杭　航，　王麗琨，　伍國鋒，　陳星宇

(貴州醫(yī)科大學(xué)附屬醫(yī)院急診醫(yī)學(xué)科/急診醫(yī)學(xué)教研室， 貴州 貴陽 550004)

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羅格列酮預(yù)處理對(duì)凝血酶激活的小膠質(zhì)細(xì)胞PPARγ、Nrf2及HO-1表達(dá)的影響*

杭航，王麗琨，伍國鋒△，陳星宇

(貴州醫(yī)科大學(xué)附屬醫(yī)院急診醫(yī)學(xué)科/急診醫(yī)學(xué)教研室， 貴州 貴陽 550004)

目的： 采用凝血酶激活新生大鼠神經(jīng)膠質(zhì)細(xì)胞，觀察羅格列酮預(yù)處理對(duì)小膠質(zhì)細(xì)胞過氧化物酶體增殖物活化受體γ(PPARγ)、核因子E2相關(guān)因子2(Nrf-2)及血紅素加氧酶-1(HO-1)表達(dá)的影響。方法: 用新生SD大鼠的腦組織，體外培養(yǎng)原代小膠質(zhì)細(xì)胞14 d左右分離收集細(xì)胞，分為：正常對(duì)照組、凝血酶刺激組、羅格列酮干預(yù)組(羅格列酮+凝血酶組)和維甲酸干預(yù)組(維甲酸+凝血酶組)進(jìn)行實(shí)驗(yàn)。分別采用免疫組化染色、real-time PCR和Western blot檢測(cè)PPARγ、Nrf2和HO-1的表達(dá)并進(jìn)行統(tǒng)計(jì)分析。結(jié)果: 免疫組化染色顯示，與對(duì)照組比較，刺激組、羅格列酮+凝血酶組及維甲酸+凝血酶組的PPARγ、Nrf2和HO-1染色細(xì)胞數(shù)均增多。Real-time PCR結(jié)果顯示羅格列酮+凝血酶組PPARγ、Nrf2及HO-1的mRNA表達(dá)均顯著高于刺激組、對(duì)照組及維甲酸+凝血酶組(P<0.01)，維甲酸+凝血酶組Nrf2及HO-1的mRNA表達(dá)均較刺激組和羅格列酮+凝血酶組降低(P<0.01)。Western blot 結(jié)果顯示，羅格列酮+凝血酶組PPARγ、Nrf2及HO-1的 蛋白表達(dá)也明顯高于刺激組、對(duì)照組及維甲酸+凝血酶組(P<0.01)，維甲酸+凝血酶組Nrf2及HO-1的蛋白表達(dá)均較刺激組和羅格列酮+凝血酶組降低(P<0.01)。結(jié)論: 羅格列酮預(yù)處理后可增加凝血酶激活的小膠質(zhì)細(xì)胞PPARγ、Nrf2及HO-1的表達(dá)，通過維甲酸預(yù)處理抑制Nrf2的表達(dá)后，其下游基因HO-1表達(dá)也受影響，說明PPARγ抗氧化作用可能是通過Nrf2調(diào)控下游基因?qū)崿F(xiàn)的。

