蜂膠黃酮對(duì)炎癥反應(yīng)單核細(xì)胞中NLRP3炎性小體活性的影響

張曉暉1，曾偉1，陳鵬2，姚樹桐2

(1泰山醫(yī)學(xué)院附屬醫(yī)院，山東泰安271000；2泰山醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)院)

摘要：目的觀察蜂膠黃酮對(duì)氧化型低密度脂蛋白(ox-LDL)誘導(dǎo)的炎癥反應(yīng)單核細(xì)胞中核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體3(NLRP3)炎性小體活性的影響。方法培養(yǎng)單核細(xì)胞THP1并分為5個(gè)組。實(shí)驗(yàn)1、2、3組及模型組加入ox-LDL 100 μg/mL制作單核細(xì)胞炎癥反應(yīng)模型；實(shí)驗(yàn)1、2、3組在加入ox-LDL前分別給予5、10、15 μg/mL蜂膠黃酮處理1 h；對(duì)照組不添加藥物。各組培養(yǎng)24 h后，采用Western blotting法檢測(cè)細(xì)胞中的NLRP3蛋白，采用ELISA法檢測(cè)細(xì)胞培養(yǎng)液上清中的IL-1β、IL-18。結(jié)果對(duì)照組、模型組、實(shí)驗(yàn)1組、實(shí)驗(yàn)2組、實(shí)驗(yàn)3組細(xì)胞中NLRP3蛋白相對(duì)表達(dá)量分別為0.26±0.04、1.12±0.15、0.98±0.11、0.65±0.07、0.52±0.06，模型組與對(duì)照組相比，P均<0.05；實(shí)驗(yàn)2組、實(shí)驗(yàn)3組與模型組相比，P均<0.05。模型組培養(yǎng)液上清中IL-1β、IL-18水平高于對(duì)照組，實(shí)驗(yàn)2組、實(shí)驗(yàn)3組培養(yǎng)液上清中IL-1β、IL-18水平低于模型組(P均<0.05)。結(jié)論 蜂膠黃酮可下調(diào)ox-LDL誘導(dǎo)的單核細(xì)胞炎癥反應(yīng)模型中NLRP3蛋白的表達(dá)，降低IL-1β、IL-18分泌水平，抑制NLRP3炎性小體的活性。

關(guān)鍵詞：蜂膠黃酮；核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體3；單核細(xì)胞；炎癥反應(yīng)；白細(xì)胞介素1β；白細(xì)胞介素18

doi：10.3969/j.issn.1002-266X.2015.39.004

中圖分類號(hào)：R541.4文獻(xiàn)標(biāo)志碼：A

基金項(xiàng)目：國(guó)家自然科學(xué)基金資助項(xiàng)目(81202979)。

作者簡(jiǎn)介：第一張曉暉(1977-)，男，本科，主治醫(yī)師，主要研究方向?yàn)閯?dòng)脈粥樣硬化的發(fā)生發(fā)展機(jī)制。E-mail: tyfyzxh@163.com

收稿日期：(2015-08-29)

Effects of propolis flavonoids on activation of NLRP3 inflammasome in monocytes

ZHANGXiao-hui1,ZENGWei,CHENPeng,YAOShu-tong

(1AffiliatedHospitalofTaishanMedicalCollege,Taian271000,China)

Abstract:ObjectiveTo explore the effects of propolis flavonoids on the activation of oxidized low-density lipoprotein (ox-LDL) induced nucleotide-binding oligomerization domain-like receptor containing protein 3 (NLRP3) inflammasome in mononuclear cells. MethodsThe monocytes THP1 were cultured and divided into five groups. The experimental groups 1, 2, 3 and the model group were treated with 100 μg/mL ox-LDL to produce mononuclear inflammatory reaction models. The monocytes of the experimental groups 1, 2 and 3 were treated with 5, 10 and 15 μg/mL propolis flavonoids before ox-LDL, and the monocytes of the control group was treated by ox-LDL in the absence of propo1is flavonoids. After 24 h, the expression of NLRP3 was measured by Western blotting. The levels of IL-1β and IL-18 were measured by ELISA. ResultsThe levels of NLRP3 protein in the control group, model group and the experimental groups 1, 2 and 3 were 0.26±0.04, 1.12±0.15, 0.98±0.11, 0.65±0.07 and 0.52±0.06, respectively. The expression of NLRP3 protein was significantly increased in the model group as compared with that in the control group (all P<0.05). The expression of NLRP3 protein in the experimental groups 2 and 3 was significantly decreased as compared with that of the model group (all P<0.05). The levels of IL-1β and IL-18 was significantly higher in the model group as compared with those of the control group, and the levels of IL-1β and IL-18 in the experimental groups 2 and 3 was significantly decreased as compared with those of the model group (all P<0.05). ConclusionPropolis flavonoids may down-regulate the expression of NLRP3 protein in the ox-LDL induced inflammatory response of the mononuclear cells, decrease the levels of IL-1β and IL-18 and inhibit the activation of NLRP3 inflammasome.

