劉興宇，甄艷鳳，崔建忠，高俊玲

(1唐山市工人醫(yī)院，河北唐山063000；2華北理工大學(xué)基礎(chǔ)醫(yī)學(xué)院)

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MAPKs阻滯劑U0126對(duì)創(chuàng)傷性腦損傷大鼠學(xué)習(xí)記憶功能的影響及機(jī)制探討

劉興宇1，甄艷鳳1，崔建忠1，高俊玲2

(1唐山市工人醫(yī)院，河北唐山063000；2華北理工大學(xué)基礎(chǔ)醫(yī)學(xué)院)

目的觀察絲裂原活化蛋白激酶(MAPKs)阻滯劑U0126對(duì)創(chuàng)傷性腦損傷 (TBI)大鼠學(xué)習(xí)記憶功能的影響，并探討其機(jī)制。方法將313只成年雄性SD大鼠隨機(jī)分為假手術(shù)組52只、模型組87只、DMSO組87只、U0126組87只，除假手術(shù)組外其余各組制備大鼠彌漫性腦創(chuàng)傷模型。U0126組將0.1 mg/kg U0126以0.1 mmol/L的PBS稀釋至300 μL，于造模前30 min尾靜脈注射。DMSO組同時(shí)點(diǎn)尾靜脈注射相同含量DMSO稀釋溶液，假手術(shù)組和模型組同時(shí)點(diǎn)尾靜脈注射生理鹽水300 μL。造模30 min、3 h、12 h、24 h、48 h、72 h、7 d取各組大鼠各5只(假手術(shù)組3只)，采用TUNEL法觀察各組海馬組織神經(jīng)細(xì)胞凋亡情況。造模30 min、3 h、12 h、24 h、48 h、72 h、7 d取各組大鼠各6只(假手術(shù)組3只)，采用Western blot法檢測各組海馬組織磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2(p-ERK1/2)蛋白表達(dá)。造模14、16、18、21 d取各組大鼠各10只，采用Morris水迷宮實(shí)驗(yàn)觀察大鼠空間學(xué)習(xí)記憶能力。結(jié)果造模48、72 h時(shí)，U0126組海馬組織神經(jīng)細(xì)胞凋亡數(shù)少于DMSO組、模型組，P均<0.05；造模24、48、72 h時(shí)，U0126組、DMSO組、模型組海馬組織神經(jīng)細(xì)胞凋亡數(shù)均多于假手術(shù)組，P均<0.05；造模48、72 h時(shí)，U0126組海馬組織p-ERK1/2蛋白相對(duì)表達(dá)量低于模型組、DMSO組，P均<0.05；造模12、24、48、72 h時(shí)，U0126組、DMSO組、模型組海馬組織p-ERK1/2蛋白相對(duì)表達(dá)量均短于假手術(shù)組，P均<0.05；造模16、18、21 d時(shí)U0126組大鼠潛伏期均短于DMSO組、模型組，P均<0.05；造模14、16、18、21 d時(shí)U0126組、DMSO組、模型組大鼠潛伏期均長于假手術(shù)組，P均<0.05。相關(guān)性分析結(jié)果顯示，海馬區(qū)神經(jīng)細(xì)胞凋亡數(shù)與p-ERK1/2表達(dá)呈正相關(guān)(r=0.468,P=0.002)。結(jié)論U0126可抑制TBI大鼠海馬神經(jīng)細(xì)胞凋亡，提高大鼠的學(xué)習(xí)記憶能力，可能與降低海馬組織中p-ERK1/2表達(dá)有關(guān)。

創(chuàng)傷性腦損傷；學(xué)習(xí)記憶功能；絲裂原活化蛋白激酶阻滯劑；磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2

