王愛桃武慶平徐建軍姚尚龍＊崔永武邱 頤

(1華中科技大學(xué)同濟醫(yī)學(xué)院附屬協(xié)和醫(yī)院麻醉科,武漢 430030;2內(nèi)蒙古醫(yī)學(xué)院第二附屬醫(yī)院,呼和浩特 010030)

小膠質(zhì)細胞在脂多糖引起的熱高敏中的作用

王愛桃1,2武慶平1徐建軍1姚尚龍1＊崔永武2邱 頤2

(1華中科技大學(xué)同濟醫(yī)學(xué)院附屬協(xié)和醫(yī)院麻醉科,武漢 430030;2內(nèi)蒙古醫(yī)學(xué)院第二附屬醫(yī)院,呼和浩特 010030)

目的 探討小膠質(zhì)細胞在脂多糖引起的熱高敏中的作用。方法 清潔級雄性昆明小鼠,隨機分成兩組,每組5只,腹腔注射LPS組和注射 PBS組,在注射前及后30、60、120、240min測量小鼠足底的熱痛閾;每組于注射前及后4h各處死5只取腦組織檢測IL-1β、TNF-α;每組于腹腔注射4h時處死動物,免疫熒光確定腦組織中小膠質(zhì)細胞的激活情況。然后分為四組,米諾環(huán)素+PBS組,米諾環(huán)素+LPS組,PBS+PBS組,PBS+LPS組,每組5只,連續(xù)三天腹腔注射米諾環(huán)素或 PBS,第三天注射LPS或PBS,在注射前及后30、60、120、240min測量小鼠足底的熱痛閾;每組于注射前及后4h各處死5只取腦組織檢測 IL-1β、TNF-α。結(jié)果 與注射 PBS相比 ,注射 LPS導(dǎo)致 IL-1β、TNF-α分泌增加 ,注射 60、120、240min小鼠的熱痛閾降低;與米諾環(huán)素+PBS組、米諾環(huán)素 +LPS組、PBS+PBS組相比,PBS+LPS組導(dǎo)致 IL-1β、TNF-α分泌增加,注射 60、120、240min小鼠的熱痛閾降低。結(jié)論 LPS激活小膠質(zhì)細胞分泌促炎細胞因子導(dǎo)致熱高敏。

小膠質(zhì)細胞; 脂多糖; 熱高敏; 白介素1β; 腫瘤壞死因子α

已有研究證實膠質(zhì)細胞在疼痛的起始、發(fā)生與發(fā)展中具有重要作用[1,2]。其中小膠質(zhì)細胞主要在疼痛的起始發(fā)揮作用,星形膠質(zhì)細胞主要參與疼痛的維持[3,4]。本研究選擇了小膠質(zhì)細胞進行研究,希望從源頭上治療疼痛。小膠質(zhì)細胞激活后可以產(chǎn)生促炎細胞因子 IL-1β、TNF-α,IL-1β、TNF-α參與疼痛的形成與發(fā)展[5],在炎性疼痛及慢性疼痛中扮演重要角色。脂多糖(lipopolysaccharide,LPS)為革蘭氏陰性細菌外膜的一種成份,是強致熱源。LPS作為炎癥形成的刺激因素已經(jīng)被廣泛應(yīng)用于基礎(chǔ)研究中。本研究將LPS的致炎效應(yīng)與炎性因子在疼痛中的作用相聯(lián)系,闡明小膠質(zhì)細胞在LPS致熱高敏效應(yīng)中的重要作用。

材料和方法

1.材料與儀器

LPS購自sigma公司。小膠質(zhì)細胞的抑制劑米諾環(huán)素購自sigma公司。小膠質(zhì)細胞的特異性標(biāo)記物抗Iba-1購自Wako公司。37370型熱痛測試儀(U GO BASILE公司,意大利)由華中科技大學(xué)同濟醫(yī)學(xué)院附屬協(xié)和醫(yī)院麻醉科提供。IL-1β、TNF-α ELISA試劑盒購自武漢博士德公司。

2.動物選擇及分組

清潔級雄性昆明小鼠95只,體重30～40g,由華中科技大學(xué)同濟醫(yī)學(xué)院動物中心提供。實驗前適應(yīng)環(huán)境3d,室溫22～24℃,周期光照,自由進食、飲水。本研究已通過動物保護協(xié)會批準(zhǔn)。

