陳佩雷,潘建春
(1.溫州醫(yī)科大學(xué)附屬樂(lè)清醫(yī)院 臨床藥學(xué)室,浙江 樂(lè)清 325600;2.溫州醫(yī)科大學(xué) 藥學(xué)院,浙江 溫州 325035)
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人參多糖對(duì)秀麗線蟲(chóng)polyQ聚集毒性和壽命的影響
陳佩雷1,潘建春2Δ
(1.溫州醫(yī)科大學(xué)附屬樂(lè)清醫(yī)院 臨床藥學(xué)室,浙江 樂(lè)清 325600;2.溫州醫(yī)科大學(xué) 藥學(xué)院,浙江 溫州 325035)
目的 探討人參多糖對(duì)秀麗線蟲(chóng)polyQ聚集毒性和壽命的影響。方法 將秀麗線蟲(chóng)HA759和AM141各分成2組,Control組和Ginseng組。Control組,不做任何特殊處理,Ginseng組將10 mg/mL中藥多糖與 OP50-1按照 1:4 的比例混合至總體積 50 mL后加入秀麗線蟲(chóng)培養(yǎng)平皿。觀察秀麗線蟲(chóng)HA759 ASH 神經(jīng)元的存活狀況;每天取樣統(tǒng)計(jì)秀麗線蟲(chóng)AM141周身聚集熒光點(diǎn)數(shù);研究2種秀麗線蟲(chóng)的壽命。結(jié)果 研究表明,培養(yǎng)3 d后Control組 HA759秀麗線蟲(chóng)ASH 神經(jīng)元存活率為53%,Ginseng組ASH 神經(jīng)元存活率為64%,2組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。Control組AM141秀麗線蟲(chóng)在生長(zhǎng)發(fā)育中,其體壁細(xì)胞中的聚集點(diǎn)逐漸增多,48、72、96 h的熒光聚集點(diǎn)數(shù)分別為(6±1)、(27±2)、(56±4)個(gè)。Ginseng組秀麗線蟲(chóng)熒光點(diǎn)數(shù)在48 h為(4±1)個(gè),72 h和96 h分別為(20±3)、(45±2)個(gè),相比對(duì)照均有所下降(均P<0.05)。2種秀麗線蟲(chóng)存活時(shí)間從第5天開(kāi)始即出現(xiàn)差異(P<0.05),Control組AM141秀麗線蟲(chóng)平均存活時(shí)間為(23±2)d,Ginseng組為(27±2)d;Control組HA759秀麗線蟲(chóng)平均存活時(shí)間為(24±2)d,Ginseng組為(27±2)d。結(jié)論 人參多糖能延長(zhǎng)秀麗線蟲(chóng)壽命,而抑制 polyQ聚集并緩解與衰老相關(guān)的 polyQ 神經(jīng)毒性。
人參多糖;秀麗線蟲(chóng);神經(jīng)毒性;壽命
在世界人口老齡化的21世紀(jì),患與衰老相關(guān)的神經(jīng)退行性疾病的人越來(lái)越多,由此給患者家庭和社會(huì)帶來(lái)沉重負(fù)擔(dān)[1]。預(yù)計(jì)2050年,世界范圍內(nèi)患有此類(lèi)疾病的患者將達(dá)到1 億,而目前人類(lèi)還不能科學(xué)系統(tǒng)的闡明此類(lèi)疾病的發(fā)病機(jī)理,也沒(méi)有有效的治療途徑,因此加強(qiáng)對(duì)衰老相關(guān)的神經(jīng)退行性疾病的研究成為醫(yī)療機(jī)構(gòu)和制藥行業(yè)高度重視的重大科學(xué)任務(wù)[2]。衰老促發(fā)了一系列衰老相關(guān)的疾病,蛋白聚集毒性是衰老相關(guān)神經(jīng)退行性疾病的主要病理特征[3]。人參多糖在抗衰老及衰老相關(guān)疾病方面具有獨(dú)特優(yōu)勢(shì),但其能否緩解神經(jīng)退行性疾病中的蛋白毒性?本文旨在通過(guò)秀麗線蟲(chóng)模型來(lái)探討人參多糖對(duì)秀麗線蟲(chóng)polyQ聚集毒性和壽命的影響。
1.1 藥材與菌株 人參(PanaxginsengC.A.mey)購(gòu)自亳州康圣藥業(yè)有限公司,產(chǎn)地東北,批號(hào)20120201。
AM141、HA759秀麗線蟲(chóng)株以及OP50-1來(lái)源于中南大學(xué)秀麗線蟲(chóng)遺傳中心。
1.2 試劑 瓊脂57-50-1 CP(日本廣野株式會(huì)社);瓊脂糖(OXOID);Trizol Agent(Invitrogen);SYBR Green1 QPK-201(TOYOBO);SYBR Green Master (Rox) 049138500, (Roche);Quanti Test SYBR Green PCR kit(Qiagen);100bp DNA ladder MD109(Tiangen);DEAE-Sepharose FAST Flow 500Ml(Amersham Biosciences);Goldview 電泳染色劑MN407 (博大泰克);其余試劑均為國(guó)產(chǎn)分析純。