Key words: propolis flavonoids; nucleotide-binding oligomerization domain-like receptor containing protein 3; monocytes; inflammatory response; interleukin-1β; interleukin-18

近年來(lái)，動(dòng)脈粥樣硬化的發(fā)病率逐年上升并呈年輕化趨勢(shì)，尋找安全有效的抗動(dòng)脈粥樣硬化藥物具有重要意義[1]。蜂膠黃酮是從蜂膠中提取出的活性物質(zhì)，有抗病毒、抗菌、抗氧化、抗腫瘤、調(diào)節(jié)免疫力及改善微循環(huán)等多種生物學(xué)活性[2~4]。此外，蜂膠黃酮還能抑制血小板黏附和聚集、保護(hù)血管內(nèi)皮細(xì)胞免受炎癥損傷，具體機(jī)制尚不明確。炎性體是細(xì)胞內(nèi)一類大分子蛋白復(fù)合體。核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體3(NLRP3)炎性小體是較為經(jīng)典的炎性體，它可被相關(guān)病原分子及缺血缺氧條件激活，促進(jìn)IL-1β、IL-18分泌，參與動(dòng)脈粥樣硬化的發(fā)生發(fā)展[5，6]。2015年2~8月，我們將氧化型低密度脂蛋白(ox-LDL)作用于單核細(xì)胞THP1，建立炎癥反應(yīng)模型，觀察蜂膠黃酮對(duì)THP1細(xì)胞中NLRP3炎性小體活性的影響。

1材料與方法

1.1實(shí)驗(yàn)材料THP1細(xì)胞購(gòu)自Scien Cell研究實(shí)驗(yàn)室；蜂膠黃酮由本實(shí)驗(yàn)室制備；ox-LDL購(gòu)自北京鼎國(guó)昌盛生物技術(shù)有限責(zé)任公司；NLRP3抗體購(gòu)自Abcam公司；IL-1β、IL-18 ELISA檢測(cè)試劑盒購(gòu)自R&D Systems公司。

1.2細(xì)胞培養(yǎng)與分組THP1細(xì)胞培養(yǎng)于添加10%胎牛血清的1640培養(yǎng)基中，在37 ℃、5%CO2的培養(yǎng)箱中進(jìn)行無(wú)菌培養(yǎng)。培養(yǎng)細(xì)胞傳3代以后，將細(xì)胞分為5個(gè)組，分別為對(duì)照組、模型組、實(shí)驗(yàn)1組、實(shí)驗(yàn)2組、實(shí)驗(yàn)3組。

1.3動(dòng)脈粥樣硬化單核細(xì)胞模型制作及蜂膠黃酮干預(yù)方法實(shí)驗(yàn)1、2、3組及模型組中加入ox-LDL 100 μg/mL制作單核細(xì)胞炎癥反應(yīng)模型；實(shí)驗(yàn)1、2、3組在加入ox-LDL前分別給予5、10、15 μg/mL蜂膠黃酮處理1 h；對(duì)照組不添加藥物。各組培養(yǎng)24 h后收集細(xì)胞，進(jìn)行后續(xù)實(shí)驗(yàn)。

1.4各組細(xì)胞中NLRP3蛋白檢測(cè)采用Western blotting法。向各組細(xì)胞中加入RIPA裂解液200 μL，冰上放置30 min后，4 ℃、12 000 r/min離心15 min。取上清液，用BCA法測(cè)定蛋白濃度。制備分離膠及濃縮膠，將蛋白樣品與Loading Buffer混合，煮沸5 min。在每個(gè)上樣孔加入蛋白樣品80 μg，進(jìn)行電泳。取出凝膠，在恒流180 mA下濕轉(zhuǎn)90 min，轉(zhuǎn)于NC膜上。5%脫脂奶粉室溫封閉1 h，加入兔抗人NLRP3抗體(1∶1 000)，4 ℃過(guò)夜，TBST洗膜3次，每次10 min；加入羊抗兔二抗，室溫孵育60 min，TBST洗膜3次，每次洗10min。用ECL化學(xué)發(fā)光液在暗室進(jìn)行顯色、拍片，以NLRP3/GAPDH灰度值表示NLRP3蛋白相對(duì)表達(dá)量。

1.5各組培養(yǎng)液上清中IL-1β、IL-18檢測(cè)收集各組細(xì)胞培養(yǎng)液上清，采用ELISA法檢測(cè)IL-1β、IL-18，按試劑盒說(shuō)明書操作。

1.6統(tǒng)計(jì)學(xué)方法采用SPSS16.0軟件進(jìn)行統(tǒng)計(jì)分析。計(jì)量資料以±s表示，組間比較采用方差分析。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2結(jié)果

