益心解毒方對(duì)NOX2亞基和NOX4亞基過(guò)表達(dá)及小干擾RNA引起的心肌細(xì)胞還原型輔酶Ⅱ氧化酶活性變化的機(jī)制研究

解華，麻春杰，郭淑貞，付幫澤，馮玄超，王偉

益心解毒方對(duì)NOX2亞基和NOX4亞基過(guò)表達(dá)及小干擾RNA引起的心肌細(xì)胞還原型輔酶Ⅱ氧化酶活性變化的機(jī)制研究

解華，麻春杰，郭淑貞，付幫澤，馮玄超，王偉

目的觀察益心解毒方含藥血清對(duì)NOX2、NOX4亞基過(guò)表達(dá)和小干擾RNA(RNAi)導(dǎo)致的H9C2心肌細(xì)胞還原型輔酶Ⅱ(NADPH)氧化酶活性的變化，探討益心解毒方的作用機(jī)制。方法采用正常雄性Sprague－Dawley大鼠16只，構(gòu)建針對(duì)NOX2、NOX4亞基的過(guò)表達(dá)質(zhì)粒和RNAi質(zhì)粒。采用轉(zhuǎn)染試劑瞬時(shí)轉(zhuǎn)染H9C2心肌細(xì)胞，流式細(xì)胞術(shù)檢測(cè)轉(zhuǎn)染率，轉(zhuǎn)染24 h后分組(正常H9C2細(xì)胞組、過(guò)表達(dá)組、H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組、RNAi質(zhì)粒組、益心解毒方高劑量組、益心解毒方中劑量組、益心解毒方低劑量組)給予不同劑量的含藥血清干預(yù)，24 h后檢測(cè)NADPH氧化酶活性。結(jié)果轉(zhuǎn)染NOX2亞基過(guò)表達(dá)質(zhì)粒的各組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。其中，過(guò)表達(dá)組、益心解毒方高劑量組、益心解毒方低劑量組與正常H9C2細(xì)胞組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組、RNAi質(zhì)粒組、益心解毒方中劑量組與過(guò)表達(dá)組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);RNAi質(zhì)粒組、益心解毒方高劑量組、益心解毒方低劑量組與H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方高劑量組、益心解毒方低劑量組與RNAi質(zhì)粒組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方中劑量組與益心解毒方高劑量組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方低劑量組與益心解毒方中劑量組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。轉(zhuǎn)染NOX4亞基過(guò)表達(dá)質(zhì)粒的各組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。其中，過(guò)表達(dá)組、益心解毒方高劑量組、益心解毒方低劑量組與正常H9C2細(xì)胞組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組、RNAi質(zhì)粒組、益心解毒方中劑量組、益心解毒方低劑量組與過(guò)表達(dá)組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);RNAi質(zhì)粒組、益心解毒方高劑量組、益心解毒方低劑量組與H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方高劑量組、益心解毒方低劑量組與RNAi質(zhì)粒組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方中劑量組與益心解毒方高劑量組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方低劑量組與益心解毒方中劑量組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。結(jié)論NOX2、NOX4亞基的過(guò)表達(dá)可以提高NADPH氧化酶活性，而RNAi表達(dá)可以沉默NOX2、NOX4亞基，從而降低NADPH氧化酶活性，益心解毒方不會(huì)直接影響NADPH氧化酶活性。實(shí)驗(yàn)研究表明干預(yù)和抑制NOX2、NOX4亞基的NADPH氧化酶活性調(diào)控環(huán)節(jié)，降低心肌活性氧(ROS)水平，保護(hù)心肌細(xì)胞，改善心功能，可能是益心解毒方發(fā)揮藥效的重要機(jī)制。

益心解毒方;肌細(xì)胞，心臟;基因表達(dá);小干擾RNA;氧化還原酶類

解華，麻春杰，郭淑貞，等.益心解毒方對(duì)NOX2亞基和NOX4亞基過(guò)表達(dá)及小干擾RNA引起的心肌細(xì)胞還原型輔酶Ⅱ氧化酶活性變化的機(jī)制研究［J］．中國(guó)全科醫(yī)學(xué)，2015，18(30):3727－3731.［www.chinagp.net］

Xie H，Ma CJ，Guo SZ，et al.Mechanism of Yixin Jiedu formula on the overexpression of NOX2 and NOX4 and the myocardial NADPH Ⅱ oxidase activity changes induced by RNAi［J］．Chinese General Practice，2015，18(30):3727－3731.

