魚藤酮誘導(dǎo)神經(jīng)毒性機(jī)制的研究進(jìn)展

周 謙, 陳 斌,曹 鵬,沈建平

(1. 南京中醫(yī)藥大學(xué)附屬中西醫(yī)結(jié)合醫(yī)院，江蘇 南京210028; 2. 江蘇省中醫(yī)藥研究院，江蘇 南京 210028;3. 南京野生植物綜合利用研究院,江蘇 南京 210042)

魚藤酮是魚藤酮類植物家族成員之一，是一種從豆科魚藤屬(Derris)和醉魚豆屬(Lonhocarpus)植物萃取的天然毒性物質(zhì)[1]。魚藤酮光下易分解。一般情況，其有毒成分5～6天分解，而陽光充足的夏天，僅需2～3天。魚藤酮在泥土和水中也易降解。半衰期僅1～3天[2]。由于魚藤酮半衰期短，易分解，不污染環(huán)境，因此在世界各地都普遍應(yīng)用于農(nóng)作物蟲害治理和魚塘清理，被視為是一種天然低毒且高效的殺蟲劑[3]。

1 魚藤酮危害人體的方式

魚藤酮作為農(nóng)藥而大規(guī)模使用，因此進(jìn)入人體的最可能途徑是呼吸攝入[1]。但是，資料顯示魚藤酮在胃腸道吸收緩慢且不完全，而肝臟又能有效解毒，魚藤酮進(jìn)入全身血液循環(huán)十分困難。因此，魚藤酮由于呼吸攝入而誘發(fā)Parkinson’s disease (PD)存在質(zhì)疑。有一個(gè)證據(jù)與這個(gè)假說相一致，小鼠通過霧化方式染毒魚藤酮24個(gè)月，并未導(dǎo)致神經(jīng)系統(tǒng)損傷相關(guān)疾病發(fā)生[4]。

圖1 魚藤酮的分子結(jié)構(gòu)[1]

2 魚藤酮的神經(jīng)毒性

魚藤酮高度親脂，能透過血腦屏障，極易跨膜且不需跨膜受體，因此極易造成中樞神經(jīng)系統(tǒng)損傷，特別是對神經(jīng)元的損傷[5-6]。數(shù)據(jù)顯示，小鼠靜脈注射魚藤酮后，僅需15 min，魚藤酮便在中樞系統(tǒng)積累并達(dá)到最高濃度[7]。此外，魚藤酮是一種與線粒體復(fù)合物Ⅰ具有高親和力的非競爭性抑制劑[3, 8]，魚藤酮進(jìn)入神經(jīng)細(xì)胞后，能夠在線粒體中集聚，損壞呼吸鏈的氧化磷酸化，抑制ATP的合成[9]。近期一個(gè)NIH的病例顯示，魚藤酮職業(yè)暴露的人患PD的風(fēng)險(xiǎn)是不接觸魚藤酮人的2.5倍[10]。此外，一項(xiàng)來自得克薩斯州的農(nóng)業(yè)調(diào)查顯示，農(nóng)業(yè)使用魚藤酮會增加PD的患病風(fēng)險(xiǎn)[11]。

3 魚藤酮誘發(fā)神經(jīng)毒性的潛在機(jī)制

研究表明，魚藤酮作為線粒體復(fù)合物Ⅰ抑制劑，能夠從多方面觸發(fā)神經(jīng)毒性。可能的機(jī)制包括抑制線粒體功能，誘導(dǎo)氧化應(yīng)激，觸發(fā)凋亡，聚集/降解蛋白，誘發(fā)興奮性中毒，抑制細(xì)胞周期等，或是以上眾多因素綜合作用的結(jié)果[3, 11-12]。

3.1 魚藤酮抑制線粒體

Kweon等學(xué)者指出，魚藤酮能夠通過抑制線粒體復(fù)合物Ⅰ，降低線粒體活力，抑制ATP的合成[13-14]。暗示著魚藤酮導(dǎo)致的線粒體功能受損可能是PD發(fā)生的誘因。Moon等學(xué)者指出，小鼠原代神經(jīng)元暴露低劑量魚藤酮，會造成線粒體去極化、激活caspase凋亡通路、導(dǎo)致DNA斷裂等觸發(fā)多巴胺能神經(jīng)元死亡[15]。此外，魚藤酮作用于復(fù)合物Ⅰ，會降低線粒體膜電位，誘導(dǎo)線粒體膜通透性轉(zhuǎn)換(MPT)，并觸發(fā)凋亡[16]。暗示魚藤酮關(guān)聯(lián)的線粒體功能受損是魚藤酮誘發(fā)神經(jīng)毒性的一個(gè)因素。