創(chuàng)傷性腦損傷 (TBI) 發(fā)生率高，后遺癥多，是創(chuàng)傷醫(yī)學(xué)、急救醫(yī)學(xué)的重要研究課題[1，2]。其中學(xué)習(xí)記憶能力的損害是 TBI最持久和最嚴(yán)重的癥狀之一，目前尚無有效治療方法。絲裂原活化蛋白激酶(MAPKs)是細(xì)胞內(nèi)的一類絲氨酸/蘇氨酸蛋白激酶，是細(xì)胞應(yīng)激和損傷反應(yīng)的主要信號(hào)通路,可將胞外刺激信號(hào)傳遞至胞核[3]。細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2(ERK1/2)是MAPKs的重要成員，磷酸化后可被激活。2014年1月～2015年11月，我們觀察了MAPKs阻滯劑U0126對(duì)TBI大鼠學(xué)習(xí)記憶功能的影響，并探討其可能的作用機(jī)制。現(xiàn)報(bào)告如下。

1　材料與方法

1.1模型制作及干預(yù)將313只健康成年清潔級(jí)雄性SD大鼠[體質(zhì)量300～350 g，購自北京維通利華公司，許可證號(hào)：SCXK(京)2002-003]隨機(jī)分為假手術(shù)組52只、模型組87只、DMSO組87只、U0126組87只，除假手術(shù)組外其余各組參照[1]制備大鼠彌漫性腦創(chuàng)傷模型：乙醚麻醉大鼠，用450 g、直徑18 mm的銅錘自2 m高度垂直落下撞擊置于大鼠冠狀縫與矢狀縫正中的不銹鋼墊(直徑10 mm厚3 mm)，造成大鼠彌漫性腦創(chuàng)傷模型，假手術(shù)組僅麻醉大鼠，頭皮切開、縫合。U0126組將0.1 mg/kg U0126(美國，Promega公司，藥物批號(hào)：粵衛(wèi)藥準(zhǔn)字第616021號(hào))溶解于PBS中，并以0.1 mmol/L的PBS稀釋為總量300 μL，于造模前 30 min自尾靜脈注射。DMSO組同時(shí)點(diǎn)尾靜脈注射相同含量DMSO稀釋溶液，假手術(shù)組和模型組同時(shí)點(diǎn)尾靜脈注射生理鹽水300 μL。

1.2大鼠海馬組織神經(jīng)細(xì)胞凋亡情況觀察采用TUNEL法。造模30 min、3 h、12 h、24 h、48 h、72 h、7 d取各組大鼠各5只(假手術(shù)組3只)，以35 mg/kg戊巴比妥鈉腹腔麻醉;仰臥位固定后開胸暴露心臟，左心室穿刺入升主動(dòng)脈，用室溫肝素生理鹽水沖洗內(nèi)臟血液10 min;4%多聚甲醛250 mL經(jīng)心灌流，固定30～40 min；待大鼠全身僵硬后斷頭取腦組織，固定、石蠟包埋、切片，TUNEL染色后鏡下觀察，實(shí)驗(yàn)重復(fù)3次。均設(shè)陰性對(duì)照，以PBS代替一抗。所有操作嚴(yán)格按照德國BM公司說明書操作。TUNEL陽性細(xì)胞呈藍(lán)紫色，細(xì)胞核著色。TUNEL陽性細(xì)胞陽性強(qiáng)弱用Image Proplus 6.0數(shù)碼醫(yī)學(xué)圖像分析系統(tǒng)定量分析測定，以積分光密度(IOD)值來表示大鼠海馬區(qū)神經(jīng)細(xì)胞數(shù)。

1.3大鼠海馬組織p-ERK1/2蛋白檢測采用Western blot法。造模30 min、3 h、12 h、24 h、48 h、72 h、7 d取各組大鼠各6只(假手術(shù)組3只)，處死大鼠后取海馬組織;4 ℃PBS充分洗滌，加入3倍體積的4 ℃全細(xì)胞裂解液，冰浴中勻漿；4 ℃離心5 min，12 000 r/min，取上清。轉(zhuǎn)膜、封閉后加入p-ERK1/2兔抗鼠單克隆抗體(美國Cell Signaling公司)，DAB顯色，實(shí)驗(yàn)重復(fù)3次。用圖像分析儀測定各組p-ERK1/2蛋白條帶的光密度,進(jìn)行半定量分析。