近年來(lái)研究發(fā)現(xiàn)，活性氧(ROS)在心力衰竭的發(fā)生和發(fā)展中起著關(guān)鍵作用，ROS主要來(lái)源于NOX2、NOX4亞基的還原型輔酶Ⅱ(NADPH)氧化酶［1］。有研究顯示，益心解毒方可以降低ROS的表達(dá)，改善心功能，延緩心力衰竭，推測(cè)該方可能是通過(guò)抑制NADPH氧化酶活化而發(fā)揮藥效［2］。為了深入研究NOX2、NOX4亞基的NADPH氧化酶調(diào)控的環(huán)節(jié)及作用機(jī)制。本研究構(gòu)建NOX2、NOX4亞基過(guò)表達(dá)模型和小干擾RNA(RNAi)模型觀察益心解毒方含藥血清對(duì)NOX2、NOX4亞基的NADPH氧化酶活性的影響，探討該方的作用機(jī)制，為研發(fā)以調(diào)節(jié)心肌ROS穩(wěn)態(tài)為切入點(diǎn)的、新的、可用于臨床冠心病防治的、低毒的、高效的NADPH氧化酶特異性抑制劑提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 動(dòng)物來(lái)源2013年9月—2014年1月，選取正常雄性Sprague－Dawley大鼠16只，體質(zhì)量(220±10)g，清潔級(jí)，常規(guī)飼料飼養(yǎng)，自由飲水。購(gòu)于北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司，許可證編號(hào):SCXK(京)2012－0001。

1.2 主要試劑和材料大鼠H9C2心肌細(xì)胞購(gòu)自北京協(xié)和細(xì)胞資源中心，DMEM高糖培養(yǎng)基購(gòu)自美國(guó)Gibco公司，南美胎牛血清(FBS)購(gòu)自美國(guó)Hyclone公司，lipofectamine 2000購(gòu)自美國(guó)Invitrogen公司，Opti－MEM?IReduced Serum Medium購(gòu)自美國(guó)Gibco公司，T4 DNA連接酶和BamHⅠ、XhoⅠ、SalⅠ、HindⅢ內(nèi)切酶購(gòu)自英國(guó)New England Biolabs公司，pEGFP－C1 Vector、pGenesil－1 Vector購(gòu)自美國(guó)BD公司，NOX2抗體、NOX4抗體、甘油醛－3－磷酸脫氫酶(GAPDH)抗體購(gòu)自英國(guó)Abcam公司，NADPH氧化酶測(cè)定試劑盒購(gòu)自美國(guó)GENMED SCIENTIFICS公司。其余生化試劑均為國(guó)產(chǎn)分析純。引物由北京鼎國(guó)昌盛生物技術(shù)有限責(zé)任公司合成。

1.3 方法

1.3.1 H9C2心肌細(xì)胞的培養(yǎng)取對(duì)數(shù)生長(zhǎng)期的H9C2心肌細(xì)胞，以細(xì)胞濃度1×105個(gè)/ml接種于96孔培養(yǎng)板或直徑10 cm的平皿中，用含10%FBS的DMEM高糖培養(yǎng)基于37℃、5%CO2培養(yǎng)箱中培養(yǎng)，待細(xì)胞長(zhǎng)至孔底或平皿80%～90%，選擇生長(zhǎng)狀態(tài)良好的細(xì)胞進(jìn)行實(shí)驗(yàn)。

1.3.2 NOX2、NOX4亞基過(guò)表達(dá)質(zhì)粒和RNAi質(zhì)粒的構(gòu)建

在GenBank中找到完整的NOX2亞基(登錄號(hào):NM023965，全長(zhǎng)1 713 bp)和NOX4亞基(登錄號(hào):NM053524，全長(zhǎng)2 176 bp)的mRNA序列，設(shè)計(jì)針對(duì)NOX2亞基或NOX4亞基的過(guò)表達(dá)質(zhì)粒和RNAi質(zhì)粒，另外設(shè)計(jì)1對(duì)陰性對(duì)照序列，對(duì)照組RNAi與大鼠mRNA和DNA序列均無(wú)同源性。用內(nèi)切酶對(duì)NOX2、NOX4亞基和質(zhì)粒分別進(jìn)行酶切，T4 DNA連接酶連接并轉(zhuǎn)至大腸埃希菌DH5a。選取聚合酶鏈反應(yīng)(PCR)鑒定為陽(yáng)性克隆的菌斑，進(jìn)行質(zhì)粒提取，經(jīng)北京鼎國(guó)昌盛生物技術(shù)有限責(zé)任公司進(jìn)行DNA測(cè)序，測(cè)序結(jié)果與GenBank中所報(bào)道的目的基因序列進(jìn)行比對(duì)和分析。

