楊 元，袁正洲，呂志宇，張淑江，李曉紅，李作孝

(西南醫(yī)科大學(xué)附屬醫(yī)院神經(jīng)內(nèi)科，四川 瀘州 646000)

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血管活性腸肽(VIP)對實(shí)驗(yàn)性自身免疫性腦脊髓炎(EAE)大鼠腦組織IL-17A含量的影響

楊 元，袁正洲，呂志宇，張淑江，李曉紅，李作孝

(西南醫(yī)科大學(xué)附屬醫(yī)院神經(jīng)內(nèi)科，四川 瀘州 646000)

目的 探討血管活性腸肽(vasoactive intestinal peptide, VIP)對實(shí)驗(yàn)性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis,EAE)大鼠腦組織IL-17A含量的影響。方法 60只健康雌性Wistar大鼠隨機(jī)分成正常對照組、EAE對照組、VIP低劑量防治組和VIP高劑量防治組。利用髓鞘堿性蛋白(MBP)+完全福氏佐劑(CFA)誘導(dǎo)建立EAE模型。自造模當(dāng)日起，每隔一日分別對VIP低、高劑量防治組大鼠腹腔注射VIP 4 nmol/kg(0.2 mL)、16 nmoL/kg(0.8 mL)，正常對照組及EAE對照組注射0.8 mL生理鹽水，連續(xù)10 d。觀察大鼠發(fā)病情況；ELISA法檢測腦組織勻漿中IL-17A因子含量變化；免疫組化技術(shù)，利用抗膠質(zhì)纖維酸性蛋白抗體(GFAP)檢測腦組織內(nèi)的星型膠質(zhì)細(xì)胞活化情況。結(jié)果 VIP各劑量防治組大鼠發(fā)病潛伏期延長、進(jìn)展期縮短、發(fā)病高峰期神經(jīng)功能障礙評分(NDS)降低，腦組織勻漿中IL-17A含量降低，活化的星型膠質(zhì)細(xì)胞即GFAP+細(xì)胞量減少，且各劑量組間存在一定劑量依賴關(guān)系。結(jié)論 VIP通過降低腦組織中IL-17A含量、抑制星型膠質(zhì)細(xì)胞活化，發(fā)揮對EAE的防治作用。

血管活性腸肽；實(shí)驗(yàn)性自身免疫性腦脊髓炎；IL-17A；GFAP+星型膠質(zhì)細(xì)胞

多發(fā)性硬化(multiple sclerosis, MS)作為一種中樞神經(jīng)系統(tǒng)(central nervous system，CNS)的自身免疫性疾病，其病因尚不明確，各種病因通過破壞Th1/Th2細(xì)胞免疫平衡，最終導(dǎo)致CNS炎細(xì)胞浸潤、白質(zhì)脫髓鞘、軸突變性等病理改變[1,2]。隨著對其動(dòng)物模型實(shí)驗(yàn)性自身免疫性腦脊髓炎(EAE)研究深入，越來越多的證據(jù)表明Th17細(xì)胞分泌的IL-17A因子在該疾病發(fā)病過程中也發(fā)揮重要的作用[3-5]。血管活性腸肽(vasoactive intestinal peptide, VIP)是一種非膽堿能非腎上腺能神經(jīng)遞質(zhì)，由28個(gè)氨基酸殘基組成，屬于胰高血糖素-胰泌素家族[6，7]。研究表明VIP通過誘導(dǎo)免疫細(xì)胞增殖、分化、活化及遷移，抑制炎癥因子(IL-17A、IFN-γ、NO等)的合成及分泌，發(fā)揮免疫調(diào)節(jié)的作用[8]。國內(nèi)外有將VIP運(yùn)用在類風(fēng)濕關(guān)節(jié)炎(RA)的治療報(bào)道[9，10]，但尚未明確闡明VIP對MS發(fā)病中各種免疫細(xì)胞因子的具體作用，因此本實(shí)驗(yàn)通過探討VIP對EAE大鼠腦組織IL-17A含量影響、星型膠質(zhì)細(xì)胞活化影響，為臨床應(yīng)用VIP治療MS提供理論依據(jù)。

