心肌缺血再灌注后CXC趨化因子配體16水平的變化及調(diào)節(jié)機(jī)制

曾 敏，魏 欣，李 偉，蒙緒卿，符秀虹，陳積雄，王 萍

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·論著·

心肌缺血再灌注后CXC趨化因子配體16水平的變化及調(diào)節(jié)機(jī)制

曾 敏，魏 欣，李 偉，蒙緒卿，符秀虹，陳積雄，王 萍

【摘要】目的探討心肌缺血再灌注后CXC趨化因子配體16(CXCL16)水平的變化及調(diào)節(jié)機(jī)制。方法將30只新西蘭大白兔，按照隨機(jī)數(shù)字表法分為假手術(shù)組、心肌缺血再灌注組和四氫吡咯二硫代氨基甲酯(PDTC)組。PDTC組經(jīng)耳緣靜脈注射20 mg/kg PDTC，30 min后PDTC組和心肌缺血再灌注組結(jié)扎冠狀動(dòng)脈，1.5 h后松解縫合線，假手術(shù)組只穿線不結(jié)扎。再灌注1 h后從左心房注入硫磺素S，無(wú)復(fù)流區(qū)無(wú)熒光著色；于原位重新結(jié)扎左回旋支，從左心房再次注入伊文斯藍(lán)，非藍(lán)染區(qū)域?yàn)槿毖獏^(qū)。處死動(dòng)物，沿平行房室溝方向?qū)⑿氖仪衅?，手?dòng)描記心肌切片左心室壁、缺血區(qū)、無(wú)復(fù)流區(qū)輪廓。于動(dòng)物結(jié)扎時(shí)和結(jié)扎后45、100、150 min時(shí)采用乙二胺四乙酸(EDTA)抗凝管自耳緣靜脈采血5 ml，應(yīng)用雙抗體夾心酶標(biāo)免疫分析法測(cè)定CXCL16水平。取左心室室間隔區(qū)、缺血區(qū)、無(wú)復(fù)流區(qū)心肌組織染色,以細(xì)胞核染陽(yáng)性率反映心肌NF-κB p65表達(dá)水平。結(jié)果PDTC組心肌缺血范圍和無(wú)復(fù)流范圍均小于心肌缺血再灌注組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。不同干預(yù)措施對(duì)動(dòng)物結(jié)扎后不同時(shí)間CXCL16水平的影響，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。心肌缺血再灌注組結(jié)扎后100 min CXCL16水平高于結(jié)扎時(shí)和結(jié)扎后45 min,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。各組缺血區(qū)和無(wú)復(fù)流區(qū)細(xì)胞核染陽(yáng)性率比較，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中，心肌缺血再灌注組缺血區(qū)和無(wú)復(fù)流區(qū)細(xì)胞核染陽(yáng)性率高于假手術(shù)組和PDTC組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論心肌缺血再灌注可誘導(dǎo)CXCL16的表達(dá)；抑制NF-κB的激活可降低CXCL16的水平，縮小心肌缺血范圍和無(wú)復(fù)流范圍。

CXC趨化因子配體16(CXCL16)是近年來(lái)新發(fā)現(xiàn)的CXC趨化因子家族穿膜細(xì)胞因子，以跨膜蛋白和可溶性蛋白兩種形式存在。有證據(jù)顯示，CXCL16與腸道炎癥、腎小球腎炎等疾病的發(fā)生及其嚴(yán)重程度密切相關(guān)[1-2]，對(duì)冠心病的炎癥效應(yīng)也日漸受到關(guān)注[3]。本研究旨在觀察心肌缺血再灌注不同時(shí)間窗內(nèi)CXCL16水平的變化，并通過(guò)NF-κB活性與CXCL16的關(guān)系來(lái)探討細(xì)胞因子在信號(hào)轉(zhuǎn)導(dǎo)途徑上的調(diào)節(jié)機(jī)制。

