慢性坐骨神經(jīng)損傷大鼠中腦導(dǎo)水管周圍灰質(zhì)MC4R的表達(dá)及HS014對(duì)熱痛覺過(guò)敏的影響

孫俊枝，解雅英，褚海辰，高 麗

(1. 內(nèi)蒙古醫(yī)科大學(xué)附屬醫(yī)院,內(nèi)蒙古 呼和浩特 010050;2. 青島大學(xué)醫(yī)學(xué)院附屬醫(yī)院，山東 青島 370200)

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慢性坐骨神經(jīng)損傷大鼠中腦導(dǎo)水管周圍灰質(zhì)MC4R的表達(dá)及HS014對(duì)熱痛覺過(guò)敏的影響

孫俊枝1，解雅英1，褚海辰2，高 麗1

(1. 內(nèi)蒙古醫(yī)科大學(xué)附屬醫(yī)院,內(nèi)蒙古 呼和浩特 010050;2. 青島大學(xué)醫(yī)學(xué)院附屬醫(yī)院，山東 青島 370200)

目的 觀察大鼠慢性坐骨神經(jīng)損傷(CCI)后不同時(shí)間點(diǎn)中腦導(dǎo)水管周圍灰質(zhì)(PAG)4型黑皮質(zhì)素受體(MC4R)mRNA表達(dá)變化及給予選擇性MC4R拮抗劑HS014對(duì)CCI大鼠疼痛行為學(xué)的影響。方法 ①將42只健康成年雄性Wistar大鼠隨機(jī)分為對(duì)照組6只、假手術(shù)組18只和CCI組18只，檢測(cè)各組大鼠術(shù)前和術(shù)后3，7，14 d熱縮足潛伏期(TWL)，假手術(shù)組和CCI組分別于術(shù)后3，7，14 d，對(duì)照組于術(shù)后14 d TWL檢測(cè)完成后處死大鼠，取PAG腦組織，用半定量RT-PCR法檢測(cè)PAG腦區(qū)MC4R mRNA表達(dá)情況。②將30只健康成年雄性Wistar大鼠隨機(jī)分為對(duì)照組6只、假手術(shù)組6只和CCI組18只，均先進(jìn)行PAG置管。置管后7 d，CCI組隨機(jī)分成CCI+NS組、CCI+HS014 5 μg組和CCI+HS014 10 μg組，每組6只。假手術(shù)組和CCI各組手術(shù)造模。術(shù)后7 d，CCI+NS組經(jīng)導(dǎo)管注射生理鹽水0.5 μL，CCI+HS014 5 μg組注射HS014 5 μg/0.5 μL，CCI+HS014 10 μg組注射HS014 10 μg/0.5 μL。檢測(cè)各組大鼠置管后7 d、術(shù)后7 d和干預(yù)后(注射后10～30 min完成檢測(cè))TWL。結(jié)果 CCI組大鼠術(shù)后3 d開始出現(xiàn)TWL縮短(P<0.05)，術(shù)后7 d和14 d TWL縮短更加明顯(P均<0.05)，與對(duì)照組和假手術(shù)組比較差異均有統(tǒng)計(jì)學(xué)意義(P均<0.05)。CCI組術(shù)后各時(shí)間點(diǎn)MC4RmRNA表達(dá)量均明顯高于對(duì)照組和假手術(shù)組(P均<0.05)，并且隨著CCI術(shù)后時(shí)間的推移，MC4RmRNA的表達(dá)量逐漸增高(P均<0.05)。CCI各組大鼠術(shù)后7 d TWL明顯低于置管后7 d(P均<0.05)；CCI+NS組干預(yù)后TWL與術(shù)后7 d比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05),而CCI+HS014 5 μg組和CCI+HS014 10 μg組干預(yù)后TWL均明顯長(zhǎng)于術(shù)后7 d和CCI+NS組(P均<0.05)，CCI+HS014 10 μg組干預(yù)后TWL明顯長(zhǎng)于CCI+HS014 5 μg組(P<0.05)。結(jié)論 CCI可引起PAG中MC4RmRNA的表達(dá)增加，其選擇性拮抗劑HS014可劑量依賴性減輕CCI大鼠的熱痛覺過(guò)敏，提示PAG中MC4R的表達(dá)增加可能參與神經(jīng)病理性疼痛的形成和維持。

