雷帕霉素對(duì)大鼠神經(jīng)病理性疼痛及脊髓背角神經(jīng)元凋亡的影響

馮 濤,陳 功,丁 潔,黃鳳貞(深圳市寶安區(qū)中心醫(yī)院麻醉科,深圳 518102;通訊作者,E-mail:374379335@qq.com)

雷帕霉素對(duì)大鼠神經(jīng)病理性疼痛及脊髓背角神經(jīng)元凋亡的影響

馮 濤*,陳 功,丁 潔,黃鳳貞
(深圳市寶安區(qū)中心醫(yī)院麻醉科,深圳 518102;*通訊作者,E-mail:374379335@qq.com)

目的 研究鞘內(nèi)注射雷帕霉素對(duì)大鼠神經(jīng)病理性疼痛及脊髓背角神經(jīng)元凋亡的影響。 方法 雄性SD大鼠30只隨機(jī)分為假手術(shù)組(sham組)、脊神經(jīng)結(jié)扎組(SNL組)和雷帕霉素組(Rap組),每組10只。各組大鼠L4-5鞘內(nèi)置管后,SNL組和Rap組大鼠結(jié)扎左側(cè)腰5脊神經(jīng)建立神經(jīng)病理性疼痛模型,sham組大鼠則僅分離暴露左側(cè)腰5神經(jīng)。Rap組大鼠于術(shù)后30 min鞘內(nèi)注射雷帕霉素0.1 μg/10 μl,sham組和SNL組則給予注射等容溶劑(5% DMSO)。每日1次,連續(xù)7 d。各組大鼠測(cè)量術(shù)后1,3,5,7 d機(jī)械痛閾(paw withdrawal threshold,PWT)和熱痛閾(paw withdrawal latency,PWL)。術(shù)后第7天處死大鼠并立即取左側(cè)L5節(jié)段脊髓背角,電鏡下觀察神經(jīng)元自噬體(n=3); Western blot法檢測(cè)自噬相關(guān)蛋白LC3、神經(jīng)元特異性蛋白NeuN以及凋亡相關(guān)蛋白a-caspase3表達(dá)(n=7)。 結(jié)果 與sham組比較,SNL組大鼠PWT和PWL在術(shù)后第1天和第3天開(kāi)始分別明顯降低,且持續(xù)到術(shù)后第7天(P<0.05),術(shù)后第7天 PWT、PWL分別為(12.5±1.0)g和(7.8±0.4)s,較sham組分別降低67%和44%(P<0.05);而脊髓背角LC3 Ⅱ和a-caspase3表達(dá)分別增高96%和359%(P<0.05), NeuN表達(dá)降低21%(P<0.05);與SNL組比較,Rap組大鼠各相應(yīng)時(shí)間點(diǎn)PWT和PWL明顯增高(P<0.05),術(shù)后第7天 PWT、PWL分別為(24.9±1.6)g和(11.8±0.8)s,較SNL組分別增加99%和51%(P<0.05);而脊髓背角LC3 Ⅱ和NeuN表達(dá)增高44%和19%(P<0.05), a-caspase3表達(dá)降低27%(P<0.05)。電鏡下Rap組脊髓背角神經(jīng)元自噬體較SNL組可見(jiàn)較多自噬體。 結(jié)論 雷帕霉素能減輕神經(jīng)病理性疼痛,其機(jī)制可能與雷帕霉素增加脊髓背角神經(jīng)元自噬,減少神經(jīng)元凋亡有關(guān)。

雷帕霉素; 神經(jīng)病理性疼痛; 自噬; 細(xì)胞凋亡

神經(jīng)病理性疼痛(neuropathic pain, NP)是指外周或中樞神經(jīng)系統(tǒng)受到損害后引起的疼痛,常表現(xiàn)為自發(fā)性疼痛、痛覺(jué)異常、痛覺(jué)過(guò)敏等[1],由于發(fā)病機(jī)制異常復(fù)雜,其治療一直是個(gè)世界性難題。近年來(lái)有學(xué)者發(fā)現(xiàn)NP與脊髓背角神經(jīng)元凋亡有關(guān),而通過(guò)減少脊髓背角神經(jīng)元凋亡能緩解NP[2, 3]。自噬是真核細(xì)胞通過(guò)溶酶體途徑降解細(xì)胞內(nèi)受損的細(xì)胞器及蛋白等物質(zhì),從而在維持細(xì)胞自身穩(wěn)態(tài)方面起著保護(hù)作用的關(guān)鍵機(jī)制[4]。雷帕霉素是常用的自噬誘導(dǎo)劑,我們的前期研究發(fā)現(xiàn)雷帕霉素能通過(guò)誘導(dǎo)脊髓背角神經(jīng)元細(xì)胞自噬及減少脊髓背角白介素1β水平從而有效減輕NP[5, 6]。然而雷帕霉素對(duì)NP脊髓背角神經(jīng)元凋亡的影響,目前少見(jiàn)報(bào)道。本實(shí)驗(yàn)旨在研究雷帕霉素對(duì)NP大鼠的鎮(zhèn)痛作用及對(duì)脊髓背角神經(jīng)元凋亡的影響,為其臨床應(yīng)用提供理論依據(jù)。