1.3 實(shí)驗(yàn)儀器 BSC 系列生物安全柜 BES-13611A2(北京東聯(lián)哈爾儀器制造有限公司);AIR TECH 潔凈工作臺(tái)SW-CJ-1FD(蘇凈集團(tuán)蘇州安泰空氣技術(shù)制造公司);PCR 儀(Tpersonal Biometra);定量 PCR BIORAD(聯(lián)想生物技術(shù)有限公司);高速離心機(jī)1-14(Sigma);AllegraX-30R centrifuge ALZ11L080(BECKMAN COULTER);酶標(biāo)儀 RS-232C MK3(Thermo Labsystems);熒光酶標(biāo)儀(Thermo);電泳儀DYY-6C,紫外儀 WD-9403D,電泳槽 DYCP-33A(北京市六一儀器廠)。
1.4 方法
1.4.1 秀麗線蟲(chóng)的培養(yǎng)、轉(zhuǎn)接和同步化:采用固體NGM培養(yǎng)基培養(yǎng)線蟲(chóng)。
傳代線蟲(chóng)采用瓊脂快轉(zhuǎn)接法;轉(zhuǎn)接大量線蟲(chóng)采用M9洗滌法;單個(gè)秀麗線蟲(chóng)的轉(zhuǎn)接用鉑金絲挑取線蟲(chóng)到新的 NGM板里。秀麗線蟲(chóng)的同步化培養(yǎng):L4期線蟲(chóng)擁有明顯的生理學(xué)特征,在實(shí)驗(yàn)中可以客觀地辨別,因此可采用挑取線蟲(chóng)法來(lái)得到同步化蟲(chóng)子。將產(chǎn)卵高峰的秀麗線蟲(chóng)置于新的含有食物菌苔的平皿中,讓其產(chǎn)卵 3~4 h,再將成蟲(chóng)移走,將帶有蟲(chóng)卵的平皿置于20 ℃培養(yǎng),待平皿中卵孵化后,便可得到同步化效果的線蟲(chóng)。
1.4.2 秀麗線蟲(chóng)的多聚谷氨酰胺聚集毒性實(shí)驗(yàn):瓊脂糖墊制備:稱(chēng)取1 g瓊脂糖,加入50 mL S·Medium,解凍狀態(tài)下微波加熱5 min,使其完全融化呈透明液體,然后將一塊載玻片(厚度約為 1 mm)放在兩塊玻璃板(厚度約為 2 mm)之間,趁熱吸取0.5 mL瓊脂糖滴在載玻片的中央,隨即從一側(cè)輕輕蓋上另一塊載玻片,待瓊脂糖冷去凝固后,小心移走上層載玻片,下層載玻片中央即成厚度約為 1 mm的圓形瓊脂糖墊,可用于固定秀麗線蟲(chóng)。
HA759秀麗線蟲(chóng)的準(zhǔn)備:按照上述方法獲得所需要的秀麗線蟲(chóng),將L1期同步化的HA759線蟲(chóng)加入到給藥平皿里,15 ℃生化培養(yǎng)箱培養(yǎng)3 d。
神經(jīng)元存活統(tǒng)計(jì):用 M9 溶液洗固體平皿,收集秀麗線蟲(chóng)于 1.5 mL離心管中,室溫3500 r/min離心1 min,棄上層溶液,重復(fù)此步驟 2次,最后用M9定容至50 μL,加入10 μL 20 mM NaN3溶液麻痹秀麗線蟲(chóng),混勻后取出 20 μL 滴加在制備好的瓊脂糖墊上,蓋上蓋玻片,置于熒光顯微鏡下觀察,ASH 和ASI神經(jīng)元位于秀麗線蟲(chóng)咽部后兩側(cè)的神經(jīng)元附件,經(jīng)過(guò)3 d孵育,理論上ASH神經(jīng)元僅存活10%[4]。而 ASI 神經(jīng)元基本不衰亡,可以通過(guò)觀察黃綠色熒光點(diǎn)來(lái)判斷 ASH神經(jīng)元狀態(tài)。ASH神經(jīng)元存活率(neuronal survival)計(jì)算公式如下:Neuronal survival(%)=[ Nsurvival/(Nsurvival+Ndead)]×100。公式中,Nsurvival和Ndead分別代表 ASH 神經(jīng)元存活和死亡的秀麗線蟲(chóng)數(shù)目。
1.4.3 人參多糖對(duì)秀麗線蟲(chóng)模型中多聚谷氨酰胺的抑制作用:AM141 秀麗線蟲(chóng)模型(rm)是在其 unc-54基因上轉(zhuǎn)入了融合YFP的40個(gè)多聚谷氨酰胺長(zhǎng)度的polyQ重復(fù)片段(polyQ)構(gòu)建而成。由于 unc-54是不協(xié)調(diào)基因,其本身表達(dá)在秀麗線蟲(chóng)肌球蛋白重鏈上,如果 polyQ 在其中表達(dá)則會(huì)導(dǎo)致polyQ 在體壁肌肉層聚集,導(dǎo)致因肌肉運(yùn)動(dòng)障礙而出現(xiàn)運(yùn)動(dòng)能力減退現(xiàn)象。由于是與 YFP融合,因而可以在熒光顯微鏡下觀察到表現(xiàn)為黃綠色的熒光點(diǎn)。