慢性坐骨神經(jīng)損傷;中腦導(dǎo)水管周圍灰質(zhì);4型黑皮質(zhì)素受體;HS014

中腦導(dǎo)水管周圍灰質(zhì)(midbrain periaqueductal gray,PAG)是機(jī)體內(nèi)源性痛覺調(diào)制系統(tǒng)中起核心作用的重要結(jié)構(gòu)，能通過(guò)一些下行通路調(diào)節(jié)脊髓的活動(dòng)[1]。黑皮質(zhì)素(melanocortin，MC)肽類參與機(jī)體多種認(rèn)知和行為的調(diào)節(jié)，包括抗炎、免疫、飲食、學(xué)習(xí)、記憶以及疼痛等，上述作用都由分布在不同組織、不同細(xì)胞上的黑皮質(zhì)素受體(melanocortin receptors,MCRs)介導(dǎo)。黑皮質(zhì)素有多種受體，其中脊髓MC4R與疼痛關(guān)系密切，但對(duì)于脊髓以上中樞，MC4R在疼痛調(diào)節(jié)中的作用研究甚少。本研究探討了慢性坐骨神經(jīng)損傷(CCI)大鼠PAG中MC4R表達(dá)量變化以及在神經(jīng)病理性疼痛調(diào)制中的作用。

1 實(shí)驗(yàn)資料

1.1 主要試劑和儀器 RNA提取試劑盒(上海飛捷生物技術(shù)有限公司)；RT試劑盒(Fermentas)；Mix Maker；聚合酶鏈反應(yīng)(polymerase chain reaction，F(xiàn)CR)(東盛生物)、RT-PCR引物及內(nèi)參由上海生物工程公司設(shè)計(jì)并合成；HS014(sigma)編號(hào)為043-25；熱板測(cè)痛儀(寧海白石電子醫(yī)藥儀器廠)；江灣立體定位儀(淮北正華生物儀器設(shè)備有限公司)。

1.2 實(shí)驗(yàn)動(dòng)物 健康成年雄性Wistar大鼠72只，體質(zhì)量220～260 g，動(dòng)物合格證號(hào)：SCXK(魯)2009-0010。

1.3 實(shí)驗(yàn)方法

1.3.1 實(shí)驗(yàn)一 取42只大鼠，隨機(jī)分為對(duì)照組6只、假手術(shù)組18只和CCI組18只。CCI組按Bennett等[2]方法造模：腹腔注射8%水合氯醛300 mg/kg麻醉后常規(guī)消毒右下肢, 切開皮膚及皮下組織, 鈍性分離肌肉, 于股骨后找到坐骨神經(jīng)主干, 在顯微鏡下用4/0的鉻制羊腸線松扎4道, 間距為1 mm,結(jié)扎強(qiáng)度以引起小腿肌肉輕度顫動(dòng)反應(yīng)為宜,然后逐層縫合；術(shù)后每日肌肉注射青霉素2次,每次8萬(wàn)IU。假手術(shù)組大鼠僅暴露坐骨神經(jīng)而不進(jìn)行結(jié)扎，其余操作同實(shí)驗(yàn)組。對(duì)照組大鼠不做任何處理。各組大鼠均置于安靜、溫暖、避強(qiáng)光環(huán)境中喂養(yǎng)。在術(shù)后14 d內(nèi)，每隔1～2 d觀察1次大鼠的行為學(xué)表現(xiàn)，包括大鼠的步態(tài)和左后肢的姿勢(shì)、局部皮膚以及肌肉張力的改變程度，以及是否存在舔咬肢體現(xiàn)象等。檢測(cè)各組大鼠術(shù)前和術(shù)后3，7，14 d熱縮足潛伏期(thermal withdraw latency，TWL),假手術(shù)組和CCI組分別于術(shù)后3，7，14 d，對(duì)照組于術(shù)后14 d TWL檢測(cè)完成后處死大鼠，取PAG腦組織檢測(cè)PAG腦區(qū)MC4R mRNA表達(dá)情況。

