祁德波,李為民

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電針對(duì)慢性內(nèi)臟痛敏大鼠延髓頭端腹內(nèi)側(cè)核NMDA-R1受體表達(dá)的影響

祁德波1,李為民2

(1.復(fù)旦大學(xué)力學(xué)與工程科學(xué)系,上海 200433;2.復(fù)旦大學(xué)上海醫(yī)學(xué)院神經(jīng)網(wǎng)絡(luò)系統(tǒng)生物學(xué)實(shí)驗(yàn)室，上海 200032)

在電針(EA)緩解腸易激綜合征(IBS)模型大鼠慢性內(nèi)臟痛敏基礎(chǔ)上,觀察電針處理對(duì)IBS模型大鼠延髓頭端腹內(nèi)側(cè)核(RVM)N-甲基-D-門冬氨酸1型(NMDA-R1)受體表達(dá)的影響。將新生9 d SD幼鼠分為兩組,第一組通過結(jié)直腸機(jī)械擴(kuò)張刺激制作IBS慢性內(nèi)臟痛敏模型,持續(xù)兩星期;另一組僅輕柔肛周皮膚作為對(duì)照。飼養(yǎng)至6～8星期后,分別觀察兩組大鼠由結(jié)直腸擴(kuò)張(CRD)誘發(fā)的腹部撤回反射(AWR)評(píng)分和痛閾壓力值(PTP)變化。繼而隨機(jī)將IBS模型大鼠分為模型組(M)、模型加電針組(EA)和模型加假電針組(SEA)。模型組不做任何治療,電針組穴位取雙側(cè)“足三里”和“上巨虛”,連續(xù)隔日治療4次,假電針不通電,余與電針組同。治療結(jié)束后,取材并用免疫組化方法觀察各組大鼠RVM中NMDA-R1受體表達(dá)變化。成年IBS大鼠AWR評(píng)分明顯升高(＜0.01),PTP值明顯降低(＜0.01);電針可以明顯降低AWR評(píng)分(＜0.05),并使PTP值明顯升高(＜0.01);假電針則沒有此作用。IBS大鼠RVM中NMDA-R1受體陽(yáng)性神經(jīng)元數(shù)目和積分光密度明顯高于正常對(duì)照大鼠(＜0.05);而電針可使其表達(dá)明顯降低(＜0.05);假電針則沒有這樣的作用。電針對(duì)IBS大鼠的慢性內(nèi)臟痛敏具有良好的治療作用,其作用機(jī)制可能通過調(diào)節(jié)腦內(nèi)RVM中NMDA-R1受體表達(dá)來(lái)發(fā)揮的。

針刺;電針;慢性內(nèi)臟痛敏;大鼠

內(nèi)臟感覺異常是腸易激綜合征(irritable bowel syndrome,IBS)患者出現(xiàn)慢性內(nèi)臟痛敏(chronic visceral hyperalgesia)現(xiàn)象的主要機(jī)制之一[1,2],而反復(fù)發(fā)作的腹痛是IBS患者頻繁就醫(yī)的重要原因。至今,對(duì)于IBS的治療仍無(wú)有效的方法,而IBS的治療耗費(fèi)了大量醫(yī)療和社會(huì)資源[3,4],因此尋找有效和經(jīng)濟(jì)的治療方法已成為一種必然趨勢(shì)。

針刺鎮(zhèn)痛在中醫(yī)學(xué)中已有數(shù)千年的歷史,并且現(xiàn)今仍活躍應(yīng)用于臨床實(shí)踐。相對(duì)于藥物治療,針刺治療某些疾病具有療效確切、操作簡(jiǎn)便、經(jīng)濟(jì)實(shí)用、副反應(yīng)少等優(yōu)點(diǎn)。自上世紀(jì)70年代以來(lái),針刺對(duì)慢性內(nèi)臟痛的良好療效[5-8]受到了國(guó)內(nèi)外的持續(xù)關(guān)注,但針刺緩解內(nèi)臟痛的機(jī)制不明,還有待進(jìn)一步的研究。

