NPY通過(guò)小膠質(zhì)細(xì)胞介導(dǎo)的神經(jīng)免疫途徑對(duì)大鼠癲癇發(fā)作行為學(xué)影響

李琦軍,吳永波,常軍英,邢兆國(guó),張淑麗,王道愛(ài),王彥志,穆衛(wèi)盧,李 炎,賈東召，陳濤平

(1. 河北省石家莊市第三醫(yī)院，河北 石家莊 050011；2. 河北省石家莊市公安局法醫(yī)損傷檢驗(yàn)鑒定室，河北 石家莊 050011；3. 河北省秦皇島市骨科醫(yī)院，河北 秦皇島 066001；4. 河北省正定縣疾病預(yù)防控制中心，河北 正定 050800；5. 河北大學(xué)附屬醫(yī)院，河北 保定 071000)

NPY通過(guò)小膠質(zhì)細(xì)胞介導(dǎo)的神經(jīng)免疫途徑對(duì)大鼠癲癇發(fā)作行為學(xué)影響

李琦軍1,吳永波2,常軍英1,邢兆國(guó)1,張淑麗3,王道愛(ài)4,王彥志1,穆衛(wèi)盧1,李 炎1,賈東召1，陳濤平5

(1. 河北省石家莊市第三醫(yī)院，河北 石家莊 050011；2. 河北省石家莊市公安局法醫(yī)損傷檢驗(yàn)鑒定室，河北 石家莊 050011；3. 河北省秦皇島市骨科醫(yī)院，河北 秦皇島 066001；4. 河北省正定縣疾病預(yù)防控制中心，河北 正定 050800；5. 河北大學(xué)附屬醫(yī)院，河北 保定 071000)

目的 探討神經(jīng)肽Y(NPY)以中樞神經(jīng)系統(tǒng)小膠質(zhì)細(xì)胞為靶點(diǎn)對(duì)大鼠癲癇發(fā)作的影響。方法 培養(yǎng)和純化原代SD大鼠皮質(zhì)小膠質(zhì)細(xì)胞,免疫細(xì)胞化學(xué)染色，鑒定小膠質(zhì)細(xì)胞純度及觀察細(xì)胞形態(tài)。將小膠質(zhì)細(xì)胞分為對(duì)照組、LPS組、NPY+LPS組和BIBP3226+NPY+LPS組。對(duì)照組細(xì)胞以無(wú)血清膠質(zhì)細(xì)胞培養(yǎng)液孵育6 h，LPS組細(xì)胞以含終濃度為100 ng/mL LPS的無(wú)血清膠質(zhì)細(xì)胞培養(yǎng)液孵育6 h，NPY+LPS組細(xì)胞是先以含NPY (終濃度為1 μmol/L)的無(wú)血清膠質(zhì)細(xì)胞培養(yǎng)液孵育0.5 h，然后加入LPS(終濃度為100 ng/mL)繼續(xù)孵育6 h。IBP3226+NPY+LPS組細(xì)胞先用含NPY Y1受體阻斷劑BIBP3226 (終濃度為1 μmol/L)的無(wú)血清膠質(zhì)細(xì)胞培養(yǎng)液孵育0.5 h，再加入終濃度為1 μmol/L的NPY孵育0.5 h，最后加入終濃度100 ng/mL的LPS繼續(xù)孵育6 h。20只SD大鼠隨機(jī)分為對(duì)照組、LPS組、NPY+LPS組、BIBP3226+NPY+LPS組，每組5只。將各組小膠質(zhì)細(xì)胞條件培養(yǎng)液離心后注入相應(yīng)各組大鼠的側(cè)腦室，觀察各組大鼠的行為學(xué)表現(xiàn)。根據(jù)Diehl分級(jí)標(biāo)準(zhǔn)對(duì)大鼠癲癇發(fā)作程度進(jìn)行評(píng)估。結(jié)果 20 min內(nèi)，LPS組和BIBP3226+NPY+LPS組5只大鼠均出現(xiàn)重度癲癇發(fā)作，NPY+LPS組有4只出現(xiàn)輕度癲癇發(fā)作，對(duì)照組未見(jiàn)癲癇發(fā)作。LPS組發(fā)作程度顯著重于對(duì)照組，NPY+LPS組發(fā)作程度顯著輕于LPS組，BIBP3226+NPY+LPS組發(fā)作程度顯著重于NPY+LPS組，LPS組和BIBP3226+NPY+LPS組發(fā)作程度比較差異均無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論 激活后的小膠質(zhì)細(xì)胞可以導(dǎo)致大鼠癲癇發(fā)作，這種作用是通過(guò)小膠質(zhì)細(xì)胞與神經(jīng)元之間的非直接接觸途徑實(shí)現(xiàn)的，可能與小膠質(zhì)細(xì)胞分泌到細(xì)胞外的生物活性物質(zhì)有關(guān)。NPY可以通過(guò)作用于小膠質(zhì)細(xì)胞上NPY Y1受體，抑制大鼠的癲癇發(fā)作。