小膠質(zhì)細(xì)胞； 腦出血； 羅格列酮； 過氧化物酶體增殖物活化受體γ； 核因子E2相關(guān)因子2； 血紅素加氧酶-1

腦出血占腦卒中發(fā)病率的8%～15%，其病理生理過程包括腦實(shí)質(zhì)中血腫的形成及繼發(fā)性腦損害，其發(fā)病率、死亡率及致殘率均高居不下，目前尚無特效的治療方案[1]。腦出血后凝血系統(tǒng)被激活，所釋放的凝血酶(thrombin,TH)主要通過破壞血腦屏障、造成神經(jīng)損傷以及參與炎癥反應(yīng)這幾方面起作用，而產(chǎn)生一些細(xì)胞毒性物質(zhì)，通過激活小膠質(zhì)細(xì)胞和巨噬細(xì)胞產(chǎn)生氧自由基，從而誘發(fā)氧化應(yīng)激反應(yīng)，造成繼發(fā)性腦損傷[2-4]。過氧化物酶體增殖物活化受體γ(peroxisome proliferator-activated receptor γ，PPARγ)在激活小膠質(zhì)細(xì)胞/巨噬細(xì)胞和降低氧化應(yīng)激方面都起著非常重要的作用[5]，增加PPARγ的作用將有助于提高小膠質(zhì)細(xì)胞/巨噬細(xì)胞的吞噬功能及降低氧化應(yīng)激反應(yīng)從而減輕繼發(fā)性腦損害。不少研究已經(jīng)發(fā)現(xiàn)PPARγ激動(dòng)劑可減輕繼發(fā)性腦損傷，同時(shí)有一定的神經(jīng)細(xì)胞保護(hù)作用[6-7]。羅格列酮(rosiglitazone,RGZ)是PPARγ的有效激動(dòng)劑[8]，腦出血后使用RGZ激活PPARγ可在轉(zhuǎn)錄水平上調(diào)抗氧化劑和銅鋅超氧化物歧化酶[9-10]，如血紅素加氧酶-1(heme oxygenase 1，HO-1)，從而增加細(xì)胞的氧化應(yīng)激能力。核因子E2相關(guān)因子2(nuclear factor E2-related factor 2，Nrf2)是氧化應(yīng)激的感受器，參與體內(nèi)氧化應(yīng)激反應(yīng)，是氧化應(yīng)激重要的轉(zhuǎn)錄激活因子。腦出血后PPARγ表達(dá)增加[8]， Nrf2表達(dá)也增高，參與腦內(nèi)氧化應(yīng)激反應(yīng)[11]。本研究組先前的工作發(fā)現(xiàn)，羅格列酮預(yù)處理可使凝血酶刺激后的小膠質(zhì)細(xì)胞PPARγ及HO-1表達(dá)增加[12]，然而這些變化是否與Nrf2有關(guān)聯(lián)尚不清楚。

本實(shí)驗(yàn)的目的在于采用羅格列酮預(yù)處理小膠質(zhì)細(xì)胞，觀察凝血酶激活的小膠質(zhì)細(xì)胞PPARγ、Nrf2和HO-1表達(dá)的影響，并初步探討其相互關(guān)系。

材　料　和　方　法

1實(shí)驗(yàn)材料

1.1動(dòng)物來源健康SD大鼠乳鼠，雌雄均可，1～3日齡，由貴州醫(yī)科大學(xué)動(dòng)物實(shí)驗(yàn)中心提供。

1.2主要試劑與材料北美胎牛血清(fetal bovine serum，F(xiàn)BS)、DMEM/F12培養(yǎng)基、胰酶(含EDTA，0.25%)、青霉素-鏈霉素溶液及GlutaMax(Gibco)；多聚L-賴氨酸、DNase I和羅格列酮(Sigma)；TH(湖南一格公司)；OX42抗體(Abcam)；4%多聚甲醛、山羊抗兔Ⅱ抗FITC、封閉山羊血清和抗熒光衰減封片劑(索萊寶公司)；DAPI染色液(泛博公司)；PPARγ、Nrf2和HO-1抗體(Proteintech)；哺乳動(dòng)物蛋白抽提試劑盒、SDS-PAGE上樣緩沖液、Tris-Glycine轉(zhuǎn)膜緩沖液和Tris-Glycine SDS電泳緩沖液(康為世紀(jì)公司)；cDNA合成試劑盒和real-time PCR試劑盒(Thermo)。

2實(shí)驗(yàn)方法

2.1原代細(xì)胞培養(yǎng)新生1～3 d的SD大鼠(110只)，由貴州醫(yī)科大學(xué)動(dòng)物實(shí)驗(yàn)中心提供。將出生1～3 d的SD大鼠無菌條件下處死，取出腦組織，剝離腦膜和血管，用0.25%的胰酶消化液消化后過濾離心，用完全培養(yǎng)基進(jìn)行細(xì)胞沉淀懸浮后計(jì)數(shù)細(xì)胞，以2×105個(gè)接種至培養(yǎng)瓶?jī)?nèi)。24 h后全量換液并根據(jù)細(xì)胞代謝情況，約3 d換液1次，繼續(xù)培養(yǎng)至14 d左右，采用恒溫?fù)u床振搖法分離細(xì)胞，將上層培養(yǎng)液收集后以4×104/cm2的密度接種至預(yù)先用0.01%的多聚賴氨酸包被好的培養(yǎng)板中培養(yǎng)。24 h后細(xì)胞貼壁完全，用小膠質(zhì)細(xì)胞特異性抗體OX42進(jìn)行免疫細(xì)胞化學(xué)染色鑒定純度。陽性率>95%即可用于后續(xù)實(shí)驗(yàn)。

