鞘內(nèi)注射LY294002緩解大鼠骨癌痛

網(wǎng)絡(luò)出版時(shí)間：2014-12-4 13:45網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/doi/10.3969/j.issn.1001-1978.2015.01.025.html

鞘內(nèi)注射LY294002緩解大鼠骨癌痛

金迪,楊建平,胡計(jì)嬅,王麗娜

(蘇州大學(xué)附屬第一醫(yī)院麻醉科，江蘇 蘇州215006)

中國圖書分類號：R-332；R322.81；R441.1；R345.57；R452；R738.1；R977.3

摘要:目的觀察鞘內(nèi)注射(intrathecal injection，i.t.)磷脂酰肌醇3-激酶(PI3K)抑制劑LY294002對骨癌痛大鼠疼痛行為學(xué)、脊髓背角磷酸化Akt(p-Akt)表達(dá)的影響。方法♀ SD大鼠40只，體質(zhì)量180~200 g，隨機(jī)分為5組(n=8)：假手術(shù)組(Ⅰ組)、假手術(shù)+LY294002組(Ⅱ組)、骨癌痛組(Ⅲ組)、骨癌痛+二甲基亞砜(DMSO)組(IV組)、骨癌痛+LY294002(V組),于大鼠左側(cè)脛骨干骺端骨髓腔內(nèi)注射Walker256乳腺癌細(xì)胞構(gòu)建脛骨癌痛模型。術(shù)后d 7~9鞘內(nèi)連續(xù)注射濃度為2.5 g·L`(-1)的LY294002 10μL或10μL的5%DMSO。觀測術(shù)前及術(shù)后d 7給藥后每小時(shí)機(jī)械痛閾(至8 h)。術(shù)后d 9處死大鼠，取各組大鼠的L4~6脊髓組織進(jìn)行免疫組化染色，檢測脊髓背角PI3K活化標(biāo)志p-Akt的表達(dá)。結(jié)果骨癌痛組大鼠(Ⅲ、Ⅳ、Ⅴ組)機(jī)械痛閾(MWT)明顯低于假手術(shù)組(Ⅰ組)(P<0.01)，V組在給藥后2~4h痛閾明顯升高(P<0.05)，3 h達(dá)到峰值(P<0.01)。與Ⅰ組比較，Ⅲ、Ⅳ組脊髓背角p-Akt陽性細(xì)胞數(shù)明顯增加，p-Akt表達(dá)增多(P<0.01)。與Ⅲ、Ⅳ組比較，Ⅴ組鞘內(nèi)注射LY294002后能明顯降低脊髓背角p-Akt的表達(dá)(P<0.05)。結(jié)論P(yáng)I3K/Akt通路可能參與大鼠骨癌痛的發(fā)生。

關(guān)鍵詞:骨腫瘤；疼痛；1-磷脂酰肌醇3-激酶；蛋白質(zhì)絲氨酸蘇氨酸激酶；小膠質(zhì)細(xì)胞；脊髓；信號通路

doi:10.3969/j.issn.1001-1978.2015.01.025

文章編號：

文獻(xiàn)標(biāo)志碼：A1001-1978(2015)01-0118-04

收稿日期：2014-09-02 修回日期：2014-11-08

基金項(xiàng)目：國家自然科學(xué)基金資助項(xiàng)目(No 81171057，81000479)；江蘇省自然科學(xué)基金(No BK2011305)；江蘇省臨床醫(yī)學(xué)科技專項(xiàng)(No BL2012024)

作者簡介：金迪(1989-),女，碩士，研究方向：麻醉學(xué)(疼痛),E-mail：kame_jindi@163.com;