1材料與方法

1.1動(dòng)物與試劑健康雄性清潔級(jí)新西蘭大白兔30只，月齡4～6個(gè)月，體質(zhì)量2.0～3.0 kg,購(gòu)自北京海淀興旺動(dòng)物養(yǎng)殖公司。四氫吡咯二硫代氨基甲酯(PDTC)、硫磺素S購(gòu)自美國(guó)Sigma公司；CXCL16 ELISA檢測(cè)試劑盒購(gòu)自美國(guó)R&D公司；小鼠抗兔NF-κB p65單克隆抗體購(gòu)自美國(guó)Santa Cruz公司；伊文斯藍(lán)購(gòu)自美國(guó)Alfa Aesar公司。

1.2方法

1.2.1心肌缺血再灌注模型按隨機(jī)數(shù)字表法將動(dòng)物分為假手術(shù)組、心肌缺血再灌注組(I/R組)和PDTC組，每組各10只。PDTC組經(jīng)耳緣靜脈注射20 mg/kg PDTC，假手術(shù)組和I/R組給予相同劑量的0.9%氯化鈉溶液。30 min后采用3%戊巴比妥鈉(30 mg/kg)耳緣靜脈注射麻醉動(dòng)物,氣管插管后接小動(dòng)物呼吸機(jī)(呼吸頻率32次/min，吸呼比1∶2，潮氣量15 ml/kg)進(jìn)行機(jī)械通氣。于胸骨正中打開胸腔，縱行切開心包膜，暴露心臟，并將心包膜縫合于胸壁呈吊籃狀，于冠狀動(dòng)脈左回旋支近段1/5處穿入3/0縫合線，再將縫合線穿入阻斷管，結(jié)扎冠狀動(dòng)脈，心肌缺血依據(jù)心電圖ST段抬高且有節(jié)段性運(yùn)動(dòng)不良確認(rèn)。I/R組和PDTC組結(jié)扎1.5 h后松解縫合線再灌注。假手術(shù)組只穿線不結(jié)扎。

1.2.2心肌缺血及無(wú)復(fù)流范圍再灌注1 h后從左心房注入1.5 ml/kg 6%硫磺素S，無(wú)復(fù)流區(qū)無(wú)熒光著色；于原位重新結(jié)扎左回旋支，從左心房再次注入2 ml 4%伊文斯藍(lán)，非藍(lán)染區(qū)域?yàn)槿毖獏^(qū)。處死動(dòng)物，迅速取出心臟,用冰冷的0.9%氯化鈉溶液沖洗心臟，沿平行房室溝方向?qū)⑿氖仪袨?～8片心肌短軸切片(6 μm,Leica 2323型石蠟切片機(jī))。對(duì)切片進(jìn)行拍照并保存圖像，按染色結(jié)果對(duì)各個(gè)區(qū)域分別取材，液氮保存。用Image Pro Plus 6.0圖像分析軟件打開圖像,分別手動(dòng)描記心肌切片左心室壁、缺血區(qū)、無(wú)復(fù)流區(qū)輪廓,確定左心室壁面積(LV)、缺血面積(RA)、無(wú)復(fù)流面積(NRA),并計(jì)算缺血范圍=RA/LV×100%，無(wú)復(fù)流范圍=NRA/LV×100%。

1.2.3CXCL16的測(cè)定于動(dòng)物結(jié)扎時(shí)和結(jié)扎后45、100、150 min時(shí)采用乙二胺四乙酸(EDTA)抗凝管自耳緣靜脈采血5 ml，以1 000 r/min離心15 min,離心半徑10 cm,取血漿于-80 ℃低溫凍存。采用CXCL16 ELISA檢測(cè)試劑盒,應(yīng)用雙抗體夾心酶標(biāo)免疫分析法測(cè)定CXCL16水平,經(jīng)徹底洗滌后用底物TMB顯色。采用酶標(biāo)儀在波長(zhǎng)450 nm處測(cè)定吸光度值(OD值)。