將培養(yǎng)成功的細(xì)胞，隨機(jī)分為4個(gè)組，分別為正常對(duì)照組、凝血酶刺激組、羅格列酮干預(yù)組(RGZ+TH組)和維甲酸干預(yù)組(RA+TH組)。給藥方案：對(duì)照組加入與刺激組所加凝血酶等體積的培養(yǎng)基，刺激組加入凝血酶(2×104U/L)處理24 h，RGZ+TH組予25 μmol/L的羅格列酮對(duì)小膠質(zhì)細(xì)胞預(yù)處理1 h,之后加入凝血酶(2×104U/L)，共同作用小膠質(zhì)細(xì)胞24 h，RA+TH組用維甲酸(1 μmol/L)預(yù)處理1 h后再用凝血酶(2×104U/L)刺激小膠質(zhì)細(xì)胞24 h。

2.2原代小膠質(zhì)細(xì)胞的鑒定將分離后的細(xì)胞接種于細(xì)胞爬片上24 h，經(jīng)PBS液洗滌，用4%的多聚甲醛固定15 min后，用0.25% Triton通透20 min，PBS洗3遍，每次3 min，10%正常山羊血清封閉；加入I抗為小鼠抗大鼠CD11b/c 單克隆抗體(1∶50)，37 ℃孵育2 h，PBS洗3遍，每次3 min；加入II抗為山羊抗小鼠FITC (1∶100)37 ℃避光孵育1 h，PBS洗3遍，每次3 min；DAPI染核5 min，最后加入抗熒光淬滅封片劑封片。用激光共聚焦顯微鏡觀察、攝片。以上染色均用PBS及正常牛血清代替I抗作為對(duì)照染色。2.3免疫細(xì)胞化學(xué)染色觀察PPARγ、Nrf2和HO-1的表達(dá) 分別將對(duì)照組、刺激組、RGZ+TH組和RA+TH組4組已爬片細(xì)胞予PBS液洗滌，4%多聚甲醛固定15 min,再用0.25% Triton通透20 min，PBS洗滌，10%正常山羊血清封閉；加入I 抗為PPARγ抗體(1∶50)，4 ℃孵育過夜，PBS洗3遍，每次3 min；加入 II抗為山羊抗小鼠FITC (1∶100)37 ℃避光孵育1 h，PBS洗3遍，每次3 min；DAPI染核5 min，最后加入抗熒光淬滅封片劑封片。用激光共聚焦顯微鏡觀察、攝片。以上染色均用PBS及正常牛血清代替I抗作為對(duì)照染色。同樣方法，將I抗換成Nrf2和HO-1抗體(1∶50)，觀察各組Nrf2和HO-1變化情況。2.4Real-time PCR檢測(cè)PPARγ、Nrf2和HO-1的mRNA表達(dá)將分離后的原代培養(yǎng)的小膠質(zhì)細(xì)胞以每孔1×106個(gè)接種于6孔板，培養(yǎng)24 h后，分為4組處理。處理后的細(xì)胞用預(yù)冷PBS洗2次后，加入TRIzol 1 mL裂解細(xì)胞，用槍頭充分吹打混勻。室溫放置后，加入氯仿，劇烈振蕩，再次室溫靜置后，4 ℃、12 000 r/min離心10 min；吸取上清加入異丙醇沉淀RNA，輕輕混勻，室溫靜置；以12 000 r/min、4 ℃離心10 min；棄上清， 75%乙醇洗滌RNA 2次， 4 ℃、7 500 r/min 離心5 min，棄上清，室溫放置干燥5 min；加入20 μL DEPC水，用槍頭吹打充分； 取2 μL RNA樣品于ND2000下測(cè)核酸濃度以及A260/A280比值以了解RNA純度，比值在1.8～2.2者可用于下一步實(shí)驗(yàn)；根據(jù)A260值計(jì)算RNA含量。將提出的RNA按逆轉(zhuǎn)錄試劑盒說明書進(jìn)行逆轉(zhuǎn)錄成cDNA，PPARγ、Nrf2及HO-1引物(上海生工設(shè)計(jì)并合成)序列見表1，以β-actin為內(nèi)參照，按照PCR反應(yīng)說明書進(jìn)行PCR反應(yīng)。