1.3.3 益心解毒方含藥血清及空白血清的制備將大鼠適應(yīng)性飼養(yǎng)1周，按體質(zhì)量分層后分別以高、中、低劑量給藥，每個(gè)劑量組4只大鼠。高劑量為2倍臨床等效劑量:1次/d，每次給予益心解毒方4.39 g/kg;中劑量為臨床等效劑量: 1次/d，每次給予益心解毒方2.20 g/kg;低劑量為1/2臨床等效劑量:1次/d，每次給予益心解毒方1.10 g/kg;給藥體積為10 m l/kg，水溶經(jīng)口灌胃。連續(xù)灌胃7 d，于末次給藥2 h后腹主動(dòng)脈取血8～10 ml/只，室溫靜置4 h，3 000×g離心10 min收集血清，于56℃滅活30 min，同時(shí)將所收集的大鼠血清混合以減少動(dòng)物的個(gè)體差異。0.22μm微孔濾膜過(guò)濾除菌，分裝，－80℃保存?zhèn)溆谩２杉嘞聼o(wú)藥物干預(yù)的4只大鼠血清作為空白血清，空白血清在實(shí)驗(yàn)前于37℃恒溫水浴復(fù)溫。1.3.4 MTT法檢測(cè)H9C2心肌細(xì)胞存活率將H9C2心肌細(xì)胞接種于96孔板、培養(yǎng)24～48 h，選擇生長(zhǎng)良好的細(xì)胞，轉(zhuǎn)至無(wú)血清培養(yǎng)基中同步化培養(yǎng)24 h后，分別加入濃度為40%、20%、10%、5%、2.5%、1%、0%的空白血清40μl、20μl、10μl、5μl、2.5μl、1μl及0μl，培養(yǎng)24 h后在490 nm波長(zhǎng)下記錄各孔的吸光度(OD值)，代表心肌細(xì)胞存活率，重復(fù)3次，取6孔OD值的平均值。

1.3.5 Western blotting法篩選NOX2、NOX4亞基RNAi表達(dá)質(zhì)粒用0.05%胰蛋白酶消化細(xì)胞制成單細(xì)胞懸液，鋪在10 cm平皿中(鋪板細(xì)胞數(shù)量使細(xì)胞次日貼壁匯合度在70%～90%)。按照l(shuí)ipofectamine 2000試劑說(shuō)明書將構(gòu)建的4號(hào)含NOX2亞基或NOX4亞基的RNAi質(zhì)粒轉(zhuǎn)入H9C2心肌細(xì)胞，每組質(zhì)粒加入量為24μg。將混勻的質(zhì)粒和lipofectamine 2000混合液滴加到平皿中，邊加邊混勻，于5%CO2培養(yǎng)箱中培養(yǎng)24 h后，吸掉培養(yǎng)基，更換新鮮的完全培養(yǎng)基。24 h后，提取蛋白，將曝光后的膠片進(jìn)行掃描，用Quantity One圖像分析軟件分析目標(biāo)條帶的OD值，重復(fù)3次。實(shí)驗(yàn)分為6組:正常H9C2細(xì)胞組、H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組、轉(zhuǎn)染1號(hào)RNAi質(zhì)粒組、轉(zhuǎn)染2號(hào)RNAi質(zhì)粒組、轉(zhuǎn)染3號(hào)RNAi質(zhì)粒組、轉(zhuǎn)染4號(hào)RNAi質(zhì)粒組。