1 材料和方法

1.1 主要試劑和儀器

雷帕霉素(Rapamycin)、二甲基亞砜(DMSO)和兔抗微管相關(guān)輕鏈蛋白3(microtubule-associated protein 1-light chain 3,LC3)多克隆抗體均購(gòu)自美國(guó)Sigma公司,兔抗神經(jīng)元特異性核蛋白(Neuronal Nuclei,NeuN))多克隆抗體(Abcam,美國(guó)),兔抗甘油醛-3-磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、兔抗活化型半胱氨酸-天冬氨酸酶3(activated caspase3,a-caspase3)多克隆抗體(Sant Cruz,美國(guó)),聚乙烯PE-10導(dǎo)管(anilab,寧波),蛋白提取試劑盒及蛋白定量試劑盒(南京,凱基)。大鼠爪機(jī)械痛閾測(cè)定儀及熱輻射痛閾測(cè)量?jī)x(UGO BASILE,意大利),DYY-6C型電泳儀(北京六一儀器廠),Bio-Rad化學(xué)發(fā)光熒光成像系統(tǒng)(美國(guó)),透射電鏡(型號(hào)Tecnai G2 12,荷蘭)。

1.2 實(shí)驗(yàn)動(dòng)物及分組

本實(shí)驗(yàn)得到華中科技大學(xué)同濟(jì)醫(yī)學(xué)院倫理委員會(huì)批準(zhǔn)。雄性清潔級(jí)SD大鼠30只,8周齡,體質(zhì)量200 -220 g,由武漢大學(xué)實(shí)驗(yàn)動(dòng)物中心提供(許可證號(hào)SCXK(鄂)2008-0004)。大鼠采用隨機(jī)數(shù)字表法分為假手術(shù)組(sham組),脊神經(jīng)結(jié)扎組(SNL組)和雷帕霉素組(Rap組)(n=10)。各組大鼠首先行腰4-5間隙經(jīng)鞘內(nèi)向頭端方向置入PE-10導(dǎo)管約3 cm,即脊髓腰膨大處。置管成功后,SNL組和Rap組大鼠行左側(cè)腰5脊神經(jīng)結(jié)扎建立神經(jīng)病理性疼痛模型,sham組大鼠則僅分離左側(cè)腰5脊神經(jīng)不結(jié)扎。Rap組大鼠于術(shù)后30 min經(jīng)鞘內(nèi)置管注射雷帕霉素0.1 μg(10 μl),SNL組大鼠給予等容溶劑(5% DMSO),各組注射完畢后再給予15 μl生理鹽水沖洗導(dǎo)管,以保證藥物或溶劑全部進(jìn)入鞘內(nèi),每日1次,連續(xù)7 d。動(dòng)物模型制備及藥物劑量的選擇參考我們的預(yù)實(shí)驗(yàn)及之前的研究[5]。大鼠自由進(jìn)食水,室溫保持20-25 ℃。

1.3 大鼠痛閾測(cè)量

各組大鼠于術(shù)前測(cè)定基礎(chǔ)痛閾(baseline),然后在術(shù)后1,3,5,7 d測(cè)量機(jī)械痛閾和熱痛閾。機(jī)械痛閾測(cè)量方法為將大鼠置入底板為網(wǎng)格的有機(jī)玻璃箱內(nèi),將金屬針頭連接測(cè)痛儀后,針頭緩慢勻速上升刺激大鼠患側(cè)(左側(cè))后肢足底,當(dāng)大鼠感到疼痛并收縮后爪時(shí)記錄此壓力值(paw withdrawal threshold,PWT)。熱痛閾測(cè)量方法為將大鼠置于3 mm厚的玻璃板上,使用熱輻射儀光源照射患側(cè)(左側(cè))后肢足底,記錄從照射開(kāi)始到大鼠因疼痛而縮爪的時(shí)間(paw withdrawal latency,PWL)。各組連續(xù)測(cè)定3次,每次間隔時(shí)間為5 min。

