陳玄晶, 鐘碧瑩, 吳煥林, 徐丹蘋
溫膽湯抑制氧化低密度脂蛋白誘導(dǎo)的巨噬細(xì)胞泡沫化*
陳玄晶1, 鐘碧瑩1, 吳煥林3△, 徐丹蘋2△
(1廣州中醫(yī)藥大學(xué)第二臨床醫(yī)學(xué)院,2廣東省中醫(yī)院,廣東 廣州 510020;3北京中醫(yī)藥大學(xué),北京 100700)
從巨噬細(xì)胞泡沫化角度探討溫膽湯抗動(dòng)脈粥樣硬化的作用機(jī)制。采用MTT法確定溫膽湯的無(wú)毒性干預(yù)濃度;藥物干預(yù)后,采用油紅O染色法檢測(cè)泡沫細(xì)胞的形成;采用酶法檢測(cè)細(xì)胞內(nèi)膽固醇水平及細(xì)胞膽固醇流出;采用Western blot檢測(cè)巨噬細(xì)胞膜蛋白CD36、A類清道夫受體(SR-A)及ATP結(jié)合盒轉(zhuǎn)運(yùn)體A1(ABCA1)和B類I型清道夫受體(SR-BI)的蛋白表達(dá)水平。溫膽湯凍干粉溶液對(duì)氧化型低密度脂蛋白(ox-LDL)刺激的THP-1細(xì)胞源性巨噬細(xì)胞的無(wú)毒性干預(yù)濃度為0~6 g/L,于是設(shè)置溫膽湯干預(yù)濃度梯度為1.5、3和6 g/L進(jìn)行后續(xù)實(shí)驗(yàn)。各濃度溫膽湯干預(yù)可抑制ox-LDL誘導(dǎo)的泡沫細(xì)胞形成,半定量分析顯示溫膽湯濃度依賴性降低細(xì)胞內(nèi)脂質(zhì)沉積(<0.05或<0.01)。溫膽湯濃度依賴性降低細(xì)胞內(nèi)總膽固醇、膽固醇酯和膽固醇酯化率(<0.05或<0.01),促進(jìn)細(xì)胞膽固醇流出(<0.01),減少CD36表達(dá)(<0.01)。6 g/L溫膽湯顯著減少SR-A的表達(dá)(<0.05); 3和6 g/L溫膽湯顯著促進(jìn)ABCA1和SR-BI的表達(dá)(<0.05或<0.01)。溫膽湯顯著抑制巨噬細(xì)胞泡沫化,相關(guān)機(jī)制可能是通過(guò)抑制巨噬細(xì)胞膜蛋白CD36和SR-A表達(dá)而減少巨噬細(xì)胞對(duì)膽固醇的內(nèi)化吞噬,同時(shí)上調(diào)巨噬細(xì)胞膜蛋白ABCA1和SR-BI表達(dá),從而促進(jìn)巨噬細(xì)胞內(nèi)膽固醇流出。
溫膽湯;動(dòng)脈粥樣硬化;巨噬細(xì)胞;清道夫受體;ATP結(jié)合盒轉(zhuǎn)運(yùn)體A1
巨噬細(xì)胞密切參與了動(dòng)脈粥樣硬化從脂質(zhì)條紋到最終斑塊破裂和血栓形成等各個(gè)階段,介導(dǎo)病變處的脂質(zhì)膽固醇代謝、炎癥反應(yīng)以及纖維降解等,對(duì)動(dòng)脈粥樣硬化性斑塊的形成、進(jìn)展以及斑塊的穩(wěn)定性有主導(dǎo)作用[1-2],對(duì)周圍環(huán)境中脂質(zhì)的吞噬清除也是巨噬細(xì)胞主要功能之一[3]。在動(dòng)脈粥樣硬化病程中,循環(huán)血流低密度脂蛋白膽固醇增加,滲透進(jìn)入血管內(nèi)皮下層,并被氧化形成氧化低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL)。巨噬細(xì)胞膜表面富含分化簇36 (cluster of differentiation 36, CD36)和A類清道夫受體(scavenger receptor class A, SR-A)等,可以脂筏的形式內(nèi)化吞噬ox-LDL,形成泡沫細(xì)胞[4]。隨著巨噬細(xì)胞內(nèi)膽固醇大量沉積,產(chǎn)生細(xì)胞毒性,導(dǎo)致細(xì)胞活性逐漸減弱甚至死亡。為了維持細(xì)胞活性,并抑制膽固醇的過(guò)度累積,巨噬細(xì)胞能夠啟動(dòng)具有保護(hù)作用的膽固醇外流機(jī)制,通過(guò)液相擴(kuò)散或ATP結(jié)合盒轉(zhuǎn)運(yùn)體A1 (ATP-binding cassette transporter A1, ABCA1)介導(dǎo)和B類I型清道夫受體(scavenger receptor class B type I, SR-BI)等轉(zhuǎn)運(yùn)膜蛋白介導(dǎo)的形式,將細(xì)胞內(nèi)過(guò)量膽固醇轉(zhuǎn)運(yùn)至外周血HDL[5-6]。研究表明,靶向作用于膽固醇攝取、外流過(guò)程對(duì)于動(dòng)脈粥樣硬化的治療具有重要價(jià)值[7]。
膽固醇代謝異常是動(dòng)脈粥樣硬化病變?cè)缙诰奘杉?xì)胞泡沫化的關(guān)鍵因素?,F(xiàn)代中醫(yī)證候本質(zhì)研究明確血脂代謝異常是中醫(yī)痰證的物質(zhì)基礎(chǔ)之一[8-10]。溫膽湯始載于唐初《千金要方》,是化痰的經(jīng)典代表方。近年,溫膽湯治療動(dòng)脈粥樣硬化性病變研究陸續(xù)開展,顯示出良好療效[11-12],但對(duì)溫膽湯抗動(dòng)脈粥樣硬化的作用機(jī)制鮮有涉及。本研究團(tuán)隊(duì)前期對(duì)溫膽湯及其類方的系列研究表明,溫膽湯能夠有效降低血甘油三酯(triglyceride, TG)、總膽固醇(total cholesterol, TC)和低密度脂蛋白膽固醇(low-density lipoprotein cholesterol, LDL-C)水平,改善動(dòng)脈粥樣硬化性心臟病患者臨床癥狀及預(yù)后[13-15]。本研究基于ox-LDL誘導(dǎo)的巨噬細(xì)胞泡沫化疾病模型,探討溫膽湯抗動(dòng)脈粥樣硬化的作用機(jī)制,以期為溫膽湯的臨床運(yùn)用提供更加全面的科學(xué)依據(jù),并進(jìn)一步為靶點(diǎn)明確、物質(zhì)基礎(chǔ)清晰的中藥新制劑研發(fā)奠定基礎(chǔ)。
