乳鐵蛋白對(duì)神經(jīng)病理性痛大鼠脊髓小膠質(zhì)細(xì)胞活化的影響研究

王 軍，薛 紅，宗川曰，宗 毅

·基礎(chǔ)醫(yī)學(xué)· ·論著·

乳鐵蛋白對(duì)神經(jīng)病理性痛大鼠脊髓小膠質(zhì)細(xì)胞活化的影響研究

王 軍，薛 紅，宗川曰，宗 毅

目的 探討乳鐵蛋白對(duì)神經(jīng)病理性痛大鼠脊髓背角小膠質(zhì)細(xì)胞活化的影響。方法 雄性SD大鼠32只，體質(zhì)量200～220 g，按照數(shù)字表法隨機(jī)分為4組(對(duì)照組、神經(jīng)病理性痛組、米諾環(huán)素組、乳鐵蛋白組)，每組8只。對(duì)照組大鼠僅分離坐骨神經(jīng)，不結(jié)扎，肌鞘內(nèi)注射生理鹽水8 μl；其余3組大鼠采用結(jié)扎坐骨神經(jīng)的方法制備神經(jīng)病理性痛模型。神經(jīng)病理性痛組鞘內(nèi)僅注射生理鹽水8 μl；乳鐵蛋白組鞘內(nèi)注射乳鐵蛋白100 μg，米諾環(huán)素組鞘內(nèi)注射米諾環(huán)素(小膠質(zhì)細(xì)胞特異性活化抑制劑)100 μg。給藥后每隔30 min以熱刺激法測(cè)試大鼠熱縮爪潛伏期，共180 min，測(cè)量7次后，常規(guī)處死大鼠取脊髓背角，采用免疫熒光法檢測(cè)小膠質(zhì)細(xì)胞特異性標(biāo)記物Iba-1的表達(dá)，并進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果 與對(duì)照組比較，神經(jīng)病理性痛組縮爪潛伏期明顯延長(zhǎng)，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；與神經(jīng)病理性痛組相比，米諾環(huán)素組和乳鐵蛋白組縮爪潛伏期延長(zhǎng)，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；而米諾環(huán)素組與乳鐵蛋白組爪潛伏期比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)，可間接證明乳鐵蛋白可通過(guò)抑制脊髓小膠質(zhì)細(xì)胞活化減輕大鼠神經(jīng)病理性痛。與對(duì)照組比較，神經(jīng)病理性痛組、米諾環(huán)素組和乳鐵蛋白組脊髓背角Iba-1表達(dá)明顯減少，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；與神經(jīng)病理性痛組相比，米諾環(huán)素組和乳鐵蛋白組脊髓背角Iba-1表達(dá)也明顯減少，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；而米諾環(huán)素組與乳鐵蛋白組縮爪潛伏期比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)，可直接證明乳鐵蛋白可通過(guò)抑制脊髓小膠質(zhì)細(xì)胞活化減輕大鼠神經(jīng)病理性痛。結(jié)論 乳鐵蛋白可通過(guò)抑制大鼠脊髓小膠質(zhì)細(xì)胞活化減輕神經(jīng)病理性痛，對(duì)臨床麻醉鎮(zhèn)痛過(guò)程有一定的指導(dǎo)價(jià)值。

乳鐵蛋白；神經(jīng)病理性痛；小膠質(zhì)細(xì)胞

乳鐵蛋白廣泛存在于哺乳動(dòng)物的體液中，具有抗炎、抗腫瘤及免疫調(diào)節(jié)等多種作用[1]。有研究[2-3]顯示，乳鐵蛋白對(duì)神經(jīng)病理性痛大鼠的鎮(zhèn)痛作用與大鼠脊髓背角一氧化氮(NO)-環(huán)GMP依賴(lài)性蛋白激酶(PKG)信號(hào)傳導(dǎo)通路相關(guān)，但具體的機(jī)制仍不清楚。NO存在于神經(jīng)元及神經(jīng)膠質(zhì)細(xì)胞中，且與小膠質(zhì)細(xì)胞的活化相關(guān)。本研究擬探討乳鐵蛋白對(duì)神經(jīng)病理性痛模型大鼠脊髓小膠質(zhì)細(xì)胞活化的影響，為研究乳鐵蛋白的鎮(zhèn)痛機(jī)制提供實(shí)驗(yàn)依據(jù)。現(xiàn)報(bào)道如下。