1.3.6 NOX2、NOX4亞基過(guò)表達(dá)質(zhì)粒及RNAi質(zhì)粒的H9C2心肌細(xì)胞模型的建立將重組質(zhì)粒pEGFP－C1－NOX2、 pEGFP－C1－NOX4與RNAi質(zhì)粒pGenesil－1－NOX2、pGenesil－1－NOX4轉(zhuǎn)入H9C2心肌細(xì)胞，方法同1.3.5，24 h后用倒置熒光顯微鏡拍照觀察，并收集轉(zhuǎn)染的細(xì)胞用流式細(xì)胞儀檢測(cè)轉(zhuǎn)染率(綠色熒光細(xì)胞數(shù)占細(xì)胞總數(shù)的百分比)，重復(fù)3次，取均值。實(shí)驗(yàn)分為7組:正常H9C2細(xì)胞組、過(guò)表達(dá)組、H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組、RNAi質(zhì)粒組、益心解毒方高劑量組、益心解毒方中劑量組、益心解毒方低劑量組。轉(zhuǎn)染后心肌細(xì)胞分別給予不同的藥物干預(yù):正常H9C2細(xì)胞組不干預(yù);過(guò)表達(dá)組和RNAi質(zhì)粒組在轉(zhuǎn)染NOX2亞基或NOX4亞基重組質(zhì)粒后再給予1%濃度空白血清100μl;益心解毒方高、中、低劑量組在轉(zhuǎn)染RNAi質(zhì)粒后再給予1%高、中、低濃度的含藥血清100μl。

1.3.7 NADPH氧化酶活性檢測(cè)細(xì)胞加藥處理24 h后，收集待測(cè)的各組細(xì)胞(約5×106個(gè)/m l)，按GENMED細(xì)胞NADPH活性光度法定量檢測(cè)試劑盒產(chǎn)品說(shuō)明書操作，測(cè)定NADPH氧化酶的特異活性，重復(fù)3次，取均值。

1.4 統(tǒng)計(jì)學(xué)方法采用SPSS 16.0統(tǒng)計(jì)學(xué)軟件對(duì)數(shù)據(jù)進(jìn)行處理，計(jì)量資料以(±s)表示，多組間比較采用單因素方差分析，組間兩兩比較采用q檢驗(yàn)。以P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 MTT法檢測(cè)H9C2心肌細(xì)胞存活率在無(wú)外源性刺激因素的作用下，選擇對(duì)心肌細(xì)胞無(wú)明顯促進(jìn)或抑制作用的血清濃度，相比較而言，1%空白血清濃度接近0%空白血清濃度H9C2心肌細(xì)胞存活率，排除因動(dòng)物血清對(duì)細(xì)胞產(chǎn)生的影響。確定各實(shí)驗(yàn)組選擇1%空白血清濃度(見表1)。

表1 MTT法檢測(cè)不同空白血清濃度H9C2心肌細(xì)胞存活率比較(±s，%，n=3)Table 1 Comparison of the survival rate of H9C2 myocardial cell among different concentration of blank serum detected by MTT

表1 MTT法檢測(cè)不同空白血清濃度H9C2心肌細(xì)胞存活率比較(±s，%，n=3)Table 1 Comparison of the survival rate of H9C2 myocardial cell among different concentration of blank serum detected by MTT

空白血清濃度心肌細(xì)胞存活率40%1.58±0.19 20%0.92±0.76 10%0.82±0.74 5%0.71±0.53 2.5%0.61±0.48 1%0.52±0.36 0%0.49±0.26 F值1.60 P值0.05

2.2 Western blotting法篩選NOX2、NOX4亞基RNAi質(zhì)粒

正常H9C2細(xì)胞組、H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組、轉(zhuǎn)染1號(hào)RNAi質(zhì)粒組、轉(zhuǎn)染2號(hào)RNAi質(zhì)粒組、轉(zhuǎn)染3號(hào)RNAi質(zhì)粒組、轉(zhuǎn)染4號(hào)RNAi質(zhì)粒組NOX2亞基表達(dá)水平比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);且轉(zhuǎn)染3號(hào)RNAi質(zhì)粒組和轉(zhuǎn)染4號(hào)RNAi質(zhì)粒組NOX2亞基表達(dá)水平均低于正常H9C2細(xì)胞組，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。各組NOX4亞基表達(dá)水平比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05，見表2)。轉(zhuǎn)染4號(hào)RNAi質(zhì)粒的蛋白表達(dá)水平最低，沉默效果最明顯，因此選擇4號(hào)RNAi表達(dá)質(zhì)粒進(jìn)行后續(xù)的實(shí)驗(yàn)。