3.2 魚藤酮誘導(dǎo)氧化應(yīng)激

體內(nèi)體外大量研究結(jié)果表明，魚藤酮會通過誘導(dǎo)活性氧簇(ROS)和活性氮簇(NOS)的高漲，線粒體氧化磷酸化失衡，脂質(zhì)過氧化等觸發(fā)神經(jīng)毒性，且毒性作用可以被抗氧化劑抑制[17-19]。資料顯示，慢性魚藤酮染毒小鼠在第30天和第60天，神經(jīng)細(xì)胞的NO和脂質(zhì)過氧化產(chǎn)物TBARS水平上升。并且，還出現(xiàn)了PD相關(guān)的行為學(xué)特征，如麻痹和強(qiáng)直[20]。數(shù)據(jù)表明，魚藤酮模型中的抗氧化物酶，如GSH和SOD活性下降[21]，而一些過氧化物如過氧硝酸鹽的含量上升[22]，證明了魚藤酮模型中氧化應(yīng)激的產(chǎn)生。此外，PD病人腦內(nèi)也會發(fā)生氧化應(yīng)激，表現(xiàn)包括GSH含量降低，DNA磷脂，蛋白的過氧化等。Tada-Oikawa指出，魚藤酮能夠刺激人源細(xì)胞HL-60和NJAB H2O2水平高漲，推測為存在一種魚藤酮誘導(dǎo)- H2O2機(jī)制誘發(fā)神經(jīng)毒性[12, 23]。以上數(shù)據(jù)暗示魚藤酮會通過氧化應(yīng)激關(guān)聯(lián)神經(jīng)系統(tǒng)損傷。

3.3 魚藤酮觸發(fā)凋亡

凋亡發(fā)生常涉及兩條經(jīng)典通路，分別是Fas介導(dǎo)的Caspase-8和應(yīng)急壓力介導(dǎo)的caspase-9，兩條通路最終于caspase-3匯聚，激活，導(dǎo)致核降解和細(xì)胞形態(tài)改變[24]。資料顯示，魚藤酮在誘導(dǎo)神經(jīng)毒性時(shí)，能夠觸發(fā)多種凋亡指標(biāo)，包括：細(xì)胞膜電位下降、caspase-3和caspase-9激活、Bax和細(xì)胞色素C釋放等[25]。如Wang等研究發(fā)現(xiàn)魚藤酮能夠以劑量依賴的方式觸發(fā)caspase-3的激活[26]。Ethell于2009年指出，魚藤酮誘導(dǎo)的氧化應(yīng)激會導(dǎo)致脂質(zhì)過氧化和蛋白錯(cuò)折疊，誘使神經(jīng)細(xì)胞通過Bcl-2家族級聯(lián)的方式死亡[18]。Abdin等人發(fā)現(xiàn)，在慢性魚藤酮染毒的小鼠動物模型中，隨著紋狀體線粒體復(fù)合物Ⅰ活性下降、ATP合成抑制、血漿和紋狀體Q10活性降低、抗凋亡蛋白Bcl-2活性卻增強(qiáng)。他給予的解釋是魚藤酮在誘導(dǎo)神經(jīng)毒性時(shí)觸發(fā)了線粒體的抗凋亡保護(hù)路徑。

3.4 魚藤酮導(dǎo)致蛋白集聚

魚藤酮能夠高度復(fù)制PD的病理特征，其一就是形成淀粉樣集聚蛋白-Lewy小體[27]，Lwey小體的主要成分是α-synuclein，為一種廣泛分布于中樞神經(jīng)系統(tǒng)突觸前末梢的小分子蛋白，其正常生理功能尚不明確，推測可能是與突觸的可塑性和多巴胺神經(jīng)遞質(zhì)傳遞等有關(guān)[28-30]。應(yīng)激條件下，α-synuclein易發(fā)生錯(cuò)誤折疊、集聚，并最終形成不溶性纖維結(jié)構(gòu)，導(dǎo)致細(xì)胞死亡[28, 31]。Uversky等人研究顯示魚藤酮能夠顯著加速α-synuclein纖維化和聚集[28]。此外Huang等人指出，魚藤酮還能夠誘導(dǎo)管家蛋白GADPH易位，使分子間二硫鍵的形成，導(dǎo)致GADPH在胞漿中聚集[32]。

3.5 魚藤酮導(dǎo)致蛋白降解

泛素-蛋白酶系統(tǒng)(UPS)和自嗜-溶酶體系統(tǒng)(ALP)是機(jī)體正常修復(fù)、清除機(jī)體錯(cuò)誤折疊或異常集聚蛋白質(zhì)的重要途徑[33]。然而近年來研究顯示，這兩條途徑在PD病理學(xué)的發(fā)生中起著重要作用[34]。Ren等人指出PD相關(guān)蛋白parkin能夠?qū)﹀e(cuò)誤折疊的微管蛋白起到泛素化降解作用，可能是機(jī)體保護(hù)神經(jīng)細(xì)胞避免PD發(fā)生的一個(gè)機(jī)制[35]。