表1　引物序列

2.5蛋白印跡法檢測(cè)PPARγ、Nrf2和HO-1的蛋白表達(dá) 根據(jù)細(xì)胞覆蓋率按照一定比例加入細(xì)胞抽提試劑，吹打均勻，吸出蛋白液至1.5 mL EP管中，冰上孵育20 min，10 000 r/min離心20 min后收集上清，-80 ℃冰箱保存。蛋白定量后取4倍體積樣品緩沖液，95 ℃變性10 min。蛋白上樣量為每孔30 μg，10% SDS-PAGE 電泳，將蛋白電轉(zhuǎn)移至0.45 μm PVDF膜上，依分子量大小切取條帶，加入封閉液室溫下震蕩2 h 后，取相應(yīng)條帶分別加入 I 抗兔抗鼠PPARγ多克隆抗體(1∶500)、兔抗鼠Nrf2多克隆抗體(1∶200)和兔抗鼠HO-1多克隆抗體(1∶200)4 ℃過夜。室溫孵育1 h后， 分別加入 II 抗(生物素標(biāo)記羊抗兔IgG，1∶200)室溫孵育1 h,將孵育好 II 抗的膜在漂洗液中洗滌5次，每次5 min。ECL反應(yīng)5 min，曝光，顯影定影。經(jīng)X膠片曝光顯影。圖片掃描保存電腦文件。

3統(tǒng)計(jì)學(xué)處理

使用SPSS 13.0 統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。計(jì)量資料數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。多樣本均數(shù)比較采用單因素方差分析，其中滿足方差齊性的采用Fisher方差分析，方差不齊的采用近似F檢驗(yàn)Welch法；多樣本均數(shù)間兩兩比較滿足方差齊性的采用SNK法檢驗(yàn)，方差不齊的采用Dunnett’s T3檢驗(yàn)。以P<0.05 為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)　　果

1細(xì)胞培養(yǎng)結(jié)果

膠質(zhì)細(xì)胞混合培養(yǎng)至9 d左右細(xì)胞分層明顯，相差顯微鏡下觀察底層細(xì)胞充分鋪展，緊密接觸，為星形膠質(zhì)細(xì)胞，其上層主要為散在分布的阿米巴樣細(xì)胞，胞體圓形，透光性較底層細(xì)胞好，經(jīng)振搖法分離出來的小膠質(zhì)細(xì)胞產(chǎn)量為1.2×106個(gè)，存活率≥95%， OX42染色陽性，陽性細(xì)胞(純度)≥95%，見圖1。

2PPARγ的表達(dá)

2.1免疫組化結(jié)果對(duì)照組、刺激組、RGZ+TH組及RA+TH組PPARγ均著色，對(duì)照組PPARγ可見胞質(zhì)均勻著色(綠色)，DAPI染核可見藍(lán)色；刺激組PPARγ染色細(xì)胞數(shù)較對(duì)照組增多，胞質(zhì)著色加深；RGZ+TH組PPARγ染色細(xì)胞數(shù)較對(duì)照組和刺激組明顯增多，且以細(xì)胞核染色為顯著，說明羅格列酮干預(yù)后可促進(jìn)PPARγ表達(dá)增多并且使得其表達(dá)向核內(nèi)轉(zhuǎn)移， RA+TH組PPARγ染色細(xì)胞數(shù)與刺激組無明顯差別，部分細(xì)胞著色在胞核加深，見圖2。

Figure 1.The expression of OX42 detected by immunofluorescence for identification of microglia (×200).

圖1免疫組化法檢測(cè)OX42的表達(dá)鑒定小膠質(zhì)細(xì)胞

Figure 2.PPARγ expression in the microglias observed by immunofluorescence (×400).