1.2.4心肌不同區(qū)域NF-κB p65表達(dá)水平取左心室室間隔區(qū)、缺血區(qū)、無(wú)復(fù)流區(qū)心肌組織，10%甲醛溶液固定,常規(guī)石蠟包埋,切片。將石蠟切片脫蠟,抗原修復(fù)后滴加小鼠抗兔NF-κB p65單克隆抗體(1∶75),左心室室間隔區(qū)使用PBS代替一抗,37 ℃濕盒孵育,4 ℃過(guò)夜,次日滴加特異性生物素化二抗及過(guò)氧化物酶復(fù)合物,DAB顯色,蘇木精復(fù)染,中性樹膠封片。每只動(dòng)物選取連續(xù)3張切片,分別以左心室室間隔區(qū)、缺血區(qū)、無(wú)復(fù)流區(qū)為目標(biāo)區(qū)域,每張切片隨機(jī)選取10個(gè)視野,200倍及400倍光鏡下拍照并保存圖像，Image Pro Plus 6.0圖像分析軟件分析圖片,以細(xì)胞核染陽(yáng)性率反映心肌NF-κB p65表達(dá)水平,細(xì)胞核染陽(yáng)性率=細(xì)胞核染色陽(yáng)性的細(xì)胞數(shù)/視野內(nèi)總細(xì)胞數(shù)×100%。

2結(jié)果

2.1I/R組和PDTC組心肌缺血范圍和無(wú)復(fù)流范圍比較PDTC組心肌缺血范圍和無(wú)復(fù)流范圍均小于I/R組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表1)。

Table1Comparisonofischemicareaandno-reflowareabetweencardiacI/RgroupandPDTCgroup

組別只數(shù)心肌缺血范圍無(wú)復(fù)流范圍I/R組1055.541±4.25369.743±6.384PDTC組1026.482±2.78135.643±3.201t值3.1722.919P值0.0180.023

2.2各組CXCL16水平比較不同干預(yù)措施對(duì)動(dòng)物結(jié)扎后不同時(shí)間CXCL16水平的影響，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。I/R組結(jié)扎后100 min CXCL16水平高于結(jié)扎時(shí)和結(jié)扎后45 min,差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表2)。

Table2ComparisonofCXCL16levelamongthethreegroupsatdifferenttimepoints

組別只數(shù)結(jié)扎時(shí)結(jié)扎后45min結(jié)扎后100min結(jié)扎后150min假手術(shù)組101.591±0.0621.893±0.0711.513±0.0821.692±0.094I/R組101.753±0.0942.342±0.0943.030±0.264ab2.321±0.180PDTC組101.671±0.1711.791±0.0541.941±0.1401.743±0.142F值F交互=4.690,F時(shí)間=7.320,F組間=10.210P值P交互=0.162,P時(shí)間=0.089,P組間=0.032

注：與組內(nèi)結(jié)扎時(shí)比較，aP<0.05；與組內(nèi)結(jié)扎后45 min比較，bP<0.05

2.3細(xì)胞核染陽(yáng)性率各組室間隔區(qū)細(xì)胞核染陽(yáng)性率比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。各組缺血區(qū)和無(wú)復(fù)流區(qū)細(xì)胞核染陽(yáng)性率比較，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中，I/R組缺血區(qū)和無(wú)復(fù)流區(qū)細(xì)胞核染陽(yáng)性率高于假手術(shù)組和PDTC組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表3)。

Table3Comparisonofpositivedyeingrateofmyocardialcellnucleusamongthethreegroups

組別只數(shù)室間隔區(qū)缺血區(qū)無(wú)復(fù)流區(qū)假手術(shù)組107.081±1.0907.232±1.1207.170±0.911I/R組108.764±1.88168.411±9.352ab35.324±6.060abPDTC組105.892±1.1216.022±0.8345.941±0.781F值1.533198.17016.874P值>0.05<0.01<0.05

