NR2A、NR2B在慢性胰腺炎大鼠大腦前扣帶回的表達(dá)變化及意義

周文 高峻 李桂香 李兆申

·論著·

NR2A、NR2B在慢性胰腺炎大鼠大腦前扣帶回的表達(dá)變化及意義

周文 高峻 李桂香 李兆申

目的檢測慢性胰腺炎(CP)大鼠大腦前扣帶回(ACC)的N-甲基-D-天冬氨酸受體亞基NR2A和NR2B基因表達(dá)水平，探討其在CP疼痛形成中的作用。方法成年雄性SD大鼠按數(shù)字表法隨機(jī)分為CP組、對照組和正常組。通過尾靜脈一次性注射二丁基二氯化物8 mg/kg體重的方法制備CP模型，對照組尾靜脈注射等容積乙醇和甘油混合液。4周后依次用不同粗細(xì)(3.85、5.50、7.05、10.4、17.8 g)的von frey纖維絲刺激大鼠腹部，記錄疼痛陽性反應(yīng)次數(shù)的百分率。然后處死大鼠，取胰腺組織行病理學(xué)檢查，采用實(shí)時(shí)PCR和蛋白質(zhì)印跡法檢測大腦ACC區(qū)NR2A、NR2B mRNA及蛋白表達(dá)量。結(jié)果CP模型成功率為60%。用Von frey纖維絲以3.85 g至17.8 g遞增刺激大鼠腹部，CP組大鼠陽性反應(yīng)百分率從(38.33±7.53)%遞增到(73.33±8.17%)，對照組大鼠從(7.80±6.70)%遞增到(34.44±5.27)%，正常組大鼠從(6.57±5.48)%遞增到(33.33±5.00)%，均隨著刺激強(qiáng)度增強(qiáng)而增加。每個(gè)刺激強(qiáng)度CP組大鼠的陽性反應(yīng)百分率均顯著高于對照組及正常組，差異有統(tǒng)計(jì)學(xué)意義(P值均<0.05)，而對照組與正常組間的差異均無統(tǒng)計(jì)學(xué)意義。CP組、對照組、正常組大鼠ACC區(qū)NR2A蛋白表達(dá)量分別為1.25±0.78、0.95±0.14、0.91±0.09，NR2B蛋白表達(dá)量為1.44±0.12、0.93±0.08、0.94±0.04，CP組顯著高于對照組及正常組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)；NR2A mRNA表達(dá)量為1.43±0.20、0.80±0.10、1.01±0.13，NR2B mRNA表達(dá)量為1.40±0.09、0.98±0.14、0.94±0.05，CP組顯著高于對照組及正常組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。結(jié)論CP大鼠大腦ACC區(qū)谷氨酸受體NR2A、NR2B的表達(dá)上調(diào)，可能參與CP疼痛的形成。

胰腺炎，慢性； 疼痛； 大腦皮質(zhì)； N-甲基-D-天冬氨酸

慢性胰腺炎(CP)是各種病因引起的胰腺組織和功能持續(xù)性損害，疼痛為其主要臨床表現(xiàn)，85%～90%的患者可發(fā)生腹痛，嚴(yán)重影響生活質(zhì)量[1]。現(xiàn)有理論認(rèn)為CP患者的腹痛可能與胰管壓力增高、神經(jīng)重塑、氧化應(yīng)激、神經(jīng)炎癥等相關(guān)，但均不能完全闡述疼痛的原因。近年來開始探討CP中樞神經(jīng)系統(tǒng)的內(nèi)臟痛覺傳導(dǎo)通路和關(guān)鍵中樞皮質(zhì)區(qū)域的功能是否發(fā)生變化[2]。大腦前扣帶回(anterior cingulate cortex，ACC)是疼痛感知的關(guān)鍵皮層區(qū)域[3-4]。谷氨酸是中樞神經(jīng)系統(tǒng)中感覺傳遞和感知的主要興奮性遞質(zhì)，N-甲基-D-天冬氨酸(N-methyl-D-aspartate recepter, NMDA)是其離子型受體之一。研究發(fā)現(xiàn)，大腦ACC區(qū)NMDA受體表達(dá)變化與痛厭惡情緒的調(diào)節(jié)相關(guān)，且在慢性內(nèi)臟痛動(dòng)物模型中表達(dá)水平明顯升高[5-6]。本研究觀察CP大鼠大腦ACC區(qū)NMDA受體亞基NR2A、NR2B基因表達(dá)的變化，探討其與CP疼痛的相關(guān)性。

