脊髓脂質(zhì)運(yùn)載蛋白-2對大鼠嗎啡耐受形成的影響

王 芬，劉清珍，劉 健，江姿潞，謝 滔，李偉彥

(1.徐州醫(yī)學(xué)院江蘇省麻醉學(xué)重點(diǎn)實(shí)驗(yàn)室，2.徐州醫(yī)學(xué)院江蘇省麻醉與鎮(zhèn)痛應(yīng)用技術(shù)重點(diǎn)實(shí)驗(yàn)室，江蘇 徐州 221004；3.南京軍區(qū)南京總醫(yī)院麻醉科，江蘇 南京 210002；4.第二軍醫(yī)大學(xué)研究生院，上海 200433)

脊髓脂質(zhì)運(yùn)載蛋白-2對大鼠嗎啡耐受形成的影響

王 芬1,2，劉清珍3，劉 健3，江姿潞1,2，謝 滔4，李偉彥3

(1.徐州醫(yī)學(xué)院江蘇省麻醉學(xué)重點(diǎn)實(shí)驗(yàn)室，2.徐州醫(yī)學(xué)院江蘇省麻醉與鎮(zhèn)痛應(yīng)用技術(shù)重點(diǎn)實(shí)驗(yàn)室，江蘇 徐州 221004；3.南京軍區(qū)南京總醫(yī)院麻醉科，江蘇 南京 210002；4.第二軍醫(yī)大學(xué)研究生院，上海 200433)

目的 探索用RNA干擾(RNAi)法沉默脊髓脂質(zhì)運(yùn)載蛋白-2(lipocalin-2, LCN2)的表達(dá)及其對正常大鼠嗎啡耐受形成的影響。方法 鞘內(nèi)置管成功的♂SD大鼠48只，體質(zhì)量180～220 g，隨機(jī)均分為4組，每組12只：Ⅰ組對照組、Ⅱ組嗎啡耐受組、Ⅲ組錯義小干擾RNA(mismatch siRNA, MM siRNA)組、Ⅳ組LCN2小干擾RNA(LCN2 siRNA)組。所有大鼠鞘內(nèi)置管術(shù)后d 6確定導(dǎo)管位置，記為d 0。d 2～8連續(xù)7 d，每天2次進(jìn)行皮下注射，每次注射劑量10 μg·g-1，Ⅰ組大鼠皮下注射生理鹽水(normal saline, NS)，Ⅱ-Ⅳ組大鼠皮下注射嗎啡建立嗎啡耐受模型。各組大鼠每天皮下給藥前分別鞘內(nèi)注射10 μL DEPC溶液、10 μL DEPC溶液、10 μL含4 μg MM siRNA的DEPC溶液和10 μL含4 μg LCN2 siRNA的DEPC溶液。d 1、d 9，測定所有大鼠的基礎(chǔ)熱縮足反應(yīng)潛伏期(paw withdrawal thermal latency, PWTL)及皮下注射嗎啡后45 min的PWTL，并計(jì)算嗎啡的最大可能鎮(zhèn)痛效應(yīng)百分比值(% maximal possible effect, %MPE)。d 9行為學(xué)測試結(jié)束后，取脊髓腰膨大，用Western blot法檢測磷酸化p38 絲裂原活化蛋白激酶(p-p38 MAPK)及LCN2蛋白表達(dá)水平，用免疫組織熒光染色法檢測小膠質(zhì)細(xì)胞標(biāo)記物Iba1的表達(dá)。結(jié)果 d 9，與Ⅰ組比，Ⅱ、Ⅲ組大鼠%MPE值明顯下降(P<0.05)，腰段脊髓LCN2、p-p38 MAPK、Iba1表達(dá)明顯增加(P<0.05)。與Ⅱ組比，Ⅳ組大鼠的%MPE值明顯增加(P<0.05)，脊髓LCN2、p-p38MAPK、Iba1表達(dá)明顯下降(P<0.05)。結(jié)論 鞘內(nèi)注射LCN2 siRNA沉默脊髓LCN2的表達(dá)能夠部分抑制嗎啡耐受的形成，該現(xiàn)象可能與其抑制脊髓背角小膠質(zhì)細(xì)胞的活化及脊髓p-p38 MAPK的表達(dá)有關(guān)。