圖2免疫組化檢測(cè)PPARγ的表達(dá)

2.2PPARγ的mRNA表達(dá)4組間PPARγ的mRNA表達(dá)量相對(duì)值差異具有統(tǒng)計(jì)學(xué)意義(P<0.01)；刺激組與RA+TH組之間差異無統(tǒng)計(jì)學(xué)顯著性，其余組兩兩間比較差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，見圖3。

2.3PPARγ蛋白的表達(dá)4組間PPARγ蛋白表達(dá)量相對(duì)值差異有統(tǒng)計(jì)學(xué)顯著性(P<0.01)；刺激組與RA+TH組之間差異無統(tǒng)計(jì)學(xué)顯著性,其余組兩兩間比較差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05),見圖4。

3Nrf2的表達(dá)

3.1免疫組化結(jié)果 對(duì)照組Nrf2染色為陰性，F(xiàn)ITC可見幾乎沒有細(xì)胞著色，DAPI染色后可見細(xì)胞核，融合后見染色細(xì)胞數(shù)低；刺激組Nrf2染色細(xì)胞數(shù)較對(duì)照組明顯增多，胞質(zhì)呈綠色，著色均勻，融合后可見細(xì)胞染色率較高；RGZ+TH組Nrf2染色細(xì)胞數(shù)較正常組、刺激組及RA+TH組增多，且胞質(zhì)深染，提示羅格列酮干預(yù)后可促進(jìn)Nrf2的表達(dá)；RA+TH組Nrf2染色細(xì)胞數(shù)及染色程度較RGZ+TH組及刺激組明顯減少，見圖5。

Figure 3.The mRNA expression of PPARγ in the microglia with different treatments. Mean±SD.n=20.**P<0.01vscontrol;##P<0.01vsRGZ+TH.

圖3Real-time PCR檢測(cè)PPARγ的mRNA表達(dá)

Figure 4.The protein expression of PPARγ in the microglia with different treatments. Mean±SD.n=10.**P<0.01vscontrol;##P<0.01vsRGZ+TH.

圖4Western blot檢測(cè)PPARγ蛋白的表達(dá)

Figure 5.Nrf2 expression in the microglia observed by immunofluorescence (×400).

圖5免疫組化檢測(cè)Nrf2的表達(dá)

3.2Nrf2的mRNA表達(dá)4組間Nrf2 mRNA表達(dá)量相對(duì)值差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.01)；各組兩兩間比較差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05),見圖6。3.3Nrf2蛋白的表達(dá)4組間Nrf2蛋白表達(dá)量相對(duì)值差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.01)；各組兩兩間比較差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，見圖7。

Figure 6.The mRNA expression of Nrf2 in the microglia with different treatments. Mean±SD.n=10.**P<0.01vscontrol;##P<0.01vsRGZ+TH.

圖6Real-time PCR 檢測(cè)Nrf2 mRNA的表達(dá)

Figure 7.The protein expression of Nrf2 in the microglia with different treatments.Mean±SD.n=10.**P<0.01vscontrol;##P<0.01vsRGZ+TH.

圖7Western blot檢測(cè)Nrf2蛋白的表達(dá)

4HO-1的表達(dá)

4.1免疫組化結(jié)果對(duì)照組HO-1染色為陰性，F(xiàn)ITC可見幾乎沒有細(xì)胞著色，DAPI染色后可見細(xì)胞核，融合后見染色細(xì)胞數(shù)低；刺激組HO-1染色細(xì)胞數(shù)較對(duì)照組明顯增多，胞質(zhì)呈綠色，融合后可見細(xì)胞染色率較高；RGZ+TH組HO-1染色細(xì)胞數(shù)較正常組、刺激組增多，胞質(zhì)、胞核均著色，部分細(xì)胞胞核深染，提示羅格列酮干預(yù)后可促進(jìn)HO-1的表達(dá)，RA+TH組細(xì)胞染色率較RGZ+TH組減少，染色程度也較淺，見圖8。

4.2HO-1的mRNA表達(dá)4組間HO-1 mRNA表達(dá)量相對(duì)值差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.01)；各組兩兩間比較差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05),見圖9。

4.3HO-1蛋白的表達(dá)4組間HO-1蛋白表達(dá)量相對(duì)值差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.01)；各組兩兩間比較差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05),見圖10。