神經(jīng)肽Y；小膠質(zhì)細(xì)胞；癲癇

癲癇是一種慢性、反復(fù)發(fā)作的以顱內(nèi)神經(jīng)元異常同步放電為基礎(chǔ)的中樞神經(jīng)系統(tǒng)疾病[1]。近年雖然隨著外科手術(shù)、伽馬刀技術(shù)的發(fā)展，很多藥物治療無(wú)效的患者得到了有效治療，但在我國(guó)估計(jì)仍有約100萬(wàn)患者為難治性癲癇[2]。過(guò)去人們研究癲癇的重點(diǎn)在神經(jīng)元，現(xiàn)在發(fā)現(xiàn)膠質(zhì)細(xì)胞在癲癇發(fā)生中也起著重要作用[3]。小膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)中體積最小的一種免疫細(xì)胞，廣泛存在于中樞神經(jīng)系統(tǒng)，它產(chǎn)生的很多細(xì)胞因子和癲癇的發(fā)生有關(guān)，它是把免疫和癲癇聯(lián)系起來(lái)的重要橋梁，它分泌的細(xì)胞因子在癲癇中起著重要作用，故其在腦內(nèi)神經(jīng)免疫功能調(diào)節(jié)和介導(dǎo)炎癥反應(yīng)方面起著重要作用[4]。癲癇也是一種與炎癥有關(guān)的中樞神經(jīng)系統(tǒng)疾病，很多細(xì)胞因子在癲癇的發(fā)病中起著重要作用[5-6]。筆者前幾部分實(shí)驗(yàn)已經(jīng)在細(xì)胞水平證明神經(jīng)肽Y(NPY)能夠抑制小膠質(zhì)細(xì)胞的免疫活性，減少小膠質(zhì)細(xì)胞來(lái)源的IL-1β等炎癥因子的產(chǎn)生，并通過(guò)抑制小膠質(zhì)細(xì)胞的免疫活性降低體外培養(yǎng)的大鼠皮質(zhì)神經(jīng)元的NMDA電流[7]。為了探討NPY是不是能夠通過(guò)神經(jīng)免疫途徑影響活體動(dòng)物的癲癇發(fā)作，本實(shí)驗(yàn)通過(guò)LPS激活后的小膠質(zhì)細(xì)胞條件培養(yǎng)液和經(jīng)過(guò)NPY處理后的小膠質(zhì)細(xì)胞條件培養(yǎng)液注射入大鼠腦室，觀察了活體大鼠癲癇發(fā)作的行為學(xué)變化，現(xiàn)將結(jié)果報(bào)道如下。

1 實(shí)驗(yàn)資料

1.1 實(shí)驗(yàn)動(dòng)物 SD大鼠20只，體質(zhì)量200～250 g，雄性。24 h內(nèi)新生SD大鼠30只。均為清潔級(jí)，河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供，動(dòng)物生產(chǎn)許可證號(hào)SCXK(冀)2013-1-03。根據(jù)科技部《關(guān)于善待實(shí)驗(yàn)動(dòng)物的指導(dǎo)性意見(jiàn)》使用動(dòng)物。