2.3 H9C2心肌細(xì)胞轉(zhuǎn)染率轉(zhuǎn)染24 h后，倒置熒光顯微鏡下可見大量發(fā)綠色熒光的H9C2心肌細(xì)胞，表明轉(zhuǎn)染成功(見圖1)。NOX2亞基過(guò)表達(dá)質(zhì)粒轉(zhuǎn)染率為58.45%，NOX4亞基過(guò)表達(dá)質(zhì)粒轉(zhuǎn)染率為61.03%，NOX2亞基RNAi質(zhì)粒轉(zhuǎn)染率為53.31%，NOX4亞基RNAi質(zhì)粒轉(zhuǎn)染率為61.11%(見圖2)。

圖1 H9C2心肌細(xì)胞轉(zhuǎn)染重組質(zhì)粒24 h后熒光顯微鏡觀察(×200)Figure 1 The results of transfection 24 h under fluorescentmicroscope after transfection H9C2 cells recombinant plasmid

圖2 H9C2心肌細(xì)胞轉(zhuǎn)染重組質(zhì)粒24 h后流式細(xì)胞儀檢測(cè)轉(zhuǎn)染率結(jié)果Figure2 The detection rate of H9C2 cell after transfection recombinant plasmid 24 h by FCM

2.4 各組NADPH氧化酶活性比較轉(zhuǎn)染NOX2亞基過(guò)表達(dá)質(zhì)粒的各組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。其中，過(guò)表達(dá)組、益心解毒方高劑量組、益心解毒方低劑量組與正常H9C2細(xì)胞組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組、RNAi質(zhì)粒組、益心解毒方中劑量組與過(guò)表達(dá)組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);RNAi質(zhì)粒組、益心解毒方高劑量組、益心解毒方低劑量組與H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方高劑量組、益心解毒方低劑量組與RNAi質(zhì)粒組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方中劑量組與益心解毒方高劑量組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方低劑量組與益心解毒方中劑量組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05，見表3)。

表2 各組NOX2亞基和NOX4亞基表達(dá)水平比較(±s，n=3)Table2 Comparison of protein expression level of NOX2 and NOX4 among different groups

表2 各組NOX2亞基和NOX4亞基表達(dá)水平比較(±s，n=3)Table2 Comparison of protein expression level of NOX2 and NOX4 among different groups

注:與正常H9C2細(xì)胞組比較，aP＜0.05

組別NOX2亞基NOX4 0.86±0.15 0.63±0.27 H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組0.98±0.05 0.93±0.50轉(zhuǎn)染1號(hào)RNAi質(zhì)粒組0.77±0.14 0.79±0.52轉(zhuǎn)染2號(hào)RNAi質(zhì)粒組0.81±0.08 0.82±0.46轉(zhuǎn)染3號(hào)RNAi質(zhì)粒組0.68±0.03a0.39±0.21轉(zhuǎn)染4號(hào)RNAi質(zhì)粒組0.57±0.05a0.24±0.15 F亞基正常H9C2細(xì)胞組值0.04 0.26 6.52 1.50 P值

轉(zhuǎn)染NOX4亞基過(guò)表達(dá)質(zhì)粒的各組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。其中，過(guò)表達(dá)組、益心解毒方高劑量組、益心解毒方低劑量組與正常H9C2細(xì)胞組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05); H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組、RNAi質(zhì)粒組、益心解毒方中劑量組、益心解毒方低劑量組與過(guò)表達(dá)組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);RNAi質(zhì)粒組、益心解毒方高劑量組、益心解毒方低劑量組與H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方高劑量組、益心解毒方低劑量組與RNAi質(zhì)粒組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方中劑量組與益心解毒方高劑量組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);益心解毒方低劑量組與益心解毒方中劑量組NADPH氧化酶活性比較，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05，見表3)。

表3 各組NADPH氧化酶活性比較(±s，nmol·m l－1·min－1，n=3)Table 3 Comparison of NADPH oxidase activity in different groups

表3 各組NADPH氧化酶活性比較(±s，nmol·m l－1·min－1，n=3)Table 3 Comparison of NADPH oxidase activity in different groups

注:與正常H9C2細(xì)胞組比較，aP＜0.05;與過(guò)表達(dá)組比較，bP＜0.05;與H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組比較，cP＜0.05;與RNAi質(zhì)粒組比較，dP＜0.05;與益心解毒方高劑量組比較，eP＜0.05;與益心解毒方中劑量組比較，fP＜0.05