注：與假手術(shù)組比較，aP<0.05；與PDTC組比較，bP<0.05

3討論

CXC趨化因子是一類小分子分泌蛋白,參與炎性反應(yīng)和免疫應(yīng)答過(guò)程。CXCL16屬于CXC趨化因子家族的膜結(jié)合蛋白,具有清道夫受體的作用和炎癥屬性，被認(rèn)為在腎小球腎炎[4]、肝炎、心內(nèi)膜炎[5]、腫瘤[6]及痛風(fēng)[7]等炎性疾病中發(fā)揮重要的促炎作用。Lehrke等[8]研究認(rèn)為，無(wú)論在體內(nèi)或體外,炎癥信號(hào)均能誘導(dǎo)CXCL16的表達(dá)，而CXCL16的高表達(dá)與冠心病的發(fā)生密切相關(guān)。CXCL16在炎性反應(yīng)中發(fā)揮趨化因子的作用,能激活T淋巴細(xì)胞[5],活化的T淋巴細(xì)胞通過(guò)分泌γ干擾素、腫瘤壞死因子2β等炎性因子,以旁分泌的方式影響其周圍細(xì)胞的功能活動(dòng)，而γ干擾素又反過(guò)來(lái)誘導(dǎo)CXCL16 mRNA的表達(dá)增加[9]。Yellon等[10]研究顯示，炎性反應(yīng)是心肌缺血的病理基礎(chǔ)，更是導(dǎo)致缺血再灌注損傷的重要病理生理機(jī)制。本研究結(jié)果顯示，I/R組大白兔再灌注后CXCL16水平明顯升高。CXCL16受到炎性刺激而表達(dá)并參與炎性反應(yīng)，其水平升高提示可能通過(guò)炎性反應(yīng)參與兔心肌缺血再灌注損傷過(guò)程。Zhao等[11]研究認(rèn)為，CXCL16缺陷小鼠具有抗缺血再灌注損傷的心肌保護(hù)作用，本研究結(jié)論與其相似。

本研究發(fā)現(xiàn)，特異性抑制劑PDTC可降低缺血區(qū)和無(wú)復(fù)流區(qū)NF-κB的激活，提示NF-κB可能參與了心肌缺血再灌注對(duì)CXCL16水平變化的調(diào)節(jié)。NF-κB是一組真核細(xì)胞轉(zhuǎn)錄因子,由5種亞單位p50(NF-κB1)、p52(NF-κB2)、p65(RelA)、RelB、Rel(c-Rel)構(gòu)成的二聚體蛋白。NF-κB最常見形式是p50-p65構(gòu)成的異源二聚體,幾乎存在于體內(nèi)所有細(xì)胞,且含量極高。NF-κB具有與DNA結(jié)合的特性,活化的NF-κB常與靶基因結(jié)合促進(jìn)其轉(zhuǎn)錄，從而通過(guò)下游分子如促炎細(xì)胞因子、黏附因子、趨化因子、凋亡調(diào)節(jié)基因、急性期蛋白血管緊張素Ⅱ及組織因子等參與調(diào)節(jié)炎性反應(yīng)、免疫及細(xì)胞凋亡等重要細(xì)胞功能[12]。本研究結(jié)果顯示,NF-κB在缺血再灌注心肌的缺血區(qū)和無(wú)復(fù)流區(qū)均明顯激活，尤以缺血區(qū)顯著。PDTC可通過(guò)抑制NF-κB的活性，降低CXCL16的表達(dá)，最終減少心肌缺血和無(wú)復(fù)流面積。因此，本研究認(rèn)為，缺血再灌注可能激活心肌細(xì)胞NF-κB的表達(dá)，從而介導(dǎo)CXCL16的分泌，同時(shí)，CXCL16也加重炎性反應(yīng)，進(jìn)一步促進(jìn)缺血再灌注損傷。

綜上所述，CXCL16在NF-κB的調(diào)節(jié)下加劇了心肌缺血再灌注中的炎性反應(yīng)，最終促進(jìn)缺血再灌注損傷。上述結(jié)論為臨床預(yù)防和治療心肌缺血再灌注損傷提供了新的理論基礎(chǔ)，但其具體信號(hào)轉(zhuǎn)導(dǎo)機(jī)制有待進(jìn)一步研究。

作者貢獻(xiàn)：曾敏進(jìn)行課題設(shè)計(jì)與實(shí)施、資料收集整理、撰寫論文、成文并對(duì)文章負(fù)責(zé)；李偉、蒙緒卿、符秀虹、陳積雄、王萍進(jìn)行課題實(shí)施、評(píng)估、資料收集；魏欣進(jìn)行質(zhì)量控制及審校。