2.1各組細(xì)胞中NLRP3蛋白表達(dá)比較對(duì)照組、模型組、實(shí)驗(yàn)1組、實(shí)驗(yàn)2組、實(shí)驗(yàn)3組細(xì)胞中NLRP3蛋白相對(duì)表達(dá)量分別為0.26±0.04、1.12±0.15、0.98±0.11、0.65±0.07、0.52±0.06，模型組NLRP3蛋白表達(dá)量高于對(duì)照組(P<0.05)；實(shí)驗(yàn)2組、實(shí)驗(yàn)3組NLRP3蛋白表達(dá)量低于模型組(P均<0.05)。

2.2各組培養(yǎng)液上清中IL-1β、IL-18水平比較模型組培養(yǎng)液上清中IL-1β、IL-18水平高于對(duì)照組，實(shí)驗(yàn)2組、實(shí)驗(yàn)3組培養(yǎng)液上清中IL-1β、IL-18水平低于模型組(P均<0.05)。見表1。

表1　 各組培養(yǎng)液上清中IL-1β、IL-18

水平比較(pg/mL， ± s)

表1　 各組培養(yǎng)液上清中IL-1β、IL-18

組別IL-1βIL-18實(shí)驗(yàn)1組155.18±15.67198.36±21.38實(shí)驗(yàn)2組129.56±11.89#171.07±18.74#實(shí)驗(yàn)3組91.77±10.57#150.92±16.31#模型組178.35±17.56*237.19±25.66*對(duì)照組56.28±9.78106.55±14.67

注：與對(duì)照組相比，*P<0.05；與模型組相比，#P<0.05。

3討論

蜂膠素是蜜蜂從植物新生芽條或愈傷組織中采集分泌物后，加入蜜蜂的舌腺、蠟腺等腺體分泌物及花粉等混合而成的具有特殊芳香氣味的黏膠狀物質(zhì)[7]。蜂膠的化學(xué)成分復(fù)雜，主要成分為黃酮化合物。蜂膠黃酮具有抗病毒、軟化血管、降血脂、調(diào)節(jié)免疫、改善微循環(huán)、抗氧化等廣泛的生物學(xué)活性[8,9]，還有抗動(dòng)脈粥樣硬化的作用。動(dòng)脈粥樣硬化發(fā)病機(jī)制復(fù)雜，目前存在幾種學(xué)說(shuō)，包括脂質(zhì)滲入學(xué)說(shuō)、內(nèi)皮細(xì)胞損傷學(xué)說(shuō)、炎癥學(xué)說(shuō)等[10,11]，其中脂代謝異常在動(dòng)脈粥樣硬化的形成中發(fā)揮重要作用。血液中高水平的低密度脂蛋白(LDL)被活性氧簇(ROS)等自由基氧化后成為ox-LDL，后者參與動(dòng)脈粥樣硬化的形成過(guò)程。ox-LDL可被血液中的單核細(xì)胞吞噬，被吞噬的ox-LDL在細(xì)胞中不易被分解，之后單核細(xì)胞將會(huì)轉(zhuǎn)變成泡沫細(xì)胞[12,13]。

NLRP3是經(jīng)典的炎性小體，由NLRP3、Caspase-1、含C-末端Caspase募集域的凋亡相關(guān)斑點(diǎn)樣蛋白(ASC)相互結(jié)合形成。通常情況下，NLRP3處于自身抑制狀態(tài)，當(dāng)與配體結(jié)合后自身發(fā)生寡聚化，進(jìn)而與ASC發(fā)生相互作用，通過(guò)ASC招募并激活Caspase-1，Caspase-1活化后可催化IL-1 及IL-18前體，使其成為有活性的形式，并分泌到細(xì)胞外發(fā)揮促炎作用[14]。已有報(bào)道顯示，NLRP3可被膽固醇結(jié)晶活化，促進(jìn)IL-1 、IL-18的分泌，發(fā)揮促動(dòng)脈粥樣硬化作用[15]。

本研究中，我們用ox-LDL作用于THP1細(xì)胞，誘導(dǎo)單核細(xì)胞炎癥反應(yīng)，觀察蜂膠黃酮對(duì)NLRP3炎性小體活性的影響。我們發(fā)現(xiàn)，ox-LDL可誘導(dǎo)NLRP3蛋白表達(dá)增多，并能提高NLRP3炎性小體下游炎癥因子IL-1 、IL-18分泌水平，這與Jiang等[16]的研究結(jié)果一致。而給予不同濃度蜂膠黃酮干預(yù)后，THP1細(xì)胞中NLRP3蛋白表達(dá)下調(diào)，細(xì)胞培養(yǎng)液上清中IL-1 、IL-18水平降低，提示蜂膠黃酮可抑制NLRP3炎性小體的活性，從而抑制ox-LDL誘導(dǎo)的炎癥反應(yīng)，起到抗動(dòng)脈粥樣硬化的作用，具體作用機(jī)制有待于深入研究。

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