組別轉(zhuǎn)染NOX2亞基轉(zhuǎn)染NOX4 7.34±1.98 7.08±1.56過(guò)表達(dá)組12.39±0.88a13.18±3.03aH9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組8.24±5.19b8.24±5.19bRNAi質(zhì)粒組4.11±0.82bc3.86±2.04bc益心解毒方高劑量組14.47±0.66acd14.32±1.84acd益心解毒方中劑量組5.92±3.26be5.18±0.61be益心解毒方低劑量組14.52±1.20acdf17.80±1.99abcdfF亞基正常H9C2細(xì)胞組值＜0.01＜0.01 8.14 10.67 P值

3 討論

NOX2、NOX4亞基NADPH氧化酶是心肌ROS的主要來(lái)源，且對(duì)ROS的其他來(lái)源途徑起著整合和調(diào)控作用［2］。因此，干預(yù)和抑制NOX2、NOX4亞基NADPH氧化酶活性，已成為心力衰竭藥物研發(fā)的新熱點(diǎn)［3］，但是到目前為止，文獻(xiàn)報(bào)道的大多數(shù)NOX抑制劑特征尚未清楚，或者顯示出許多其他的藥理作用，或者缺乏成藥性，目前還沒有可用于臨床的NOX特異性抑制劑［4］。因此，研發(fā)針對(duì)NOX2亞基和/或NOX4亞基NADPH氧化酶的、特異性高的藥物仍然是目前心力衰竭藥物研究的熱點(diǎn)。

本研究發(fā)現(xiàn)，過(guò)表達(dá)組NADPH氧化酶活性高于正常H9C2細(xì)胞組，提示構(gòu)建的NOX2亞基和NOX4亞基過(guò)表達(dá)質(zhì)粒引起H9C2心肌細(xì)胞中NOX2亞基和NOX4亞基的過(guò)度表達(dá)，進(jìn)而誘導(dǎo)NADPH氧化酶的活化;同時(shí)本研究發(fā)現(xiàn)，RNAi質(zhì)粒組NADPH氧化酶活性低于過(guò)表達(dá)組和H9C2轉(zhuǎn)陰性對(duì)照質(zhì)粒組，說(shuō)明RNAi可以沉默NOX2、NOX4亞基，進(jìn)而降低NADPH氧化酶活性。RNAi是生命體內(nèi)存在的一種轉(zhuǎn)錄后基因沉默的分子機(jī)制［6］，針對(duì)心力衰竭的NADPH氧化酶主要來(lái)源于NOX2、NOX4亞基［5］，調(diào)控環(huán)節(jié)主要為NOX2、NOX4亞基的表達(dá)，因此筆者通過(guò)抑制NOX2亞基和NOX4基因的表達(dá)使NADPH氧化酶活性降低，進(jìn)而有效抑制ROS的產(chǎn)生，而NOX2亞基和NOX4亞基的過(guò)度表達(dá)可以使NADPH氧化酶過(guò)度活化，引起心肌細(xì)胞損傷。

本課題組在臨床工作中采用溫陽(yáng)益氣法治療心力衰竭的同時(shí)輔以活血解毒法，發(fā)現(xiàn)可以起到明顯的治療效果，益心解毒方正是以益氣溫陽(yáng)、活血解“毒”立法，適用于治療心力衰竭陽(yáng)(氣)虧虛、瘀“毒”內(nèi)阻證者，由黃芪、銀花、炙甘草等藥味組成。本研究運(yùn)用該復(fù)方的血清藥作用于轉(zhuǎn)染RNAi質(zhì)粒的H9C2心肌細(xì)胞發(fā)現(xiàn)，益心解毒方中劑量組NADPH氧化酶活性低于過(guò)表達(dá)組，而與RNAi質(zhì)粒組比較無(wú)差異，說(shuō)明在RNAi沉默了NOX2亞基和NOX4亞基后，益心解毒方可能不會(huì)直接影響NADPH氧化酶活性，揭示其通過(guò)干預(yù)NOX2亞基或NOX4亞基進(jìn)而調(diào)控NADPH氧化酶活性。本研究中益心解毒方高劑量組和益心解毒方低劑量組NADPH與過(guò)表達(dá)組比較無(wú)差異，中藥血清學(xué)實(shí)驗(yàn)復(fù)雜，故還需做進(jìn)一步研究加以驗(yàn)證。