1.4 透射電鏡觀察脊髓背角自噬

術(shù)后7 d各組大鼠各取3只采用水合氯醛350 mg/kg腹腔注射麻醉,用含0.25%戊二醛和4%多聚甲醛的0.1 mol/L磷酸緩沖液經(jīng)心臟灌注后取脊髓L4-5節(jié)段左側(cè)脊髓背角,2.5%戊二醛過(guò)夜,1%四氧化鋨固定,丙酮脫水,環(huán)氧樹脂包埋。標(biāo)本制成超薄切片后以3%枸櫞酸鉛染色,然后在透射電鏡下觀察神經(jīng)元自噬情況。

1.5 蛋白標(biāo)本采集及檢測(cè)

1.6 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 大鼠機(jī)械痛閾和熱痛閾

所有大鼠均未見(jiàn)明顯傷口感染。sham組大鼠活動(dòng)未見(jiàn)明顯異常,痛閾無(wú)明顯變化(P>0.05)。SNL組和Rap組大鼠術(shù)后出現(xiàn)術(shù)側(cè)(左側(cè))后爪呈內(nèi)收畸形,行走輕度異常。與sham組大鼠相比,SNL組大鼠術(shù)后1 d出現(xiàn)機(jī)械痛閾(PWT)明顯降低(P<0.05),術(shù)后3 d熱痛閾(PWL)明顯降低(P<0.05),且持續(xù)到7 d;與SNL組相比,Rap組大鼠術(shù)后各相應(yīng)時(shí)間點(diǎn)PWT和PWL明顯增高(P<0.05,見(jiàn)圖1,2)。

2.2 透射電鏡觀察脊髓背角自噬體

術(shù)后7 d,透射電鏡下sham組神經(jīng)元細(xì)胞核、內(nèi)質(zhì)網(wǎng)、線粒體和溶酶體形態(tài)基本正常,自噬體少見(jiàn)。SNL和Rap組神經(jīng)元可見(jiàn)細(xì)胞核皺縮,線粒體變形腫脹,溶酶體形態(tài)異常,在胞質(zhì)中可見(jiàn)雙層膜組成的自噬囊泡,有的自噬囊泡中可見(jiàn)高電子密度的自噬體。與SNL組相比,Rap組自噬體較多,且細(xì)胞器受損情況好于SNL組(見(jiàn)圖3)。

與sham組比較,#P<0.05;與SNL組比較,*P<0.05圖1 各組大鼠術(shù)后7 d機(jī)械痛閾比較Figure 1 Comparison of mechanical pain(PWT) of rats between three groups (±s,n=10)

與sham組比較,#P<0.05;與SNL組比較,*P<0.05圖2 各組大鼠術(shù)后7 d熱痛閾(PWL)比較Figure 2 Comparison of thermal pain(PWL) of rats between three groups (±s,n=10)

圖3 各組大鼠術(shù)后7 d脊髓背角神經(jīng)元透射電鏡下形態(tài) (scale bars=1 μm,箭頭為自噬體)Figure 3 Morphology of autophagosomes in neurons of the spinal cord at 7 d after surgery in each group under transmission electron microscope (scale bars=1 μm)

2.3 大鼠脊髓背角LC3蛋白水平比較

與sham相比,SNL組大鼠在術(shù)后7 d脊髓背角LC3 Ⅱ和a-caspase3表達(dá)明顯增高(P<0.05),NeuN表達(dá)明顯下降(P<0.05);與SNL組比較,Rap組LC3 Ⅱ和NeuN表達(dá)明顯增高(P<0.05),而a-caspase3表達(dá)明顯下降(P<0.05,見(jiàn)圖4-6)。

與sham組比較,#P<0.05;與SNL組比較,*P<0.05圖4 各組大鼠術(shù)后7 d脊髓背角LC3表達(dá)比較Figure 4 The expression of LC3 in L5 spinal cord dorsal horn at 7 d after surgery in each group (±s,n=7)

與sham組比較,#P<0.05;與SNL組比較,*P<0.05圖5 各組大鼠術(shù)后7 d脊髓背角NeuN表達(dá)比較Figure 5 The expression of NeuN in L5 spinal cord dorsal horn at 7 d after surgery in each group (±s,n=7)