1 材料和方法

1.1 主要實(shí)驗(yàn)動(dòng)物、藥物及試劑

健康雌性Wistar大鼠60只(200 g～250 g/只、6～8周齡)、健康豚鼠10只(250 g～300 g)/只，均購買于西南醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心，均為一級合格動(dòng)物【生產(chǎn)許可證號：SCXK(川)2013-17】(喂食清潔飼料和自來水，每日按時(shí)更換墊料，保持動(dòng)物房的清潔、適宜的溫度和濕度等，使用許可證號【SYXK(川)2013-181】)；血管活性腸肽(北京博奧森生物技術(shù)有限公司)；完全弗氏佐劑CFA(美國Sigma公司產(chǎn)品)；IL-17A ELISA免疫試劑盒(廣州雅怡生物科技有限公司 )；兔抗GFAP多克隆抗體(1:160)(上海雅吉生物科技有限公司)；SP免疫組化試劑盒(武漢博士德生物技術(shù)公司)。

1.2 實(shí)驗(yàn)方法

1.2.1 免疫抗原的制備：斷頸處死10只豚鼠后，在無菌條件下迅速分離出脊髓，去脊膜，稱重后移入玻璃勻漿器，與等量的0℃生理鹽水混合后研磨成50%的全脊髓勻漿(WSCH)。將完全弗氏佐劑(CFA)與WSCH等體積混合，玻璃注射器抽打至油包水乳狀，以滴在水面不散即為合格抗原。

1.2.2 實(shí)驗(yàn)動(dòng)物分組、制模及藥物干預(yù)：將60只雌性Wistar大鼠隨機(jī)分為四組(15只/組)：正常對照組、EAE對照組、VIP低劑量防治組、VIP高劑量防治組。將免疫抗原注入EAE對照組、VIP低、高劑量防治組大鼠雙側(cè)后肢足掌皮下(0.2 mL/100 g)進(jìn)行主動(dòng)免疫，正常對照組給予等量生理鹽水+CFA。自造模當(dāng)日起，VIP低、高劑量防治組每隔一日分別腹腔注射VIP 4 nmoL/kg (0.2 mL)、16 nmoL/kg (0.8 mL)，正常對照組及EAE對照組給予腹腔注射0.8 mL NS，連續(xù)10 d。

1.2.3 動(dòng)態(tài)觀察實(shí)驗(yàn)動(dòng)物：自造模日起，每日早晨10 時(shí) 由同一觀察者采用Kono’s評分標(biāo)準(zhǔn)對大鼠進(jìn)行神經(jīng)功能讓障礙評分(NDS)。在發(fā)病高峰期(將連續(xù)3 d評分無加重、四肢癱瘓或死亡作為大鼠發(fā)病高峰期)處死大鼠，未發(fā)病大鼠飼養(yǎng)8周后處死。

1.2.4 檢測腦組織勻漿中IL-17A含量變化：采用雙抗體夾心(ABC-ELISA)法檢測腦組織勻漿中IL-17A含量。

1.2.5 檢測腦組織中星型膠質(zhì)細(xì)胞的活化情況：利用免疫組織化學(xué)(SP)技術(shù)，采用抗膠質(zhì)纖維酸性蛋白抗體(GFAP)檢測各組大鼠腦組織內(nèi)的星型膠質(zhì)細(xì)胞活化情況。

將大鼠腦組織行石蠟切片后常規(guī)脫蠟、水化、清除內(nèi)源性過氧化物酶活性、封閉組織蛋白后，逐次滴加一抗(以1∶160 稀釋的兔抗GFAP多克隆抗體)、生物素標(biāo)記的二抗 (山羊抗兔IgG抗體)、鏈霉卵白素(辣根酶標(biāo)記)，DAB顯色液，復(fù)染，脫水，透明，封片。400倍光鏡下觀察大鼠腦組織GFAP+細(xì)胞表達(dá)情況，胞質(zhì)呈棕黃色為陽性細(xì)胞。采用ImagePro Plus 軟件分析GFAP+細(xì)胞的平均光密度值(IOD)，對GFAP+表達(dá)行半定量測定。

2 統(tǒng)計(jì)學(xué)方法

3 結(jié)果

3.1 各組大鼠的發(fā)病情況

正常對照組大鼠未發(fā)病，EAE對照組、VIP各劑量組大鼠均出現(xiàn)不同程度的臨床癥狀：發(fā)病大鼠先后表現(xiàn)為食量下降，精神萎靡，后肢無力，尾部拖地，在免疫后第14～17天病情癥狀最為典型：大鼠出現(xiàn)雙側(cè)后肢無力拖地、行走畫圈、爬行困難、自傷后肢、精神極度衰弱、抽搐、甚至死亡。但與EAE對照組比較，VIP低、高劑量組大鼠癥狀明顯緩解，表現(xiàn)為發(fā)病潛伏期時(shí)間延長、進(jìn)展期時(shí)間縮短、發(fā)病高峰期NDS降低。見表1。