Al-Chaer[9]于2001年報(bào)道了一種能夠模擬臨床IBS內(nèi)臟痛敏的大鼠模型。我們?cè)诔晒?fù)制了該IBS模型大鼠后,觀察了電針對(duì)腸易激綜合征的治療作用,結(jié)果發(fā)現(xiàn)電針能明顯緩解模型大鼠慢性內(nèi)臟痛敏并可能是通過NMDA受體發(fā)揮作用的[10-12],然而目前對(duì)于針刺治療IBS模型大鼠慢性內(nèi)臟痛敏作用的機(jī)制研究還有待于進(jìn)一步的開展。鑒于慢性內(nèi)臟痛的形成和發(fā)展與延髓頭端腹內(nèi)側(cè)核(rostral ventromedial medulla, RVM)中谷氨酸遞質(zhì)受體系統(tǒng)有著密切聯(lián)系[13-16],本研究選擇了RVM中NMDA-R1受體為研究對(duì)象,觀察和分析NMDA-R1受體在針刺緩解IBS模型大鼠慢性內(nèi)臟痛敏中的參與機(jī)制。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物

雄性Sprague-Dawley(SD)新生大鼠(出生后5 d),由中科院上海實(shí)驗(yàn)動(dòng)物中心提供。每窩幼鼠與1只雌性哺乳大鼠共同飼養(yǎng)在1個(gè)塑料籠內(nèi),哺乳大鼠自由飲食,飼養(yǎng)環(huán)境為12 h～12 h晝夜節(jié)律變換,室溫控制在18～24℃,濕度控制在50%～70%。飼養(yǎng)3 d后用于實(shí)驗(yàn)。實(shí)驗(yàn)時(shí)室溫維持在20～24℃。所有對(duì)動(dòng)物的實(shí)驗(yàn)處理符合國(guó)際疼痛研究會(huì)制定的準(zhǔn)則。

1.2 IBS慢性內(nèi)臟痛敏模型制作

IBS模型制作方法如前我們文章所述[10,12,17],新生的SD雄性大鼠自出生后9 d起,在清醒狀態(tài)下,使用自制的刺激球囊,蘸少許甘油,自肛門輕柔插入,深度2 cm,大致達(dá)到大鼠的降結(jié)腸部位。球囊充氣0.2 mL,持續(xù)1 min,然后撤氣,緩慢抽出刺激球囊。30 min后,重復(fù)一次同樣的刺激。1 d內(nèi)給予以上操作1次,連續(xù)2星期。正常對(duì)照組大鼠是將新生乳鼠與IBS造模鼠一起飼養(yǎng),只用球囊輕揉肛周皮膚,但不做球囊擴(kuò)張刺激。造模結(jié)束后,飼養(yǎng)大鼠至6～8周齡,體重達(dá)到200～250 g時(shí),進(jìn)行下一步的實(shí)驗(yàn)。動(dòng)物隨機(jī)分為正常對(duì)照組(N),模型組(M),模型加電針組(EA)和模型加假電針組(SEA),每組8只。

1.3 腹部撤回反射(AWR)評(píng)分

大鼠飼養(yǎng)6～8星期后,對(duì)大鼠進(jìn)行結(jié)直腸擴(kuò)張刺激(colorectal distension,CRD),觀察腹部撤回反射(abdominal withdraw reflex,AWR)評(píng)分。于清醒狀態(tài)下從大鼠肛門置入擴(kuò)張球囊,深度為6 cm,并置大鼠于平臺(tái)上,限制其于一20 cm×8 cm×8 cm透明塑料盒內(nèi),讓大鼠適應(yīng)15～20 min后進(jìn)行CRD刺激和AWR評(píng)分。開始正式實(shí)驗(yàn)前,將連接臺(tái)式血壓計(jì)和一個(gè)10 mL注射器的三通管與大鼠球囊相接,后分別給予20、40、60、80 mmHg四個(gè)不同壓力分級(jí)的CRD刺激。每次CRD刺激持續(xù)20 s,間隔5 min后給下一次刺激;每個(gè)刺激強(qiáng)度重復(fù)3次,取觀察指標(biāo)AWR評(píng)分的平均值作為每次最后的評(píng)分值。相應(yīng)的評(píng)分標(biāo)準(zhǔn)參照Al-Chaer的方法和我們的改進(jìn)[9,10,17],即對(duì)CRD無(wú)行為反應(yīng),評(píng)為0分;給予刺激大鼠有頭部或軀體動(dòng)作短暫停頓,評(píng)為1分;刺激期間,見有腹部肌肉的收縮,評(píng)為2分;如有腹部抬起行為,評(píng)為3分;身體拱起,并抬起盆腔和陰囊者,評(píng)為4分。