人THP-1單核細(xì)胞購(gòu)于中國(guó)科學(xué)院細(xì)胞庫(kù)。常規(guī)方法復(fù)蘇后凍存于液氮,以含有10%胎牛血清的RPMI-1640培養(yǎng)基置于37℃、5% CO2細(xì)胞培養(yǎng)箱內(nèi)培養(yǎng),每日觀察細(xì)胞生長(zhǎng)狀態(tài),取對(duì)數(shù)生長(zhǎng)期,生長(zhǎng)至80%以上融合的THP-1細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)。
根據(jù)唐代孫思邈《千金要方》中所載溫膽湯,按原經(jīng)方比例配伍。每克生藥加入6 mL超純水,浸泡30 min,加熱煮沸后文火繼續(xù)煎煮30 min,過(guò)濾至濾液自然滴盡,收集藥液;每克藥渣加入4 mL超純水,再次煎煮,方法同前,合并2次收集的濾液;使用旋轉(zhuǎn)蒸發(fā)儀,將濾液濃縮成浸膏;預(yù)冷冷凍干燥機(jī),放入浸膏,制成凍干粉。
油紅O、ProteoPrep?Membrane Extraction Kit、佛波酯(phorbol myristate acetate, PMA)、二甲基亞砜(dimethyl sulfoxide, DMSO)和脫脂奶粉購(gòu)自Sigma-Aldrich; RPMI-1640培養(yǎng)基購(gòu)自Gibco; Cholesterol Efflux Assay試劑盒、anti-ABCA1 antibody、anti-scavenging receptor SR-BI antibody和anti-CD36 antibody購(gòu)自Abcam; GAPDH rabbit mAb、anti-rabbit IgG及HRP-linked antibody、anti-rat IgG及HRP-linked antibody購(gòu)自Cell Signaling Technology; anti-SRA antibody購(gòu)自Santa Cruz;蘇木素染液和MTT購(gòu)自碧云天生物技術(shù)有限公司;胎牛血清(fetal bovine serum, FBS)購(gòu)自HyClone; ECL發(fā)光液(Immobilon ECL Ultra Western HRP Substrate)購(gòu)自Millipore; BCA蛋白濃度檢測(cè)試劑盒購(gòu)自Thermo Fisher Scientific;總膽固醇酶法檢測(cè)試劑盒和游離膽固醇酶法檢測(cè)試劑盒購(gòu)自北京普利萊基因技術(shù)有限公司; ox-LDL購(gòu)自廣州奕源公司。
TE2000-S熒光倒置顯微鏡(NIKON);自動(dòng)細(xì)胞計(jì)數(shù)儀(Costar); Multiskan FC自動(dòng)酶標(biāo)儀(Thermo Fisher Scientific); Milli-Q Integral 3型超純水系統(tǒng)(Millipore);垂直電泳儀、蛋白轉(zhuǎn)膜系統(tǒng)、ChemiDoc XRS凝膠成像系統(tǒng)(Bio-Rad); MB-102振蕩恒溫金屬?。ê贾莶┤湛萍加邢薰荆?/p>
5.1巨噬細(xì)胞泡沫化模型的建立[16-17]取對(duì)數(shù)生長(zhǎng)期的THP-1細(xì)胞,計(jì)數(shù),移入離心管, 800 r/min,離心5 min;吸棄上清液,加入含160 nmol/L PMA和10% FBS的RPMI-1640培養(yǎng)基,調(diào)整細(xì)胞密度后接種于細(xì)胞板,置于37℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)24 h;次日鏡下觀察細(xì)胞形態(tài), THP-1細(xì)胞由懸浮生長(zhǎng)轉(zhuǎn)為貼壁生長(zhǎng),體積增大,形態(tài)由圓形轉(zhuǎn)變?yōu)椴灰?guī)則形態(tài),伸出偽足,提示THP-1單核細(xì)胞已分化為巨噬細(xì)胞。
誘導(dǎo)分化為THP-1細(xì)胞源性巨噬細(xì)胞后,加入含50 mg/L ox-LDL和3% FBS的RMPI-1640培養(yǎng)基,置于37℃、5% CO2細(xì)胞培養(yǎng)箱繼續(xù)培養(yǎng)24 h;次日鏡下觀察細(xì)胞形態(tài),鏡下觀察細(xì)胞體積進(jìn)一步增大,胞漿內(nèi)脂質(zhì)增多,部分聚集呈透亮狀脂滴;油紅O染色后,細(xì)胞內(nèi)出現(xiàn)大量亮紅色圓形脂滴,呈戒環(huán)樣,提示THP-1細(xì)胞源性巨噬細(xì)胞轉(zhuǎn)變?yōu)榕菽?xì)胞。