3. 2 各組大鼠腦組織中IL-17的含量變化

同正常對照組比較，EAE對照組、VIP各劑量組大鼠腦組織勻漿中IL-17A含量升高；與EAE對照組比較，VIP各劑量組大鼠腦組織勻漿中IL-17A含量明顯降低；與VIP低劑量組比較，VIP高劑量組腦組織勻漿中IL-17A含量明顯降低。說明在IL-17A在EAE發(fā)病過程中起促進(jìn)作用；同時(shí)VIP可以降低EAE大鼠腦組織勻漿中IL-17A含量，且呈一定劑量依賴關(guān)系。見表2。

3.3 各組大鼠腦組織中星型膠質(zhì)細(xì)胞活化情況

正常對照組大鼠腦組織未見GFAP+細(xì)胞表達(dá)；EAE對照組大鼠腦組織內(nèi)活化的GFAP+細(xì)胞呈彌漫性分布；VIP各劑量組大鼠腦組織GFAP+細(xì)胞明顯減少，主要延血管周圍分布。見表3，圖1。

Tab.1 Comparison of the incubation period, progression period and peak neurological dysfunction scores in the EAE control group and each dose VIP treatment group

組別Groups潛伏期(d)incubationperiod進(jìn)展期(d)ProgressionperiodNDS(min)NDSscoresEAE對照組Contol10.34±2.137.85±1.654.16±0.77VIP低劑量組LowdoseVIP14.17±1.67★6.20±1.97☆3.75±0.98ΔVIP高劑量組HighdoseVIP20.73±1.98★●3.99±1.57★○2.59±0.58★●F37.4828.5418.95P<0.01<0.05<0.01

注： 與EAE對照組比較，★P<0.01、☆P<0.05、ΔP>0.05; 與VIP低劑量組比較，●P<0.01、○P<0.05.

Note.★P<0.01,☆P<0.05 andΔP>0.05, Compared with the control group.●P<0.01,○P<0.05, Compared with the low-dose VIP group.

Tab.2 Content of IL-17A in the brain tissue of each group rats

組別Groups動(dòng)物數(shù)nIL-17A(pg/mL)正常對照組Normalcontrol1522.84±3.51EAE對照組EAEcontrol1578.26±5.87★VIP低劑量組Low-doseVIP1557.49±4.85★●VIP高劑量組High-doseVIP1530.01±5.59★●▲F101.99P<0.01

注：與正常對照組比較，★P<0.01；與EAE對照組比較，●P<0.01；與VIP低劑量組比較，▲P<0.01。

Note.★P<0.01, compared with the normal control group.●P<0.01, compared with the EAE control group.▲P<0.01, compared with the low-dose VIP group.

Tab.3 The average IOD of GFAP+cells in the brain tissue of EAE control group and each dose VIP group by immunohistochemistry(×10-2)

組別Groups動(dòng)物數(shù)nGFAP+細(xì)胞平均IOD(×10-2)MeanIODofGFAP+cellsEAE對照組EAEcontrol1515.78±1.92VIP低劑量組Low-doseVIP1512.40±1.79☆VIP高劑量組High-doseVIP158.32±1.82★▲F48.09P<0.01

注: 與EAE對照組比較，★P<0.01、☆P<0.05；與VIP低劑量組比較，▲P<0.01。

Note.★P<0.01,☆P<0.05, compared with the EAE control group.▲P<0.01, compared with the low-dose VIP group.

圖1 各組大鼠側(cè)腦室旁腦組織GFAP+細(xì)胞表達(dá)。免疫組化染色，(標(biāo)尺=100 μm)。Fig.1 Expression of GFAP+ cells in the rat lateral ventricle of all group rats. Immunohistochemical staining.

3.4 相關(guān)性分析顯示

利用Pearson 直線相關(guān)軟件分析,結(jié)果顯示發(fā)病大鼠腦組織勻漿中IL-17A因子含量與與發(fā)病高峰期NDS呈正相關(guān) (r=0.798,P<0.05)，與腦組織勻漿中GFAP+細(xì)胞平均IOD呈正相關(guān)(r=0.825,P<0.05)。

4 討論

IL-17A作為一種具強(qiáng)促炎作用的細(xì)胞因子，其主要作用包括：①調(diào)節(jié)固有免疫[11，12]：IL-17A作為炎癥介質(zhì)，參與自身免疫性炎性疾病的發(fā)生和發(fā)展，通過作用于基因靶點(diǎn)產(chǎn)生急性反應(yīng)蛋白、炎癥趨化因子等物質(zhì)，同時(shí)上調(diào)MHC-II抗原、集落共刺激因子、及T細(xì)胞刺激因子、促使T細(xì)胞活化，導(dǎo)致免疫失衡；②信號轉(zhuǎn)導(dǎo)作用[13，14]：IL-17A作為細(xì)胞內(nèi)及細(xì)胞間信使，參與信號的轉(zhuǎn)導(dǎo)過程，可促進(jìn)多種炎性蛋白的轉(zhuǎn)錄及合成，加速免疫紊亂。