1.4大鼠學(xué)習(xí)記憶能力檢測采用Morris水迷宮實(shí)驗(yàn)。造模14、16、18、21 d,取10只各組大鼠進(jìn)行Morris水迷宮實(shí)驗(yàn)。將安全島隨機(jī)置于水迷宮四個(gè)象限(N、S、E、W)中的任一象限，加水(奶粉沖呈乳白色)至高過安全島10 mm，水溫22～25 ℃，由攝象機(jī)及計(jì)算機(jī)自動(dòng)跟蹤、拍攝和統(tǒng)計(jì)大鼠分別由其他3個(gè)象限入水后尋找到安全島的時(shí)間(潛伏期)。如在180 s內(nèi)未找到安全島，則潛伏期值記為180 s。水迷宮視頻分析系統(tǒng)由中國醫(yī)學(xué)科學(xué)院藥物研究所提供。實(shí)驗(yàn)重復(fù)3次，取平均值。以搜索安全島潛伏期表示其學(xué)習(xí)記憶能力。

2　結(jié)果

2.1各組海馬組織神經(jīng)細(xì)胞凋亡情況比較造模后不同時(shí)點(diǎn)各組海馬組織神經(jīng)細(xì)胞凋亡情況比較見表1。

表1　造模后不同時(shí)點(diǎn)各組海馬組織神經(jīng)細(xì)胞凋亡情況比較±s)

注：與模型組和DMSO組比較,#P<0.05；與假手術(shù)組比較，*P<0.05。

2.2各組海馬組織p-ERK1/2蛋白相對(duì)表達(dá)量比較造模后不同時(shí)點(diǎn)各組海馬組織p-ERK1/2蛋白相對(duì)表達(dá)量比較見表2。

2.3各組大鼠學(xué)習(xí)記憶能力比較造模后不同時(shí)點(diǎn)各組大鼠學(xué)習(xí)記憶能力比較見表3。

2.4海馬區(qū)神經(jīng)細(xì)胞凋亡與p-ERK1/2表達(dá)的關(guān)系相關(guān)性分析結(jié)果顯示，海馬區(qū)神經(jīng)細(xì)胞凋亡情況與p-ERK1/2相關(guān)性呈正相關(guān)(r=0.468,P=0.002)。

表2　造模后不同時(shí)點(diǎn)各組海馬組織p-ERK1/2蛋白相對(duì)表達(dá)量

注：與模型組、DMSO組比較,#P<0.05；與假手術(shù)組比較，*P<0.05。

表3　造模14、16、18、21 d各組大鼠學(xué)習(xí)記憶能力比較

注：與DMSO組、模型組比較,﹟P<0.05; 與假手術(shù)組比較,*P<0.01。

3　討論

MAPKs是細(xì)胞內(nèi)的一類絲氨酸/蘇氨酸蛋白激酶，是細(xì)胞應(yīng)激和損傷反應(yīng)的主要信號(hào)通路,可將胞外刺激信號(hào)傳遞至胞核。細(xì)胞外信號(hào)調(diào)節(jié)激酶ERK是MAPKs重要成員[3]。研究顯示，腦缺血損傷后，激活的ERK1/2對(duì)神經(jīng)元具有保護(hù)作用[4]，但有更多文獻(xiàn)報(bào)道與之相反。Mori等[5]研究顯示，腦損傷后ERK1/2的激活與細(xì)胞凋亡密切相關(guān)，其特異性抑制劑可明顯減少神經(jīng)細(xì)胞死亡數(shù)目，減輕創(chuàng)傷后的腦損傷面積，并改善了損傷后的神經(jīng)功能。Clausen等[6]在閉合性腦損傷的模型中，應(yīng)用共聚焦顯微鏡觀測到損傷中心及周邊區(qū)活化的ERK1/2與TUNEL陽性細(xì)胞共同定位，為ERK通路活化可導(dǎo)致細(xì)胞凋亡提供最為直觀的證據(jù)。本研究發(fā)現(xiàn)，海馬區(qū)神經(jīng)細(xì)胞凋亡與p-ERK1/2表達(dá)呈正相關(guān)。這一方面反應(yīng)了創(chuàng)傷后ERK1/2的激活可加重腦損傷的病理過程，同時(shí)也提示ERK通路可能是腦損傷后治療的新靶點(diǎn)。