5.2藥物毒性實(shí)驗(yàn)取對(duì)數(shù)增長(zhǎng)期、生長(zhǎng)狀態(tài)良好的THP-1細(xì)胞,調(diào)整細(xì)胞密度為每孔1×104個(gè),接種于96孔板,每個(gè)濃度組設(shè)置2個(gè)復(fù)孔; PMA誘導(dǎo)分化為巨噬細(xì)胞后,使用含3% FBS的RPMI-1640培養(yǎng)基配制濃度梯度為0.375、0.75、1.5、3、6、12和24 g/L溫膽湯凍干粉溶液,協(xié)同50 mg/L ox-LDL共同干預(yù);無(wú)施加藥物和ox-LDL干預(yù)的THP-1細(xì)胞源性巨噬細(xì)胞為對(duì)照組;培養(yǎng)24 h后,加入MTT溶液,置于細(xì)胞培養(yǎng)箱中避光培養(yǎng)4 h;加入DMSO,置于水平低速搖床振蕩10 min;使用酶標(biāo)儀測(cè)量490 nm波長(zhǎng)處的吸光度(490),計(jì)算細(xì)胞相對(duì)活力。細(xì)胞相對(duì)活力(%)=實(shí)驗(yàn)組490/對(duì)照組490×100%。
5.3藥物干預(yù)使用含3% FBS的RPMI-1640培養(yǎng)基溶解溫膽湯凍干粉,微孔濾膜過(guò)濾后, -20℃存儲(chǔ)備用。取對(duì)數(shù)生長(zhǎng)期、狀態(tài)良好的THP-1細(xì)胞,接種于細(xì)胞板,按前述操作誘導(dǎo)為THP-1細(xì)胞源性巨噬細(xì)胞。根據(jù)藥物毒性實(shí)驗(yàn)檢測(cè)結(jié)果,分別稀釋調(diào)整溫膽湯凍干粉濃度為1.5、3和6 g/L,并加入終濃度為50 mg/L ox-LDL共同培養(yǎng)。單純使用50 mg/L ox-LDL刺激,不施加藥物干預(yù)的THP-1細(xì)胞源性巨噬細(xì)胞為對(duì)照(control)組。培養(yǎng)24 h后進(jìn)行后續(xù)實(shí)驗(yàn)。
5.4油紅O染色檢測(cè)泡沫細(xì)胞形成取對(duì)數(shù)增長(zhǎng)期、生長(zhǎng)狀態(tài)良好的THP-1細(xì)胞,以每孔2×105個(gè)接種于24孔細(xì)胞板中,每組設(shè)置2個(gè)復(fù)孔;按前述方法誘導(dǎo)分化為巨噬細(xì)胞后進(jìn)行藥物干預(yù),并將無(wú)添加ox-LDL刺激和藥物干預(yù)的THP-1細(xì)胞源性巨噬細(xì)胞設(shè)為空白對(duì)照組;使用加入4%多聚甲醛固定10 min;加入60%異丙醇10 min;加入油紅O工作液,室溫染色30 min;吸棄油紅O工作液,加入超純水,倒置顯微鏡下觀察、拍照留存;加入60%異丙醇2 min;加入異丙醇水平搖床振蕩30 min;取上清液于96孔板,異丙醇為調(diào)零孔,酶標(biāo)儀測(cè)量每孔的492值以進(jìn)行半定量分析。
5.5酶法檢測(cè)細(xì)胞內(nèi)膽固醇含量取對(duì)數(shù)增長(zhǎng)期、生長(zhǎng)狀態(tài)良好的THP-1細(xì)胞,以每孔2×106個(gè)接種于6孔細(xì)胞板,每組設(shè)置1個(gè)復(fù)孔;按前述方法誘導(dǎo)分化為巨噬細(xì)胞后進(jìn)行藥物干預(yù);按照膽固醇酶法檢測(cè)試劑盒說(shuō)明書操作,并使取適量細(xì)胞上清液用于BCA蛋白濃度測(cè)定;使用酶標(biāo)儀測(cè)量各孔的550值;將各個(gè)標(biāo)準(zhǔn)品和待測(cè)樣品的550值減去空白標(biāo)準(zhǔn)品的550值即為校正后的550值;使用校正后的標(biāo)準(zhǔn)品550值與其對(duì)應(yīng)的濃度繪制標(biāo)準(zhǔn)曲線,并計(jì)算各待測(cè)樣品的膽固醇濃度;使用每mg蛋白濃度校正膽固醇含量,計(jì)算膽固醇濃度。(1)膽固醇酯(cholesterol ester, CE)=總膽固醇含量-游離膽固醇含量;(2)膽固醇酯化率(cholesterol esterification rate, CER)=CE含量/總膽固醇含量×100%。
5.6膽固醇流出實(shí)驗(yàn)取對(duì)數(shù)增長(zhǎng)、狀態(tài)良好的THP-1細(xì)胞,以每孔4×104個(gè)接種于96孔白色細(xì)胞板,每組設(shè)置2個(gè)復(fù)孔;按前述使用PMA誘導(dǎo)THP-1細(xì)胞源性巨噬細(xì)胞后,加入標(biāo)記膽固醇試劑,于細(xì)胞培養(yǎng)箱中培養(yǎng)16 h;分別加入含1.5、3和6 g/L溫膽湯凍干粉及10% FBS的RPMI-1640培養(yǎng)基,無(wú)施加藥物干預(yù)含10% FBS的RPMI-1640培養(yǎng)基為對(duì)照組,與細(xì)胞培養(yǎng)箱中繼續(xù)培養(yǎng)6 h;轉(zhuǎn)移細(xì)胞上清液至新的96孔白色細(xì)胞板,使用熒光分光光度計(jì)測(cè)量培養(yǎng)基熒光值(激發(fā)波長(zhǎng)482 nm,發(fā)射波長(zhǎng)515 nm);在含有細(xì)胞的96孔板中加入細(xì)胞裂解液,室溫水平搖床振蕩30 min;測(cè)量細(xì)胞裂解液熒光值(激發(fā)波長(zhǎng)和發(fā)射波長(zhǎng)同前)。膽固醇流出率(%)=培養(yǎng)基熒光值/(培養(yǎng)基熒光值+細(xì)胞裂解液熒光值)×100%。
5.7Western blot檢測(cè)巨噬細(xì)胞膜蛋白CD36、SR-A、ABCA1和SR-BI的表達(dá)水平取對(duì)數(shù)增長(zhǎng)、狀態(tài)良好THP-1細(xì)胞,以每孔2×106個(gè)接種于6孔細(xì)胞板;按前述方法誘導(dǎo)分化為巨噬細(xì)胞后進(jìn)行藥物干預(yù);收集各組細(xì)胞,加入細(xì)胞裂解液, 4℃、12 000 r/min離心15 min;根據(jù)BCA蛋白濃度測(cè)量結(jié)果調(diào)整各組樣品濃度,100℃恒溫金屬浴變性;每組取30~50 μg蛋白,于10% SDS-PAGE分離蛋白;轉(zhuǎn)膜后, 5%脫脂奶粉封閉2 h,加入一定稀釋比例I抗, 4℃孵育過(guò)夜;TBST洗膜3次,加入對(duì)應(yīng)II抗溶液,室溫孵育2 h;ECL化學(xué)發(fā)光試劑顯影曝光;以GAPDH為內(nèi)參照,使用Image Lab 5.1.0軟件進(jìn)行蛋白條帶灰度分析。