1.4 痛閾壓力值(PTP)測(cè)定

痛閾壓力值(pain threshold pressure,PTP)測(cè)定前準(zhǔn)備工作與AWR評(píng)分相同。CRD刺激以5 mmHg的間隔逐級(jí)增加,當(dāng)大鼠腹壁出現(xiàn)收縮時(shí)的壓力值即為PTP值,然后迅速撤氣。間隔5 min后給下一次刺激;每個(gè)刺激強(qiáng)度重復(fù)5次,取觀察指標(biāo)PTP平均值作為每次最后的評(píng)分值。

1.5 選穴和電針參數(shù)

電針穴位取雙側(cè)“足三里”和“上巨虛”。電針治療儀為華佗牌電針儀(SDZ-IV型),采用疏密波(密波頻率5～100 Hz可調(diào),疏波頻率是密波頻率的1/5,疏波時(shí)間為5 s,密波時(shí)間為10 s),以動(dòng)物下肢發(fā)生輕度抖動(dòng)為宜,共持續(xù)30 min,隔日治療1次。假電針組不通電,余與治療組同。在第4次電針治療后第2天對(duì)大鼠進(jìn)行CRD刺激,對(duì)AWR變化評(píng)分,評(píng)估電針的治療作用。

1.6 固定和冰凍切片的制備

大鼠在5%戊巴比妥麻醉下,迅速打開胸腔,暴露心臟,經(jīng)左心室插管至升主動(dòng)脈,剪開右心耳?？焖俟嗳腩A(yù)冷的生理鹽水300 mL沖洗,隨后換用4%多聚甲醛(pH 7.4)300 mL,約30 min內(nèi)灌注完畢。鉗斷大鼠顱骨,取出延髓(RVM取材部位及橫截面示意圖見Fig.1[18]),放入含20%蔗糖的4%多聚甲醛液中固定24 h后,再浸入30%蔗糖磷酸緩沖液保存,后將其制成30mm連續(xù)冰凍切片(CM-1900恒冷切片機(jī),德國(guó)徠卡公司),分別置于不同標(biāo)記的24孔板中。

1.7 免疫組織化學(xué)檢測(cè)

先將切片置于羊血清中室溫下孵育30 min,再將切片浸于兔抗大鼠NMDA-R1抗體(武漢博士德公司,工作濃度1:100)溶液中,4℃放置48 h,然后相繼用羊抗兔生物素化二抗和HRP標(biāo)記鏈親和素(SABC免疫組化試劑盒,武漢博士德公司)室溫中各孵育30 min,DAB顯色(DAB試劑盒,武漢博士德公司),無(wú)水乙醇脫水,二甲苯透明,中性樹膠封片。分別用正常羊血清代替一抗和省略一抗作陰性對(duì)照。每只大鼠取3張切片,對(duì)NMDA-R1受體免疫陽(yáng)性細(xì)胞進(jìn)行計(jì)數(shù),取算術(shù)平均值;每只大鼠取3張切片,每張切片取5個(gè)視野,用Image Pro Plus(IPP)軟件計(jì)算積分光密度(integrated optical density,IOD)。

1.8 統(tǒng)計(jì)學(xué)方法

數(shù)據(jù)均以平均值±標(biāo)準(zhǔn)誤(Mean±SEM)表示。用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,多組間的比較采用單因素方差分析,并用SNK進(jìn)行兩兩比較。以＜0.05表示差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 IBS模型大鼠與正常大鼠PTP值及AWR評(píng)分比較