1 材料與方法

1.1 材料 米諾環(huán)素(小膠質(zhì)細(xì)胞特異性活化抑制劑)、牛乳鐵蛋白(美國(guó)Sigma公司)，兔抗Iba-1抗體(日本W(wǎng)ako公司)，F(xiàn)ITC標(biāo)志的羊抗兔抗體(美國(guó)Cruz公司)；熱刺激儀7370(意大利Basile公司)，熒光定量PCR儀(美國(guó)ABI公司)，凝膠成像系統(tǒng)(美國(guó)Image公司)。冰凍切片機(jī)、激光共聚焦顯微鏡(中國(guó)Leica分公司)。

1.2 動(dòng)物選擇及分組 健康雄性SD大鼠32只，體質(zhì)量200～220 g，由徐州醫(yī)學(xué)院動(dòng)物中心提供，動(dòng)物證編號(hào)：20151123291。單籠飼養(yǎng)，飼養(yǎng)室溫度為20～26℃，光照時(shí)間8:00-22:00，大鼠自由進(jìn)食及飲水。按照數(shù)字表法隨機(jī)分為4組，每組8只，對(duì)照組大鼠僅分離坐骨神經(jīng)，不結(jié)扎，肌鞘內(nèi)注射生理鹽水8 μl，其余3組大鼠采用結(jié)扎坐骨神經(jīng)的方法制備神經(jīng)病理性痛大鼠模型。神經(jīng)病理性痛組鞘內(nèi)僅注射生理鹽水8 μl；乳鐵蛋白組肌鞘內(nèi)注射乳鐵蛋白100 μg，米諾環(huán)素組鞘內(nèi)注射米諾環(huán)素100 μg。給藥后每隔30 min以熱刺激法測(cè)試大鼠熱縮爪潛伏期，共180 min，測(cè)量7次后，處死大鼠取脊髓背角，采用免疫熒光法檢測(cè)大鼠脊髓背角小膠質(zhì)細(xì)胞標(biāo)記物Iba-1的表達(dá)。

1.3 神經(jīng)病理性大鼠模型的制備 大鼠神經(jīng)病理性痛模型的制備參照文獻(xiàn)[4]的方法。腹腔注射戊巴比妥鈉(50 mg/kg)麻醉大鼠后，常規(guī)消毒左后肢，切開(kāi)皮膚、皮下組織，鈍性分離肌肉，于股骨后暴露坐骨神經(jīng)主干，光學(xué)顯微鏡下用4-0鉻制腸線松扎5處，間隔1 mm，結(jié)扎線松緊度以引起大鼠腿部輕微抽搐但不影響坐骨神經(jīng)主干神經(jīng)外膜的血運(yùn)為度，然后逐層依次縫合，依照參照文獻(xiàn)[4]介紹的方法行鞘內(nèi)置管。

1.4 大鼠縮爪潛伏期的測(cè)定 參照文獻(xiàn)[5]介紹的縮爪潛伏期測(cè)定方法，將SD大鼠放置于熱測(cè)試儀玻璃表面，測(cè)試前讓大鼠微適應(yīng)25 min后，移動(dòng)玻璃下方的熱刺激源(強(qiáng)光柱)，采用微電子計(jì)時(shí)器記錄大鼠后爪光源聚焦至由于疼痛而回縮爪的時(shí)間，即為一個(gè)縮爪潛伏期。整個(gè)實(shí)驗(yàn)過(guò)程中，光柱照射強(qiáng)度不變，并調(diào)整后爪回縮潛伏期為10 s。為避免大鼠足底被燙傷，熱刺激源刺激最長(zhǎng)時(shí)間為30 s。分別在給藥后0、30、60、90、120、150、180 min時(shí)記錄大鼠縮爪潛伏期，每一時(shí)間點(diǎn)重復(fù)測(cè)定3次，取平均值。