U0126是高選擇性和高效的MAPKs阻滯劑，可通過直接抑制MEK活性而阻滯其下游分子ERK1 /2的磷酸化[7]。本研究結(jié)果顯示，與假手術(shù)組比較，模型組創(chuàng)傷后30 min海馬區(qū)p-ERK1/2相對(duì)表達(dá)量升高，且隨創(chuàng)傷時(shí)間增加其相對(duì)表達(dá)量逐漸升高，于創(chuàng)傷后24 h達(dá)到峰值，U0126組p-ERK1/2相對(duì)表達(dá)量低于模型組，可能原因?yàn)镸APKs通路被阻斷。TUNEL檢測結(jié)果顯示,隨著創(chuàng)傷時(shí)間延長，海馬區(qū)神經(jīng)細(xì)胞凋亡數(shù)增多；而U0126組較模型組海馬區(qū)細(xì)胞凋亡數(shù)明顯減少；說明腦損傷后， ERK的活化增加，導(dǎo)致了神經(jīng)細(xì)胞凋亡的增多，加重了腦損傷，與以往文獻(xiàn)報(bào)道一致。

海馬作為學(xué)習(xí)記憶信息通路的必要結(jié)構(gòu)，在空間學(xué)習(xí)記憶的鞏固方面發(fā)揮著重要作用[8]。海馬神經(jīng)細(xì)胞凋亡可能是導(dǎo)致TBI后學(xué)習(xí)記憶功能障礙、特別是空間學(xué)習(xí)記憶能力下降的機(jī)制之一。p-ERK參與認(rèn)知功能，如學(xué)習(xí)與記憶形成[9～12]。最新研究證實(shí)，Ras/ERK通路參與了人類的認(rèn)知過程。Silva等[13]利用基因工程技術(shù)，在研究Ⅰ型神經(jīng)纖維母細(xì)胞瘤與精神發(fā)育遲緩的關(guān)系中發(fā)現(xiàn)，學(xué)習(xí)功能的降低與海馬Ras/ERK信號(hào)通路的缺損密切相關(guān)。目前臨床普遍認(rèn)為ERK通路參與了突觸可塑性變化，進(jìn)而影響了大腦信息存儲(chǔ)及記憶鞏固等過程。Mazzucchelli等[14]研究發(fā)現(xiàn),ERK1基因敲除小鼠長時(shí)記憶功能顯著增強(qiáng),而短時(shí)記憶力保持。Zhang等[15]研究發(fā)現(xiàn),經(jīng)過氣味休克訓(xùn)練1 h的正常幼鼠ERK1和ERK2磷酸化水平顯著增強(qiáng)，從而激活ERK通路，而MAPK/ERK抑制劑PD98059可明顯降低ERK磷酸化水平，進(jìn)而影響大鼠的學(xué)習(xí)記憶功能。

本研究中，U0126可減少TBI大鼠海馬神經(jīng)細(xì)胞凋亡數(shù)，提高大鼠的學(xué)習(xí)記憶能力，可能與降低海馬組織中p-ERK1/2表達(dá)有關(guān)。但ERK通路激活后，通過作用于哪些因子發(fā)揮腦保護(hù)作用還有待進(jìn)一步研究。