通訊作者楊建平(1957-)，男，博士，博士生導(dǎo)師，研究方向：麻醉學(xué)(疼痛),,E-mail：szyangjp@hotmail.com

Abstract:AimTo investigate the effect of intrathecal injection of PI3K inhibitor LY294002 on pain behaviour and expression of p-Akt in spinal dorsal horns in bone cancer pain(BCP) rats. MethodsForty female SD rats weighing 180~200g were randomly divided into five groups (n=8 each):(Ⅰ)sham group;(Ⅱ)sham+LY294002 group;(Ⅲ)BCP group;(Ⅳ)BCP+DMSO group;(V)BCP+LY294002 group. BCP rat model was induced by inoculating Walker 256 mammary gland carcinoma cells into the medullary cavity of the left tibia.Rats received i.t.injections of either PI3K inhibitor LY294002 10μL(2.5g·L`(-1))or 5%DMSO 10 μL at the time of d 7~9 after the operation. Mechanical withdrawal threshold(MWT) test was performed before and after i.t.injections on d7(till 8h).The rats were sacrificed after inoculation and the L4~6 segments of the spinal cords were removed for immunohistochemistry to determinate the expression changes of spinal p-Akt.ResultsCompared with I group,the rats in Ⅲ,Ⅳ,Ⅴ group showed obvious mechanical hyperalgesia.The MWT of V group increased apparently from 2nd hour to 4th hour(P<0.05),and reached the peak in 3rd hour(P<0.01). Compared with I group,the expression of p-Akt in the spinal cord in Ⅲ,Ⅳ group increased obviously(P<0.01).Compared with Ⅲ,Ⅳ group,i.t.injections of LY294002 obviously cut down the expression of p-Akt in the spinal cord(P<0.05).ConclusionPI3K/Akt singaling pathway may take part in the development of bone cancer pain.

癌性痛是一種病理性疼痛，而骨癌痛又是癌性痛中最主要的一種。既往的研究觀察到，高達(dá)85%的肺、乳腺、前列腺癌癥患者都存在骨轉(zhuǎn)移，三分之一的患者都有骨痛癥狀[1]。雖然骨癌痛嚴(yán)重影響患者的生活質(zhì)量，但由于其發(fā)生機(jī)制尚未明了，臨床治療效果不佳。研究發(fā)現(xiàn)，PI3K/Akt信號通路參與神經(jīng)病理痛[2-3]和炎性痛的發(fā)生[4-6]，但其是否參與骨癌痛的發(fā)生發(fā)展尚未可知。本研究擬通過構(gòu)建大鼠脛骨癌痛模型，鞘內(nèi)給予PI3K抑制劑LY294002后觀察大鼠機(jī)械痛閾、脊髓p-Akt表達(dá)情況，探討骨癌痛發(fā)生的脊髓機(jī)制。

1材料與方法

1.1主要試劑和儀器Walker256細(xì)胞(南京中醫(yī)藥大學(xué)新藥與海洋藥物研究中心藥理毒理研究室惠贈(zèng))，PI3K抑制劑LY294002(Calbiochem，美國)，兔抗p-Akt一抗(Cell Signaling，美國)，SP試劑盒(北京中杉生物，中國)，DAB顯色試劑盒(北京中杉生物，中國)，照相機(jī)Olympus-C7070wz(Olympus，日本)，倒置顯微鏡Olympus-CKX41(Olympus，日本)，von-frey針刺觸覺測量儀(stoelting，美國)。

2結(jié)果

2.1機(jī)械痛閾測定結(jié)果各組大鼠的MWT基礎(chǔ)值差異均無統(tǒng)計(jì)學(xué)意義(P>0.05)。Ⅰ，Ⅱ組大鼠d 7 MWT值比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)，Ⅲ，Ⅳ組大鼠d 7 MWT值均低于Ⅰ組(P<0.01)，且該兩組間MWT值差異亦無統(tǒng)計(jì)學(xué)意義(P>0.05)。與Ⅲ組比較，術(shù)后d 7給藥后V組大鼠2~4 h的MWT值差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，給藥后3 h達(dá)到峰值(P<0.01),見Fig 1,2。

Fig 1The MWT of rats in all groups before

**P<0.01vsI group(d7)

Fig 2Effect of PI3K inhibitor LY294002 on MWT

*P<0.05，**P<0.01vsⅠ group,#P<0.05，##P<0.01vsⅢ group

2.2免疫組化染色結(jié)果與Ⅰ組比較，術(shù)后d 9，Ⅲ組、Ⅳ組和V組大鼠脊髓背角p-Akt陽性細(xì)胞數(shù)增加，表達(dá)上調(diào)(P<0.01)；與Ⅲ組和Ⅳ組比較，Ⅴ組脊髓背角p-Akt陽性細(xì)胞數(shù)降低，表達(dá)下調(diào)(P<0.01)；Ⅰ組和Ⅱ組、Ⅲ組和Ⅳ組p-Akt的陽性細(xì)胞數(shù)及表達(dá)比較差異無統(tǒng)計(jì)學(xué)意義(P<0.01)，見Fig 3，Tab 1。

Tab 1Effect of PI3K inhibitor LY294002 on

GroupNumMODⅠ13.63±2.670.011±0.0058Ⅱ13.75±3.010.012±0.0053Ⅲ64.25±5.57**0.047±0.0078**Ⅳ65.38±10.65**0.046±0.0072**Ⅴ37.25±6.79**##0.024±0.0089*##