1.5 大鼠脊髓背角Iba-1表達(dá)水平的測(cè)定 在最后1次大鼠縮爪潛伏期測(cè)定結(jié)束后，腹腔注射戊巴比妥鈉(50 mg/kg)麻醉大鼠后，從主動(dòng)脈依次灌注多聚甲醛和生理鹽水，取大鼠脊髓L4-5置于多聚甲醛溶液中固定3.5 h，轉(zhuǎn)入25%蔗糖溶液中，4℃恒溫過(guò)夜。待組織沉底后，沉淀，連續(xù)冰凍切片，片厚25 μm，浸泡在0.01 mol/L PB液中。PBS液清洗3次后，在5%小牛血清中加入0.3% TritonX-100，16℃孵育35 min。加入兔抗Iba-1抗體(一抗，1∶100)，4℃孵育24～48 h，PBS液再?zèng)_洗3次后，滴加的羊抗兔IgG(FITC標(biāo)記,二抗，1：100)，16℃孵育2.0 h，PBS液再?zèng)_洗3次。三酰甘油與PBS(1：1)混合液封片，對(duì)照組用PBS液代替一、二抗。每只大鼠取L4-5節(jié)段脊髓切片3張，熒光染色后，采用Leica激光共聚焦顯微鏡進(jìn)行觀察，并測(cè)量切片中部每0.4 mm×0.4 mm范圍的像素密度，重復(fù)測(cè)定3次，取平均值。采用Image Pro P 5.0圖像統(tǒng)計(jì)軟件，計(jì)算切片的熒光光密度，轉(zhuǎn)化成定量數(shù)據(jù)后表達(dá)Iba-1在大鼠脊髓背角小膠質(zhì)細(xì)胞的活性。

1.6 統(tǒng)計(jì)學(xué)處理 采用SPSS 15.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)處理，計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示，組間均數(shù)比較采用t檢驗(yàn)。以P<0.05表示為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 各組大鼠熱刺激縮爪潛伏期的比較 與對(duì)照組比較，神經(jīng)病理性痛組縮爪潛伏期明顯延長(zhǎng)，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；與神經(jīng)病理性痛組相比，米諾環(huán)素組和乳鐵蛋白組部分時(shí)間點(diǎn)縮爪潛伏期明顯延長(zhǎng)，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；米諾環(huán)素組與乳鐵蛋白組縮爪潛伏期比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。見(jiàn)表1。

表1 各組大鼠熱刺激縮爪潛伏期的比較(s，x±s)

組別大鼠數(shù)0min30min60min90min120min150min180min假手術(shù)組89.9±1.110.1±0.810.4±1.69.8±1.310.3±2.19.7±1.410.5±1.5神經(jīng)病理性痛組85.5±1.1a4.7±0.9a4.6±1.1a5.4±0.8a4.9±1.2a5.2±0.7a4.8±0.6a米諾環(huán)素組85.6±0.87.2±1.6b8.7±1.6b8.5±1.8b8.4±1.4b6.1±0.65.4±0.8乳鐵蛋白組85.2±0.98.1±1.2b9.6±1.8b9.3±1.5b6.4±1.26.3±0.76.1±1.3

注：與假手術(shù)組比較aP<0.05；與神經(jīng)病理性痛組比較bP<0.05化。本研究結(jié)果顯示，與對(duì)照組比較，神經(jīng)病理性痛組縮爪潛伏期明顯延長(zhǎng)，差異有統(tǒng)計(jì)學(xué)意義；與神經(jīng)病理性痛組相比，米諾環(huán)素組和乳鐵蛋白組縮爪潛伏期延長(zhǎng)，而米諾環(huán)素組與乳鐵蛋白組爪潛伏期延長(zhǎng)比較差異無(wú)統(tǒng)計(jì)學(xué)意義，可間接證明乳鐵蛋白可通過(guò)抑制脊髓小膠質(zhì)細(xì)胞活化減輕大鼠神經(jīng)病理性痛。與對(duì)照組比較，神經(jīng)病理性痛組、米諾環(huán)素組和乳鐵蛋白組脊髓背角Iba-1表達(dá)明顯減少，差異有統(tǒng)計(jì)學(xué)意義；與神經(jīng)病理性痛組相比，米諾環(huán)素組和乳鐵蛋白組脊髓背角Iba-1表達(dá)也明顯減少，差異有統(tǒng)計(jì)學(xué)意義，而米諾環(huán)素組與乳鐵蛋白組縮爪潛伏期比較，差異無(wú)統(tǒng)計(jì)學(xué)意義，提示乳鐵蛋白可通過(guò)抑制脊髓小膠質(zhì)細(xì)胞活化減輕大鼠神經(jīng)病理性痛。