將同步化的 L1 期線蟲(chóng)加入到給藥的平皿中(10 mg/mL人參多糖),生化培養(yǎng)箱 20 ℃培養(yǎng)4 d。第2天開(kāi)始,每天收集1個(gè)給藥平皿中的秀麗線蟲(chóng);在熒光顯微鏡下統(tǒng)計(jì)秀麗線蟲(chóng)周身的熒光點(diǎn)數(shù),如此連續(xù)統(tǒng)計(jì)3 d。
1.4.4 秀麗線蟲(chóng)的固體壽命實(shí)驗(yàn) :取10 mg/mL 的人參多糖,在旋轉(zhuǎn)器上旋轉(zhuǎn)促溶3 h,使其充分溶解后,過(guò)0.45 μm微孔濾膜除菌及不溶雜質(zhì)。用S·Medium稀釋成不同的人參多糖溶液:每次實(shí)驗(yàn)現(xiàn)用現(xiàn)配。實(shí)驗(yàn)分2組,Control組和Ginseng組。Control組,不做任何特殊處理,Ginseng組將藥物(10 mg/mL人參多糖)與 OP50-1按照1:4的比例混合體積至 50 mL,將混合液加入平皿,輕輕晃動(dòng),使均勻分布于平皿中央。操作臺(tái)中放置,使菌液充分被吸收,至不再流動(dòng)。
準(zhǔn)備實(shí)驗(yàn)所需L4期秀麗線蟲(chóng):用鉑金絲將10條產(chǎn)卵期的秀麗線蟲(chóng)轉(zhuǎn)移到35 mm的玻璃平皿上,產(chǎn)卵3 h后,將蟲(chóng)子移走,將有蟲(chóng)卵的平皿20 ℃培養(yǎng)48 h 達(dá)到L4期,根據(jù)實(shí)驗(yàn)樣本量,確定產(chǎn)卵期蟲(chóng)子個(gè)數(shù)。產(chǎn)卵期蟲(chóng)子平均每個(gè)蟲(chóng)子產(chǎn)卵6個(gè)/h。
上樣:將L4期的蟲(chóng)子挑到鋪好的平皿上,35~40條/板,每個(gè)組別分別做 3個(gè)平行平皿,使每組的樣本量約為100條。20 ℃培養(yǎng)箱中培養(yǎng)。
培養(yǎng)和數(shù)據(jù)統(tǒng)計(jì):產(chǎn)卵期的秀麗線蟲(chóng),每天都要用鉑金絲將線蟲(chóng)轉(zhuǎn)移到新的平皿上;之后每隔1天轉(zhuǎn)移1次。詳細(xì)記錄線蟲(chóng)的死亡個(gè)數(shù)及死亡狀況(機(jī)械損傷、爬墻、內(nèi)孵)。用鉑金絲觸之尾部,頭部均不動(dòng)者判斷為死亡,每天統(tǒng)計(jì),直至秀麗線蟲(chóng)全部死亡。
2.1 人參多糖對(duì)多聚谷氨酰胺聚集毒性的影響 將同步化的L1期 HA759秀麗線蟲(chóng)加入到對(duì)照和給藥平皿中,15 ℃培養(yǎng)3 d,熒光顯微鏡下統(tǒng)計(jì)熒光點(diǎn)。未經(jīng)多糖處理的Control組 HA759秀麗線蟲(chóng)在培養(yǎng)3d后,ASH 神經(jīng)元存活率為53%。Ginseng組HA759秀麗線蟲(chóng)ASH 神經(jīng)元存活率有所提高,達(dá)到了64%,2組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05),見(jiàn)圖1。表明人參多糖具有緩解 polyQ 聚集介導(dǎo)的神經(jīng)毒性作用。
圖1 人參多糖對(duì)HA759秀麗線蟲(chóng) ASH 神經(jīng)元存活的影響*P<0.05,與control組相比Fig.1 Effect of ginseng on ASH neuron survival in HA759 model*P<0.05,compared with control group
2.2 人參多糖對(duì)多聚谷氨酰胺聚集的抑制作用 未經(jīng)多糖處理的AM141秀麗線蟲(chóng),在生長(zhǎng)發(fā)育中其體壁細(xì)胞中的聚集點(diǎn)逐漸增多,48~96 h的熒光聚集點(diǎn)數(shù)分別為(6±1)、(27±2)、(56±4)個(gè)。加入100 μg/plate的人參多糖處理后,熒光點(diǎn)數(shù)在48 h為(4±1)個(gè),在72 h和96 h分別為(20±3)、(45±2)個(gè),相比對(duì)照均有所下降(P<0.05),見(jiàn)圖2、圖3。結(jié)果表明,人參多糖對(duì) AM141PolyQ 的聚集有一定的緩解作用。
圖2 AM141秀麗線蟲(chóng)體壁細(xì)胞熒光點(diǎn)的表達(dá)情況Fig.2 AM141 C.elegans somatic cell fluorescence expression