嗎啡耐受；脂質(zhì)運(yùn)載蛋白-2；小干擾RNA；小膠質(zhì)細(xì)胞；磷酸化p38 絲裂原活化蛋白激酶；脊髓

嗎啡(morphine)耐受是指長期反復(fù)使用嗎啡后，其鎮(zhèn)痛作用逐漸減退甚至消失，需加大劑量才能產(chǎn)生既定藥理效應(yīng)的現(xiàn)象。大量研究顯示，膠質(zhì)細(xì)胞活化是嗎啡耐受的重要機(jī)制，其中小膠質(zhì)細(xì)胞活化主要參與了嗎啡耐受的形成，星型膠質(zhì)細(xì)胞活化則參與了嗎啡耐受的維持[1-2]。脂質(zhì)運(yùn)載蛋白-2(lipocalin-2, LCN2)，也被稱作24p3或中性粒細(xì)胞明膠酶相關(guān)脂質(zhì)運(yùn)載蛋白(neutrophil gelatinase-associated lipocalin, NGAL)，是一個分子質(zhì)量為25 ku的糖蛋白，最初在中性粒細(xì)胞顆粒中發(fā)現(xiàn)。研究發(fā)現(xiàn)，LCN2能夠誘導(dǎo)小膠質(zhì)細(xì)胞和p38 MAPK活化，進(jìn)而誘導(dǎo)炎癥因子釋放而導(dǎo)致神經(jīng)病理性疼痛[3]，而小膠質(zhì)細(xì)胞、p38 MPAK通路均參與了嗎啡耐受的形成，其中p-p38MAPK主要表達(dá)于小膠質(zhì)細(xì)胞[4,5]，因此，我們推測LCN2亦可能參與了嗎啡耐受的發(fā)生。本實(shí)驗(yàn)擬用鞘內(nèi)注射LCN2 siRNA選擇性抑制LCN2表達(dá)的方法，探討LCN2對大鼠嗎啡耐受形成的影響，并探索相關(guān)機(jī)制，為防治嗎啡耐受提供新的依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)動物與分組清潔級♂SD大鼠，體質(zhì)量180～220 g(南京軍區(qū)南京總醫(yī)院比較醫(yī)學(xué)科提供)。鹽酸嗎啡注射液(130704-2，東北制藥集團(tuán)沈陽第一制藥有限公司，中國)，DEPC溶液，LCN2 siRNA序列：sense: 5′-GCCUCAAGGAUAACAACAUTT-3′; antisense: 5′-AUGUUGUUAUCCUUGAGGCTT-3′; 錯義siRNA序列：sense: 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense: 5′- ACGUGACACGUUCGGAGAATT-3′(上海吉瑪制藥技術(shù)有限公司，中國)。所有大鼠鞘內(nèi)置管成功后，采用隨機(jī)數(shù)字表法將其隨機(jī)分為4組(n=12)：Ⅰ組，對照組(DEPC+NS組)；Ⅱ組，嗎啡耐受組(DEPC+morphine組);Ⅲ組,錯義 siRNA組(MM siRNA+morphine組);Ⅳ組,LCN2 siRNA組(LCN2 siRNA+morphine組)。

1.2 鞘內(nèi)置管參考Milligan等[6]的方法，大鼠腹腔注射質(zhì)量分?jǐn)?shù)為2 %的戊巴比妥鈉50 μg·g-1麻醉，以L5-6為中心作縱切口，分離椎旁肌肉，暴露椎間隙，以稍鈍針頭穿破黃韌帶及硬脊膜，可見大鼠出現(xiàn)甩尾反射及腦脊液外溢。將一段20 cm的PE-10導(dǎo)管(寧波市科技園區(qū)安來軟件科技有限公司，中國)經(jīng)椎間隙向頭端插入2 cm。經(jīng)皮下自頸部將其引出后縫合固定導(dǎo)管，熱熔封口。術(shù)后分籠飼養(yǎng)，自由飲水和攝食，恢復(fù)5 d。置管d 6經(jīng)導(dǎo)管注射質(zhì)量分?jǐn)?shù)為2%的利多卡因15 μL，如注射后30 s內(nèi)大鼠出現(xiàn)雙后肢麻痹現(xiàn)象說明導(dǎo)管位置正確，記為d 0。置管不成功大鼠予以麻醉處死。