目前尚無直接證據(jù)表明自噬直接參與了魚藤酮關(guān)聯(lián)PD。但是，Pan等指出自噬增強(qiáng)子雷帕霉素(Rapamycin)能夠通過加強(qiáng)自噬，削弱魚藤酮誘導(dǎo)的神經(jīng)細(xì)胞凋亡。暗示Rapamycin觸發(fā)的自噬或因參與清除不需要的集聚蛋白或損傷的線粒體起到神經(jīng)保護(hù)作用[36]。

3.6 魚藤酮導(dǎo)致興奮中毒

興奮中毒是指各種原因引起腦損傷時(shí)，興奮性神經(jīng)遞質(zhì)從神經(jīng)末梢釋放增加，受體過度激活，引起興奮毒性，興奮毒性參與了多種神經(jīng)系統(tǒng)疾病的發(fā)生，如中風(fēng)、腦損傷、多發(fā)性硬化和神經(jīng)變性疾病的發(fā)生壞死和凋亡[37]。谷氨酸是機(jī)體中一種重要的興奮性的神經(jīng)遞質(zhì)，存在于50%以上的神經(jīng)組織，并在神經(jīng)元興奮中起到扮演重要角色。在病理狀態(tài)如能量不足、氧化應(yīng)激、線粒體功能紊亂、鈣超載等時(shí)，谷氨酸釋放過多，產(chǎn)生興奮毒性，對神經(jīng)元細(xì)胞產(chǎn)生損傷[37-38]。Wu等人分別于2007年和2009年報(bào)道，魚藤酮能夠加強(qiáng)谷氨酸興奮中毒誘導(dǎo)多巴胺神經(jīng)元產(chǎn)生毒性。這個(gè)結(jié)果出現(xiàn)，暗示魚藤酮觸發(fā)的中腦多巴胺能神經(jīng)元毒性可能與興奮中毒有關(guān)[39-40]。

3.7 魚藤酮耗竭神經(jīng)元營養(yǎng)因子

資料顯示，神經(jīng)營養(yǎng)因子在魚藤酮誘發(fā)的神經(jīng)變性疾病中起著關(guān)鍵作用[41-43]。Ming等人在2009年發(fā)現(xiàn)人視網(wǎng)膜內(nèi)皮細(xì)胞會分泌源于神經(jīng)膠質(zhì)細(xì)胞的和大腦的神經(jīng)營養(yǎng)因子以保護(hù)多巴胺能神經(jīng)元免于魚藤酮誘導(dǎo)的神經(jīng)毒性[43]。之后，Xiong等人發(fā)現(xiàn)魚藤酮會導(dǎo)致血管內(nèi)皮生長因子(VEGF)在中腦的表達(dá)顯著降低[44]。這兩個(gè)結(jié)果的出現(xiàn)，暗示了魚藤酮誘導(dǎo)的神經(jīng)毒性涉及了相關(guān)神經(jīng)營養(yǎng)因子耗竭。

3.8 魚藤酮與衰老

PD是三大神經(jīng)變性疾病之一，其發(fā)病率僅次于AD。證據(jù)表明，PD是一個(gè)與年齡相關(guān)的神經(jīng)變性疾病[45]。而Cannon 2009年報(bào)道，不同年齡的雄性小鼠(3，7，12～14個(gè)月)，以腹腔注射的方式攝入魚藤酮，中年鼠相比于年輕小鼠對魚藤酮更加敏感，更易產(chǎn)生神經(jīng)毒性[46]。暗示衰老參與魚藤酮誘導(dǎo)的神經(jīng)毒性并關(guān)聯(lián)PD的發(fā)生。

3.9 魚藤酮與Ca2+過載

資料顯示，胞內(nèi)Ca2+穩(wěn)態(tài)失衡，會造成急性神經(jīng)損傷，誘導(dǎo)神經(jīng)毒性和神經(jīng)凋亡，并參與神經(jīng)系統(tǒng)疾病的發(fā)生[47-48]。Wang等學(xué)者發(fā)現(xiàn)，魚藤酮在誘導(dǎo)SH-SY5Y細(xì)胞凋亡時(shí)相伴著 Ca2+通道開放，胞內(nèi)Ca2+水平高漲[26]。表明Ca2+穩(wěn)態(tài)失衡可能是魚藤酮誘導(dǎo)的神經(jīng)毒性的因素之一。