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Protective effects of MAPKs inhibitor U0126 on learning and memory function of rats with traumatic brain injury

LIUXingyu1,ZHENYanfeng,CUIJianzhong,GAOJunling

(1TangshanGongrenHospital,Tangshan063000,China)

ObjectiveTo observe the protection of mitogen-activated protein kinase (MAPK) inhibitor U0126 on learning and memory function of rats with traumatic brain injury (TBI) and to investigate its mechanism. MethodsMale Sprague-Dawley rats (n=313) were randomly divided into four groups: the sham operation group (n=52), TBI group (n=87), DMSO group (n=87) and U0126 group (n=87). Except the TBI group, diffused brain injury rat models were established in the other groups. In the U0126 group, 0.l mg/kg U0126 was dissolved in PBS, and 0.1 mM PBS was diluted to the total amount of 300 μL, then, this solution was injected into the rats 30 min before modeling by caudal vein. Rats in the DMSO group were injected with the same amount of DMSO dilute solution, and rats in the sham operation group and TBI group were injected with 300 μL normal saline. The in situ apoptosis assay was used to observe the nerve apoptosis of hippocampus of each group at 30 min, 3 h, 12 h, 24 h, 48 h, 72 h and 7 d after modeling (n=5 respectively, except for sham operation groupn=3). The phosphorylated extracellular signal-regulated kinase (ERK) 1/2 (p-ERK1/2) protein was detected in each group by Western blotting at 30 min, 3 h, 12 h, 24 h, 48 h, 72 h and 7 d after modeling (n=6 respectively, except for sham operation groupn=3). The learning and memory function was evaluated by Morris water maze on the 14th, 16th, 18th and 21rd day after modeling in each group (eachn=10). ResultsThe number of nerve cell apoptosis of hippocampus in the U0126 group was lower than that of DMSO and TBI groups at 48 h and 72 h after modeling, allP<0.05; the number of nerve cell apoptosis of hippocampus in the U0126, DMSO and TBI groups was all higher than that of the sham operation group at 24 h, 48 h and 72 h, allP<0.05; the relative expression of p-ERK1/2 in hippocampus of the U0126 group was lower than that of the DMSOand TBL groups at 48 h and 72 h after modeling, allP<0.05; the relative expression of p-ERK1/2 in hippocampus of the U0126、DMSO and TBI groups was all higler than that of the sham group at 12 h, 24 h, 48 h and 72 h after modeling, allP<0.05; the incubation period in the U0126 group was shorter than that of the DMSO and TBI groups on the 16th, 18th and 21rd day, allP<0.05; the incubation period in the U0126, DMSO and TBI groups was longer than that of the sham operation group on the 16th, 18th and 21rd day, allP<0.05; correlation analysis showed that the number of nerve cell apoptosis in hippocampus was positively correlated with p-ERK1/2 expression (r=0.468,P=0.002). ConclusionU0126 can inhibit neuronal apoptosis in hippocampus and improve learning and memory in rats, which may be correlated with the decreased p-ERK1/2 expression.

traumatic brain injury; learning and memory function; mitogen-activated protein kinase inhibitor; phosphorylated extracellular signal-regulated kinase 1/2

河北省自然科學(xué)基金資助項(xiàng)目(H2012401071)；河北省引進(jìn)留學(xué)人員資助項(xiàng)目(2012-02)。

劉興宇(1980-)，男，碩士，主治醫(yī)師，研究方向?yàn)槟X創(chuàng)傷機(jī)制。E-mail: 15931588358@126.com

簡介：高俊玲(1960-)，女，博士，教授，研究方向?yàn)槟X損傷與腦保護(hù)單位。E-mail: junlinggao@163.com

10.3969/j.issn.1002-266X.2016.18.007

R651.1

A

1002-266X(2016)18-0021-04

2015-11-08)