1.3 給藥方案參照Raghavendra等[4]方法，d 2～8，連續(xù)7 d，每天8 ∶00和16 ∶00，Ⅱ～Ⅳ組大鼠皮下注射10 μg·g-1嗎啡建立嗎啡耐受模型，Ⅰ組大鼠皮下注射10 μg·g-1生理鹽水。各組大鼠每天皮下給藥前分別鞘內(nèi)注射10 μL DEPC溶劑(Ⅰ組、Ⅱ組)、4 μg MM siRNA(Ⅲ組)、4 μg LCN2 siRNA的DEPC溶液(Ⅳ組)，siRNA均用10 μL DEPC溶劑稀釋。

1.4 行為學(xué)測試d 1、d 9，參照Hargreaves等[7]方法，使用足底輻射熱痛覺測試儀(Model 390, IITC Life ScienceInc, 美國)，測定所有大鼠的基礎(chǔ)熱縮足反應(yīng)潛伏期(paw withdrawal thermal latency, PWTL)，皮下注射嗎啡后45 min的PWTL，每只大鼠重復(fù)測量5次，單次光照時間不超過20 s(cut-off time)，去掉最大及最小值，取平均值即為大鼠的PWTL，并計(jì)算嗎啡的最大可能鎮(zhèn)痛效應(yīng)百分比值(% maximal possible effect, MPE/%)，MPE/%=(給藥后PWTL-基礎(chǔ)PWTL) / (cut-off time-基礎(chǔ)PWTL)×100%。

1.5 標(biāo)本采集d 9行為學(xué)測試結(jié)束后，大鼠腹腔注射質(zhì)量分?jǐn)?shù)為2%的戊巴比妥鈉80 μg·g-1麻醉。取8只大鼠快速截取脊髓腰膨大于液氮中保存，留待Western blot法檢測蛋白表達(dá)。另取4只大鼠開胸灌注，留取脊髓腰膨大用于免疫熒光染色。

1.6 Western blot用含蛋白酶抑制劑、磷酸酶抑制劑和PMSF的RIPA裂解液將L4-6脊髓組織進(jìn)行勻漿。蛋白定量煮樣后，配置SDS-PAGE凝膠，每孔上樣量50 μg(10 μL)，電泳，轉(zhuǎn)膜，質(zhì)量分?jǐn)?shù)為5 %的脫脂牛奶室溫封閉1 h，加一抗：兔抗大鼠LCN2(1 ∶200，Millipore，美國)或兔抗大鼠p-p38 MAPK(1 ∶800, Cell Signaling Technology，美國),4 ℃孵育過夜。d 2，棄一抗，TBST洗5 min×6次，加辣根過氧化物酶標(biāo)記的羊抗兔二抗(1 ∶1 000，Cell Signaling Technology，美國)，室溫孵育1 h，TBST洗5 min × 6次，增強(qiáng)型化學(xué)發(fā)光試劑顯色曝光，顯影，定影。 內(nèi)參為GAPDH(1 ∶1 000，Cell Signaling Technology，美國)或β-actin(1 ∶1 000，Cell Signaling Technology，美國 )。掃描后蛋白條帶用Image J軟件分析，以目的條帶灰度值與內(nèi)參蛋白的比值反映目的蛋白表達(dá)的相對水平，進(jìn)行半定量分析。

1.7 免疫組織熒光染色取L4-6脊髓，置入質(zhì)量分?jǐn)?shù)為4 %的多聚甲醛溶液，4 ℃固定4～6 h。然后轉(zhuǎn)入質(zhì)量分?jǐn)?shù)為15 %的蔗糖溶液，待組織沉底后再轉(zhuǎn)入質(zhì)量分?jǐn)?shù)為30 %的蔗糖溶液，4 ℃放置48 h。OCT包埋組織后，放入-22 ℃冰凍切片機(jī)(CM1900, Leica，美國)，40 min后行冰凍切片，片厚35 μm。PBST洗5 min×3次，質(zhì)量分?jǐn)?shù)為5 %的驢血清封閉2 h，加山羊抗大鼠Iba1一抗(1 ∶100, Abcam，美國)，4 ℃孵育過夜，PBST洗3次，加入熒光標(biāo)記二抗(1 ∶1 000, Life Technologies，美國)室溫避光孵育2 h，PBST洗5 min×3次。避光貼片、甘油封片。普通熒光顯微鏡下觀察攝片。