首先將昆明小鼠隨機分成兩組,腹腔注射LPS 0.33mg/kg[6]組和注射等量的 PBS組,在注射前及后 30、60、120、240min 測量小鼠足底的熱痛閾;每組于注射前及后4h各處死5只取腦組織勻漿檢測IL-1β、TNF-α。每組于注射4h處死取腦組織做免疫熒光確定小膠質(zhì)細胞激活的情況。再將昆明小鼠隨機分為4組,米諾環(huán)素+PBS組,米諾環(huán)素+LPS組,PBS+PBS組,PBS+LPS組,每組 5只,連續(xù)三天腹腔注射米諾環(huán)素 50mg/kg(前兩組)或等量PBS(后兩組),第三天還要再注射LPS 0.33mg/kg(米諾環(huán)素+LPS組和 PBS+LPS組)或等量 PBS(米諾環(huán)素+PBS組和 PBS+PBS組),在注射前及后 30、60、120、240min 測量小鼠足底的熱痛閾;每組于注射前及后4h各處死5只取腦組織勻漿檢測IL-1β、TNF-α。

3.熱痛閾的測定

在室溫(20-25℃)、安靜環(huán)境下檢測,采用熱痛測試儀參照文獻[7]介紹的方法測定小鼠右后爪熱痛閾。從光熱開始照射小鼠足底到縮足反應(yīng)的時間即為小鼠熱傷害縮腿反應(yīng)潛伏期(PWL),以此反映其熱痛閾。連續(xù)測3次,每次間隔5min,取平均值。最長的熱輻射時間設(shè)定為20s。單次光照射時間超過20s仍未出現(xiàn)縮足反應(yīng)為痛覺遲鈍,予以剔除。

4.ELISA 檢測 IL-1β、TNF-α

腦組織80g加入磷酸鹽緩沖液(p H7.4)勻漿,3000rpm/min離心20min,取上清液。根據(jù)試劑盒說明進行操作。

5.免疫熒光

腹腔注射生理鹽水組和注射LPS組注射4h時,處死動物在冰上快速地取腦組織做冰凍切片,厚度為10μm,丙酮固定15min,晾干后儲存于-80℃。用時取出晾干四十分鐘,PBS洗5min,5%BSA室溫封閉 2h,5%BSA稀釋一抗(兔單克隆 antimouse Iba1),4℃孵育過夜。PBS洗10min×3次。5%BSA稀釋二抗FITC-labeled anti-rabbit IgG Ab(Southern Biotechnology Associates)室溫孵育90min,PBS洗5min×8次。熒光顯微鏡觀察。

6.統(tǒng)計學(xué)處理

使用Excel 2003軟件進行數(shù)據(jù)錄入,數(shù)據(jù)管理和分析使用 SAS9.13(SAS Institute Inc. ,Cary,NC,USA)英文版。根據(jù)資料的特征分別采用了成組t檢驗、配對t檢驗、Wilcoxon秩和檢驗、兩因素方差分析、多元線性回歸分析及重復(fù)測量的方差分析等,P≤0.05被認(rèn)為有統(tǒng)計學(xué)意義。

結(jié) 果

1.注射PBS組各個時間點熱痛閾差異無統(tǒng)計學(xué)意義(P>0.05);注射LPS組0～120min熱痛閾逐漸降低,在120min時將到最低,此后逐漸回升(P<0.01);重復(fù)測量資料的方差分析結(jié)果顯示,注射LPS組小鼠的熱痛閾顯著低于注射PBS組,其中注射LPS 60、120、240min小鼠的熱痛閾降低(P<0.01),見表 1。

表1 腹腔注射LPS不同時間熱痛閾的比較 (n=5,±s)Table 1 Comparison of the change in thermal nociceptive threshold at each time point after injections(i.p.)of LPS(n=5,±s)

表1 腹腔注射LPS不同時間熱痛閾的比較 (n=5,±s)Table 1 Comparison of the change in thermal nociceptive threshold at each time point after injections(i.p.)of LPS(n=5,±s)

aP<0.05,compared with the PBS group.

