肖衛(wèi)華,陳佩杰,王 茹,董靜梅

過度訓(xùn)練及補(bǔ)充二聯(lián)甲苯或谷氨酰胺對大鼠腹膜巨噬細(xì)胞活性氧和誘導(dǎo)型一氧化氮合酶的影響

肖衛(wèi)華1,2,陳佩杰1,王 茹1,董靜梅1

巨噬細(xì)胞廣泛分布于肝臟、肺、腹膜、脂肪組織、血液、骨骼和肌肉等機(jī)體多個(gè)組織和器官。它是機(jī)體抵抗病原微生物入侵的第一道防線,可通過“呼吸爆發(fā)”形式,即:在病原體刺激時(shí)由NADPH氧化酶介導(dǎo)產(chǎn)生大量活性氧(reactive oxygen species,ROS),殺滅入侵的病原微生物,這是巨噬細(xì)胞殺菌的主要機(jī)制,在病菌防御中發(fā)揮重要作用[15]。ROS過量生成可導(dǎo)致細(xì)胞損傷,誘導(dǎo)凋亡,但巨噬細(xì)胞內(nèi)生理水平 ROS有重要功能,它是促進(jìn)巨噬細(xì)胞存活和誘導(dǎo)巨噬細(xì)胞分化的重要信號分子[17,26]。最新研究成果表明,巨噬細(xì)胞與肥胖、動(dòng)脈粥樣硬化等多種疾病密切相關(guān),巨噬細(xì)胞可浸潤脂肪組織、動(dòng)脈粥樣硬化斑塊等處,提高氧化應(yīng)激水平,釋放炎性因子,導(dǎo)致機(jī)體的慢性炎癥反應(yīng),有氧運(yùn)動(dòng)則有助于改善上述疾病癥狀,運(yùn)動(dòng)的主要效應(yīng)在于降低脂肪組織和動(dòng)脈粥樣硬化斑塊處氧化應(yīng)激水平和炎癥水平,從而使癥狀得以緩解[7,13,27],這一過程可能與NO的抗氧化效應(yīng)密切相關(guān)[3]。

研究表明,抗氧化劑二聯(lián)甲苯(DPI)可特異性抑制NADPH氧化酶活性從而降低ROS生成[8],營養(yǎng)素谷氨酰胺(Gln)則有利于提高免疫細(xì)胞活性[12],但過度訓(xùn)練及補(bǔ)充DPI或 Gln是否影響巨噬細(xì)胞 ROS生成,未見相關(guān)報(bào)道。ROS與NO的關(guān)系是一個(gè)有爭議的問題,有人認(rèn)為ROS生成增加可減少NO產(chǎn)量[24],也有人認(rèn)為NO生成增加可降低氧化應(yīng)激水平[4]。過度訓(xùn)練引起巨噬細(xì)胞ROS改變過程中NO是否發(fā)揮作用仍不得而知。因此,本研究通過建立過度訓(xùn)練模型,觀察 ROS及誘導(dǎo)型一氧化氮合酶(iNOS,催化合成NO的酶)的變化,以了解過度訓(xùn)練對巨噬細(xì)胞ROS的影響、補(bǔ)充抗氧化劑和營養(yǎng)素能否改善過度訓(xùn)練效應(yīng),以及過度訓(xùn)練發(fā)生時(shí)巨噬細(xì)胞內(nèi) ROS與NO的相互關(guān)系。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物及分組

8周齡健康雄性wistar大鼠56只,體重180±10 g,購于上海實(shí)驗(yàn)動(dòng)物中心。動(dòng)物飼養(yǎng)環(huán)境溫度為22℃±2℃,相對濕度50%～70%,每天光照時(shí)間為12 h,自由飲食。將大鼠隨機(jī)分為安靜對照組(C)、過度訓(xùn)練組(E)、過度訓(xùn)練補(bǔ)充DPI組(ED)、過度訓(xùn)練補(bǔ)充谷氨酰胺組(EG)。后3組根據(jù)取材時(shí)間不同分為2組,運(yùn)動(dòng)后36h取材組(E1、ED1、EG1),運(yùn)動(dòng)后 7 天取材組 (E2、ED2、EG2)?？傆?jì) 7組,每組8只。

1.2 過度訓(xùn)練方案及給藥方案

動(dòng)物適應(yīng)性飼養(yǎng)1周后,E、ED、EG組開始進(jìn)行遞增負(fù)荷跑臺(tái)訓(xùn)練,每周5次,周二、周六休息,共訓(xùn)練11周。過度訓(xùn)練方案參考文獻(xiàn)方法[5](表1)。

ED組腹腔注射DPI:第5周開始至第8周每天 1次(0.1 mg/kg),第9周每天1次(0.2 mg/kg),第10周每天2次(0.2 mg/kg),第11周每天 2次(0.3 mg/kg)。