2 結(jié)果

2.1 行為學(xué)測試d 1、d 9各組大鼠基礎(chǔ)PWTL(Basic PWTL)差異均無顯著性(Fig 1)。d 1，各組大鼠%MPE差異無顯著性，d 9，與Ⅰ組比較，Ⅱ、Ⅲ、Ⅳ組大鼠%MPE明顯降低(P<0.05)。與Ⅱ組比較，Ⅲ組大鼠的%MPE無明顯變化，而Ⅳ組大鼠的%MPE明顯升高(P<0.05)見Fig 2。

Fig 1 Basic PWTL of rats on d 1 and d 9(n=8)

On d 1 and d 9, there was no significant difference in the basic PWTL among four groups (P>0.05).

Fig 2 MPE of rats on d 1 and d 9(n=8)

On d 1, there was no significant difference in the %MPE among four groups (P>0.05), while on d 9, the MPE significantly declined in group Ⅱ-Ⅳ compared to group I (P<0.05) and the MPE significantly increased in group Ⅳ compared to group Ⅱ (P<0.05).*P<0.05vsgroup Ⅰ;#P<0.05vsgroup Ⅱ

2.2 脊髓腰膨大LCN2與p-p38 MAPK蛋白的表達(dá)變化與Ⅰ組相比，其余各組大鼠脊髓腰膨大的LCN2表達(dá)量均升高(P<0.05)。與Ⅱ組相比，Ⅲ組大鼠脊髓腰膨大的LCN2表達(dá)無明顯變化，而Ⅳ組大鼠脊髓腰膨大的LCN2表達(dá)明顯下降(P<0.05)(Fig 3)。與Ⅰ組相比，Ⅱ、Ⅲ組p-p38 MAPK表達(dá)量均升高(P<0.05)。與Ⅱ組相比，Ⅲ組p-p38 MAPK表達(dá)無明顯變化，而Ⅳ組p-p38 MAPK表達(dá)明顯下降(P<0.05),見Fig 4。

Fig 3 Expression of LCN2 in L5 spinal cord of rats on d 9(n=3)

On d 9, compared with group Ⅰ, the expression of LCN2 in the L5 spinal cord of rats was significantly increased in group Ⅱ-Ⅳ (P<0.05), while compared with group Ⅱ, the expression of LCN2 was significantly decreased in group Ⅳ (P<0.05).*P<0.05vsgroup Ⅰ;#P<0.05vsgroup Ⅱ.

Fig 4 Expression of p-p38 MAPK in L5 spinal cord of rats on d 9(n=3)

On d 9, compared with group Ⅰ, the expression of p-p38 MAPK in the L5 spinal cord of rats was significantly increased in group Ⅱ and Ⅲ (P<0.05), while compared with group Ⅱ, the expression of p-p38 MAPK was significantly decreased in group Ⅳ (P<0.05).*P<0.05vsgroup Ⅰ;#P<0.05vsgroup Ⅱ.

2.3 腰段脊髓背角小膠質(zhì)細(xì)胞標(biāo)記物Iba1的表達(dá)變化免疫熒光染色結(jié)果顯示，Ⅰ組大鼠腰段脊髓背角Iba1表達(dá)量較少。與Ⅰ組相比，Ⅱ組、Ⅲ組Iba1表達(dá)量明顯增加，小膠質(zhì)細(xì)胞胞體變大，分支增加。與Ⅱ組相比，Ⅲ組Iba1表達(dá)無明顯變化，Ⅳ組Iba1表達(dá)量明顯降低，小膠質(zhì)細(xì)胞胞體較小，分支也減少(Fig 5)。