圖3 人參多糖對(duì)AM141秀麗線蟲(chóng)體壁熒光聚集點(diǎn)的影響*P<0.05,與control組相比Fig.3 Effect of ginseng polysaccharide on AM141 C.elegans somatic fluorescence point*P<0.05, compared with control group
2.3 人參多糖對(duì)Poly Q模型秀麗線蟲(chóng)壽命的影響 對(duì)AM141、HA759秀麗線蟲(chóng)以100 μg/plate劑量的人參多糖處理,觀察其對(duì)秀麗線蟲(chóng)壽命的影響。從第5天開(kāi)始,Control組和Ginseng組就出現(xiàn)明顯差別;Control組AM141秀麗線蟲(chóng)平均存活時(shí)間為(23±2)d,Ginseng組AM141秀麗線蟲(chóng)平均存活時(shí)間為(27±2)d;Control組HA759秀麗線蟲(chóng)平均存活時(shí)間為(24±2)d,Ginseng組HA759秀麗線蟲(chóng)平均存活時(shí)間為(27±2)d,2組比較差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)圖4。
圖4 人參多糖對(duì) AM141(A)和HA759(B)秀麗線蟲(chóng)壽命的影響Fig.4 Effect of ginseng polysaccharide on the lifespan of C.elegans AM141 (A) and HA759 (B)
衰老的特點(diǎn)是細(xì)胞內(nèi)大分子,包括被自由基損傷蛋白質(zhì)的不斷積累[5]。隨著機(jī)體衰老,細(xì)胞內(nèi)氧化自由基的含量呈指數(shù)增加,蛋白質(zhì)的這種不可逆氧化損傷也隨著衰老惡化,相應(yīng)的由此引發(fā)的神經(jīng)毒性也隨之加劇[6-8]。衰老是神經(jīng)退行性疾病的高危因子,那么具有延緩衰老的藥物是否可以緩解神經(jīng)退行性疾病蛋白的毒性?文獻(xiàn)報(bào)道,在秀麗線蟲(chóng)polyQ 蛋白聚集產(chǎn)生的毒性是有壽命依賴(lài)性的,同時(shí),聚集引起的毒性又進(jìn)一步加速衰老,此時(shí),蛋白動(dòng)態(tài)平衡總體表現(xiàn)出失調(diào)和紊亂的發(fā)展趨勢(shì)[9-11]。胰島素樣信號(hào)是線蟲(chóng)壽命的主要調(diào)控者,對(duì)此通路進(jìn)行遺傳學(xué)操作以延長(zhǎng)線蟲(chóng)壽命,polyQ聚集也會(huì)隨之延遲[12]。研究表明,淀粉樣蛋白結(jié)合染料硫磺素T能夠延長(zhǎng)秀麗線蟲(chóng)的壽命,穩(wěn)定突變的亞穩(wěn)態(tài)蛋白質(zhì)和緩解Aβ 聚集毒性,還可以通過(guò)調(diào)控分子伴侶和蛋白質(zhì)降解途徑以減緩polyQ聚集[13]。
本研究發(fā)現(xiàn),人參多糖緩解了polyQ 模型HA759線蟲(chóng)株中由polyQ異常聚集介導(dǎo)的神經(jīng)毒性,減少了AM141 體壁細(xì)胞中PolyQ蛋白的聚集,說(shuō)明人參多糖是通過(guò)減緩多聚谷氨酰胺的聚集起到神經(jīng)保護(hù)作用的。最近研究表明,蛋白動(dòng)態(tài)失衡不僅是經(jīng)退行性疾病發(fā)生和發(fā)展的重要因素,也是衰老的主要特征,壽命調(diào)節(jié)相關(guān)信號(hào)是聯(lián)系衰老、蛋白動(dòng)態(tài)平衡以及神經(jīng)退行性疾病這3者的重要橋梁[14-15]。而本文中人參多糖分別不同程度的延長(zhǎng) polyQ 秀麗線蟲(chóng)模型AM141及HA759的壽命,也證實(shí)了其增強(qiáng)機(jī)體維護(hù)蛋白平衡的能力。
本文以天然人參多糖為研究對(duì)象,利用模式生物秀麗線蟲(chóng)及現(xiàn)代生物學(xué)技術(shù)和方法,針對(duì)與衰老及衰老相關(guān)的神經(jīng)退行性疾病進(jìn)行生物活性和作用機(jī)理的研究。研究結(jié)果表明,人參多糖不僅能夠延長(zhǎng)秀麗線蟲(chóng)的壽命,而且還能抑制 polyQ 聚集并緩解與衰老相關(guān)的 polyQ 神經(jīng)毒性,說(shuō)明人參多糖可以通過(guò)延緩衰老,維護(hù)蛋白質(zhì)平衡,起到神經(jīng)保護(hù)作用。這些研究結(jié)果為抗衰老中藥在治療衰老相關(guān)的神經(jīng)退行性疾病提供了理論參考。
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(編校:吳茜)
Effect of ginseng polysaccharides on polyQ accumulation toxicity and lifes pan ofCaenorhabditiselegans