星型膠質(zhì)細(xì)胞作為中樞神經(jīng)系統(tǒng)一種非特異性抗原提呈細(xì)胞(APC)，參與復(fù)雜的免疫病理過程[15]。在EAE的炎癥反應(yīng)中，IL-17A通過Act1轉(zhuǎn)錄因子激活星型膠質(zhì)細(xì)胞促進(jìn)釋放炎癥因子，加速髓鞘脫失的發(fā)生發(fā)展[16，17]，同時(shí)活化的星型膠質(zhì)細(xì)胞通過上調(diào)GFAP+的表達(dá)，形成活化的膠質(zhì)斑塊[18]。因此抑制LI-17A產(chǎn)生、干預(yù)星型膠質(zhì)細(xì)胞介導(dǎo)的炎癥過程，是治療EAE的一種有效方法。

本實(shí)驗(yàn)研究發(fā)現(xiàn)：與正常對照組相比，EAE對照組、VIP各劑量組大鼠腦組織勻漿中IL-17A含量升高(P均 <0.01)、腦組織中GFAP+細(xì)胞的平均光密度值(IOD)增加(P<0.01、P<0.05)，表明IL-17A在EAE發(fā)生發(fā)展過程中起促進(jìn)作用。與EAE對照組比較，VIP各劑量組大鼠腦組織勻漿中IL-17A含量均明顯降低(P<0.01)、腦組織中GFAP+細(xì)胞的IOD明顯降低(P<0.01)，且兩者呈正相關(guān)關(guān)系(r=0.825,P<0.05)。表明VIP通過降低發(fā)病大鼠腦組織勻漿中IL-17A含量，可明顯緩解EAE大鼠腦組織星型膠質(zhì)細(xì)胞活化的病理改變,從而減輕EAE的臨床表現(xiàn)。

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Effect of vasoactive intestinal peptide (VIP) on the content of IL-17A in the brain tissue of rats with experimental autoimmune encephalomyelitis (EAE)

YANG Yuan, YUAN Zheng-zhou, LV Zhi-yu, ZHANG Shu-jiang, LI Xiao-hong, LI Zuo-xiao

(Department of Neurology. Affiliated Hospital of Southwest Medical University，Luzhou Sichuan 646000, China)

Objective To explore the effect of vasoactive intestinal peptide (VIP) on the content of IL-17A in the brain tissue of rat models of experimental autoimmune encephalomyelitis (EAE). Methods Sixty healthy female Wistar rats were randomly divided into normal control group, EAE control group, low-dose VIP group and high-dose VIP group. Ten healthy guinea pigs were used to prepare anti- IL-17A antibody. Myelin basic protein (MBP) + complete adjuvant (CFA) were used to establish the EAE model. Since the first day of modelling, the low-dose and high-dose VIP groups received intraperitoneal injection of VIP 4 nmol/kg (0.2 mL) and 16 nmol/kg (0.8 mL), respectively, every other day for 10 consecutive days. The normal control group and EAE group were injected with 0.8 mL saline instead of VIP. The incubation period, progression and the peak of neurological dysfunction scores (NDS) of the rats were recorded. The levels of IL-17A in the brain tissue was determined by ELISA assay, and the GFAP+astrocyte activation in brain at morbidity peak in the rats was examined using anti-GFAP (glial fibrillary acidic protein) antibodies. Results The incubation period were extended, the progression period was shortened and the peak neuological dysfunction score (NDS) was decreased in the VIP-treated groups, in a dose-response relationship. The cytokine levels of IL-17A and the astrocyte activation degree in brain tissue were reduced in each VIP dose group, in a dose-response relationship. Conclusions VIP exerts therapeutic effect on experimental autoimmune encephalomyelitis through lowering the IL-17A content and inhibition of astrocyte activation in the brain tissue.

Vasoactive intestinal peptide; Experimental autoimmune encephalomyelitis; IL-17A; GFAP+astrocytes; Rats; Guinea pigs

楊元，女，碩士，E-mail: lsh880801@163.com，電話：18715760908。

李作孝，男，教授，碩士生導(dǎo)師，E-mail: lzx3235@sina.cn，電話：13882794776。

R-33

A

1671-7856(2016)10-0032-04

10.3969.j.issn.1671-7856. 2016.10.007

2016-05-06