1.2 主要儀器和試劑 超凈工作臺(tái)(日本Sanyo公司)，恒溫培養(yǎng)箱(德國(guó)Heraeus公司)，光學(xué)顯微鏡(日本OLYMPUS公司)，熒光顯微鏡MI3000B+DFC450C型(德國(guó)Leica公司)，TLL-C臺(tái)式高速低溫離心機(jī)(北京四環(huán)科學(xué)儀器廠)，立體定向儀(深圳市瑞沃德生命科技有限公司)，微量注射器(上海光正醫(yī)療儀器有限公司)，實(shí)驗(yàn)動(dòng)物電子秤(蘇杭科技器材有限公司)，醫(yī)用止血鉗(福州宜欣醫(yī)療器械公司)，手術(shù)剪(福州宜欣醫(yī)療器械公司)，醫(yī)用磨鉆(張家港市華新醫(yī)療器械廠)，醫(yī)用縫合線(浙江億人醫(yī)療器械有限公司)，小鼠單克隆抗體IBA-1 (美國(guó)Sigma公司)，F(xiàn)ITC標(biāo)記的山羊抗小鼠IgG(美國(guó)Proteintech公司)，脂多糖(LPS)(美國(guó)Sigma公司)，胎牛血清(美國(guó)Gibco公司)，DMEMF12培養(yǎng)液(美國(guó)Gibco公司)，BIBP3226(美國(guó)Tocris Bioscience公司)，水合氯醛 (分析純)(上海生工公司),75%醫(yī)用乙醇(浙江一佳醫(yī)療器械有限公司),生理鹽水 (石家莊四藥有限公司)。

1.3 原代大鼠皮質(zhì)混合膠質(zhì)細(xì)胞的培養(yǎng)和小膠質(zhì)細(xì)胞的分離純化 參照Nakajima等[8]所述方法，先將24 h內(nèi)新生大鼠頭部置于碎冰中2 min降溫，然后乙醇消毒頭部，無(wú)菌環(huán)境下開(kāi)顱取腦，將腦組織放入裝有DMEM/F12培養(yǎng)液的培養(yǎng)皿中，培養(yǎng)皿放置于冰板上保持低溫。在800倍解剖顯微鏡下剝除腦膜及血管，取部分大腦皮質(zhì)，剪碎后用0.125%胰蛋白酶37 ℃消化15 min，加入含10%血清的DMEM/F12培養(yǎng)液終止消化，1 000 r/min離心5 min后，棄上清，在沉淀物中加入膠質(zhì)細(xì)胞培養(yǎng)液，膠質(zhì)細(xì)胞培養(yǎng)液為含10%胎牛血清、1 IU/mL青霉素、100 μg/mL鏈霉素的DMEM/F12培養(yǎng)液，制成單細(xì)胞懸液，以15×106個(gè)細(xì)胞/瓶接種于750 mL培養(yǎng)瓶中，放置于37 ℃、5%CO2培養(yǎng)箱中培養(yǎng)。第2天全量換液1次，以后每3 d更換1/2體積培養(yǎng)液,光學(xué)顯微鏡下觀察混合膠質(zhì)細(xì)胞生長(zhǎng)情況。培養(yǎng)至第14天，細(xì)胞充分分層生長(zhǎng)后，置于37 ℃恒溫?fù)u床中180 r/min振搖2 h，收集細(xì)胞懸液，1 000 r/min離心5 min，去上清，用DMEM/F12膠質(zhì)細(xì)胞培養(yǎng)液吹打成單細(xì)胞懸液，以1×104個(gè)細(xì)胞/皿接種于預(yù)先放置經(jīng)多聚賴氨酸處理過(guò)的蓋玻片的3.5 cm培養(yǎng)皿，用于形態(tài)學(xué)觀察和細(xì)胞免疫化學(xué)染色。

