1 mg·mL-13-羥基丁酸甲酯組0.149±0.007*1,χ2=3.3231 mg·mL-13-羥基丁酸乙酯組0.122±0.003*1,χ2=3.634 0.1 mg·mL-13-羥基丁酸甲酯組0.106±0.004,χ2=0.5680.1 mg·mL-13-羥基丁酸乙酯組0.086±0.003,χ2=0.115 0.01 mg·mL-13-羥基丁酸甲酯組0.127±0.003*1,χ2=2.1190.01 mg·mL-13-羥基丁酸乙酯組0.105±0.003*1,χ2=2.616 0.001 mg·mL-13-羥基丁酸甲酯組0.141±0.003*1,χ2=3.1350.001 mg·mL-13-羥基丁酸乙酯組0.118±0.004*1,χ2=3.213

PC12細(xì)胞是大鼠腎上腺髓質(zhì)嗜鉻細(xì)胞瘤克隆化的細(xì)胞株,在正常情況下PC12細(xì)胞在形態(tài)上和生理生化上與腎上腺髓質(zhì)細(xì)胞相似,而在培養(yǎng)物中添加神經(jīng)生長因子(nerve growth factor,NGF)后,PC12細(xì)胞具有神經(jīng)細(xì)胞的形態(tài)和相類似的代謝產(chǎn)物形成[9],目前PC12細(xì)胞正被廣泛地應(yīng)用于研究各種作用于神經(jīng)細(xì)胞的分化、受體及神經(jīng)遞質(zhì)釋放等的中樞神經(jīng)系統(tǒng)藥物的作用機(jī)制。細(xì)胞凋亡在20世紀(jì)70年代首先由HERR等[10]提出,細(xì)胞凋亡在組織和器官的發(fā)育或者多種病變過程中起著重要的作用。神經(jīng)細(xì)胞凋亡是許多中樞神經(jīng)系統(tǒng)疾病發(fā)生的最終原因,諸如老年癡呆癥、帕金森病和低氧缺血性腦損傷等,而在體外細(xì)胞培養(yǎng)過程中模型細(xì)胞凋亡的方式有很多種,缺血清誘導(dǎo)細(xì)胞凋亡是其中比較簡便且行之有效的方法之一,具體做法是去除培養(yǎng)液中的血清,使細(xì)胞在一定時(shí)間內(nèi)處于無血清培養(yǎng)狀態(tài),細(xì)胞因缺乏必要的生長因子和營養(yǎng)物質(zhì)而發(fā)生凋亡[11]。本研究發(fā)現(xiàn),PC12細(xì)胞缺血清誘導(dǎo)凋亡組與正常的含血清培養(yǎng)組比較,細(xì)胞形態(tài)變化明顯,活性降低,凋亡增加,而在添加了不同濃度3-羥基丁酸甲酯或者3-羥基丁酸乙酯的實(shí)驗(yàn)組中,極低濃度的3-羥基丁酸甲酯和3-羥基丁酸乙酯都可以抑制缺血清誘導(dǎo)的PC12細(xì)胞凋亡,細(xì)胞數(shù)目增加,對(duì)細(xì)胞形態(tài)具有一定的修復(fù)作用,細(xì)胞活性顯著增強(qiáng),高濃度組細(xì)胞的活性可達(dá)到與陰性對(duì)照組接近。推測(cè)這兩種小分子可能在低濃度時(shí)作為某一種信號(hào)分子啟動(dòng)了細(xì)胞內(nèi)抑制細(xì)胞凋亡基因的表達(dá),在較高濃度時(shí)同時(shí)啟動(dòng)了凋亡基因的表達(dá)而表現(xiàn)出對(duì)細(xì)胞的保護(hù)作用減弱,但是隨著濃度的進(jìn)一步升高,可作為營養(yǎng)成分促進(jìn)細(xì)胞的生存,詳細(xì)機(jī)制還有待進(jìn)一步研究。

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DOI 10.3870/yydb.2014.11.007

Protective Effect of 3-hydroxybutyrate Derivate on the Apoptosis of PC12

ZOU Xiang-hui1,LIU Bo-cong1,2,ZHUANG Dong-hong1,ZHA Guang-cai1,WU Yun-ying1
(1.Department of Biology,Hanshan Normal University,Chaozhou 521041,China;2.Department of Biology,Shantou University, Shantou 515063,China)

ObjectiveTo investigate the protective effect of 3-hydroxybutyratederivate(methyl-3-hydroxybutyrateand ethyl-3-hydroxybutyrate)on the apoptosis of PC12 induced by FBS deficiency.MethodsMethyl-3-hydroxybutyrateand ethyl-3-hydroxybutyratewere prepared by chemical degradation.PC12 cells were divided into different groups:medium with FBS as negative control,medium without FBS as positive control,medium with different concentrations of methyl-3-hydroxybutyrateor ethyl-3-hydroxybutyratewithout FBS as sample groups.The shape and viability of cells in each group were analyzed by optical microscope and MTT.ResultsCompared with the negative control,cells in the positive control group demonstrated a large number of apoptosis,smaller and thinner morphology,and lower activity.However,the activity of cells was improved in the sample groups.Low concentration(0.01 and 0.001 mg·mL-1)of methyl-3-hydroxybutyrateand ethyl-3-hydroxybutyrateshowed a protective effect on cell apoptosis,and 1 mg·mL-1had the best protection effect.ConclusionHigh purity methyl-3-hydroxybutyrateand ethyl-3-hydroxybutyratecan be produced by chemical method,and both chemicals present good effect on protecting the PC12 from apoptosis.

3-hydroxybutyrate;Methyl-3-hydroxybutyrate;Ethyl-3-hydroxybutyrate;PC12 cell;Apoptosis

R977;R965

A

1004-0781(2014)11-1427-04

2013-09-27

2013-10-25

*廣東省自然科學(xué)基金博士啟動(dòng)項(xiàng)目(10452104101004655);韓山師范學(xué)院青年基金項(xiàng)目(LQ200801)

鄒湘輝(1976-),男,湖南衡陽人,副教授,博士,主要研究方向:細(xì)胞生物學(xué)。E-mail:zxh11043@126.com。