Group 0 min 30 min 60 min 120 min 240 min PBS 12.63 ±0.17 12.70 ±0.30 13.03 ±0.23 13.33 ±0.18 12.50 ±0.14 LPS 13.10 ±0.39 11.67 ±0.08 7.95 ±0.21a 3.29 ±0.23a 6.32 ±0.31a

2.PBS組注射前、后 IL-1 和 TNF-α含量差異無統(tǒng)計學(xué)意義(P>0.05);與注射前相比,LPS組注射 4h 引起 IL-1β、TNF-α分泌增加(P<0.01);與注射 PBS組相比,LPS組注射4h引起 IL-1β、TNF-α分泌增加(P<0.01),見表2。

表2 腹腔注射LPS前及后4h腦組織勻漿測量IL-1 、TNF-α含量的比較 (n=5,±s)Table 2 Comparison of the change in IL-1 and TNF-αsecretion at 0,4 h after injections(i.p.)of LPS in the brain tissues

表2 腹腔注射LPS前及后4h腦組織勻漿測量IL-1 、TNF-α含量的比較 (n=5,±s)Table 2 Comparison of the change in IL-1 and TNF-αsecretion at 0,4 h after injections(i.p.)of LPS in the brain tissues

aP<0.05,compared with‘0 h’;bP<0.05,compared with the PBS group

Index Group 0 h 4 h IL-1 PBS 20.62 ±1.28 26.83 ±3.38 LPS 20.97 ±2.77 309.73 ±10.80abTNF-α PBS 45.38 ±4.09 48.65 ±4.78 LPS 45.13 ±3.89 404.65 ±12.33ab

3.米諾環(huán)素+PBS組、米諾環(huán)素+LPS組、PBS+PBS組各個時間點熱痛閾差異無統(tǒng)計學(xué)意義(P>0.05);PBS+LPS組0～120min熱痛閾逐漸降低,在120min時將到最低,此后逐漸回升(P<0.01);與米諾環(huán)素+PBS組、米諾環(huán)素+LPS組、PBS+PBS組相比,PBS+LPS組 60、120、240min小鼠的熱痛閾降低(P<0.01),見表3。

表 3 四組小鼠注射前、后 30、60、120、240min 熱痛閾的比較 (n=5,±s)Table 3 Comparison of the change in thermal nociceptive threshold at each time point in the four groups(n=5,±s)

表 3 四組小鼠注射前、后 30、60、120、240min 熱痛閾的比較 (n=5,±s)Table 3 Comparison of the change in thermal nociceptive threshold at each time point in the four groups(n=5,±s)

aP<0.05,compared with minocycline+PBS group;bP<0.05,compared with minocycline+LPS group;cP<0.05,compared with PBS+PBS group

Group Minocycline PBS PBS LPS PBS LPS 0 12.73 ±0.31 12.88 ±0.16 12.55 ±0.14 13.03 ±0.37 30 12.71 ±0.57 12.09 ±0.39 12.65 ±0.31 11.59 ±0.21 60 12.67 ±0.24 11.45 ±0.33 12.91 ±0.28 7.93 ±0.28abc120 12.55 ±0.17 10.99 ±0.47 13.37 ±0.31 3.33 ±0.30abc240 12.65 ±0.41 11.33 ±0.71 12.71 ±0.37 6.35 ±0.33abc

4.各組注射前 IL-1β、TNF-α含量差異無統(tǒng)計學(xué)意義(P>0.05);與注射前比較,米諾環(huán)素+LPS組、PBS+LPS組注射4h IL-1β、TNF-α分泌增加(P<0.01);與米諾環(huán)素+PBS組、PBS+PBS組相比,米諾環(huán)素+LPS組注射4h IL-1β、TNF-α分泌增加(P<0.01);與米諾環(huán)素+PBS組、米諾環(huán)素+LPS組、PBS+PBS組相比,PBS+LPS組注射4h IL-1β、TNF-α分泌增加(P<0.01),見表 4。

表4 四組小鼠腦組織勻漿 IL-1β、TNF-α含量的比較(n=5,±s)Table 4 Comparison of the change in IL-1 and TNF-α secretion in the brain tissues(n=5,±s)

表4 四組小鼠腦組織勻漿 IL-1β、TNF-α含量的比較(n=5,±s)Table 4 Comparison of the change in IL-1 and TNF-α secretion in the brain tissues(n=5,±s)

aP<0.05,compared with‘0 h’;bP<0.05,compared with minocycline+PBS group;cP<0.05,compared with minocycline+LPS group;dP<0.05,compared with PBS+PBS group.