討　　論

綜合上述實(shí)驗(yàn)結(jié)果，提示凝血酶刺激小膠質(zhì)細(xì)胞可誘導(dǎo)PPARγ、Nrf2和HO-1的表達(dá)，而羅格列酮預(yù)處理后PPARγ、Nrf2和HO-1的表達(dá)增加更為顯著，維甲酸預(yù)處理后Nrf2和HO-1的表達(dá)下降。Nrf2和PPARγ均參加腦內(nèi)氧化應(yīng)激反應(yīng)，Nrf2有助于神經(jīng)保護(hù)和腦缺血后腦損傷的改善[13-14]，而腦出血后PPARγ則主要通過減少氧化應(yīng)激、炎癥和解毒的分子的生成，從而中和許多有毒物質(zhì)介導(dǎo)的繼發(fā)性腦傷害。PPARγ在小膠質(zhì)細(xì)胞的激活和降低氧化應(yīng)激方面都起著至關(guān)重要的作用[9]。PPARγ被激活后，一方面可通過轉(zhuǎn)錄水平上調(diào)細(xì)胞表面清道夫受體CD36，來幫助小膠質(zhì)細(xì)胞和巨噬細(xì)胞通過吞噬或內(nèi)吞作用來清除壞死或凋亡的細(xì)胞殘骸[15-16]；另一方面，激活的PPARγ可通過轉(zhuǎn)錄水平上調(diào)抗氧化劑和銅鋅超氧化物歧化酶[5]，如HO-1、SOD等，從而增加細(xì)胞的抗氧化功能。噻唑烷二酮類藥物(如羅格列酮)是PPARγ有效的激動(dòng)劑[17]。本課題組前期研究表明，腦出血后血腫周圍腦組織發(fā)生一系列的病理生理變化，包括谷氨酸含量增加、基質(zhì)金屬蛋白酶表達(dá)增多、血腦屏障通透性升高等，而且使用羅格列酮激活PPARγ從而減輕繼發(fā)性腦損害方面的研究取得了一定的實(shí)驗(yàn)成果[18-23]。然而，對(duì)于使用羅格列酮激活PPARγ減輕氧化應(yīng)激反應(yīng)從而降低腦損傷程度的機(jī)制尚不明確。本實(shí)驗(yàn)中，使用羅格列酮后PPARγ表達(dá)增加，其下游HO-1 mRNA及蛋白表達(dá)均增加，這樣便增加了腦細(xì)胞的抗氧化應(yīng)激能力。

在腦出血后的抗氧化應(yīng)激反應(yīng)過程中，Nrf2可以誘導(dǎo)抗氧化應(yīng)激有關(guān)組件的產(chǎn)生、減少過氧化物的形成、增加CD36的吞噬功能以及增加對(duì)紅細(xì)胞碎片的吞噬作用。近期研究表明，Nrf2在抗氧化應(yīng)激能力的提升、增加吞噬功能以及促進(jìn)血腫吸收方面發(fā)揮重要作用[7]。Nrf2-ARE通路是體內(nèi)內(nèi)源性抗氧化系統(tǒng)中非常重要的一條通路，其中抗氧化反應(yīng)元件(antioxidant response element，ARE)與Nrf2結(jié)合并起著關(guān)鍵作用[24]，而PPARγ和Nrf2的相互作用可能涉及多種機(jī)制。首先，PPAR反應(yīng)元件(PPAR response element, PPRE)和ARE共存在相同的基因，如CD36[25-27]和過氧化氫酶；其次，Nrf2和PPARγ之間存在著相互的轉(zhuǎn)錄調(diào)控，如Nrf2基因包含著PPRE[28]，而PPARγ基因也包含著ARE[29-30]。前期有研究發(fā)現(xiàn)維甲酸可以抑制Nrf2的表達(dá)[31-32]。 本實(shí)驗(yàn)中，凝血酶激活后的小膠質(zhì)細(xì)胞PPARγ和Nrf2增加，其下游基因HO-1也增加，使用羅格列酮后見PPARγ、Nrf2和HO-1增加更為顯著，然而使用維甲酸后，PPARγ表達(dá)與凝血酶激活表達(dá)差異無統(tǒng)計(jì)學(xué)顯著性，Nrf2和HO-1的表達(dá)下降明顯，PPARγ的下游基因是HO-1，因此推斷PPARγ有可能通過Nrf2調(diào)控后者的產(chǎn)生，從而發(fā)揮抗氧化應(yīng)激作用。

Figure 8.HO-1 expression in the microglia observed by immunofluorescence (×400).