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(本文編輯：吳立波)

【關(guān)鍵詞】心肌再灌注；趨化因子CXCL16；信號(hào)傳導(dǎo)；NF-κB

曾敏，魏欣，李偉，等.心肌缺血再灌注后CXC趨化因子配體16水平的變化及調(diào)節(jié)機(jī)制[J].中國(guó)全科醫(yī)學(xué)，2016，19(5)：542-544，548.[www.chinagp.net]

Zeng M,Wei X,Li W,et al.Change and regulatory mechanism of the level of plasma CXC-chemokine ligand 16 after myocardial ischemia reperfusion[J].Chinese General Practice，2016，19(5)：542-544，548.

Change and Regulatory Mechanism of the Level of Plasma CXC-chemokine Ligand 16 After Myocardial Ischemia ReperfusionZENGMin,WEIXin,LIWei,etal.HainanProvincialPeople′sHospital,Haikou570311,China

【Abstract】ObjectiveTo investigate the change and regulation mechanism of the level of plasma CXC-chemokine ligand 16 (CXCL16).MethodsWe divided 30 New Zealand white rabbits into 3 groups:sham-operation group,cardiac ischemia/reperfusion(I/R) group and pyrrolidine dithiocarbamate(PDTC) group.The PDTC group was administrated with 20 mg/kg PDTC by ear marginal vein injection,and 30 minutes later,the PDTC group and I/R group were administrated with coronary artery ligation;1.5 hours later,suture line was unbound;and the sham-operation group was only sutured without ligation.One hour after reperfusion,Thioflavine S was injected into atrium sinistrum,leaving no fluorescence staining in no reflow area.Ligation of left circumflex artery was made again at the primary site,and Evans blue was injected into atrium sinistrum again,with no blue stain area in ischemic area.After the animals were killed,ventricular slice was taken parallel to the atrioventricular groove,and the outlines of left ventricular wall,ischemic region and no reflow area were made manually on myocardial biopsy.During ligation,and 45 minutes,100 minutes and 150 minutes after ligation,ethylenediamine tetraacetic acid(EDTA) anticoagulative tube was employed to sample 5 ml blood from ear marginal vein,and double antibody sandwich enzyme immunoassay was used to determine CXCL16 level.Myocardial tissue staining results of left ventricular septal area,ischemic region and no flow area were obtained,and the dyeing positive rate of cell nucleus was used to reflect the expression of NF-κB p65.ResultsThe area of myocardial ischemia and the no-flow area of PDTC group were both smaller than I/R group(P<0.05).Different intervention measures were significantly different in the influence on the level of CXCL16 at different time points (P<0.05).In I/R group,the level of CXCL16 at 100 minutes after ligation was higher (P<0.05) than that during ligation and 45 minutes after ligation.The three groups were significantly different in the dyeing positive rate of ischemic area and no-flow area (P<0.05);the ischemic area and no-flow area of the I/R group were higher than sham-operation group and PDTC group in the dying positive rate of cell nucleus (P<0.05).ConclusionMyocardial ischemia reperfusion could induce the expression of CXCL16,and the inhibition of the activation of NF-κB could reduce the level of CXCL16 and reduce the myocardial ischemia area and no flow area.

【Key words】Myocardial reperfusion；Chemokine CXCL16；Signal transduction；NF-kappa B

(收稿日期：2015-06-05；修回日期：2015-10-25)

【中圖分類號(hào)】R 816.2

【文獻(xiàn)標(biāo)識(shí)碼】A

doi：10.3969/j.issn.1007-9572.2016.05.011

通信作者：曾敏，570311海南省?？谑?，海南省人民醫(yī)院；E-mail：hndzm6@126.com

基金項(xiàng)目：國(guó)家自然科學(xué)基金資助項(xiàng)目(81260043;81100153)；海南省應(yīng)用技術(shù)研發(fā)與示范推廣專項(xiàng)(ZDXM2014065)
作者單位：570311海南省?？谑?，海南省人民醫(yī)院