EG組補(bǔ)充 Gln:第 5周至第 8周灌胃(0.8 g/kg/day),以后幾周加至飲用水,劑量逐周加大到1.1 g/kg/day。

1.3 腹膜巨噬細(xì)胞分離與純化

常規(guī)方法,腹腔灌洗液分離,貼壁培養(yǎng)2 h純化,重懸細(xì)胞后用細(xì)胞計(jì)數(shù)板計(jì)數(shù)并調(diào)細(xì)胞濃度至107個(gè)/ml備用。

1.4 活性氧的測定

取100μl上述細(xì)胞懸液(106個(gè)細(xì)胞),按活性氧測定試劑盒 (上海杰美基因科技有限公司)說明書,用流式細(xì)胞儀(Bechman Coulter Epics XL)檢測30 000個(gè)以上細(xì)胞,測定平均熒光強(qiáng)度。

1.5 RNA抽提與cDNA合成

取上述剩余的細(xì)胞懸液,離心去上清收集細(xì)胞。使用異硫氫酸胍-氯仿經(jīng)典法抽提總 RNA。按 Fermentas公司第一鏈cDNA合成試劑盒說明,在 Eppendorf梯度PCR儀進(jìn)行反轉(zhuǎn)錄,20μl體系,合成的 cDNA儲(chǔ)存在 -20℃?zhèn)溆谩?/p>

作者單位:1.上海體育學(xué)院運(yùn)動(dòng)科學(xué)學(xué)院,上海200438;2.湘南學(xué)院體育系,湖南郴州423000

表1 本研究大鼠過度訓(xùn)練方案一覽表Table 1 Overtraining Protocol of Rat

1.6 熒光定量PCR

設(shè)計(jì)目的基因誘導(dǎo)型一氧化氮合酶(iNOS)(AY211532.1)和內(nèi)參β-actin熒光定量引物(上海生工生物工程技術(shù)服務(wù)有限公司合成)。iNOS:Forward-ctc act gtg gct gtg gtc acc ta,Reverse-ggg tct tcg ggc ttc agg tta,產(chǎn)物 101 bp。β-actin:Forward-gga gat tac tgc cct ggc tcc ta,Reverse-gac tca tcg tac tcc tgc ttg ctg,產(chǎn)物 150 bp。使用RG3000型定量 PCR儀進(jìn)行雙標(biāo)曲法相對定量 PCR(SY BR Green試劑盒購自 Fermentas公司)。iNOS基因在1～104 U(units)范圍內(nèi),檢測閾值(CT)與拷貝數(shù)對數(shù)呈線性關(guān)系,兩者相關(guān)系數(shù) r2＞0.99(圖1)。熔解曲線分析顯示:iNOS呈單一產(chǎn)物峰(圖2),PCR產(chǎn)物電泳只見101 bp處單一條帶。

圖1 本研究iNOS標(biāo)準(zhǔn)曲線圖Figure 1. iNOS Standard Curve

1.7 統(tǒng)計(jì)方法

實(shí)驗(yàn)數(shù)據(jù)由SPSS 17.0統(tǒng)計(jì)軟件處理,單因素方差分析和相關(guān)分析,結(jié)果以平均值±標(biāo)準(zhǔn)差表示,統(tǒng)計(jì)學(xué)顯著性水平定為0.05,非常顯著性水平定為0.01。

圖2 本研究iNOS熔解曲線圖Figure 2. iNOS Melt Curve

2 結(jié)果

2.1 活性氧生成量

各組ROS生成量見表2。運(yùn)動(dòng)后36 h取材組中,E1組ROS生成量顯著低于C組(P＜0.05);ED1組與 E1組相比ROS生成量顯著降低(P＜0.05),與C組相比極顯著降低(P＜0.01);EG1組與 E1組相比沒有顯著差異(P＞0.05),與C組相比極顯著降低(P＜0.01)。運(yùn)動(dòng)后第7天取材組中,E2組、ED2組分別與 E1組、ED1組相比,ROS生成量極顯著增加(P＜0.01);EG2組與 EG1組相比ROS生成量顯著增加(P＜0.05);E2組、ED2組、EG2組、C組4組之間比較無顯著差異(圖3)。

表2 本研究各組活性氧生成量一覽表Table 2 The Production of ROS

圖3 本研究 ROS生成量示意圖Figure 3. The Production of ROS

2.2 誘導(dǎo)型一氧化氮合酶mRNA表達(dá)