1.3.2 實(shí)驗(yàn)二 取剩余30只大鼠，隨機(jī)分為對(duì)照組6只、假手術(shù)組6只和CCI組18只。均先進(jìn)行PAG置管：大鼠在8%水合氯醛300 mg/kg麻醉下， 固定于立體定位儀。 以H2O2溶液腐蝕皮下組織，暴露顱骨， PAG(AP6.5，L 0.5，H 6.0 mm) 埋植不銹鋼套管(長(zhǎng)11 mm，外徑0.6 mm)，套管與矢狀面成10°角，尖端高于注射部位2.0 mm，用牙托粉固定于顱骨，套管內(nèi)插入等長(zhǎng)不銹鋼管芯，以保證套管通暢。置管后7d，CCI組隨機(jī)分成CCI+NS組、CCI+HS014 5 μg組和CCI+HS014 10 μg組，每組6只。假手術(shù)組和CCI各組按“實(shí)驗(yàn)一”方法造模。術(shù)后7 d，CCI+NS組經(jīng)導(dǎo)管注射生理鹽水0.5 μL，CCI+HS014 5 μg組注射HS014 5 μg/0.5 μL，CCI+HS014 10 μg組注射HS014 10 μg/0.5 μL，對(duì)照組和假手術(shù)組不做任何干預(yù)。檢測(cè)各組大鼠置管后7 d、術(shù)后7 d和干預(yù)后(注射后10～30 min完成檢測(cè))TWL。

1.4 檢測(cè)方法

1.4.1 熱刺激痛反應(yīng)閾值的測(cè)定 室溫保持在25～30 ℃，將待測(cè)大鼠放入觀測(cè)盒內(nèi)的同時(shí)開始計(jì)時(shí)(及時(shí)清除大鼠排出的糞便，保持玻璃平板的清潔與干燥)，記錄開始測(cè)試到引起大鼠舔足的時(shí)間(潛伏期)。以30 s為最大閾值，如超過(guò)30 s則停止測(cè)試，以防止局部燙傷，每一足底測(cè)定3次，時(shí)間間隔為10 min，取平均值代表TWL。

1.4.2 PAG腦區(qū)MC4R mRNA表達(dá)檢測(cè)方法 采用RT-PCR法檢測(cè)。大鼠處死后，取PAG腦組織-70 ℃冰箱保存待用。按RNA提取試劑盒說(shuō)明操作提取組織總RNA，用分光光度計(jì)測(cè)定RNA濃度，每份標(biāo)本取等量RNA行逆轉(zhuǎn)錄，按試劑盒說(shuō)明書操作合成cDNA及PCR擴(kuò)增。MC4R引物序列(399 bp)：sense 5’-CTTGCACAGTATCGGGCGTTCT-3’, antisense 5’-GTTCTTGACTCCGCAGGGCATA-3’; 反應(yīng)條件：94 ℃ 充分變性5 min；94 ℃變性30 s，50 ℃退火30 s，70 ℃延伸30 s，39個(gè)循環(huán)；70 ℃充分延伸10 min。取5 μL擴(kuò)增產(chǎn)物于瓊脂糖凝膠電泳，PCR產(chǎn)物鑒定及半定量分析照相于凝膠圖像分析系統(tǒng)進(jìn)行掃描分析，計(jì)算檢測(cè)指標(biāo)的mRNA與β-actin mRNA比值。