1.4 小膠質(zhì)細(xì)胞分組及處理方法 將分離純化好的小膠質(zhì)細(xì)胞以5×105個(gè)細(xì)胞/孔接種于6孔培養(yǎng)板。培養(yǎng)3 d后換新鮮無(wú)血清膠質(zhì)細(xì)胞培養(yǎng)液培養(yǎng)12 h使細(xì)胞同步化，然后將細(xì)胞分為對(duì)照組、LPS組、NPY+LPS組和BIBP3226+NPY+LPS組。對(duì)照組細(xì)胞以無(wú)血清膠質(zhì)細(xì)胞培養(yǎng)液孵育6 h，LPS組細(xì)胞以含終濃度為100 ng/mL LPS的無(wú)血清膠質(zhì)細(xì)胞培養(yǎng)液孵育6 h，NPY+LPS組細(xì)胞先以含終濃度為1 μmol/L NPY的無(wú)血清膠質(zhì)細(xì)胞培養(yǎng)液孵育0.5 h，然后加入終濃度為100 ng/mL LPY繼續(xù)孵育6 h。IBP3226+NPY+LPS組細(xì)胞先用含終濃度為1 μmol/L NPY Y1受體阻斷劑BIBP3226的無(wú)血清膠質(zhì)細(xì)胞培養(yǎng)液孵育0.5 h，再加入終濃度為1 μmol/L的NPY孵育0.5 h，最后加入終濃度100 ng/mL的LPS繼續(xù)孵育6 h。取各組培養(yǎng)液離心后備用。

1.5 動(dòng)物分組及處理 將SD大鼠隨機(jī)分為對(duì)照組、LPS組、NPY+LPS組、BIBP3226+NPY+LPS組，每組5只。將各組小膠質(zhì)細(xì)胞條件培養(yǎng)液離心后注入相應(yīng)各組動(dòng)物的側(cè)腦室。具體方法：根據(jù)大鼠體質(zhì)量，將配置好的10%水合氯醛以 3.5 mg/kg劑量行腹腔注射麻醉，麻醉起效后，將大鼠固定于立體定向儀上，在頭部切口區(qū)域剪毛，顯露頭部皮膚，用75%乙醇消毒，沿頭頂部正中線做縱向切口，約3 cm，切開(kāi)頭皮，用頭皮拉鉤將頭皮向兩側(cè)牽開(kāi)固定,在顱骨上涂抹少量雙氧水，以清晰顯示人字縫。根據(jù)George Painos大鼠腦立體定位圖譜，確定右側(cè)腦室三維坐標(biāo):X=-1.0 mm,Y=1.5 mm,Z=-3.8 mm，以此為注射點(diǎn)，電動(dòng)磨鉆磨開(kāi)顱骨,顯露腦膜，在定位器引導(dǎo)下用微量注射器注入10 μL小膠質(zhì)細(xì)胞條件培養(yǎng)液。骨蠟封閉顱骨缺損，縫合皮膚，觀察各組動(dòng)物行為學(xué)表現(xiàn)，根據(jù)Diehl的分級(jí)標(biāo)準(zhǔn)[9]對(duì)大鼠的癲癇發(fā)作程度進(jìn)行評(píng)估。

1.6 統(tǒng)計(jì)學(xué)方法 利用SPSS 10.0統(tǒng)計(jì)分析軟件進(jìn)行統(tǒng)計(jì)學(xué)處理，采用Kruskal Wallis秩和檢驗(yàn)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 原代小膠質(zhì)細(xì)胞形態(tài)學(xué)觀察 分離純化后的小膠質(zhì)細(xì)胞，可見(jiàn)體積較小、折光性強(qiáng)，有著細(xì)長(zhǎng)突起(箭頭)，偶可見(jiàn)體積較大的星形膠質(zhì)細(xì)胞，神經(jīng)元已經(jīng)很難看到，見(jiàn)圖1。分離純化后的小膠質(zhì)細(xì)胞采用IBA-1免疫細(xì)胞化學(xué)熒光染色方法進(jìn)行鑒定，可見(jiàn)細(xì)胞呈分支狀或者梭形，有細(xì)長(zhǎng)的突起(箭頭)，IBA-1將胞體和突起染成紅色，Hoechst 33258將胞核染成藍(lán)色，純度大于95%，滿足實(shí)驗(yàn)需要，見(jiàn)圖2。