3 討論

NP的發(fā)病機(jī)制非常復(fù)雜,近年來(lái)的一些研究逐步揭示了NP與脊髓背角神經(jīng)元的凋亡之間的關(guān)系。Scholz等[7]在坐骨神經(jīng)結(jié)扎、脊神經(jīng)結(jié)扎、選擇性外周神經(jīng)損傷三種外周神經(jīng)損傷引起的NP動(dòng)物模型中,均發(fā)現(xiàn)伴有脊髓背角γ氨基丁酸(γ-ami-nobutyric acid,GABA)能神經(jīng)元凋亡。GABA能神經(jīng)元為抑制性中間神經(jīng)元,由于其凋亡導(dǎo)致GABA釋放減少,從而使神經(jīng)興奮性增加,進(jìn)而參與了NP;而使用caspase廣泛抑制劑zVAD,能減少GABA能神經(jīng)元凋亡,并明顯減輕神經(jīng)病理性疼痛。此外有研究發(fā)現(xiàn)坐骨神經(jīng)結(jié)扎模型大鼠給予caspase3的特異性抑制劑Z-DEVD-FMK后能明顯緩解疼痛,其機(jī)制與減少脊髓背角神經(jīng)元凋亡和抑制神經(jīng)生長(zhǎng)相關(guān)蛋白(GAP-43)有關(guān)[8]。GAP-43是神經(jīng)元軸突生長(zhǎng)標(biāo)記物,與神經(jīng)元軸突芽生和神經(jīng)元可塑性有關(guān),而神經(jīng)元軸突芽生和神經(jīng)元可塑性是神經(jīng)病理性疼痛的重要機(jī)制。此外另有研究也發(fā)現(xiàn)通過(guò)減少脊髓背角神經(jīng)元凋亡能緩解神經(jīng)病理性疼痛[9,10]，阻斷或減輕脊髓背角神經(jīng)元凋亡,有望成為治療NP的一個(gè)新思路。

與sham組比較,#P<0.05;與SNL組比較,*P<0.05圖6 各組大鼠術(shù)后7 d脊髓背角a-caspase3表達(dá)比較Figure 6 The expression of a-caspase3 in L5 spinal cord dorsal horn at 7 d after surgery in each group

與sham組比較,SNL大鼠機(jī)械痛閾和熱痛閾明顯降低,說(shuō)明我們成功建立了神經(jīng)病理性疼痛模型。為了研究神經(jīng)病理性疼痛時(shí)自噬的變化,本課題組檢測(cè)了脊髓背角蛋白微管相關(guān)輕鏈蛋白3(microtubule-associated protein light chain 3, LC3),并在電鏡下觀察脊髓背角神經(jīng)元自噬體。LC3是自噬體的形成的關(guān)鍵蛋白,當(dāng)自噬被激活時(shí),細(xì)胞質(zhì)里的LC3(LC3 Ⅰ)在泛素樣反應(yīng)酶的作用下與磷脂酰乙醇胺生成LC3 Ⅱ,LC3 Ⅱ由于特異定位于自噬體的內(nèi)外膜,因此LC3 Ⅱ是反映自噬的一個(gè)可靠的指標(biāo)[5]。與sham組大鼠相比,SNL組大鼠術(shù)后第7天脊髓背角LC3 Ⅱ表達(dá)增高,說(shuō)明其自噬水平增加,這與其電鏡下發(fā)現(xiàn)脊髓背角神經(jīng)元自噬體輕度增多相一致,反映了脊髓背角對(duì)脊神經(jīng)結(jié)扎這一病理刺激的保護(hù)性反應(yīng);而反映凋亡指標(biāo)的蛋白a-caspase3表達(dá)明顯增高,神經(jīng)元的特異性標(biāo)志物神經(jīng)特異核蛋白(NeuN)表達(dá)明顯減少,說(shuō)明脊髓背角神經(jīng)元發(fā)生了凋亡并參與了NP,這與已有報(bào)道相符[7]。與SNL組大鼠相比,Rap組大鼠機(jī)械痛閾和熱痛閾明顯增加,說(shuō)明雷帕霉素能明顯緩解神經(jīng)病理性疼痛。Rap組大鼠LC3 Ⅱ表達(dá)較SNL組明顯增加,電鏡下觀察可見(jiàn)脊髓背角神經(jīng)元自噬增加,說(shuō)明鞘內(nèi)給予雷帕霉素確實(shí)增加了Rap組大鼠脊髓背角神經(jīng)元自噬水平。同時(shí)Rap組大鼠a-caspase3表達(dá)下降,NeuN表達(dá)增加,說(shuō)明雷帕霉素減少了Rap組大鼠脊髓背角神經(jīng)元凋亡。近來(lái)有多項(xiàng)研究發(fā)現(xiàn)當(dāng)中樞系統(tǒng)受損如腦缺血時(shí),增加自噬能減少神經(jīng)元的凋亡從而起到神經(jīng)保護(hù)作用[11,12],由于神經(jīng)病理性疼痛的根本原因在于中樞或外周神經(jīng)受損,因此雷帕霉素減輕神經(jīng)病理性疼痛的機(jī)制可能與其增加脊髓背角自噬,減少神經(jīng)元凋亡有關(guān)。