綜上所述，益心解毒方針對(duì)心力衰竭的核心病機(jī)立法，具有益氣溫陽(yáng)，活血解毒功效，前期的臨床研究和動(dòng)物實(shí)驗(yàn)均表明［7－9］該方在改善心功能、減緩心室重構(gòu)等方面藥效顯著。本研究提示益心解毒方通過(guò)抑制NOX2、NOX4亞基NADPH氧化酶活性調(diào)控環(huán)節(jié)來(lái)降低NADPH氧化酶活性，進(jìn)而降低心肌ROS水平可能是其發(fā)揮藥效的重要機(jī)制，具有良好的臨床開發(fā)前景。

［1］Sciarretta S，Yee D，Ammann P，et al.Role of NADPH oxidase in the regulation of autophagy in cardiomyocytes［J］．Clin Sci(Lond)，2015，128(7):387－403.

［2］Yang CM，Lee IT，Hsu RC，et al.NADPH oxidase/ROS－dependent PYK2 activation is involved in TNF－α－induced matrix metalloproteinase－9 expression in rat heart－derived H9c2 cells［J］．Toxicol Appl Pharmacol，2013，272(2):431－442.

［3］Shah AM.Parsing the role of NADPH oxidase enzymes and reactive oxygen species in heart failure［J］．Circulation，2015，131(7): 602－604.

［4］Jaquet V，Scapozza L，Clark RA，et al.Small－molecule NOX inhibitors:ROS－generating NADPH oxidases as therapeutic targets［J］．Antioxid Redox Signal，2009，11(10):2535－2525.

［5］Raghunathan S，Patel BM.Therapeutic implications of small interfering RNA in cardiovascular diseases［J］．Fundam Clin Pharmacol，2013，27(1):1－20.

［6］Cao Y，Zhao DC，Zhao XY.Contrasting roles of NADPH oxidase isoforms in cardiac remodeling［J］．Chin Heart J，2013，25(3): 383－386.(in Chinese)曹妍，趙德超，趙曉宇.NADPH氧化酶亞基在心臟重構(gòu)中作用的比較［J］．心臟雜志，2013，25(3):383－386.

［7］Li C，Wang Y，Ouyang YL，et al.Effects of a compound Chinese herbal medicine Yixin Jiedu Formula on haemodynamic in ratswith heart failureof qi－deficiency and blood stasis syndrome［J］．Journal of Chinese Integrative Medicine，2012，10(5):577－583.(in Chinese)李春，王勇，歐陽(yáng)雨林，等.益心解毒方對(duì)心力衰竭氣虛血瘀證大鼠血流動(dòng)力學(xué)影響的實(shí)驗(yàn)研究［J］．中西醫(yī)結(jié)合學(xué)報(bào)，2012，10(5):577－583.

［8］Wang Y，LiC，CuoWJ，et al.Experimental study on role of AngIINADPH oxidase－ROS pathway in syndrome of blood stasis based on chronic myocardial ischemia model［J］．China Journal of Traditional Chinese Medicine and Pharmacy，2011，26(10):2265－2268. (in Chinese)王勇，李春，啜文靜，等.血管緊張素Ⅱ－NADPH氧化酶－活性氧通路在慢性心肌缺血血瘀證中作用的實(shí)驗(yàn)研究［J］．中華中醫(yī)藥雜志，2011，26(10):2265－2268.

［9］Feng XC，Guo SZ，Lian HJ，et al.Study of Yixin Jiedu Formula on the effects on NOX2 and NOX4 in myocardial tissue of heart failure rats with qideficiency and blood stasis syndrome［J］．China Journal of Traditional Chinese Medicine and Pharmacy，2015，30(7):2535－2538.(in Chinese)馮玄超，郭淑貞，廉洪建，等.益心解毒方對(duì)氣虛血瘀證心力衰竭大鼠心肌組織中NOX2和NOX4的影響［J］．中華中醫(yī)藥雜志，2015，30(7):2535－2538.