圖1 原代培養(yǎng)的大鼠皮質(zhì)小膠質(zhì)細(xì)胞 (400×)

2.2 各組大鼠癲癇發(fā)作情況 LPS組和BIBP3226+NPY+LPS組大鼠注射相應(yīng)培養(yǎng)液后20 min內(nèi)出現(xiàn)前肢或后肢間斷性抽搐、四肢節(jié)律性抽搐，發(fā)作等級(jí)為Ⅲ～Ⅳ級(jí)(重度發(fā)作)；NPY+LPS組有4只大鼠出現(xiàn)點(diǎn)頭、咀嚼、須動(dòng)、頭面部抽搐，發(fā)作等級(jí)為Ⅰ～Ⅱ級(jí)(輕度發(fā)作)；對(duì)照組未見(jiàn)點(diǎn)頭、豎毛、抽搐等表現(xiàn)。LPS組和BIBP3226+NPY+LPS組發(fā)作等級(jí)明顯高于對(duì)照組和NPY+LPS組(P均<0.05)。

圖2 IBA-1抗體染色后的大鼠皮質(zhì)小膠質(zhì)細(xì)胞 (600×)

3 討 論

癲癇是一種以腦部神經(jīng)元反復(fù)過(guò)度同步放電為特征的疾病，臨床上表現(xiàn)為短暫、反復(fù)的神經(jīng)系統(tǒng)功能失常。癲癇的發(fā)生與很多因素有關(guān)，包括遺傳因素、腦內(nèi)疾病、外傷、全身疾病等，如先天性腦畸形、先天性顱內(nèi)積水、嬰兒的產(chǎn)傷及成人的顱內(nèi)損傷、細(xì)菌或病毒性顱內(nèi)感染、顱內(nèi)腫瘤等。癲癇發(fā)作時(shí)，患者意識(shí)突然喪失會(huì)導(dǎo)致跌倒，發(fā)生外傷性損害，長(zhǎng)期癲癇也會(huì)導(dǎo)致精神障礙、智力衰退，甚至?xí)?dǎo)致患者死亡。雖然有伽馬刀、手術(shù)切除癲癇灶等新的治療方法出現(xiàn)，但藥物治療仍是治療癲癇的主要方法之一。很多癲癇經(jīng)過(guò)藥物或者手術(shù)治療后癥狀能夠明顯緩解，但也有些病例經(jīng)過(guò)長(zhǎng)期(2～3年)使用多種抗癲癇藥后癥狀仍不能緩解，這種癲癇稱為頑固性癲癇。尋找新的治療方法及有效的治療藥物是人們攻克癲癇頑疾的關(guān)鍵。