運(yùn)用SPSS 19.0軟件進(jìn)行統(tǒng)計(jì)分析。每個(gè)實(shí)驗(yàn)均獨(dú)立重復(fù)3次以上,計(jì)量資料服從正態(tài)分布采用均數(shù)±標(biāo)準(zhǔn)差(Mean±SD)表示。多組間比較采用單因素方差分析,組間多重比較采用Bonferroni校正檢驗(yàn)或Tamhane's T2檢驗(yàn)。以<0.05為差異有統(tǒng)計(jì)學(xué)意義。
當(dāng)溫膽湯濃度升至12 g/L時(shí)的細(xì)胞相對(duì)活力降至(16.52±4.76)%,與對(duì)照組比較有顯著差異(<0.01),說(shuō)明溫膽湯濃度≥12 g/L時(shí),協(xié)同50 mg/L ox-LDL干預(yù)會(huì)對(duì)THP-1細(xì)胞源性泡沫細(xì)胞產(chǎn)生明顯毒性作用,溫膽湯安全干預(yù)濃度為0~6 g/L,見圖1。
Figure 1. The viability of THP-1 cell-derived macrophages treated with ox-LDL at 50 mg/L and Wendan decoction at different concentrations was measured by MTT assay. Mean±SD. n=6. **P<0.01 vs 0 g/L group.
油紅O染色結(jié)果顯示,空白對(duì)照組細(xì)胞胞漿內(nèi)僅有少量細(xì)小紅色顆粒;對(duì)照組細(xì)胞經(jīng)ox-LDL刺激后體積增大,胞漿內(nèi)有明顯亮紅色脂滴,呈典型戒環(huán)樣泡沫細(xì)胞形態(tài);與對(duì)照組比較,1.5、3和6 g/L溫膽湯組細(xì)胞胞漿內(nèi)紅色脂滴均明顯減少、變小,半定量分析結(jié)果顯示,溫膽湯劑量依賴性降低細(xì)胞內(nèi)脂質(zhì)沉積,與對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(<0.05或<0.01),見圖2。
Figure 2. The formation of foam cells induced by ox-LDL was examined by oil red O staining (×200). A: negative control group; B: 0 g/L Wendan decoction+50 mg/L ox-LDL group; C: 1.5 g/L Wendan decoction+50 mg/L ox-LDL group; D: 3 g/L Wendan decoction+50 mg/L ox-LDL group; E: 6 g/L Wendan decoction+50 mg/L ox-LDL group; F: semi-quantitative analysis of intracellular lipid. Mean±SD. n=5. *P<0.05, **P<0.01 vs 0 g/L group.
1.5、3和6 g/L溫膽湯干預(yù)后細(xì)胞內(nèi)TC、CE和CER均顯著降低,與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)顯著性(0.05或<0.01),而各濃度溫膽湯組FC水平與對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)顯著性(>0.05); 6 g/L溫膽湯降低CE的效果優(yōu)于1.5 g/L溫膽湯(<0.05), 6 g/L溫膽湯降低CER的效果優(yōu)于1.5和3 g/L溫膽湯(<0.05),見表1。
表1 細(xì)胞內(nèi)膽固醇水平
TC: total cholesterol; FC: free cholesterol; CE: cholesterol ester; CER: cholesterol esterification rate.*<0.05**<0.01control group;?<0.051.5 g/L Wendan decoction group;?<0.053 g/L Wendan decoction group.
對(duì)照組細(xì)胞膽固醇流出率為(7.70±0.93)%;1.5、3和6 g/L溫膽湯干預(yù)后,細(xì)胞膽固醇流出率分別為(10.23±0.94)%、(12.12±1.19)%和(13.42±0.81)%,與對(duì)照組比較顯著升高(<0.01);溫膽湯各濃度組間比較結(jié)果顯示, 3 g/L和6 g/L溫膽湯促進(jìn)細(xì)胞膽固醇流出率藥效優(yōu)于1.5 g/L溫膽湯(<0.05或<0.01),見圖3。
Figure 3. Cholesterol efflux in THP-1 cell-derived macrophages. Mean±SD. n=5. **P<0.01 vs 0 g/L group;#P<0.05, ##P<0.01 vs 1.5 g/L group.