2 結(jié) 果

2.1 CCI組大鼠行為學(xué)變化 術(shù)后1 d開始，CCI組大鼠手術(shù)側(cè)足趾并攏輕度外翻，下肢行走無(wú)力，步態(tài)呈現(xiàn)跛行，經(jīng)常左后足懸空或不敢著地；站立時(shí)以右后肢持重，左后肢抬起并緊貼于腹部，左后肢懸空時(shí)間常超過(guò)25 s，但未出現(xiàn)自噬肢體現(xiàn)象。

2.2 3組大鼠不同時(shí)間點(diǎn)TWL比較 3組大鼠術(shù)前TWL比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P均>0.05)；假手術(shù)組大鼠術(shù)后各時(shí)間點(diǎn)TWL比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P均>0.05)；CCI組大鼠術(shù)后3 d開始出現(xiàn)TWL縮短(P<0.05)，術(shù)后7 d和14 d TWL縮短更加明顯(P均<0.05)，與對(duì)照組和假手術(shù)組比較差異均有統(tǒng)計(jì)學(xué)意義(P均<0.05)。見表1。

表1 3組大鼠不同時(shí)間點(diǎn)TWL比較

注：①與CCI組比較，P<0.05;②與術(shù)前1 d比較，P<0.05。

2.3 3組大鼠術(shù)后不同時(shí)間點(diǎn)PAG中MC4RmRNA表達(dá)情況 CCI組術(shù)后各時(shí)間點(diǎn)MC4RmRNA表達(dá)量均明顯高于對(duì)照組和假手術(shù)組(P均<0.05)，并且隨著CCI術(shù)后時(shí)間的推移，MC4RmRNA的表達(dá)量逐漸增高(P均<0.05)。見表2及圖1。

表2 3組大鼠術(shù)后不同時(shí)間點(diǎn)PAG中MC4RmRNA表達(dá)情況

注：①與CCI組比較，P<0.05;②與術(shù)后3 d比較，P<0.05。

2.4 各組大鼠置管后7 d、術(shù)后7 d和干預(yù)后TWL比較 各組大鼠置管后7 d TWL比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P均>0.05)；CCI各組大鼠術(shù)后7 d TWL明顯低于置管后7 d(P均<0.05)，對(duì)照組和假手術(shù)組TWL沒有明顯變化(P>0.05)；CCI+NS組干預(yù)后TWL與術(shù)后7 d比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05),而CCI+HS014 5 μg組和CCI+HS014 10 μg組干預(yù)后TWL均明顯長(zhǎng)于術(shù)后7 d和CCI+NS組(P均<0.05)，CCI+HS014 10 μg組干預(yù)后TWL明顯長(zhǎng)于CCI+HS014 5 μg組(P<0.05)。見表3。

1為對(duì)照組；2為假手術(shù)組術(shù)后3 d；3為CCI組術(shù)后3 d；4為假手術(shù)組術(shù)后7 d；5為CCI組術(shù)后7 d；6為假手術(shù)組術(shù)后14 d；7為CCI組術(shù)后14 d

組別置管后7d術(shù)后7d干預(yù)后對(duì)照組16.50±2.7417.17±1.7215.67±1.51假手術(shù)組16.83±3.4317.83±1.4716.67±1.86CCI+NS組17.17±3.194.67±1.63①4.17±1.17CCI+HS0145μg組17.67±1.634.17±1.72①8.83±1.47②③CCI+HS01410μg組18.83±2.045.00±1.41①11.17±2.56②③④

注：①與置管后7 d比較，P<0.05； ②與術(shù)后7 d比較，P<0.05；③與CCI+NS組比較，P<0.05；④與CCI+HS014 5 μg組比較，P<0.05。

3 討 論

本研究中大鼠CCI后出現(xiàn)跛行，足呈輕度外翻狀，且出現(xiàn)如舔足、后足懸空等后肢保護(hù)現(xiàn)象，熱縮足潛伏期明顯下降，與Bennett等[2]報(bào)道一致，說(shuō)明CCI所致的神經(jīng)病理性疼痛模型制備成功。