癲癇發(fā)病機(jī)制涉及很多方面，其中神經(jīng)機(jī)制是其發(fā)病的主要因素，但很多其他因素如免疫和內(nèi)分泌因素對(duì)神經(jīng)元的功能起著調(diào)節(jié)作用，這種調(diào)節(jié)功能的失調(diào)會(huì)加重癲癇的發(fā)生、發(fā)展。目前研究發(fā)現(xiàn)中樞神經(jīng)系統(tǒng)內(nèi)的IL-1β和TNF-α主要由小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞產(chǎn)生，側(cè)腦室注射IL-1β能夠促進(jìn)熱性驚厥的發(fā)生，而注射IL-1β受體拮抗劑則起到相反的作用。在癲癇發(fā)生后，腦內(nèi)IL-1β、TNF-α明顯上升，說(shuō)明IL-1β、TNF-α與癲癇發(fā)生有著密切關(guān)系[10-11]，二者增加都起到促進(jìn)癲癇發(fā)生的作用[12]。Vezzani等[13]研究證實(shí)給予IL-1β后，動(dòng)物的腦電活動(dòng)增強(qiáng)，這種效應(yīng)可被NMDA受體阻斷劑阻斷，說(shuō)明IL-1β是通過(guò)NMDA受體起作用的。朱曉琴等[14]研究證明，腦室內(nèi)注射IL-1β能夠誘發(fā)癲癇，其作用機(jī)制可能與其激活谷氨酸受體有關(guān)。NMDA受體是一種谷氨酸受體，其介導(dǎo)的興奮性神經(jīng)毒性在癲癇的發(fā)病發(fā)展過(guò)程中起著關(guān)鍵作用[15]。此受體被過(guò)度激活后，可以導(dǎo)致神經(jīng)元損傷[16-17]。IL-1β和TNF-α促進(jìn)癲癇發(fā)作的機(jī)制可能是激活谷氨酸NMDA受體，促進(jìn)Ca2+內(nèi)流，導(dǎo)致神經(jīng)元細(xì)胞內(nèi)Ca2+超載，從而產(chǎn)生細(xì)胞毒性作用,誘發(fā)癲癇的產(chǎn)生[18-19]。另外小膠質(zhì)細(xì)胞是腦內(nèi)起著監(jiān)視作用的免疫細(xì)胞，對(duì)刺激非常敏感。當(dāng)內(nèi)環(huán)境發(fā)生變化時(shí)，小膠質(zhì)細(xì)胞被激活，激活的小膠質(zhì)細(xì)胞釋放大量的炎性因子，對(duì)神經(jīng)元產(chǎn)生毒性作用，導(dǎo)致癲癇發(fā)生或加重癲癇發(fā)作程度[20-21]。Rodgers等[22]研究表明，LPS可以誘發(fā)局部癲癇樣放電，其機(jī)制可能是LPS激活膠質(zhì)細(xì)胞，通過(guò)膠質(zhì)細(xì)胞的激活，產(chǎn)生大量IL-1β，誘發(fā)癲癇。加入IL-1β受體拮抗劑后，阻斷了IL-1β的作用，這種放電明顯減弱或消失，說(shuō)明膠質(zhì)細(xì)胞來(lái)源的IL-1β是誘發(fā)放電的主要因素之一，而并非LPS 本身影響神經(jīng)元導(dǎo)致的癲癇。

LPS可以導(dǎo)致小膠質(zhì)細(xì)胞大量產(chǎn)生IL-1β和TNF-α，這可能是經(jīng)LPS處理后的膠質(zhì)細(xì)胞培養(yǎng)液注入大腦側(cè)腦室后誘發(fā)動(dòng)物癲癇的機(jī)制之一。而NPY可以抑制小膠質(zhì)細(xì)胞的激活和IL-1β、TNF-α的產(chǎn)生。本實(shí)驗(yàn)結(jié)果顯示，經(jīng)LPS激活的膠質(zhì)細(xì)胞培養(yǎng)液注入大鼠腦室并未出現(xiàn)癲癇發(fā)作，經(jīng)過(guò)LPS激活后的小膠質(zhì)細(xì)胞培養(yǎng)液注入大鼠側(cè)腦室時(shí)可以很快誘發(fā)大鼠癲癇的發(fā)生，且發(fā)作程度較重，將使用NPY抑制小膠質(zhì)細(xì)胞生物活性的條件培養(yǎng)液注入大鼠側(cè)腦室后癲癇發(fā)作明顯減輕。本實(shí)驗(yàn)中，體外培養(yǎng)的小膠質(zhì)細(xì)胞與腦組織并未直接接觸，說(shuō)明小膠質(zhì)細(xì)胞分泌到培養(yǎng)液中的生物活性物質(zhì)是造成癲癇發(fā)作的主要原因。當(dāng)用BIBP3226阻斷NPY Y1受體后，NPY的作用消失，說(shuō)明NPY是通過(guò)Y1受體起作用的。這為NPY以小膠質(zhì)細(xì)胞為治療靶點(diǎn)，通過(guò)神經(jīng)-免疫途徑治療癲癇提供了實(shí)驗(yàn)依據(jù)。