Mechanism of Yixin Jiedu Formula on the Overexpression of NOX2 and NOX4 and the Myocardial NADPH Ⅱ Oxidase Activity Changes Induced by RNAi

XIE Hua，MAChun－jie，GUO Shu－zhen，et al.Beijing University of Chinese Medicine，Beijing 100029，China

Objective To observe the influence of Yixin Jiedu formula on NOX2 and NOX4 overexpression and H9C2 myocardial NADPH Ⅱ oxidase activity changes induced by RNAi and investigate its mechanism.Methods We selected 16 normal male Sprague－Dawley rats and constructed the overexpression plasmids of NOX2 and NOX4 and RNAi plasmids.Transfection reagents were employed to conduct transient transfection of H9C2 myocardial cells，flow cytometry was undertaken to examine transfection efficiency.At 24 hours after transfection，the subjects were divided into normal H9C2 cell group，overexpression group，H9C2 negative－turning control plasmid group，RNAi plasmid group，high－dose Yixin Jiedu formula group，middle－dose Yixin Jiedu formula group and low－dose Yixin Jiedu formula group.All the groups were intervened with different doses of drug serum，and NADPH oxidase activity was detected 24 hours after drug administration.Results The groups in which overexpression plasmids of NOX2 was transfected were significantly different in NADPH oxidase activity(P＜0.05);overexpression group，high－dose Yixin Jiedu formula group and low－dose Yixin Jiedu formula group were significantly different from normal H9C2 cell group in NADPH oxidase activity(P＜0.05);H9C2 negative－turning control plasmid group，RNAi plasmid group，middle－dose Yixin Jiedu formula group were significantly different from normal H9C2 cell group in NADPH oxidase activity(P＜0.05);RNAi plasmid group，high－dose Yixin Jiedu formula group and low－dose Yixin Jiedu formula group were significantly different from H9C2 negative－turning control plasmid group in NADPH oxidase activity(P＜0.05);high－dose Yixin Jiedu formula group and low－dose Yixin Jiedu formula group were significantly different from RNAi plasmid group in NADPH oxidase activity(P＜0.05);midd le－dose Yixin Jiedu formula group and high－dose Yixin Jiedu formula group were significantly different in NADPH oxidase activity(P＜0.05);low－dose Yixin Jiedu formula group and middle－dose Yixin Jiedu formula group were significantly different in NADPH oxidase activity(P＜0.05).The groups in which overexpression plasmids of NOX4 was transfected were significantly different in NADPH oxidase activity(P＜0.05); overexpression group，high－dose Yixin Jiedu formula group and low－dose Yixin Jiedu formula group were significantly different from normal H9C2 cell group in NADPH oxidase activity(P＜0.05);H9C2 negative－turning control plasmid group，RNAi plasmid group，middle－dose Yixin Jiedu formula group and low－dose Yixin Jiedu formula group were significantly different from overexpression group in NADPH oxidase activity(P＜0.05);RNAi plasmid group，high－dose Yixin Jiedu formula group and low－dose Yixin Jiedu formula group were significantly different from H9C2 negative－turning group in NADPH oxidase activity (P＜0.05);high－dose Yixin Jiedu formula group and low－dose Yixin Jiedu formula group were significantly different from RNAi plasmid group in NADPH oxidase activity(P＜0.05);middle－dose Yixin Jiedu formula group and high－dose Yixin Jiedu formula group were significantly different in NADPH oxidase activity(P＜0.05);low－dose Yixin Jiedu formula group andmiddle－dose Yixin Jiedu formula group were significantly different in NADPH oxidase activity(P＜0.05).ConclusionThe overexpression of NOX2 and NOX4 can significantly improve NADPH oxidase activity，and RNAi expression can silence NOX2 and NOX4，thereby significantly reducing NADPH oxidase activity.Yixin Jiedu formula doesn't directly influence NADPH oxidase activity.The study shows the importantmechanism of Yixin Jiedu formulamay be that itcan intervene and inhibit NADPH oxidase activity of NOX2 and NOX4，reduce the ROS level ofmyocardialmuscle，protectmyocardial cell and improve cardiac function.

Yixin jiedu formula;Myocytes，cardiac;Gene expression;RNAi;Oxidoreductases

R 542.2

A

10.3969/j.issn.1007－9572.2015.30.020

2015－03－21;

2015－07－13)

(本文編輯:李婷婷)

國(guó)家自然科學(xué)基金資助項(xiàng)目(81102839);科技部重大新藥創(chuàng)制(2012ZX09103－201－011);北京中醫(yī)藥大學(xué)自主選題(2015－JYB－JSMS012)

100029北京市，北京中醫(yī)藥大學(xué)(解華，郭淑貞，付幫澤，馮玄超，王偉);內(nèi)蒙古醫(yī)科大學(xué)(麻春杰)

王偉，100029北京市，北京中醫(yī)藥大學(xué);E－mail:wangwei26960@126.com