由表1可見,與正常對(duì)照組比較,模型組的PTP值明顯下降(＜0.01)。AWR評(píng)分結(jié)果顯示,在不同強(qiáng)度(20、40、60、80 mmHg)的CRD誘發(fā)內(nèi)臟痛刺激下,模型組相比于正常對(duì)照組,AWR評(píng)分明顯升高(＜0.05)。

2.2 電針組與假電針組治療前后PTP值及AWR評(píng)分比較

由表2可見,電針組治療后與治療前及假電針組治療后相比,AWR評(píng)分明顯降低(＜0.05),PTP明顯升高(＜0.01),而假電針組治療后與治療前AWR評(píng)分的差異無(wú)統(tǒng)計(jì)學(xué)意義(＞0.05)。

2.3 各組RVM中NMDA-R1受體表達(dá)比較

由表3可見,模型組NMDA-R1受體免疫陽(yáng)性細(xì)胞的計(jì)數(shù)和IOD與正常對(duì)照組相比明顯升高(＜0.05),而電針組NMDA-R1受體免疫陽(yáng)性細(xì)胞的計(jì)數(shù)和IOD明顯下降(＜0.05),但假電針組與模型組相比無(wú)明顯變化(＞0.05)。

表1 IBS模型大鼠與正常大鼠PTP值及AWR評(píng)分比較

表2 電針組與假電針組治療前后PTP值及AWR評(píng)分比較

注:與同組治療前比較1)＜0.01,2)＜0.05;與假電針組比較3)＜0.05

表3 各組RVM中NMDA-R1受體表達(dá)比較

注:與正常對(duì)照組比較1)＜0.05;與模型組比較2)＜0.05

3 討論

內(nèi)臟痛敏是IBS患者主要臨床癥狀之一,嚴(yán)重影響著他們的生活質(zhì)量。有研究表明,幼年和童年時(shí)期受到身體、言語(yǔ)、性等方面的虐待所造成的心理創(chuàng)傷,改變了中樞神經(jīng)系統(tǒng)可塑性,這可能與成年后IBS內(nèi)臟痛敏的發(fā)病密切相關(guān)[19]。大鼠在新生期受到結(jié)直腸炎癥[9,20]、機(jī)械擴(kuò)張[9-12]等傷害刺激,以及母嬰分離[21]等精神因素困擾,可使大鼠成年后機(jī)體產(chǎn)生內(nèi)臟痛覺超敏(allodynia)和痛覺過敏(hyperalgesia)狀態(tài),前者為對(duì)正常狀態(tài)下非傷害性刺激產(chǎn)生疼痛反應(yīng),后者為對(duì)傷害性刺激產(chǎn)生的疼痛反應(yīng)增強(qiáng)。

如前所述,本研究中的模型大鼠在出生后9～21 d,每天給予大鼠結(jié)直腸擴(kuò)張刺激,恢復(fù)2星期后,行為學(xué)測(cè)試結(jié)果表明,成年模型大鼠明顯處于痛覺超敏和痛覺過敏狀態(tài)。PTP數(shù)據(jù)顯示,IBS模型大鼠的痛閾值明顯下降;IBS模型大鼠在20 mmHg(非傷害性),40、60、80 mmHg(傷害性)CRD刺激時(shí)AWR評(píng)分明顯高于正常對(duì)照組。這表明于出生早期給予連續(xù)傷害性機(jī)械刺激,可誘發(fā)動(dòng)物持續(xù)至成年的痛覺過敏,能較好地模擬IBS的臨床表現(xiàn)。

延髓頭端腹內(nèi)側(cè)核(RVM)是脊髓上神經(jīng)中樞對(duì)內(nèi)臟傷害刺激進(jìn)行下行調(diào)制的共同通路。RVM對(duì)內(nèi)臟痛具有雙重調(diào)節(jié)作用,抑制和易化傷害輸入。根據(jù)大量研究成果,Porreca等[21]提出假設(shè),RVM對(duì)于疼痛信息的下行調(diào)節(jié)功能紊亂,可能是疼痛感覺異常如IBS,纖維肌痛和神經(jīng)性疼痛的重要原因,而RVM中谷氨酸遞質(zhì)及其受體,特別是NMDA受體,在內(nèi)臟痛敏的調(diào)節(jié)中發(fā)揮了重要作用[13-16]。本研究的免疫組化結(jié)果顯示,模型組大鼠與正常對(duì)照組大鼠相比,NMDA-R1受免疫陽(yáng)性細(xì)胞的計(jì)數(shù)和IOD與正常對(duì)照組相比明顯升高,這表明RVM中NMDA-R1受體表達(dá)升高,是IBS模型大鼠內(nèi)臟痛敏現(xiàn)象發(fā)生的重要原因。