MC4R是G-蛋白耦聯(lián)受體，其分布于脊髓與傷害性信息傳遞關(guān)系密切的Ⅰ層、Ⅱ?qū)雍廷鷮右约氨掣窠?jīng)節(jié)[3]，在脊髓以上結(jié)構(gòu)包括大腦皮質(zhì)、丘腦、下丘腦、海馬和PAG中也發(fā)現(xiàn)有MC4R的表達(dá)[4]，由于以上分布部位均與疼痛信號(hào)的傳遞與調(diào)制有關(guān)，提示MC4R在脊髓及脊髓以上結(jié)構(gòu)都可能參與對(duì)疼痛的調(diào)節(jié)。近年來(lái)，已有很多研究證明，脊髓MC4R在神經(jīng)病理性疼痛的發(fā)生和發(fā)展過(guò)程中起了重要作用[5]。另外，MC4R也可以與其他經(jīng)典的疼痛調(diào)節(jié)神經(jīng)遞質(zhì)產(chǎn)生協(xié)同作用，共同參與對(duì)疼痛信息的調(diào)節(jié)。研究發(fā)現(xiàn)，MC4R參與阿片鎮(zhèn)痛機(jī)制，脊髓MC4R拮抗劑可以增強(qiáng)嗎啡在神經(jīng)病理性疼痛中的鎮(zhèn)痛作用[6]，顯著延緩嗎啡耐受的進(jìn)程[7]，且其作用機(jī)制可能是通過(guò)抑制神經(jīng)膠質(zhì)細(xì)胞激活和神經(jīng)炎癥反應(yīng)來(lái)實(shí)現(xiàn)的[8-9]。Beltramo等[10]研究表明，神經(jīng)病理性疼痛大鼠脊髓MC4R表達(dá)上調(diào)，且其與觸覺過(guò)敏和熱覺過(guò)敏的出現(xiàn)相平行，而不同選擇性MC4R拮抗劑SHU9119和JKC363具有抗痛覺過(guò)敏作用，而其激動(dòng)劑MTⅡ則可增強(qiáng)痛覺過(guò)敏[11-12]，提示MC4R的激活可能是導(dǎo)致神經(jīng)病理性疼痛的原因之一，但這些研究?jī)H限于脊髓水平，對(duì)神經(jīng)病理性疼痛過(guò)程中脊髓以上腦區(qū)MC4R表達(dá)量的研究甚少。本研究則表明神經(jīng)病理性疼痛癥狀與脊髓以上腦區(qū)PAG的MC4R表達(dá)量相關(guān)。

PAG是機(jī)體內(nèi)源性疼痛調(diào)制的重要結(jié)構(gòu)，在PAG中，同時(shí)存在下行疼痛抑制系統(tǒng)[13]和下行疼痛易化系統(tǒng)[14]，那么神經(jīng)病理性疼痛大鼠PAG中MC4R mRNA表達(dá)增加在疼痛調(diào)節(jié)中究竟是促進(jìn)還是抑制作用，還不明確。通過(guò)研究部位注射MC4R選擇性拮抗劑或激動(dòng)劑，進(jìn)而明確MC4R作用是研究中常用的方法。HS014是MC4R選擇性拮抗劑，在損傷神經(jīng)周圍、鞘內(nèi)給予HS014均可產(chǎn)生抗痛覺過(guò)敏作用[15]，提示MC4R在外周和脊髓可產(chǎn)生致痛作用。本研究結(jié)果發(fā)現(xiàn)，神經(jīng)病理性疼痛大鼠PAG給予HS014注射后可提高熱痛閾，產(chǎn)生鎮(zhèn)痛作用，提示PAG中MC4R 功能活化可以對(duì)神經(jīng)病理性疼痛大鼠產(chǎn)生致痛作用。