材料和方法

一、實(shí)驗(yàn)動(dòng)物及主要試劑

健康雄性SD大鼠20只，由第二軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心提供，體重180～220 g，飼養(yǎng)在室溫26℃左右、相對濕度60%～70%、晝夜周期12 h/12 h的環(huán)境中，水和食物供給充足。DBTC購自Sigma公司，逆轉(zhuǎn)錄試劑盒、PCR試劑盒購自TAKARA公司。兔抗鼠NR2A多抗購自Millipore公司、兔抗鼠NR2B多抗購自Abcam公司，辣根過氧化物標(biāo)記的山羊抗兔二抗購自美國Millipore公司，內(nèi)參Tubulin購自上海碧云天生物技術(shù)有限公司。

二、動(dòng)物模型建立及分組

20只SD大鼠按隨機(jī)數(shù)字表法分為CP組(10只)，對照組(5只)，正常組(5只)。CP模型采用尾靜脈一次注射DBTC 8 mg/kg體重的方法制備[7-8]。對照組注射等容積乙醇和甘油混合液，正常組未予處理。實(shí)驗(yàn)前禁食24 h，不禁水。

三、機(jī)械痛測定

建模4周后大鼠腹部備皮，在大致相同的位置做標(biāo)記。將大鼠置于懸掛的帶罩網(wǎng)狀架子上適應(yīng)環(huán)境30 min。依次用不同粗細(xì)(3.85、5.50、7.05、10.4、17.8 g)的von frey纖維絲從下方刺激大鼠腹部標(biāo)記處。每個(gè)規(guī)格使用10次，每次1～2 s，間隔15 s，以纖毛彎曲90°為準(zhǔn)。大鼠舔舐腹部、收腹或身體收縮為陽性反應(yīng)[9-10]。記錄陽性反應(yīng)次數(shù)的百分率。

四、胰腺組織病理學(xué)檢查

4周后處死大鼠，取胰腺組織，4%甲醛溶液固定，常規(guī)行病理學(xué)檢查。

五、蛋白質(zhì)印跡法檢測NR2A、NR2B蛋白表達(dá)

4周后腹腔注射7%的水合氯醛麻醉大鼠，斷頭后迅速將頭置于冰上取出ACC區(qū)腦組織，置-80℃冰箱保存。用裂解液提取蛋白，定量后行蛋白質(zhì)印跡法檢測NR2A、NR2B蛋白表達(dá)。抗NR2A抗體1∶500稀釋，抗NR2B抗體1∶1 000稀釋、抗Tubulin抗體1∶2 000稀釋。山羊抗兔二抗1∶5 000稀釋。最后通過凝膠成像系統(tǒng)采集圖像，ImageJ軟件掃描，以目的條帶與內(nèi)參條帶灰度值比表示蛋白相對表達(dá)量。

六、實(shí)時(shí)RT-PCR法檢測NR2A、NR2B mRNA表達(dá)

使用Trazol提取大鼠大腦ACC區(qū)組織的總RNA，先反轉(zhuǎn)錄成cDNA，再行實(shí)時(shí)PCR反應(yīng)擴(kuò)增產(chǎn)物。根據(jù)GeneBank目的基因的全長序列，應(yīng)用Primer3 Plus軟件設(shè)計(jì)引物。NR2A 上游引物 5′-GCCAGTTACACAGCCAACCT-3′，下游引物 5′-CAAATCGGAAAGGTGGAGAA-3′；NR2B 上游引物 5′-CGGATGTGTCCTGAGACTGA-3′，下游引物 5′-ATTCCTCATGCAGGTTCCAC-3′；內(nèi)參GAPDH 上游引物 5′-CCCCCAATGTATCCGTTGTG-3′，下游引物 5′-TAGCCCAGGATGCCCTTTAGT-3′。引物合成由上海生工生物工程有限公司完成。反轉(zhuǎn)錄反應(yīng)條件：37℃ 15 min，80℃ 30 s。PCR反應(yīng)條件：95℃ 10 min，95℃ 15 s、60℃ 1 min，48個(gè)循環(huán)。通過儀器自帶軟件獲取Ct值，采用公式2-△△Ct計(jì)算mRNA的相對表達(dá)量。實(shí)驗(yàn)重復(fù)3次，取均值。