Western blot結(jié)果顯示,與對(duì)照組比較,各濃度溫膽湯干預(yù)均抑制CD36蛋白表達(dá)水平(<0.05或<0.01),溫膽湯各濃度組間比較, 1.5、3和6 g/L溫膽湯干預(yù)后CD36表達(dá)量依次降低,但差異無(wú)統(tǒng)計(jì)學(xué)顯著性(>0.05); 1.5和3 g/L溫膽湯干預(yù)后SR-A表達(dá)水平雖有降低,但與對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)顯著性(>0.05),而6 g/L溫膽湯干預(yù)顯著抑制SR-A表達(dá)(<0.05),溫膽湯各濃度組間比較,溫膽湯抑制SR-A蛋白表達(dá)水平呈一定濃度依賴性,但組間差異無(wú)統(tǒng)計(jì)學(xué)顯著性(>0.05),見圖4。
Figure 4. The protein levels of CD36 and SR-A in THP-1 cell-derived macrophages. Mean±SD. n=5. *P<0.05, **P<0.01 vs 0 g/L group.
Western blot結(jié)果顯示, 1.5 g/L溫膽湯干預(yù)后ABCA1蛋白表達(dá)水平略有升高,但與對(duì)照組比較差異無(wú)統(tǒng)計(jì)顯著性(>0.05),而3 g/L和6 g/L溫膽湯組ABCA1蛋白表達(dá)水平與對(duì)照組比較顯著升高(<0.05或<0.01),溫膽湯各濃度組間比較, ABCA1蛋白表達(dá)水平隨著溫膽湯濃度升高依次提高,但各濃度組間差異無(wú)統(tǒng)計(jì)學(xué)顯著性(>0.05); SR-BI蛋白水平檢測(cè)結(jié)果顯示,與對(duì)照組比較, 1.5 g/L溫膽湯組SR-BI蛋白表達(dá)水平略微升高,但與對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)顯著性(>0.05),而3 g/L和6 g/L溫膽湯組細(xì)胞膜SR-BI蛋白表達(dá)水平與對(duì)照組比較(<0.01),溫膽湯各濃度組間比較, SR-BI蛋白水平的差異無(wú)統(tǒng)計(jì)學(xué)顯著性(>0.05),見圖5。
Figure 5. The protein levels of ABCA1 and SR-BI in THP-1 cell-derived macrophages. Mean±SD. n=5. *P<0.05, **P<0.01 vs 0 g/L group.
本研究發(fā)現(xiàn),溫膽湯濃度依賴性抑制ox-LDL誘導(dǎo)的THP-1細(xì)胞源性巨噬細(xì)胞泡沫化,降低細(xì)胞內(nèi)膽固醇水平以及膽固醇酯化率,并促進(jìn)細(xì)胞膽固醇流出率;同時(shí)溫膽湯干預(yù)有效降低THP-1細(xì)胞源性巨噬細(xì)胞清道夫受體CD36和SR-A蛋白表達(dá)水平,并提高膽固醇逆轉(zhuǎn)運(yùn)相關(guān)膜蛋白ABCA1和SR-BI表達(dá)。
膽固醇在動(dòng)脈壁上沉積以及泡沫細(xì)胞形成是早期動(dòng)脈粥樣硬化的典型病理改變[18]。調(diào)節(jié)巨噬細(xì)胞內(nèi)膽固醇代謝和轉(zhuǎn)運(yùn)的平衡,從而抑制泡沫細(xì)胞的形成,已成為防治動(dòng)脈粥樣硬化的重要靶點(diǎn)[7]。ox-LDL是巨噬細(xì)胞泡沫化過(guò)程中膽固醇的主要來(lái)源,并且能夠抑制巨噬細(xì)胞膽固醇外流,加劇膽固醇在巨噬細(xì)胞內(nèi)沉積,從而促進(jìn)動(dòng)脈粥樣硬化病變進(jìn)展[19]。本研究使用ox-LDL刺激巨噬細(xì)胞,誘導(dǎo)泡沫細(xì)胞形成,從而模擬動(dòng)脈粥樣硬化病變過(guò)程中膽固醇沉積和泡沫細(xì)胞形成。不同濃度溫膽湯干預(yù)后,采用油紅O染色及半定量分析檢測(cè)干預(yù)后的泡沫細(xì)胞內(nèi)的脂質(zhì)沉積以及泡沫細(xì)胞的形成情況,鏡下觀察表明溫膽湯能夠有效減少泡沫細(xì)胞的形成,并且減少細(xì)胞內(nèi)脂滴的數(shù)量、大小,半定量分析顯示溫膽湯濃度依賴性抑制細(xì)胞內(nèi)脂質(zhì)沉積。
溫膽湯可顯著抑制細(xì)胞內(nèi)脂質(zhì)沉積和泡沫細(xì)胞形成,故推測(cè)溫膽湯也可能減少細(xì)胞內(nèi)膽固醇含量。使用酶法膽固醇試劑盒,進(jìn)一步檢測(cè)溫膽湯對(duì)細(xì)胞內(nèi)膽固醇水平的影響。結(jié)果顯示,溫膽湯干預(yù)可降低細(xì)胞內(nèi)膽固醇含量,呈一定劑量依賴性,以6 g/L溫膽湯干預(yù)藥效最顯著。與對(duì)照組比較,6 g/L溫膽湯組細(xì)胞TC、CE和CER分別降低了約52%、70%和40%,其中以降低CE效果最為顯著。CE是泡沫細(xì)胞內(nèi)脂質(zhì)沉積的主要成分, ox-LDL被巨噬細(xì)胞吞噬后,經(jīng)細(xì)胞內(nèi)酸性脂肪酶和中性膽固醇水解酶作用降解為具有細(xì)胞毒性的FC,再經(jīng)由酰基輔酶A的酯化作用形成CE沉積在細(xì)胞內(nèi),當(dāng)CE在細(xì)胞內(nèi)累計(jì)超過(guò)TC的60%時(shí)可認(rèn)為形成泡沫細(xì)胞[20]。減少CE在細(xì)胞內(nèi)沉積可抑制泡沫細(xì)胞形成,從而延緩動(dòng)脈粥樣硬化斑塊的進(jìn)展[21]。我們研究發(fā)現(xiàn)溫膽湯及其藥效成分單體不但降低細(xì)胞內(nèi)TC,更能有效減少細(xì)胞內(nèi)CE水平,降低CER。