圖8免疫組化檢測(cè)HO-1的表達(dá)

Figure 9.The mRNA expression of HO-1 in the microglia with different treatments. Mean±SD.n=10.**P<0.01vscontrol;##P<0.01vsRGZ+TH.

圖9Real-time PCR檢測(cè)HO-1的mRNA表達(dá)

Figure 10.The protein expression of HO-1 in the microglia with different treatments. Mean±SD.n=10.**P<0.01vscontrol；##P<0.01vsRGZ+TH.

圖10Western blot檢測(cè)HO-1蛋白的表達(dá)

綜上所述，凝血酶激活小膠質(zhì)細(xì)胞可以誘導(dǎo)PPARγ、Nrf2和HO-1表達(dá)增加；羅格列酮預(yù)處理后，凝血酶激活的小膠質(zhì)細(xì)胞PPARγ、Nrf2和HO-1的表達(dá)明顯增加；使用維甲酸預(yù)處理后Nrf2下降，其下游基因HO-1也下降；推測(cè)PPARγ可能部分通過Nrf2作用其下游基因，從而發(fā)揮其抗氧化作用，這一課題有待進(jìn)一步研究。

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(責(zé)任編輯： 盧萍， 羅森)

Rosiglitazone pretreatment influences expression of PPARγ, Nrf2 and HO-1 in thrombin-activated microglia

HANG Hang, WANG Li-kun, WU Guo-feng, CHEN Xing-yu

(DepartmentofEmergencyMedicine,TheAffiliatedHospitalofGuizhouMedicalUniversity,Guiyang550004,China.E-mail:wuguofeng3013@sina.com)

AIM: To observe the effect of rosiglitazone (RGZ) pretreatment on the expression of peroxisome proliferator-activated receptor γ (PPARγ), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the microglia cells activated by thrombin. METHODS: Microglia cells were obtained from the brain tissues of the newborn rats and were primarily culturedinvitro. After cultured for 14 d, the microglia cells were used in the experiment. The isolated microglia cells were randomly divided into normal control group, thrombin stimulation group (TH group), rosiglitazone intervention group (RGZ+TH group) and retinoic acid intervention group (RA+TH group). The expression of PPARγ, Nrf2 and HO-1 was observed by immunocytochemistry, real-time PCR and Western blot.RESULTS: The number of positive staining cells of PPARγ, Nrf2 and HO-1 in TH group, RGZ+TH group and RA+TH group were increased remarkably as compared with control group. The significant increases in PPARγ, Nrf2 and HO-1 were observed in RGZ+TH group compared with other groups. The mRNA expression of PPARγ, Nrf2 and HO-1 in RGZ+TH group was increased significantly as compared with TH group, control group or RA+TH group (P<0.01), Besides, the mRNA expression of Nrf2 and HO-1 in RA+TH group was decreased as compared with TH group or RGZ+TH group (P<0.01). The protein levels of PPARγ, Nrf2 and HO-1 in RGZ+TH group were significantly increased as compared with TH group, control group or RA+TH group (P<0.01). The protein expression of Nrf2 and HO-1 in RA+TH group was decreased as compared with TH group or RGZ+TH group (P<0.01).CONCLUSION: Rosiglitazone pretreatment might increase the expression of PPARγ, Nrf2 and HO-1 in the microglia cells activated by thrombin. By inhibiting the expression of Nrf2 after RA pretreatment, the expression of the downstream geneHO-1 is also influenced. The anti-oxidative stress effects of rosiglitazone might be achieved partly by modulating Nrf2 to control the downstream geneHO-1.

Microglia; Cerebral hemorrhage; Rosiglitazone; Peroxisome proliferator-activated receptor γ; Nuclear factor E2-related factor 2; Heme oxygenase-1

1000- 4718(2016)04- 0671- 09

2015- 11- 03

2016- 01- 28

國家自然科學(xué)基金資助項(xiàng)目(No. 81460185/H09106)；貴州省科技基金資助項(xiàng)目(No. 2013-2043)

Tel: 0851-86752545; E-mail: wuguofeng3013@sina.com

R459.7

A

10.3969/j.issn.1000- 4718.2016.04.015

雜志網(wǎng)址： http://www.cjpp.net