綜上所述，乳鐵蛋白可通過(guò)抑制NO的合成，減少炎性介質(zhì)，進(jìn)一步減少脊髓背角小膠質(zhì)細(xì)胞的活化水平，減輕大鼠神經(jīng)病理性痛，可對(duì)臨床麻醉鎮(zhèn)痛有一定的借鑒意義。

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(本文編輯：王映紅)

2.2 各組大鼠脊髓背角Iba-1表達(dá)水平比較 與對(duì)照組比較，神經(jīng)病理性痛組、米諾環(huán)素組和乳鐵蛋白組脊髓背角Iba-1表達(dá)明顯減少，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；與神經(jīng)病理性痛組比較，米諾環(huán)素組和乳鐵蛋白組脊髓背角Iba-1表達(dá)也明顯減少，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。而米諾環(huán)素組與乳鐵蛋白組Iba-1得表達(dá)量比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。見(jiàn)圖1、表2。

注：A為對(duì)照組，B為神經(jīng)病理性痛組，C為米諾環(huán)素組，D為乳鐵蛋白組圖1 各組大鼠脊髓背角Iba-1表達(dá)的熒光圖(熒光染色×100)

組別大鼠數(shù)OD值假手術(shù)組80.41±0.08神經(jīng)病理性痛組80.97±0.12a米諾環(huán)素組80.49±0.11ab乳鐵蛋白組80.51±0.13ab

注：與假手術(shù)組比較aP<0.05；與神經(jīng)病理性痛組比較bP<0.05

3 討論

乳鐵蛋白的鎮(zhèn)痛效果與神經(jīng)病理性痛大鼠脊髓背角環(huán)GMP依賴(lài)性PKG信號(hào)傳導(dǎo)導(dǎo)通路有關(guān)，但確切作用機(jī)制有待于進(jìn)一步研究。NO廣泛存在于神經(jīng)元及神經(jīng)小膠質(zhì)細(xì)胞中，且與脊髓背角小膠質(zhì)細(xì)胞活化密切相關(guān)。本實(shí)驗(yàn)參照文獻(xiàn)[5]，采用結(jié)扎坐骨神經(jīng)的方法制備大鼠神經(jīng)病理性痛模型已經(jīng)廣泛應(yīng)用于疼痛學(xué)實(shí)驗(yàn)，此模型作用效果與人類(lèi)周?chē)窠?jīng)損傷誘發(fā)的神經(jīng)病理性疼痛的臨床癥狀和行為學(xué)相似，因此，在實(shí)驗(yàn)中該方法制備大鼠神經(jīng)病理性痛模型具有較好的可行性。同時(shí)本研究結(jié)果顯示，與對(duì)照組比較，神經(jīng)病理性痛組結(jié)扎坐骨神經(jīng)后PWL明顯縮短，提示大鼠神經(jīng)病理性痛模型制備成功。

