柳四新 何丹 余孝君 王家祺
[摘要] 目的 觀察度洛西汀對小腦顆粒神經(jīng)元谷氨酸毒性作用的保護作用及機制。 方法 體外培養(yǎng)CD-1小鼠小腦顆粒神經(jīng)元,神經(jīng)元細胞隨機分為8組:對照組,谷氨酸組,度洛西汀組,度洛西汀+谷氨酸組,SB204741處理組,SB204741+谷氨酸組,LY294002處理組,LY294002+谷氨酸組。采用TUNEL法檢測神經(jīng)元細胞凋亡程度,采用共聚焦顯微鏡fura-2探針檢測神經(jīng)元內(nèi)Ca2+水平。 結(jié)果 與對照組比較,谷氨酸毒性能明顯增加神經(jīng)元凋亡[(1.62±0.96)比(13.98±3.49)](P < 0.05),度洛西汀顯著抑制由谷氨酸毒性引起的神經(jīng)元凋亡。度洛西汀能降低由谷氨酸毒性激活的神經(jīng)元內(nèi)Ca2+升高。5-HT2B SB204741能抑制由谷氨酸毒性引起的神經(jīng)元凋亡和神經(jīng)元內(nèi)Ca2+升高,而LY294002只能抑制谷氨酸毒性引起的神經(jīng)元凋亡,對于谷氨酸毒性引起的神經(jīng)元內(nèi)Ca2+升高無作用。 結(jié)論 度洛西汀具有抵抗神經(jīng)元谷氨酸毒性作用,其機制主要是通過5-HT2B受體活化細胞內(nèi)鈣庫釋放,從而引起EGFR間接激活PI3K/AKT/mTOR通路,抑制由谷氨酸毒性引起的神經(jīng)元凋亡。
[關鍵詞] 度洛西汀;谷氨酸毒性;神經(jīng)元
[中圖分類號] R971 [文獻標識碼] A [文章編號] 1673-7210(2016)11(c)-0030-04
Protective effect of Duloxetine on glutamate toxicity of cerebellar granule neurons and its mechanism
LIU Sixin HE Dan YU Xiaojun WANG Jiaqi
The Second Department of Neurology, the First Hospital of Changsha City, Hu'nan Province, Changsha 410005, China
[Abstract] Objective To observe the protective effect of Duloxetine on glutamate toxicity of cerebellar granule neurons and its mechanism. Methods The cerebellar granule neurons derived from CD-1 mice were cultured in vitro, all the neurons were randomly divided into eight groups: control group, glutamate group, Duloxetine group, Duloxetine+glutamate group, SB204741 treatment group, SB204741+glutamate group, LY294002 treatment group, LY294002+glutamate group. The apoptosis degrees of neurons were detected by TUNEL method, the levels of Ca2+ in neurons were detected by confocal microscopy fura-2. Results Compared with control group, glutamate toxicity could significantly increase the neuron apoptosis [(1.62±0.96) vs (13.98±3.49)] (P < 0.05), and Duloxetine could inhibit the neuron apoptosis caused by glutamate toxicity. Duloxetine could significantly decrease the increasing concentration of Ca2+ in neurons activated by glutamate toxicity. 5-HT2B SB204741 could significantly inhibit the neuron apoptosis and the increasing concentration of Ca2+ in neurons caused by glutamate toxicity, while LY294002 could only inhibit the neuron apoptosis caused by glutamate toxicity, which had no effects on the increasing concentration of Ca2+ in neurons activated by glutamate toxicity. Conclusion Duloxetine has the effects of inhibiting the glutamate toxicity of neurons, the main mechanism may be associated with the release of intracellular calcium store activated by the receptor of 5-HT2B, which cause EGFR activating PI3K/AKT/mTOR signal pathway indirectly, so as to inhibit the neuron apoptosis caused by glutamate toxicity.