綜上所述,本實(shí)驗(yàn)發(fā)現(xiàn)SNL大鼠給予雷帕霉素后,脊髓背角神經(jīng)元自噬增加,凋亡明顯減少,雷帕霉素可能通過(guò)上調(diào)影響脊髓背角神經(jīng)元自噬、減少神經(jīng)元凋亡從而減輕神經(jīng)病理性疼痛。

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Effectsofrapamycinonneuropathicpainandapoptosisofneuronsinspinalcordofratsafterspinalnerveligation

FENG Tao*,CHEN Gong,DING Jie,HUANG Fengzhen
(DepartmentofAnesthesiology,BaoanCentralHospital,Shenzhen518102,China;*Correspondingauthor,E-mail:374379335@qq.com)

ObjectiveTo investigate the effects of rapamycin on neuropathic pain and apoptosis of neurons in the spinal cord of rats after spinal nerve ligation.MethodsThirty male SD rats were randomly divided into three groups(n=10): sham group, spinal nerve ligation group(SNL group) and rapamycin group(Rap group). After intrathecal catheter implantation between L4-5 vertebrae, the rats in SNL group and Rap group underwent L5 spinal nerve ligation. The surgical procedure in sham group was identical except nerve ligation.The rats were injected with rapamycin(0.1 μg/10 μl) per day for 7 d via intrathecal catheter in Rap group after L5 spinal nerve ligation, while the rats were injected intrathecally with the same volume of vehicle(5%DMSO) in sham group and SNL group. Paw withdrawal threshold(PWT) and paw withdrawal latency(PWL) were measured at 1, 3, 5, and 7 d after surgery. At day 7 after surgery, the rats were sacrificed. Ipsilateral L5 spinal cord dorsal horn was examined using transmission electron microscope. Expression of LC3, NeuN and a-caspase3 in the L5 cord dorsal horn were measured by Western blot.ResultsCompared with sham group, PWT and PWL of rats were significantly decreased at 1, 3 day after SNL in SNL group, respectively, and lasted for 7 d(P<0.05). At day 7 after SNL, PWT[(12.5±1.0)g] and PWL[(7.8±0.4)s] were decreased by 67% and 44%, respectively(P<0.05), the levels of LC3 Ⅱ and a-caspase3 expression increased by 96% and 359%, respectively(P<0.05), while the levels of NeuN expression decreased by 21%. Compared to SNL group, PWT and PWL of rats were significantly increased in Rap group(P<0.05). At day 7 after SNL, PWT[(24.9±1.6)g]and PWL[(11.8±0.8)s]were increased by 99% and 51%, respectively(P<0.05), the levels of LC3 Ⅱ and NeuN expression increased by 44% and 19%, respectively(P<0.05), while the levels of a-caspase3 expression decreased by 27%(P<0.05). More autophagosomes were found in Rap group than in SNL group.ConclusionIntrathecal rapamycin may ameliorate SNL-induced neuropathic pain by increasing the autophagy and inhibiting the apoptosis of neurons.

rapamycin; neuropathic pain; autophagy; apoptosis

廣東省深圳市衛(wèi)生計(jì)生系統(tǒng)暨深圳市寶安區(qū)科技創(chuàng)新局科研項(xiàng)目(201507067)

馮濤,男,1979-09生,博士,副主任醫(yī)師,E-mail:374379335@qq.com

2017-08-10

R365

A

1007-6611(2017)12-1236-05

10.13753/j.issn.1007-6611.2017.12.008