細(xì)胞內(nèi)膽固醇的過(guò)量累積可產(chǎn)生細(xì)胞毒性,巨噬細(xì)胞內(nèi)膽固醇逆向轉(zhuǎn)運(yùn)至外周是為維持自身活性而啟動(dòng)的保護(hù)性過(guò)程[6]。通過(guò)對(duì)THP-1細(xì)胞源性巨噬細(xì)胞中標(biāo)記膽固醇的流出率進(jìn)行測(cè)量,我們發(fā)現(xiàn)溫膽湯能夠顯著促進(jìn)巨噬泡沫細(xì)胞內(nèi)膽固醇的外流,提高細(xì)胞膽固醇流出率,呈一定劑量依賴性。因此,我們認(rèn)為溫膽湯能夠抑制細(xì)胞內(nèi)膽固醇和CE的沉積,同時(shí)提高細(xì)胞內(nèi)膽固醇流出率,從而有效抑制泡沫細(xì)胞的形成。
CD36和SR-A均為清道夫受體家族成員,介導(dǎo)了絕大部分巨噬細(xì)胞對(duì)氧化或乙?;揎桳DL的攝取代謝,可達(dá)到75%~90%,其中乙?;疞DL主要由SR-A調(diào)節(jié), ox-LDL則優(yōu)先由CD36調(diào)節(jié)[4],并且CD36和SR-A所介導(dǎo)的巨噬細(xì)胞膽固醇吞噬不受機(jī)體膽固醇負(fù)反饋調(diào)節(jié)控制,使細(xì)胞內(nèi)正常膽固醇轉(zhuǎn)運(yùn)平衡被破壞。大量動(dòng)物和細(xì)胞實(shí)驗(yàn)顯示, CD36和SR-A是調(diào)節(jié)巨噬細(xì)胞內(nèi)膽固醇代謝的有效靶點(diǎn)[22-23]。本研究結(jié)果顯示,溫膽湯能夠顯著降低細(xì)胞CD36和SR-A蛋白表達(dá)水平,呈一定劑量依賴性。其中6 g/L溫膽湯干預(yù)降低CD36和SR-A表達(dá)水平效果最佳,與對(duì)照組比較分別降低了54%和47%。溫膽湯各濃度組均能顯著降低細(xì)胞CD36蛋白表達(dá)水平,而僅6 g/L溫膽湯表現(xiàn)出顯著降低SR-A的作用,說(shuō)明溫膽湯可能主要通過(guò)抑制CD36蛋白表達(dá)而抑制巨噬細(xì)胞對(duì)膽固醇的吞噬。有研究顯示,在對(duì)修飾性LDL的代謝中,乙?;疞DL主要由SR-A調(diào)節(jié),而ox-LDL則優(yōu)先由CD36調(diào)節(jié)[24]。鑒于此,我們認(rèn)為與抑制SR-A的作用相比,溫膽湯對(duì)CD36的顯著抑制能力將更加有助于抑制巨噬細(xì)胞內(nèi)的膽固醇沉積。
巨噬細(xì)胞內(nèi)膽固醇的外流主要通過(guò)4種方式,包括液相擴(kuò)散、ABCA1介導(dǎo)、ABCG1介導(dǎo)和SR-BI介導(dǎo),分別占35%、35%、21%和9%[25]。研究顯示,在膽固醇代謝正常的小鼠巨噬細(xì)胞中,膽固醇外流主要通過(guò)液相擴(kuò)散,而在過(guò)量負(fù)載膽固醇的巨噬細(xì)胞中,膽固醇外流主要通過(guò)轉(zhuǎn)運(yùn)蛋白介導(dǎo)。此外,在膽固醇過(guò)載的人巨噬細(xì)胞中,膽固醇外流主要由ABCA1介導(dǎo),其次為SR-BI,而ABCG1對(duì)人巨噬細(xì)胞的膽固醇外流幾乎沒有貢獻(xiàn)[5-6]。前期研究發(fā)現(xiàn)溫膽湯能夠顯著促進(jìn)細(xì)胞內(nèi)膽固醇外流,于是我們進(jìn)一步對(duì)THP-1細(xì)胞源性巨噬細(xì)胞膽固醇外流相關(guān)的膜轉(zhuǎn)運(yùn)蛋白進(jìn)行Western blot檢測(cè),以挖掘溫膽湯調(diào)節(jié)巨噬細(xì)胞膽固醇逆轉(zhuǎn)運(yùn)的機(jī)制。本研究結(jié)果顯示,溫膽湯干預(yù)后巨噬細(xì)胞ABCA1和SR-BI蛋白表達(dá)水平均顯著增加,溫膽湯最佳藥效組與對(duì)照組比較分別增加了約59%和63%,提示溫膽湯可能通過(guò)影響ABCA1和SR-BI蛋白表達(dá)而促進(jìn)巨噬細(xì)胞的膽固醇外流。
綜上所述,我們認(rèn)為溫膽湯能夠有效抑制ox-LDL誘導(dǎo)的巨噬細(xì)胞泡沫化。相關(guān)機(jī)制可能是通過(guò)抑制巨噬細(xì)胞清道夫受體CD36和SR-A的表達(dá),從而減少巨噬細(xì)胞對(duì)膽固醇的內(nèi)化吞噬,降低細(xì)胞內(nèi)膽固醇蓄積和膽固醇酯化;同時(shí)上調(diào)巨噬細(xì)胞膽固醇逆轉(zhuǎn)運(yùn)相關(guān)膜蛋白ABCA1和SR-BI表達(dá),從而促進(jìn)巨噬細(xì)胞內(nèi)膽固醇流出。本研究結(jié)果從巨噬細(xì)胞泡沫化角度明確了溫膽湯抗動(dòng)脈粥樣硬化的作用機(jī)制,為溫膽湯防治動(dòng)脈粥樣硬化性疾病的臨床運(yùn)用提供了有力證據(jù)。
[1] Linton MF, Fazio S. Macrophages, inflammation, and atherosclerosis[J]. Int J Obesity, 2003, 27:35-40.