[Key words] Duloxetine; Glutamate toxicity; Neuron
谷氨酸是中樞神經(jīng)系統(tǒng)中最重要的興奮性神經(jīng)遞質(zhì)之一[1-2],谷氨酸毒性是指谷氨過度釋放引起的神經(jīng)元過度興奮,造成神經(jīng)元不同程度的損傷和死亡[3]。腦卒中、腦缺血、阿爾茨海默病和亨延頓病等多種中樞神經(jīng)系統(tǒng)疾病的共同特征是谷氨酸過度釋放引起的神經(jīng)毒性作用[4]。鹽酸度洛西?。―uloxetine)是新一代抗抑郁藥物,屬于5-羥色胺(5-HT)和去甲腎上腺素(NE)再攝取的雙重抑制劑[5]。體內(nèi)體外實驗證明,度洛西汀具有抑制神經(jīng)毒性作用[6],但具體分子機制鮮見報道。本研究采用度洛西汀干預,在體外培養(yǎng)的小腦顆粒神經(jīng)元谷氨酸毒性作用后,觀察度洛西汀對谷氨酸引起的神經(jīng)元毒性作用的效應,并聯(lián)合5-HT2B SB204741和LY294002探討谷氨酸引起神經(jīng)元凋亡和細胞內(nèi)Ca2+水平變化,探討其作用機制。
1 材料與方法
1.1 材料
7 d齡新生CD-1小鼠[雄性,12 h光照/12 h避光,河南醫(yī)科大學實驗動物中心,SCXK(豫)2005-0001];谷氨酸、多聚賴氨酸、LY294002、SB204741和MTT均購自于Sigma公司,馬血清、DMEM培養(yǎng)液和胰蛋白酶購自于Gibco公司,熒光探針fura-2購自于invitrogen公司。
1.2 神經(jīng)元原代培養(yǎng)
CD-1小鼠小腦顆粒神經(jīng)元原代培養(yǎng)方法。取出生7 d的CD-1小鼠,斷頭后小心剝離小腦顆粒神經(jīng)元,去除表面和溝回內(nèi)的血膜,將純化后的小腦組織進行反復剪磨,加1.25 g/L胰酶消化37℃,20 min,含血清培養(yǎng)基終止消化后,將小腦組織DMEM沖洗后后過100目篩,吹打細胞接種于24孔板和96孔板及多聚賴氨酸孵育4 h的蓋玻片上,無血清DMEM培養(yǎng)基37℃,5%CO2培養(yǎng)箱24 h。換成含10%馬血清的DMEM培養(yǎng)基,37℃,5%CO2培養(yǎng)箱7 d待用。
1.3 細胞處理
檢測神經(jīng)元凋亡,小腦顆粒神經(jīng)元細胞共隨機分為8組:對照組,谷氨酸組,度洛西汀組,度洛西汀+谷氨酸組,SB204741處理組,SB204741+谷氨酸組,LY294002處理組,LY294002+谷氨酸組。檢測鈣離子水平,小腦顆粒神經(jīng)元細胞共隨機分為3個4組:對照組,谷氨酸組,度洛西汀組,度洛西汀+谷氨酸組;或?qū)φ战M,谷氨酸組,SB204741處理組,SB204741+谷氨酸組;或?qū)φ战M,谷氨酸組,LY294002處理組,LY294002+谷氨酸組。其中度洛西汀、5-HT2B SB204741和LY294002預處理20 min后,再進行對照生理鹽水處理或者刺激因素50 mol/L谷氨酸。
1.4 細胞凋亡測定
細胞凋亡采用北京中山生物技術(shù)公司的TUNEL細胞凋亡試劑盒,按照試劑盒說明書操作。細胞處理后進行細胞凋亡測定,采用多功能酶標儀在波長490 nm處進行光度值檢測,并分別進行計數(shù)和統(tǒng)計分析。
1.5 細胞內(nèi)Ca2+水平的檢測
采用成熟分化的神經(jīng)元細胞于蓋玻片上以fura-2比例為1∶1000孵育30 min,等滲液沖洗去除熒光背景后,于共聚焦顯微鏡成像,激發(fā)光550 nm,發(fā)射光340 nm和380 nm,間隔20 s激光掃描1次,至刺激因素處理開始共觀察15 min,檢測45個循環(huán)。采用340/380比值計算細胞內(nèi)Ca2+水平動態(tài)變化。為了降低細胞間差異,將所有檢測點的340/380比值除以刺激處理時間點的340/380比值,刺激處理時間點的340/380比值標準化為1,相同處理組內(nèi)各取20個細胞進行統(tǒng)計分析。
1.6 統(tǒng)計學方法
所有數(shù)據(jù)采用SPSS 17.0統(tǒng)計學軟件進行統(tǒng)計學分析,計量資料以均數(shù)±標準差(x±s)表示,符合正態(tài)分布的使用方差分析,不符合正態(tài)分布的采用非參數(shù)秩和檢驗,以P < 0.05為差異有統(tǒng)計學意義。
2 結(jié)果
2.1 神經(jīng)元細胞凋亡