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The inhibiting effect of NPY on the epileptic seizures of rats by acting on microglia

LI Qijun1, WU Yongbo2,CHANG Junying1,XING Zhaoguo1,ZHANG Shuli3, WANG Daoai4,WANG Yanzhi1,MU Weilu1, LI Yan1,JIA Dongzhao1，Chen Taoping5

(1.The Third Hospital of Shijiazhuang, Shijiazhuang 050011,Hebei, China; 2.Forensic Damage Identification Room,the Public Security Bureau of Shijiazhuang, Shijiazhuang 050011,Hebei, China; 3.Orthopedic Hospital of Qinghuangdao, Qinhuangdao 066001, Hebei, China; 4.Zhengding Centers for Disease Control, Zhengding 050800, Hebei, China；5. Affiliated Hospital of Hebei University,Baoding 071000,Hebei，China)

Objective It is to explore the influence of NPY on the epileptic seizure of rats by acting on microglia.Methods The primary cortex microglia of rats was cultured and purified, morphology of microglia were observed through immunocytochemistry staining, Primary cerebral cortical microglia of rats was divided into control group, LPS group, NPY+LPS group, NPY group and BIBP3226+NPY+LPS group. Microglia cells in control group were incubated with serum-free medium for 6 h; microglia cells in LPS group were incubated with serum-free medium including LPS(final concentration 100 ng/mL)for 6 h; microglia cells in NPY+LPS group were incubated with serum-free medium including NPY(final concentration 1 μmol/L) for 0.5 h firstly, then continued the incubation for 6 h after adding LPS (final concentration 100 ng/mL); microglia cells in NPY group were incubated in serum-free medium including NPY (final concentration 1 μmol/L) for 6 h; microglia cells in BIBP3226+NPY+LPS group were incubated in serum-free medium including BIBP3226 (final concentration 1 μmol/L) which was NPY Y1 receptor blocking reagent for 0.5 h, then we incubated them for 0.5 h after adding NPY (final concentration 1 μmol/L),at last the cells were incubated for 6 h after adding LPS with the final concentration 100 ng/mL. 20 adult SD rats were divided into control group,LPS group,NPY+LPS group and BIBP3226+NPY+LPS group,every group included five rats. The levels of epileptic seizure of rats in each group were observed after microglia conditioned mediums were injected respectively into ventricle of the adult rats. Behaviors of the rats in every group were observed, and epilepsy degree was evaluated based on Diehl’s method.Results In 20 minutes, all the 5 rats in LSP group and BIBP3226+NPY+LPS group appeared severe epileptic seizure. There were 4 rats in NPY+LPS group appeared mild epileptic seizures in the same time.There was no rat appeared epileptic seizure in control group. The degrees of epileptic seizure of LPS group were significantly higher than that of the control group. the degrees of NPY+LPS group were significantly lower than that of LPS group. The degrees of BIBP3226+NPY+LPS group were significantly higher than that of NPY+LPS group. There was no difference in the degrees between LPS group and BIBP3226+NPY+LPS group. Conclusion LPS could stimulate microglia cells to cause epileptic seizure of rats. NPY can restrain epileptic seizure of rats through acting on NPYY1 receptor on microglia. This may be related to something with biological activity.

neuropeptide Y; microglia; epileptic seizur

李琦軍，男，副主任醫(yī)師，博士，從事骨科診治研究工作。

陳濤平，E-mail：18603121976@163.com

10.3969/j.issn.1008-8849.2015.11.004

R-332

A

1008-8849(2015)11-1150-04

2014-09-14