Index Group Minocycline PBS PBS LPS PBS LPS IL-1β 0 h 25.8 ±3.3 23.9 ±3.1 23.5 ±3.2 20.7 ±1.3 4 h 24.2 ±2.0 72.4 ±2.7acd 24.0 ±2.1 308.9 ±13.8abcdTNF-α 0 h 45.8 ±3.3 45.6 ±3.4 45.2 ±4.3 45.4 ±3.8 4 h 44.7 ±2.6 110.7 ±13.1acd 46.4 ±3.3 405.0 ±12.7abcd

5.免疫熒光表明,腹腔注射脂多糖后引起腦部小膠質(zhì)細胞的激活,表現(xiàn)為細胞數(shù)目的增多和細胞形態(tài)的改變。C為注射PBS組,E為注射LPS組,下面兩幅圖是上面方框圖的放大,見圖1。圖例50μm。

圖1 免疫熒光顯示腹腔注射LPS 4h時腦組織中的小膠質(zhì)細胞被激活。標(biāo)尺50μmFig.1 Immunofluorescence indicated that microglia was activated at 4 h after injections(i.p. )of LPS in the brain tissues.

討 論

疼痛困擾著大量的人群,日益成為社會的負擔(dān),由于其發(fā)病機制復(fù)雜,至今仍無有效的治療措施。大量研究顯示:膠質(zhì)細胞在疼痛中扮演重要角色,如弗氏佐劑引起的關(guān)節(jié)炎[8]、骨癌[9]、坐骨神經(jīng)炎癥[10]、脊神經(jīng)根損傷[11]和坐骨神經(jīng)損傷[12],證實在不同的條件下膠質(zhì)的活化導(dǎo)致了神經(jīng)病理痛和炎性痛[13]。這些研究給疼痛的治療帶來了新的希望。小膠質(zhì)細胞主要分布在中樞神經(jīng)系統(tǒng),占中樞神經(jīng)系統(tǒng)中細胞總數(shù)的5%-12%,對外界刺激非常敏感,被激活時釋放 IL-1β、TNF-α等炎性因子。這些炎性因子是一類小分子多肽,它們刺激傷害感受器的末端,引起疼痛敏感,并且互相促進,彼此協(xié)同,在病理性疼痛中起著舉足輕重的作用,Wu CT等研究發(fā)現(xiàn)促炎細胞因子中 IL-1β、TNF-α可能是與疼痛加速作用最為密切的炎性因子[14]。疼痛機制的研究中發(fā)現(xiàn),外周神經(jīng)損傷所引發(fā)的疼痛,是由于初級傳入神經(jīng)元釋放降鈣素基因相關(guān)肽(CGRP)激活星形膠質(zhì)細胞和小膠質(zhì)細胞MAPK信號通路后分泌促炎細胞因子所致[15,16];還發(fā)現(xiàn)在完全弗氏佐劑(CAF)所致炎性痛大鼠模型中,神經(jīng)生長因子(NGF)在炎性組織中表達,沿外周神經(jīng)末梢逆行運輸至DRG內(nèi)細胞體,NGF激活膠質(zhì)細胞和神經(jīng)元內(nèi)p38MAPK,后者可增加VR1蛋白和促炎細胞因子的表達,增加的VR1順行轉(zhuǎn)運至外周末端傷害感受器,增加對熱的敏感性;增加的細胞因子促進神經(jīng)細胞和膠質(zhì)細胞激活,形成炎癥級聯(lián)反應(yīng),維持疼痛的擴大和高敏[17];且給予促炎細胞因子拮抗劑能夠逆轉(zhuǎn)神經(jīng)病理疼痛狀態(tài)[18,19]。本研究顯示腹腔注射LPS可以引起昆明小鼠腦組織中 IL-1β、TNF-α的分泌,并引起熱高敏。