[2] Shibata N, Glass CK.Macrophages, oxysterols and atherosclerosis[J]. Circ J, 2010, 74(10):2045-2051.
[3] Liu X, Ntambi JM.Atherosclerosis: Keep your macrophages in shape[J]. Nat Med, 2009, 15(12):1357-1358.
[4] Parthasarathy S, Fong LG, Otero D, et al. Recognition of solubilized apoproteins from delipidated, oxidized low density lipoprotein (LDL) by the acetyl-LDL receptor[J]. Proc Natl Acad Sci U S A, 1987, 84(2):537-540.
[5] Ohashi R, Mu H, Wang X, et al. Reverse cholesterol transport and cholesterol efflux in atherosclerosis[J]. QJM, 2005, 98(12):845-856.
[6] Pennings M, Meurs I, Ye D, et al.Regulation of cholesterol homeostasis in macrophages and consequences for atherosclerotic lesion development[J]. FEBS Lett, 2006, 580(23):5588-5596.
[7] Wilson HM, Barker RN, Erwig LP. Macrophages: promising targets for the treatment of atherosclerosis[J]. Curr Vasc Pharmacol, 2009, 7(2):234-243.
[8]陳玄晶,徐丹蘋,陳小光,等. 冠心病中醫(yī)痰證與客觀化指標(biāo)關(guān)系研究概述[J]. 中華中醫(yī)藥雜志, 2017, 32(7):3089-3092.
Chen XJ, Xu DP, Chen XG, et al. Overview of the relationship between phlegm syndrome and objective indexes in coronary heart disease[J]. Chin J Tradit Chin Med Pharm, 2017, 32(7):3089-3092.
[9]吳煥林,呂渭輝,潘桂娟,等. 中醫(yī)痰證診斷標(biāo)準(zhǔn)[J]. 中國(guó)中西醫(yī)結(jié)合雜志, 2016, 31(7):776-780.
Wu HL, Lv WH, Pan GJ, et al. Diagnosis standards for phlegm syndrome of Chinese medicine[J]. Chin J Integr Tradit West Med, 2016, 31(7):776-780.
[10] 徐濟(jì)民,蔡沛源,李龍官,等. 痰濁型冠心病與血脂水平關(guān)系的探討[J]. 中西醫(yī)結(jié)合雜志, 1984, 4(5):265-267.
Xu JM, Cai PY, Li LG, et al. The relationship between coronary heart disease of turbid-phlegm type and blood lipid levels[J]. Chin J Integr Tradit West Med, 1984, 4(5):265-267.
[11] Xu JH, Huang YM, Ling W, et al. Wen Dan decoction for hemorrhagic stroke and ischemic stroke[J]. Complement Ther Med, 2015, 23(2):298-308.
[12]何皓颋. 加味溫膽湯對(duì)氣虛痰瘀型冠狀動(dòng)脈粥樣硬化性心臟病多支病變不完全血運(yùn)重建患者心絞痛的影響[J]. 中醫(yī)臨床研究, 2019, 11(10):73-75.
He HT. Effects of the Wendan decoction on angina of incomplete revascularization patients with coronary heart disease of the QixuTanyu type[J]. Clin J Chin Med, 2019, 11(10):73-75.
[13] 陳玄晶,陳小光,徐丹蘋,等. 溫膽湯調(diào)節(jié)血脂異常系統(tǒng)評(píng)價(jià)[J]. 中國(guó)中醫(yī)藥信息雜志, 2017, 24(8):93-98.
Chen XJ, Chen XG, Xu DP, et al. Systematic assessment on effects of Wendan decoction on dyslipidemia[J]. Chin J Inf Traditi Chin Med, 2017, 24(8): 93-98.
[14] 吳煥林,徐丹蘋,羅文杰. 鄧鐵濤調(diào)脾護(hù)心法治療冠心病心絞痛方案抗心肌缺血作用的臨床隊(duì)列研究[J]. 遼寧中醫(yī)藥雜志, 2012, 39(3):385-387.
Wu HL, Xu DP, Luo WJ. Clinical cohort study of anti-myocardial ischemia effect of DENG Tie-taotreating angina pectoris with spleen-regulating-and-heart-nourishing theory[J]. Liaoling J Tradit Chin Med, 2012, 39(3): 385-387.
[15] 徐丹蘋,王俠,盛小剛,等. 參術(shù)冠心方治療冠心病穩(wěn)定型心絞痛臨床研究[J]. 廣州中醫(yī)藥大學(xué)學(xué)報(bào), 2014, 31(2):173-177.