3 討論

近年來研究發(fā)現(xiàn)，LCN2在膠質(zhì)細(xì)胞活化及神經(jīng)病理性痛中發(fā)揮重要作用。靜止的小膠質(zhì)細(xì)胞受炎癥刺激后釋放LCN2，而LCN2能進(jìn)一步激活小膠質(zhì)細(xì)胞，使小膠質(zhì)細(xì)胞變?yōu)榉种钚∧z質(zhì)細(xì)胞，且導(dǎo)致小膠質(zhì)細(xì)胞凋亡敏感性增加[8]。而LCN2主要通過激活小鼠或大鼠脊髓水平小膠質(zhì)細(xì)胞及其p-p38 MAPK信號通路誘導(dǎo)痛覺過敏的產(chǎn)生，從而參與神經(jīng)病理性疼痛及慢性炎癥性痛[3,9]。已有研究顯示，嗎啡耐受能夠促進(jìn)大鼠腰段脊髓水平小膠質(zhì)細(xì)胞活化及p-p38 MAPK的表達(dá)，且p-p38MAPK主要表達(dá)于活化的小膠質(zhì)細(xì)胞中，這表明小膠質(zhì)細(xì)胞及其內(nèi)的p-p38 MAPK信號通路參與了嗎啡耐受的形成過程，且多項(xiàng)研究表明，趨化因子也參與了嗎啡耐受形成[10-11]。因此，我們推測LCN2可能通過激活脊髓小膠質(zhì)細(xì)胞及其內(nèi)p-p38 MAPK信號通路，進(jìn)而誘導(dǎo)趨化因子及炎癥因子的釋放，從而參與了嗎啡耐受的形成。

Fig 5 Expression of Iba1 in dorsal horn of L5 spinal cord of rats on d 9

On d 9, compared with group Ⅰ, the immunoreactivity of Iba1 in the dorsal horn of L5 spinal cord of rats was markedly increased in group Ⅱ and Ⅲ, while compared with group Ⅱ, the immunoreactivity was markedly decreased in group Ⅳ.

最近，小干擾RNA(siRNA)基因沉默技術(shù)在分子生物學(xué)領(lǐng)域迅速發(fā)展。siRNA是一段長度約21～22個堿基的雙鏈RNA，可以特異性地誘導(dǎo)轉(zhuǎn)錄后基因沉默，即RNAi[12]。近年來，LCN2的研究主要采用基因敲除動物[3]、短發(fā)夾RNA(short hairpin)[13]或siRNA[14]等技術(shù)達(dá)到抑制LCN2表達(dá)的作用，本實(shí)驗(yàn)采用鞘內(nèi)注射特異性LCN2 siRNA的方法進(jìn)行研究，參照文獻(xiàn)[15-17]，在每天給予嗎啡前鞘內(nèi)注射LCN2 siRNA 4 μg，連續(xù)7 d，并檢測脊髓內(nèi)LCN2 mRNA及蛋白表達(dá)水平，結(jié)果顯示此種給藥方法及給藥劑量能產(chǎn)生較好的基因沉默效果而無明顯不良反應(yīng)。本實(shí)驗(yàn)參照Raghavendra等[4]方法建立慢性嗎啡耐受模型，d 9嗎啡耐受組MPE值較對照組明顯下降，表示嗎啡鎮(zhèn)痛效能下降，嗎啡耐受已經(jīng)形成。與嗎啡耐受組比較，LCN2 siRNA處理組大鼠MPE值明顯升高，即嗎啡的鎮(zhèn)痛效能得到改善，提示LCN2 siRNA能夠緩解嗎啡耐受的形成。進(jìn)一步檢測顯示，與對照組相比，嗎啡耐受組大鼠脊髓內(nèi)Iba1及p-p38 MAPK的表達(dá)明顯增加，而LCN2 siRNA處理后兩者表達(dá)均明顯降低。

本研究表明，嗎啡耐受能夠促進(jìn)脊髓水平LCN2表達(dá)，進(jìn)而促進(jìn)小膠質(zhì)細(xì)胞活化及其內(nèi)p-p38 MAPK的表達(dá)。而通過向大鼠鞘內(nèi)注射LCN2 siRNA后，脊髓水平的LCN2表達(dá)減少，進(jìn)而小膠質(zhì)細(xì)胞活化及其p-p38 MAPK表達(dá)水平亦下降，而相應(yīng)地，大鼠的嗎啡耐受部分緩解。因此，我們推測，脊髓水平LCN2可能通過激活小膠質(zhì)細(xì)胞及其p-p38 MAPK信號通路，進(jìn)一步誘導(dǎo)趨化因子及炎癥因子的釋放，從而參與嗎啡耐受的形成，而鞘內(nèi)注射LCN2 siRNA特異性抑制LCN2表達(dá)后，可能通過抑制小膠質(zhì)細(xì)胞活化及其p-p38 MAPK信號通路，進(jìn)而提高嗎啡鎮(zhèn)痛效能，達(dá)到緩解嗎啡耐受形成的作用。