先前的研究認(rèn)為LPS很少越過血腦屏障進人中樞神經(jīng)系統(tǒng)直接作用于神經(jīng)組織,但近來的研究證實外周給予小劑量的LPS并不引起全身的炎癥反應(yīng),這些LPS經(jīng)血循環(huán)改變腦血管的直徑和通透性,引起腦來源的神經(jīng)元和小膠質(zhì)細胞釋放TNF-α、NO等炎癥介質(zhì)[21]。本研究外周給予LPS,應(yīng)用Iba-1特異性標(biāo)記小膠質(zhì)細胞,免疫熒光檢測發(fā)現(xiàn)腦組織中小膠質(zhì)細胞被激活,同樣證實外周給予LPS可以激活腦源性的小膠質(zhì)細胞。

本研究首次證實LPS可以引起昆明小鼠熱高敏,并且發(fā)現(xiàn)應(yīng)用米諾環(huán)素可以降低LPS引起昆明小鼠的熱高敏,米諾環(huán)素是一種二代四環(huán)素類藥物,其分子量小,可以透過血腦屏障,抑制小膠質(zhì)細胞的活化而對星形膠質(zhì)細胞和神經(jīng)元無明顯作用,且本身無直接鎮(zhèn)痛作用[20]。本研究顯示腹腔注射米諾環(huán)素可以降低LPS引起昆明小鼠 IL-1β、TNF-α分泌,減低熱高敏。由此我們認(rèn)為小膠質(zhì)細胞在LPS引起的熱高敏中起關(guān)鍵性作用。活化的小膠質(zhì)細胞增加促炎細胞因子分泌,引起痛覺高敏,其可能機制與多條信號轉(zhuǎn)導(dǎo)通路相關(guān)。小膠質(zhì)細胞表面表達大量受體,受刺激時多個信號通路被激活,其中磷酸化的p38MAPK可能通過激活其下游的轉(zhuǎn)錄因子進入細胞核,啟動炎癥因子mRNA合成,最終生成并釋放大量包括 IL-1β、TNF-α等細胞因子,IL-1β、TNF-α等細胞因子一方面直接刺激傷害感受器的末端引發(fā)疼痛高敏,另一方面進一步激活神經(jīng)元、膠質(zhì)細胞p38MAPK通路及其他信號轉(zhuǎn)導(dǎo)通路如ERK、JN K,釋放更多的疼痛介質(zhì),從而形成疼痛級聯(lián)反應(yīng)的不良循環(huán),造成痛敏。

綜上所述,LPS激活小膠質(zhì)細胞分泌促炎細胞因子導(dǎo)致熱高敏。米諾環(huán)素可以減輕LPS作用于昆明小鼠引起的 IL-1β、TNF-α分泌和熱高敏。

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Role of Microglia in Lipopolysaccharide-induced Thermal Hyperalgesia

Wang Aitao1,2,Wu Qingping1,Xu Jianjun1,Yao Shanglong1,＊,Cui Yongwu2,Qiu Yi2

(1Department of A nesthesiology,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,W uhan430030;2Department of A nesthesiology,The Second A f f iliated Hospital of Inner Monglia Medical College,Huhhot010030,China)

Objective To explore the role of microglia in the lipopolysaccharide(LPS)-induced thermal hyperalgesia.Methods After adult kunming mice

an intraperitoneal injection of PBS or LPS,thermal nociceptive thresholds,IL-1βand TNF-α were determined;activation of microglia in the brain was determined by immunofluorescence.Then,according to the different agents,injected mice were divided into 4 groups:minocycline+PBS,minocycline+LPS,PBS+PBS,and PBS+LPS.The mice received an intraperitoneal injection of PBS or minocycline(the former agent of each group)for 3 consecutive days.On the 3 day,PBS or LPS(the latter agent of each group was added).The thermal nociceptive thresholds and the expressions of brain IL-1β and TNF-α were assayed. Results LPS injection led to proinflammatory cytokine secretion and thermal hyperalgesia in kunming mice.Minocycline blocked LPS-stimulated inflammatory cytokine secretion and thermal hyperalgesia in kunming mice.Conclusion These data indicate that IL-1β and TNF-α are induced by LPS through activating brain-derived microglia,hence modulate thermal hyperalgesia.

Microglia; Lipopolysaccharide; Hyperalgesia; IL-1β; TNF-α

R329

A

10.3870/zgzzhx.2011.02.017

2010-08-10

2011-02-15

王愛桃,女(1977年),漢族,主治醫(yī)師。

＊通訊作者(To whom correspondence should be addressed)