七、統(tǒng)計(jì)學(xué)方法

結(jié) 果

一、大鼠胰腺組織病理變化

建模4周后，CP組大鼠胰腺的腺泡萎縮，出現(xiàn)空泡狀細(xì)胞，小葉結(jié)構(gòu)破壞，膠原纖維散在分布于血管和小葉間，炎性細(xì)胞浸潤(圖1A)。CP制備成功率為60%。對照組與正常組大鼠胰腺組織未見明顯病理改變(圖1B、1C)。

圖1 CP組(1A)、對照組(1B)、正常組(1C)大鼠胰腺組織病理改變(HE ×100)

二、大鼠機(jī)械痛反應(yīng)百分率

用von frey纖維絲以3.85 g至17.8 g遞增刺激大鼠腹部，CP組大鼠陽性反應(yīng)次數(shù)百分率從(38.33±7.53)%遞增到(73.33±8.17)%，對照組大鼠從(7.80±6.70)%遞增到(34.44±5.27)%，正常組大鼠從(6.57±5.48)%遞增到(33.33±5.0)%，均隨著刺激強(qiáng)度增強(qiáng)而增加。每個(gè)刺激強(qiáng)度CP組大鼠的陽性反應(yīng)次數(shù)百分率均顯著高于對照組及正常組，差異有統(tǒng)計(jì)學(xué)意義(P值均<0.05)，而對照組與正常組間的差異均無統(tǒng)計(jì)學(xué)意義。

三、大鼠大腦ACC區(qū)NR2A、NR2B蛋白表達(dá)量的變化

CP組、對照組、正常組大鼠大腦ACC區(qū)NR2A蛋白表達(dá)量分別為1.25±0.78、0.95±0.14、0.91±0.09；NR2B蛋白表達(dá)量為1.44±0.12、0.93±0.08、0.94±0.04。CP組顯著高于對照組及正常組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)，而對照組與正常組的差異無統(tǒng)計(jì)學(xué)意義(P>0.05，圖2)。

圖2 正常組(1)、CP組(2)、對照組(3)大鼠大腦ACC區(qū)NR2A和NR2B蛋白表達(dá)

四、大鼠大腦ACC區(qū)NR2A、NR2B mRNA表達(dá)量的變化

CP組、對照組、正常組大鼠大腦ACC區(qū)NR2A mRNA表達(dá)量分別為1.43±0.20、0.80±0.10、1.01±0.13；NR2B mRNA表達(dá)量為1.40±0.09、0.98±0.14、0.94±0.05。CP組顯著高于對照組及正常組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)，而對照組與正常組的差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。

討 論

CP為臨床常見胰腺疾病，疼痛作為其主要臨床表現(xiàn)之一，是部分患者就診的首發(fā)癥狀并反復(fù)就醫(yī)的原因，但目前公認(rèn)的CP疼痛機(jī)制尚未明確。近年來的研究發(fā)現(xiàn)，CP患者大腦杏仁核和ACC等處的血流增加，參與疼痛加工的大腦皮質(zhì)厚度顯著變薄，且皮質(zhì)厚度與疼痛評分呈正相關(guān)[11-12]。

ACC區(qū)是疼痛感知的關(guān)鍵皮層區(qū)域，其本身可參與疼痛傳遞和調(diào)節(jié)過程，還可通過調(diào)節(jié)多種疼痛相關(guān)受體參與痛覺行為的反應(yīng)過程。NMDA受體在ACC區(qū)分布廣泛，可興奮突觸后傳遞,并在神經(jīng)可塑性中起重要作用，與記憶形成、慢性疼痛等多種中樞功能有關(guān)，其中NR2A、NR2B在多種慢性疼痛中表達(dá)明顯增高[13-15]。有研究表明，通過慢病毒感染沉默ACC區(qū)NR2B表達(dá)可減弱ACC神經(jīng)元的興奮性，顯著緩解疼痛反應(yīng)[16]。NMDA受體依賴長時(shí)程增強(qiáng)效應(yīng)(long-termpotentiation, LTP)是ACC長期可塑性的主要形式，與感覺傳遞和感知相關(guān)的中樞區(qū)域的突觸傳遞一起被認(rèn)為是慢性疼痛的關(guān)鍵細(xì)胞機(jī)制。在外周刺激長時(shí)間作用下，突觸后NMDA受體觸發(fā)ACC的錐體神經(jīng)元中的LTP，通過激活谷氨酸受體，增加鈣離子通透性。突觸后神經(jīng)元內(nèi)鈣離子濃度升高，激活鈣依賴的信號(hào)通路，正反饋引起谷氨酸及谷氨酸受體的表達(dá)上調(diào)，增強(qiáng)興奮性突觸傳遞，形成LTP，導(dǎo)致慢性內(nèi)臟痛的形成與增強(qiáng)[17-18]。