Xu DP, Wang X, Sheng XG, et al. Double-blind, randomized, controlled clinical trial of ShenzhuGuanxin Prescription for treatment of stable angina due to coronary heart disease[J]. J Guangzhou Univ Tradit Chin Med, 2014, 31(2):173-177.
[16] 陳亮,孫毅凡,陳濤,等. 佛波酯誘導(dǎo)THP-1細(xì)胞分化條件的優(yōu)化及自噬模型的建立[J].中國(guó)防癆雜志, 2013, 35(12):997-1001.
Chen L, Sun YF, Chen T, et al. Optimization of culture for THP-1 derived macrophages induced by PMA and establishment of autophagy model[J].Chin J Antitubercul, 2013, 35(12):997-1001.
[17] Hundal RS, Gómez-Mu?oz A, Kong JY, et al. Oxidized low density lipoprotein inhibits macrophage apoptosis by blocking ceramide generation, thereby maintaining protein kinase B activation and Bcl-XL levels[J]. J Biol Chem, 2013, 278(27):24399-24408.
[18] Liu X, Ntambi JM. Atherosclerosis: keep your macrophages in shape[J]. Nat Med, 2009, 15(129):1357-1358.
[19] Yuan Y, Li P, Ye J. Lipid homeostasis and the formation of macrophage-derived foam cells in atherosclerosis[J]. Protein Cell, 2012, 3(3):173-181.
[20] Ghosh S. Early steps in reverse cholesterol transport[J]. Curr Opin Endocrinol Diabetes Obes, 2012, 19(2):136-141.
[21] Zhao B, Song J, Chow WN, et al. Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr mice[J]. J Clin Invest, 2007, 117(10):2983-2992.
[22] Makinen PI, Lappalainen JP, Heinonen SE, et al. Silencing of either SR-A or CD36 reduces atherosclerosis in hyperlipidaemic mice and reveals reciprocal upregulation of these receptors[J]. Cardiovasc Res, 2010, 88(3):530-538.
[23] 肖銘甲,陳衛(wèi)紅,吳煒,等. 虎杖苷抑制ox-LDL誘導(dǎo)的脂質(zhì)過(guò)氧化并下調(diào)巨噬細(xì)胞清道夫受體CD36表達(dá)[J]. 中國(guó)病理生理雜志, 2010, 26(7):1280-1284.
Xiao MJ, Chen WH, Wu W, et al. Polydatin inhibits lipid peroxidation induced by ox-LDL and down-regulates CD36 expression in macrophages[J]. Chin J Pathophysiol, 2010, 26(7):1280-1284.
[24] Kunjathoor VV, Febbraio M, Podrez EA, et al. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages[J]. J Biol Chem, 2002, 277(51):49982-49988.
[25] Yancey PG, Bortnick AE,Kellner WG, et al. Importance of different pathways of cellular cholesterol efflux[J]. Arterioscler Thromb Vasc Biol, 2003, 23(5):712-719.
Inhibitory effect of Wendan decoction on formation of foam cells induced by ox-LDL
CHEN Xuan-jing1, ZHONG Bi-ying1, WU Huan-lin3, XU Dan-ping2
(1,2,510020,;3,100700,)
To explore the anti-atherosclerotic mechanism of Wendan decoction based on formation of foam cells.The optimal concentrations of Wendan decoction without cytotoxity to cells were selected by MTT assay. After Wendan decoction treatment, the formation of foam cells was examined by oil red O staining. The cholesterol efflux, cholesterol level, free cholesterol level and cholesterol esterification rate were analyzed using cholesterol efflux assay, total cholesterol assay and free cholesterol assay. The expression levels of macrophage membrane proteins, including CD36, scavenger receptor class A (SR-A), ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI), were quantified by Western blot.The optimal concentrations of Wendan decoction without cytotoxity to the cells were 0~6 g/L. Wendan decoction at the concentrations of 1.5, 3 and 6 g/L were selected for the experiments. Wendan decoction at these concentrations inhibited the formation of foam cells induced by oxidized low-density lipoprotein (ox-LDL), and reduced the accumulation of intracellular lipids in a concentration-dependent manner (<0.05 or<0.01). Wendan decoction also reduced intracellular total cholesterol level, cholesterol ester level and cholesterol esterification rate (<0.05 or<0.01), promoted efflux of intracellular cholesterol (<0.01), and decreased the protein level of CD36 in THP-1 cell-derived macrophages (<0.01) in a concentration-dependent manner. Wendan decoction at the concentration of 6 g/L significantly reduced the protein level of SR-A in THP-1 cell-derived macrophages (<0.05). At the concentrations of 3 and 6 g/L, Wendan decoction significantly increased the protein levels of ABCA1 and SR-BI in THP-1 cell-derived macrophages (<0.05 or<0.01).Wendan decoction significantly inhibits ox-LDL-induced formation of foam cells by reducing cholesterol deposition and promoting cholesterol efflux, and its mechanism may be related to the down-regulation of CD36 and SR-A and the up-regulation of ABCA1 and SR-BI.
Wendan decoction; Atherosclerosis; Macrophages; Scavenger receptor; ATP-binding cassette transporter A1
R543.5; R363.2
A
10.3969/j.issn.1000-4718.2020.11.005
1000-4718(2020)11-1952-08
2020-04-12
2020-05-20
國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81774219);中醫(yī)藥科學(xué)技術(shù)研究專項(xiàng)(No.YN2019MJ13)
Tel: 020-81887233; E-mail: heart-center2@gzucm.edu.cn(徐丹蘋); wuhuanlinboshi@aliyun.com(吳煥林)
(責(zé)任編輯:宋延君,羅森)