*P<0.05，**P<0.01vsⅠ or Ⅱ group;##P<0.01vsⅢ or Ⅳ group

3討論

本研究建立的脛骨癌痛模型是目前進(jìn)行癌痛研究的常用模型[7]，能模擬人類骨癌痛基本特征，且造模技術(shù)純熟，模型穩(wěn)定。本研究在造模后d 7，造模大鼠出現(xiàn)機(jī)械痛閾降低，且活動(dòng)度下降，提示脛骨癌痛模型已建立。

磷脂酰肌醇-3-激酶(phosphoinositide 3-kinase，PI3K)是一種可使肌醇環(huán)D-3位羥基磷酸化的磷脂酰肌醇激酶，哺乳類的PI3K可分為3個(gè)亞型：Class I、ClassII和ClassIII，能激活下游Akt的是ClassI亞型。Akt又稱蛋白激酶B(protein kinase B，PKB)是一種絲氨酸/蘇氨酸蛋白激酶，屬于AGC蛋白激酶家族。PI3K活化后與Akt結(jié)合，使Akt從胞液轉(zhuǎn)移至胞膜內(nèi)側(cè)，同時(shí)引起其構(gòu)象變化并發(fā)生磷酸化，即磷酸化Akt(p-Akt)[10-12]，因此Akt是PI3K信號通路中的中心環(huán)節(jié)，p-Akt常作為PI3K活化的標(biāo)志[13-14]。

既往研究證明，PI3K/Akt信號通路參與了慢性疼痛的發(fā)生和發(fā)展。在神經(jīng)病理痛大鼠模型上，身體同側(cè)背根神經(jīng)節(jié)和脊髓背角的p-Akt陽性神經(jīng)元明顯增加，而鞘內(nèi)注射PI3K抑制劑wortmannin或LY294002，以及Akt抑制劑IV或(-)-魚藤素可以明顯緩解大鼠的機(jī)械痛敏[2]。此外，Guedes等[3]發(fā)現(xiàn)，在坐骨神經(jīng)橫斷術(shù)(sciatic nerve transection ，SNT)后7 d，總Akt和p-Akt的表達(dá)均增高。在皮內(nèi)注射辣椒素構(gòu)建的炎性痛模型中，p-Akt在腰段脊髓背角和背根神經(jīng)節(jié)的表達(dá)都明顯增高[4,15]，而預(yù)先給予PI3K抑制劑則可以預(yù)防由辣椒素引起的大鼠的機(jī)械痛敏，且Wortmannin和LY294002這兩種PI3K的抑制劑均對此有效[4,15]。Pezet等[6]還發(fā)現(xiàn)，鞘內(nèi)給予LY294002后可以緩解由足底注射福爾馬林引發(fā)的炎性痛，該作用有劑量依賴性，且對第一時(shí)相和第二時(shí)相的炎性痛均有緩解作用。

本實(shí)驗(yàn)研究觀察，骨癌痛造模成功的大鼠產(chǎn)生明顯的機(jī)械痛覺超敏，脊髓背角L4~6水平的p-Akt表達(dá)增加，PI3K/Akt通路被激活；而連續(xù)3 d鞘內(nèi)給予PI3K抑制劑LY294002阻斷PI3K/Akt信號通路后，脊髓p-Akt表達(dá)降低，PI3K/Akt活化受到抑制。術(shù)后7 d的MWT值顯示，大鼠已產(chǎn)生的機(jī)械痛覺過敏，在鞘內(nèi)注射LY294002后2~4 h被部分逆轉(zhuǎn)，骨癌痛得到部分緩解，3 h時(shí)達(dá)到峰值。因此我們有理由推測，大鼠骨癌痛模型中脊髓水平PI3K/Akt信號通路得到激活，且主要在骨癌痛早期發(fā)生作用。

Fig 3Different expressions of p-Akt in spinal dorsal horn in rats Intrathecal LY294002 significantly decreased the expression of p-Akt in BCP rats.Scale Bar:50μm.

綜上所述，脊髓PI3K/Akt信號通路的激活可能參與了大鼠骨癌痛的形成。

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Intrathecal injection of LY294002 attenuates bone cancer pain

JIN Di, YANG Jian-ping, HU Ji-hua, WANG Li-na

(DeptofAnesthesiology,theFirstAffiliatedHospitalofSoochowUniversity,SuzhouJiangsu215006,China)

Key words: bone neoplasms; pain; 1-phosphatidylinositol 3-kinase;protein-serine-threonine kinases; microglia;spinal cord;signaling pathway