神經(jīng)元由神經(jīng)膠質(zhì)細(xì)胞對(duì)提供營(yíng)養(yǎng)作用和支撐其結(jié)構(gòu)，傷害性刺激信號(hào)產(chǎn)生后，中樞神經(jīng)系統(tǒng)產(chǎn)生部分免疫因子參與多信號(hào)的傳遞，免疫因子的生成和釋放導(dǎo)致神經(jīng)病理性痛的形成，如脊髓背角小膠質(zhì)細(xì)胞的活化可引起炎癥，可進(jìn)一步對(duì)神經(jīng)病理性痛進(jìn)行調(diào)節(jié)，乳鐵蛋白與神經(jīng)病理性痛也密切相關(guān)[5-6]。多項(xiàng)研究[2-3，7-8]表明，NO合酶缺失的大鼠神經(jīng)損傷后脊髓背角小膠質(zhì)細(xì)胞活化明顯減少，神經(jīng)病理性痛大鼠臨床癥狀也相應(yīng)減輕，從而抑制NO的合成，進(jìn)一步減少脊髓背角小膠質(zhì)細(xì)胞的活化，從而減輕神經(jīng)病理性痛。而乳鐵蛋白在神經(jīng)病理性痛大鼠的鎮(zhèn)痛作用機(jī)制與脊髓背角NO-環(huán)GMP依賴(lài)性PKG信號(hào)傳導(dǎo)通路密切相關(guān)，因此，可以推測(cè)，在神經(jīng)病理性痛大鼠的鎮(zhèn)痛作用中，乳鐵蛋白通過(guò)與NO相關(guān)信號(hào)傳導(dǎo)通路抑制脊髓背角小膠質(zhì)細(xì)胞活

Effects of lactoferrin on microglial activation in the spinal cord in the rat model of neuropathic pain

Wang Jun, Xue Hong, Zong Chuanyue, Zong Yi

(DepartmentofAnesthesiology,SecondPeople′sHospitalofHuaianCity,Huaian223002,China)

Objective To investigate the effects of lactoferrin on microglial activation in the spinal cord in the rat model of neuropathic pain (NP).MethodsThirty-two male SD rats with the weight of 200-220 g were randomly divided into 4 groups, i.e. the control group, the neuropathic pain group, the minocycline group and the lactoferrin group, each consisting of 8 animals. The sciatic nerve was only exposed but not ligated in the animals of the control group, and 8 μl of normal saline were injected intrathecally. The neuropathic pain model was produced by placing loosely constrictive ligatures around the common sciatic nerve in the animals of the other groups. Eight μl of normal saline were injected intrathecally in the animals of the neuropathic pain group. One hundred μl of lactoferrin were given intrathecally in the animals of the lactoferrin group, while the same amount of minocycline was also given intrathecally in the animals of the minocycline group. Following medication, the paw withdrawal latency (PWL) was detected every 30 minutes by using thermal nociceptive stimulus with a total of 180 minutes. After detections for 7 times, the rats were sacrificed for the collection of spinal dorsal horn. The expression of Iba-1 was determined by immunofluorescence, and then statistical analyses were made accordingly.ResultsPWL for the animals of the neuropathic pain group was obviously prolonged, as compared with that of the control group, and statistical significance could be seen, when comparisons were made between them(P<0.05). PWL for the animals of both the minocycline group and the lactoferrin group was prolonged, as compared with that of the neuropathic pain group, also with statistical significance(P<0.05). No statistical significance could be noted in PWL for the animals of the minocycline group and the lactoferrin group, when comparisons were made between them(P>0.05). This was an indirect indication that lactoferrin could alleviate neuropathic pain through the inhibition of the activation of spinal microglia. The expression levels of Iba-1 in the animals of the minocycline group and the lactoferrin group, as well as the neuropathic pain group, were significantly decreased, and statistical significance could be seen, when it was compared with that of the control group(P<0.05). The expression levels of Iba-1 in the animals of the minocycline group and the lactoferrin group were also significantly decreased, as compared with that of the neuropathic pain group, also with statistical significance(P<0.05). No statistical significance was shown in PWL, when comparisons were made between the minocycline group and the lactoferrin group(P>0.05). This was also an indirect indication that lactoferrin could alleviate neuropathic pain through the inhibition of the activation of spinal microglia.ConclusionLactoferrin could alleviate neuropathic pain through the inhibition of the activation of spinal microglia, which had certain medical value in the clinical practice of anesthesia.

Lactoferrin; Neuropathic pain; Microglia

223002 江蘇 淮安，淮安市第二人民醫(yī)院麻醉科

R614

A

10.3969/j.issn.1009-0754.2016.06.007

2016-03-22)