對照組含有少量的TUNEL陽性細胞,50 mol/L谷氨酸后,TUNEL陽性細胞顯著增加,即細胞凋亡率顯著升高。度洛西汀能顯著抑制由谷氨酸引起的細胞凋亡增多。抑制劑包括5-HT2B SB204741處理組和AKT抑制劑LY294002處理組,與實驗組50 mol/L谷氨酸比較,細胞凋亡率明顯減少,差異有統(tǒng)計學意義(P < 0.05)。谷氨酸組的凋亡率與其他組比較,差異均有統(tǒng)計學意義(P < 0.05)。見表1。
2.2 細胞內(nèi)Ca2+水平
共聚焦熒光倒置顯微鏡檢測結(jié)果顯示,對照組細胞內(nèi)Ca2+水平隨時間變化無明顯變化,15 min之間差異無統(tǒng)計學意義(P > 0.05)。谷氨酸組在給予谷氨酸刺激時間點開始細胞內(nèi)Ca2+水平明顯上升,約80%,15 min恢復基線水平。在處理后的1~10 min之間,谷氨酸組與對照組的Ca2+水平差異均有統(tǒng)計學意義(P < 0.05)。度洛西汀能顯著抑制谷氨酸引起的細胞內(nèi)Ca2+水平升高。見圖1。
2.3 可能相關信號轉(zhuǎn)導通路驗證
采用5-HT2B抑制劑SB204741和AKT抑制劑LY294002聯(lián)合50 mol/L谷氨酸,細胞內(nèi)Ca2+水平變化與對照組比較,差異無統(tǒng)計學意義(P > 0.05)。見圖2~3。
3 討論
研究表明,腦缺血再灌注、腦卒中和老年性癡呆是常見的中樞神經(jīng)系統(tǒng)疾病,具有發(fā)生率高、致殘率高、致死率高的特點,已經(jīng)成為嚴重威脅人類生活質(zhì)量的重要原因[4]。腦缺血再灌注、腦卒中和老年性癡呆的共同發(fā)病特征是興奮性谷氨酸毒性,這個谷氨酸毒性在疾病的病理進程中扮演著重要角色。谷氨酸毒性作用易造成神經(jīng)元不同程度的損傷和死亡,而神經(jīng)元不可再生特性決定了使用藥物治療和預防谷氨酸毒性作用的重要性[3]。谷氨酸是中樞神經(jīng)系統(tǒng)重要的興奮性神經(jīng)遞質(zhì)[1,5-6]。谷氨酸無法直接透過血腦屏障,在腦內(nèi)谷氨酸和谷氨酰胺循環(huán)中星形膠質(zhì)細胞發(fā)揮了重要的作用。谷氨酸受體包括離子型谷氨酸受體和代謝型谷氨酸受體,其中包括AMPA受體在內(nèi)的離子型谷氨酸受體是參與腦缺血和腦損傷的神經(jīng)元死亡的重要活化受體[7]。腦出血等病理因素導致神經(jīng)元細胞外液的谷氨酸濃度劇增,通過與突觸后谷氨酸受體結(jié)合,引起細胞膜上的鈉離子通道開放,大量鈉離子進入細胞內(nèi)引起細胞腫脹,造成神經(jīng)元死亡[8]。另有研究報道,谷氨酸可以通過與NMDA谷氨酸受體結(jié)合,導致細胞膜上L-型鈣離子通路開放,細胞內(nèi)鈣庫釋放大量鈣離子,引起細胞內(nèi)鈣離子超載,重要體現(xiàn)在線粒體功能不全引起的延遲性神經(jīng)元死亡[9-10]。細胞內(nèi)鈣離子超載具有多種原因,可能是由于細胞膜上的L-型鈣通道活化而增加細胞外的鈣離子進入到細胞內(nèi),細胞內(nèi)鈣離子升高后可以介導TRPC受體激活線粒體內(nèi)的敏感性,增加由TRPC介導的細胞內(nèi)鈣庫的釋放,細胞內(nèi)鈣離子瞬間釋放增加,導致細胞內(nèi)鈣超載[11-12]。從臨床上應用安全的藥物中發(fā)展和挖掘具有神經(jīng)保護作用的藥物成為臨床上治療腦缺血和腦損傷等疾病的重要策略[13]。
度洛西汀是一種5-HT和NE再攝取抑制劑,其主要作用機制是通過直接抑制5-HT攝取和NE攝取來增加細胞間隙的5-HT和NE的濃度[14]。本研究結(jié)果顯示谷氨酸具有激活NMDA等谷氨酸受體作用,造成細胞內(nèi)鈣超載出現(xiàn)大量神經(jīng)元細胞凋亡,度洛西汀通過與5-HT2B受體特異性結(jié)合,受體酪氨酸激活介導磷脂酰肌醇-3激酶(PI3K)/絲氨酸-蘇氨酸蛋白激酶(AKT)通路引起神經(jīng)元壞死[15-17]。另有研究表示度洛西汀與多巴胺能、腎上腺能、膽堿能、谷氨酸受體等幾乎無親和力,但未見報道度洛西汀直接結(jié)合5-HT受體[18],這與Ray等[19]報道氟西汀結(jié)合5-HT受體發(fā)揮抗抑郁作用相類似。動物實驗表明,度洛西汀能減少大鼠大腦皮層5-HT受體密度和NE受體密度。度洛西汀推薦用于治療化療所致周圍神經(jīng)炎病變,止疼效果較好,但需要用藥5周才能起效,與治療抑郁癥起效慢一致[6],因此度洛西汀治療谷氨酸毒性作用神經(jīng)元壞死也可能需要長期治療起效[20]。
本研究應用度洛西汀處理能顯著降低細胞內(nèi)鈣超載引起的神經(jīng)元凋亡,在臨床實踐中度洛西汀具有良好的安全性,值得臨床推廣。
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(收稿日期:2016-05-25 本文編輯:張瑜杰)