CHEN Pei-lei1,PAN Jian-chun2Δ
(1.Department of Clinical Pharmacy, Yueqing Hospital Affiliated to Wenzhou Medical University, Yueqing 325600, China; 2.College of Pharmacy, Wenzhou Medical University, Wenzhou 325035, China)
ObjectiveTo study the ginseng polysaccharides for prolonging the life span of theC.elegansand inhibit the toxic effects of polyQ accumulation.MethodsCaenorhabditis elegans of HA759 and AM141 were divided into control group and Ginseng group, seprately.Control group didn’t do any special treatment, Ginseng group were given 10 mg/mL polysaccharide and OP50-1 in the proportion of 1:4 mixed volume to 50 mL.C.elegansof glutamine (polyQ) polymer HA759 neurotoxicity model test of glutamine protein polymer toxicity experiment were done.The ASH neuron survival condition were tested.After sampling statistics gathered nematodes in the whole fluorescent points every day, study ginseng polysaccharide on polymers glutamine aggregation inhibition.Finally the solid life of twoC.elegans were studied.ResultsThe survival rate of ASH neurons inCaenorhabditiselegansHA759 of control group after 3 days culture was 53%.which of Ginseng group was 64% (P<0.05).The fluorescence of 48~96h aggregation points inCaenorhabditiselegansAM141 of control group were(6±1), (27±2), (56±4), which of Ginseng group were (4±1) in 48 h, (20±3) in 72 h and (45±2) in 96 h, the differences between two groups were all significant(P<0.05).The average survival time ofCaenorhabditiselegansAM141 of control group was (23±2)days, which of Ginseng group was (27±2)days; average survival time ofCaenorhabditiselegansHA759 of control group was (24±2)days, which of Ginseng group was (27±2)days,the difterences were all signiyicant(P<0.05).ConclusionGinseng polysaccharides can not only prolong the lifespan of theC.elegans, but also can restrain polyQ gathered and ease the polyQ neurotoxicity associated with aging.
ginseng polysaccharide;C.elegans;neurotoxicity;life span
陳佩雷,女,碩士在讀,主管藥師,研究方向:臨床藥學(xué)與藥物分析,E-mail:C13868847229@163.com;潘建春,通訊作者,男,本科,教授,研究方向:神經(jīng)藥理學(xué),E-mail:pjc13857750765@163.com。
R9
A
1005-1678(2015)03-0069-03