3.10 魚藤酮與多巴胺

多巴胺是中樞系統(tǒng)的一種神經(jīng)遞質(zhì)，能夠幫助調(diào)節(jié)機(jī)體運(yùn)動和機(jī)械行為，此外，它又是下丘腦神經(jīng)內(nèi)分泌軸的一個(gè)重要成分，對合成去甲腎上腺素、腎上腺素和中樞神經(jīng)系統(tǒng)都起著重要作用[12]。資料顯示，多巴胺神經(jīng)傳遞畸參與了多種神經(jīng)系統(tǒng)疾病，如AD、PD的發(fā)生[49]。然而，有關(guān)魚藤酮關(guān)聯(lián)多巴胺導(dǎo)致神經(jīng)毒性的假說存在分歧。比如Abdin等人報(bào)道魚藤酮能夠以劑量依賴的方式導(dǎo)致中腦紋狀體電壓降低、線粒體去極化、氨基酸和多巴胺的釋放增加，誘發(fā)神經(jīng)毒性[14]。而Bayersdorfer指出，胞內(nèi)多巴胺水平降低，會減小神經(jīng)毒性，并對多巴胺神經(jīng)能元起到保護(hù)作用[50]。暗示多巴胺內(nèi)功能穩(wěn)態(tài)對多巴胺能神經(jīng)元活性起著關(guān)鍵作用。

3.11 魚藤酮與細(xì)胞周期

近年來，迷亂的細(xì)胞周期被認(rèn)為與神經(jīng)死亡和神經(jīng)系統(tǒng)疾病的發(fā)生有關(guān)[51]。證據(jù)表明魚藤酮會導(dǎo)致細(xì)胞周期紊亂，誘導(dǎo)細(xì)胞凋亡[52-53]。如Wang等人發(fā)現(xiàn)，魚藤酮能以濃度依賴的方式，使SH-SY5Y細(xì)胞停滯停留在G2/M期，而用胞內(nèi)Ca2+螯合劑BAPTA能夠抑制魚藤酮導(dǎo)致的G2/M期停滯，并起到抑制凋亡，保護(hù)神經(jīng)的作用[54]。

3.12 其他機(jī)制

成人腦神經(jīng)元的新生主要發(fā)生在兩個(gè)區(qū)域，腦室下區(qū)和海馬齒狀回顆粒下層，這兩個(gè)腦區(qū)同樣也是神經(jīng)干細(xì)胞的來源，神經(jīng)干細(xì)胞能夠遷移到黑質(zhì)致密部并分化成多巴胺能神經(jīng)元[3]。而Ishido等人證明魚藤酮暴露能以濃度依賴的方式抑制體外模型中神經(jīng)干細(xì)胞的遷移和增殖，誘導(dǎo)神經(jīng)細(xì)胞凋亡[55]。這個(gè)證據(jù)也首次證明了魚藤酮的神經(jīng)毒性涉及了功能受損的神經(jīng)干細(xì)胞。

此外，魚藤酮還能通過激活MAPK信號級聯(lián)的c-Jun,，JNK和P38誘導(dǎo)神經(jīng)細(xì)胞的凋亡，關(guān)聯(lián)PD的發(fā)生[56-57]，如Wu等人發(fā)現(xiàn)魚藤酮能夠激活p38(MAPK), P-p38(MAPK), p53, 和Bax蛋白能誘發(fā)神經(jīng)毒性[57]。這些結(jié)果表明魚藤酮觸發(fā)的神經(jīng)毒性涉及了信號通路的參與。

4 小 結(jié)

魚藤酮原本是農(nóng)業(yè)上廣泛運(yùn)用的植物源殺蟲劑，現(xiàn)在卻被認(rèn)為具有神經(jīng)毒性，能夠高度復(fù)制PD的病理學(xué)和臨床學(xué)相關(guān)特征，如多巴胺能神經(jīng)元凋亡以及淀粉蛋白Lwey小體的集聚，關(guān)聯(lián)PD的發(fā)生。然而，目前關(guān)于魚藤酮誘發(fā)神經(jīng)毒性的機(jī)制并不全面，有些問題尚為查明。比半衰期短，降解快，難以進(jìn)入全身血液循環(huán)的魚藤酮是以何種機(jī)制進(jìn)入人體、如何抑制線粒體功能復(fù)合物Ⅰ、如何誘導(dǎo)氧化應(yīng)激、誘導(dǎo)氧化應(yīng)激的具體成分、其中是否存在重要信號通路交叉共同作用等。因而，更加深入研究魚藤酮誘發(fā)神經(jīng)毒性的分子機(jī)制，可為更好尋找魚藤酮相關(guān)的神經(jīng)系統(tǒng)疾病治療提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)，對人類健康起著重要意義。

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