綜上所述，CCI可引起PAG中MC4RmRNA的表達(dá)增加，其選擇性拮抗劑HS014可劑量依賴性減輕CCI大鼠的熱痛覺過(guò)敏，提示PAG中MC4R的表達(dá)增加可能參與神經(jīng)病理性疼痛的形成和維持，但MC4R表達(dá)增加的具體部位(是神經(jīng)元還是膠質(zhì)細(xì)胞)以及MC4R活化后引起哪些神經(jīng)遞質(zhì)的釋放變化等還需要進(jìn)一步研究。

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Change of MC4R expression in periaqueductal gray in rats with CCI and effect of selective antagonist HS014 on their thermal hyperalgesia

SUN Junzhi1, XIE Yaying1, CHU Haichen2, GAO Li1

(1.The Affiliated Hospital of Inner Mongolia Medicial University,Hohhot 010050, Inner Mongolia, China;2.The Affiliated Hospital of Qingdao Medical College, Qingdao 370200,Shandong,China)

Objective It is to investigate the change of melanocortin 4 receptor (MC4R) expression in the periaqueductal gray (PAG) in rats with chronic sciatic nerve compression injury(CCI)and the effect of HS014 (a selective antagonist of MC4R) on the thermal hyperalgesia induced by CCI. Methods 42 healthy male Wistar rats were randomly divided into 3 groups, including the control group (n=6), sham operation group(n=18)and CCI group (n=18). Thermal withdraw latency (TWL) were measured before operation and 3,7,14 d after operation. Rats in the CCI group and sham operation group were killed on postoperative 3,7,14 d after measuring TWL and rats in the control group were killed on postoperative 14 d after measuring TWL, the PAG was taken for detecting the expreesion of MC4R by using RT-PCR. Another 30 rats were randomly divided into control group (n=6), sham operation group(n=6)and CCI group (n=18), rats in the three groups were all received PAG cannulation before surgery. 7 days after PAG cannulation, rats in CCI group were randomly divided into CCI+NS group,CCI+HS014 5 μg group and CCI+HS014 10 μg group (n=6 each),then the sham operation group and CCI group received operation.14 days after operation, CCI+NS group received PAG injection of 0.5 μL normal saline, CCI+HS014 5 μg group received PAG injection of HS014 5 μg/0.5 μL and CCI+HS014 10 μg received PAG injection of HS014 10 μg/0.5 μL. TWL were measured at 7 days after PAG cannulation,14 days after operation and 10～30 min after drug administration. Results The behavior tests showed that TWL started to decrease at 3 days after CCI(P<0.05), and obviously decreased at 7 and 14 days after surgery(P<0.05). It was statistically significant comparing with the corresponding time point of sham group and control group (allP<0.05). RT-PCR analysis revealed that the mRNA expression of MC4R in PAG was significantly higher at each time point in CCI group than that in the sham group and control group (allP<0.05), and the MC4RmRNA expression was gradually increased with time in the CCI group (allP<0.05). TWL was significantly decreased in the whole CCI group after operation compared with 7 days after PAG cannulation(allP<0.05); There was no significant difference in TWL before and after NS treatment in the CCI group (P>0.05). Compared with CCI+NS group and 7 d after PAG cannulation group,TWL was significantly prolonged after PAG injection in the CCI+HS014 5 μg group and CCI+HS014 10 μg group (allP<0.05) and TWL in the CCI+HS014 10 μg group was more longer than that in the CCI+HS014 5 μg group after PAG injection (P<0.05). Conclusion The present study suggests that damage to peripheral neurons can cause the increased expression of MC4R mRNA in PAG and the selective MC4R inhibitor HS014 can dose dependently reduce the thermal hyperalgesia induced by CCI, prompting that the increased expression of MC4R in PAG may be involved in the development and maintenance of neuropathic pain induced by the peripheral nervel injury.

CCI; PAG; MC4R; HS014

孫俊枝，女，主治醫(yī)師，碩士，主要從事臨床麻醉和神經(jīng)病理性疼痛方面的研究。

解雅英，E-mail：190269233@qq.com

10.3969/j.issn.1008-8849.2016.35.006

R-332

A

1008-8849(2016)35-3892-04

2016-05-31