總之，鞘內(nèi)注射LCN2 siRNA能特異性沉默脊髓LCN2的表達(dá)，進(jìn)而部分抑制嗎啡耐受的形成。該效應(yīng)可能與其抑制嗎啡誘導(dǎo)的腰段脊髓內(nèi)小膠質(zhì)細(xì)胞活化及其p-p38 MAPK信號通路有關(guān)。本研究提示，阻斷脊髓LCN2，有望為嗎啡耐受的機(jī)制研究提供新思路，并為嗎啡耐受的臨床防治提供新的途徑。

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Effect of spinal cord lipocalin-2 on development of morphine tolerance in normal rats

WANG Fen1,2, LIU Qing-zhen3, LIU Jian3, JIANG Zi-lu1,2, XIE Tao4, LI Wei-yan3

(1.KeyLaboratoryofAnesthesiologyofJiangsuProvince; 2.KeyLaboratoryofAnesthesiaandAnalgesiaApplicationTechnologyofJiangsuProvince,XuzhouMedicalCollege,XuzhouJiangsu221004,China;3.DeptofAnesthesiology,NanjingGeneralHospitalofNanjingMilitaryCommand,Nanjing210002,China;4.GraduateSchoolofSecondMilitaryMedicalUniversity,Shanghai200433,China)

Aim To explore the effect of knockdown spinal cord LCN2 by RNAi on the development of morphine tolerance in normal rats. Methods After successful intrathecal implantation, fourty-eight male Sprague-Dawley rats weighing 180 - 220 grams were randomly divided into 4 groups (n=12): group I: control group, group II: morphine tolerance group, group Ⅲ: mismatch siRNA group, group IV: LCN2 siRNA group. The sixth day after intrathecal implantation, rats were tested to ensure the position of catheters, and it was recorded as d 0. On d 2 - 8, rats were subcutaneously (s.c) injected of normal saline (NS) (group I) or morphine (group Ⅱ,Ⅲ,Ⅳ) 10 μg·g-1twice a day at 8:00 and 16:00. Before everyday s.c injection, rats were intrathecally injected of 10 μL DEPC solution (group Ⅰ,Ⅱ), 10 μL DEPC solution containing 4 μg mismatch siRNA (group III) and 4 μg LCN2 siRNA solution (group IV). Paw withdrawal latencies to thermal stimuli (PWTL) were tested before morphine injection and 45 minutes after morphine injection on d 1 and d 9. The percentage of maximal possible effect (%MPE) was calculated later. Animals were sacrificed on d 9 after the behavioral test and the lumbar enlargement segments of the spinal cord were removed for detecting the expression of phosphorylated-p38 mitogen-activated protein kinase (p-p38 MAPK) and LCN2 by Western blot and microglia marker Iba1 by immunofluorecence. Results On d 1, there was no significant difference in %MPE among four groups. On d 9, compared to group Ⅰ, %MPE was significantly reduced (P<0.05) while p-p38MAPK, LCN2 and Iba1 were markedly up-regulated in group Ⅱ and Ⅲ (P<0.05). On d 9, compared to group Ⅱ, %MPE was significantly increased while p-p38MAPK, LCN2 and Iba1 were markedly reduced in group IV (P<0.05). Conclusion Using LCN2 siRNA to knockdown spinal LCN2 relieves the development of morphine tolerance in normal rats possibly through inhibiting the activation of microglia and p38 MAPK in the spinal cord.

morphine tolerance; LCN2; siRNA; microglia; p-p38 MAPK; spinal cord

時間：2015-3-16 15:41 網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/34.1086.R.20150316.1541.011.html

2014-12-25,

2015-02-26

江蘇省自然科學(xué)基金面上研究項(xiàng)目(No BK20131328)

王 芬(1989-)女，碩士，研究方向：麻醉與鎮(zhèn)痛的基礎(chǔ)與臨床，E-mail：DocWangFen@126.com； 李偉彥(1965-)，男，博士，主任醫(yī)師，碩士生導(dǎo)師，研究方向：麻醉與鎮(zhèn)痛的基礎(chǔ)與臨床，Tel：025-80860147，E-mail：weiyanlee@sina.com

10.3969/j.issn.1001-1978.2015.04.025

A

1001-1978(2015)04-0565-05

R-332；R322.81；R341；R342.2；R345.57； R971.2