本研究在成功制作了IBS大鼠模型后,重新驗(yàn)證了電針對(duì)模型大鼠的作用。研究顯示,隔日電針治療4次后,發(fā)現(xiàn)電針治療組AWR評(píng)分明顯下降,PTP值明顯上升,而假電針則無(wú)此效應(yīng),這顯示電針能明顯緩解模型大鼠的內(nèi)臟痛癥狀。我們?cè)陔娽樦委熤笸ㄟ^免疫組織化學(xué)方法來(lái)觀察電針對(duì)RVM NMDA-R1受體表達(dá)的影響,以探討電針緩解內(nèi)臟痛的神經(jīng)生物學(xué)機(jī)制。實(shí)驗(yàn)顯示,電針可以下調(diào)NMDA-R1受體的表達(dá),而電針可能通過下調(diào)RVM中NMDA-R1受體的表達(dá)來(lái)實(shí)現(xiàn)其治療作用。

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Effect of Electroacupuncture on Expression of NMDA-R1 Receptor in the Rostral Ventromedia Medulla of Rats with Chronic Visceral Hyperalgesia

-1,-2.

1.,,200433,; 2.,200032,

To investigate the effect of electroacupuncture (EA) on the expression of N-methyl-D-aspartate receptor 1 (NMDA-R1) in the rostral ventromedia medulla (RVM) on the basis of EA relieving chronic visceral hyperalgesia in a rat model of irritable bowel syndrome (IBS).Nine-day-old SD rats were allocated to two groups. In one group, a model of IBS chronic visceral hyperalgesia was made by 2 weeks’ mechanical dilation of colorectum. In the other group as a control, perianal skin was kneaded lightly. Colorectal distention (CRD)-induced abdominal withdraw reflex (AWR) was scored and pain threshold pressure (PTP) was measured in the two groups at 6-8 weeks after feeding. Then, IBS rats were randomly allocated to model, electroacupuncture and sham electroacupuncture groups. The model group was not given treatment. In the electroacupuncture group, bilateral points Zusanli and Shangjuxu were selected and treatment was given once every other day for 4 times. The sham electroacupuncture group was given the same treatment as in the electroacupuncture group except no electrification. After treatment was finished, the specimen was taken and the expression of NMDA-R1 in RVM was examined by an immunohistochemical method in each group of rats.The AWR score increased significantly (＜0.01) and PTP decreased significantly (＜0.01) in the adult IBS rats. Electroacupuncture increased the AWR score significantly (＜0.05) and decreased PTP significantly (＜0.01). Sham electroacupuncture had no such effects. The number and integrated optical density of RVM NMDA-R1-positive neurons were significantly higher in the IBS rats than in the normal control rats (＜0.05). Electroacupuncture increased its expression significantly (＜0.05). Sham electroacupuncture had no such effect.Electroacupuncture has a good therapeutic effect on chronic visceral hyperalgesia in IBS rats. The mechanism of its action may be that it regulates the expression of NMDA-R1 in RVM.

Acupuncture; Electroacupuncture; Chronic visceral hyperalgesia; Rats;NMDA-R1receptor expression

1005-0957(2011)07-0491-04

R2-03

A

10.3969/j.issn.1005-0957.2011.07.490

2011-04-20

祁德波(1983 - ),男,2008 級(jí)碩士生

李為民(1959 - ),男,教授,博士生導(dǎo)師,研究方向?yàn)獒槾绦?yīng)的神經(jīng)生物學(xué)機(jī)制及其復(fù)雜網(wǎng)絡(luò)系統(tǒng),植物神經(jīng)功能調(diào)控機(jī)制,E-mail:wmli9110@fudan.edu.cn