本研究結(jié)果顯示，CP大鼠中樞ACC區(qū)域NR2A、NR2B表達(dá)上調(diào)，可增強(qiáng)ACC神經(jīng)元的興奮性，促進(jìn)傷害性信號(hào)傳遞，參與CP疼痛的形成。至于NMDA受體依賴LTP的具體機(jī)制有待進(jìn)一步研究。

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NR2AandNR2Bexpressionchangesinanteriorcingulatecortexofchronicpancreatitisratsanditssignificance

ZhouWen,GaoJun,LiGuixiang,LiZhaoshen.

DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

LiZhaoshen,Email:zhsli@81890.net

ObjectiveTo observe the changes of expression levels of NMDA receptor NR2A and NR2B in anterior cingulate cortex (ACC) of chronic pancreatitis(CP) rats and explore its roles in the pathogenesis of pain in CP.MethodsMale adult SD rats were randomly divided into CP group, control group and normal group using random number method. CP rat model was established by injecting 8mg/kg dibutyltin dichloride (DBTC) through tail vein. Control group was injected with a equal volume mixture of ethanol and glycerol via tail vein. After 4 weeks, von Frey hair of different sizes (3.85, 5.50, 7.05, 10.4, 17.8 g) were used to stimulate the abdomen of the rats and the percentage of the positive pain response was recorded. Then the rats were sacrificed. The pancreas was collected for pathological examination. NR2A and NR2B subunit mRNA and protein expression in ACC was detected by realtime-PCR and Western Blotting, respectively.ResultsThe success rate of CP model establishment was 60%. As Van Frey hair used to stimulate the ratabdomen increased from 3.85 g to 17.8 g, the percentage of positive pain response increased from (38.33±7.53)% to(73.33±8.17)% in CP group，from (7.80±6.70)% to (34.44±5.27)% in control group，from(6.57±5.48)% to(33.33±5.00)% in normal group, which was increased with the increase of the stimulation intensity. For each stimulation intensity, the percentage of positive pain response in CP group was all obviously higher than those in control and normal group, and the differences were all statistically significant (allP<0.01), but there was no statistical difference between control group and normal group. The protein expression of NR2A of ACC in CP group, control group and normal group was 1.25±0.78, 0.95±0.14 and 0.91±0.09, respectively. The protein of NR2B in three groups were 1.44±0.12, 0.93±0.08 and 0.94±0.04, respectively.NR2A and NR2B protein expressions in the CP group were both significantly higher than those in control group and normal group, and the difference was statistically significant (P<0.01). The mRNA expression of NR2A in three groups were 1.43±0.20, 0.80±0.10 and 1.01±0.13, respectively. The mRNA expression of NR2B in three groups were 1.40±0.09, 0.98±0.14 and 0.94±0.05, respectively. The mRNA expressions of NR2A and NR2B in CP group were significantly higher than those in normal and control group, and the difference was statistically significant (P<0.01).ConclusionsThe expression of glutamate receptor NR2A and NR2B in ACC were upregulated in CP rats and may be involved in the development of the pain.

Pancreatitis, chronic; Pain; Cerebral cortex; Receptors, N-methyl-D-aspartate

FundprogramNational Natural Science Foundation of China(81270540)

10.3760/cma.j.issn.1674-1935.2017.06.003

200433 上海，第二軍醫(yī)大學(xué)長海醫(yī)院消化內(nèi)科

李兆申，Email:zhsli@81890.net

國家自然科學(xué)基金(81270540)

2017-04-26)

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