運(yùn)動(dòng)后36 h取材組中,與 C組相比,E1組iNOS mRNA表達(dá)增加,但無統(tǒng)計(jì)學(xué)差異(P＞0.05);ED1組相對E1組顯著增加(P＜0.05),相對C組極顯著增加(P＜0.01);EG1組與 E1組、C組相比無顯著差異。運(yùn)動(dòng)后第7天取材組中,E2組、EG2組分別與 E1組、EG1組相比無顯著差異,ED2組相對 ED1組顯著降低(P＜0.05)。E2組、ED2組、EG2組、C組4組之間比較無顯著差異。

2.3 ROS與iNOS相互關(guān)系

對巨噬細(xì)胞內(nèi)ROS與iNOS mRNA表達(dá)量進(jìn)行相關(guān)分析,結(jié)果顯示,二者呈直線負(fù)相關(guān),相關(guān)系數(shù) r=-0.45(P=0.004)。

圖4 本研究iNOS mRNA表達(dá)量示意圖Figure 4. The mRNA level of iNOS

以各組ROS和iNOS表達(dá)量相對C組的倍比關(guān)系作折線圖,發(fā)現(xiàn)ROS生成量較高時(shí),iNOS mRNA表達(dá)處于較低水平(E2組);而隨著iNOS mRNA表達(dá)水平增加,ROS生成量呈現(xiàn)降低趨勢(圖5)。

圖5 本研究巨噬細(xì)胞內(nèi) ROS與iNOS mRNA相互關(guān)系圖Figure 5. Relationship betw een ROS and iNOS of macrophage

3 討論

一般認(rèn)為,ROS過量生成可通過間接損傷DNA、類脂和蛋白,或直接通過 ROS介導(dǎo)激活相關(guān)信號途徑來誘導(dǎo)細(xì)胞凋亡[6]。組織或器官中ROS含量異常增加表示機(jī)體氧化應(yīng)激水平提高,是一種負(fù)向效應(yīng),動(dòng)脈粥樣硬化、肥胖、糖尿病、高血壓等常見疾病的發(fā)生發(fā)展都與ROS有密切的聯(lián)系[11,21-23]。而生理水平的ROS對巨噬細(xì)胞功能的維持是非常重要的,ROS能誘導(dǎo)蛋白激酶B激活,促進(jìn)巨噬細(xì)胞存活,同時(shí)ROS也是一種重要的胞內(nèi)信號介質(zhì),在介導(dǎo)巨噬細(xì)胞分化過程中發(fā)揮關(guān)鍵作用。此外,ROS還能調(diào)控巨噬細(xì)胞炎性因子分泌,即生理水平 ROS對調(diào)控巨噬細(xì)胞存活、分化、炎性因子分泌等都有重要作用[17,26]。

從理論上推測,運(yùn)動(dòng)應(yīng)能增加機(jī)體氧化應(yīng)激水平,從而提高ROS生成。但是本研究發(fā)現(xiàn),過度訓(xùn)練使巨噬細(xì)胞ROS生成量降低,顯著低于生理水平(P＜0.05,圖3),勢必對ROS介導(dǎo)的生理功能造成負(fù)面影響,這可能是高頻率大強(qiáng)度反復(fù)運(yùn)動(dòng)應(yīng)激使機(jī)體處于免疫抑制的表現(xiàn)。停訓(xùn)1周后,過度訓(xùn)練組巨噬細(xì)胞內(nèi)ROS水平恢復(fù)到安靜組水平。

過度訓(xùn)練補(bǔ)充抗氧化劑DPI使ROS生成量進(jìn)一步降低,顯著低于過度訓(xùn)練組(P＜0.05),且與安靜對照組相比極顯著降低(P＜0.01,圖3),表明過度訓(xùn)練時(shí)應(yīng)用DPI會(huì)使ROS生成受抑狀況進(jìn)一步惡化,不利其行使正常生理功能。這與DPI抑制巨噬細(xì)胞ROS生成的文獻(xiàn)報(bào)道是一致的[8]。停訓(xùn)1周后,過度訓(xùn)練補(bǔ)充DPI組巨噬細(xì)胞內(nèi)ROS生成量也恢復(fù)至正常水平。

谷氨酰胺是巨噬細(xì)胞重要的能量來源,運(yùn)動(dòng)時(shí)補(bǔ)充谷氨酰胺是否能帶來好的效應(yīng),仍是一個(gè)有爭議的問題[18]。本研究發(fā)現(xiàn),過度訓(xùn)練補(bǔ)充谷氨酰胺時(shí) ROS生成量與過度訓(xùn)練組相比,沒有顯著差異(E1:179.10±29.10;EG1:172.58±49.62),與安靜對照相比極顯著降低(P＜0.01),表明過度訓(xùn)練時(shí)補(bǔ)充谷氨酰胺并不能改變因過度訓(xùn)練引起的ROS生成受抑,對巨噬細(xì)胞內(nèi)ROS介導(dǎo)的生理功能無改善作用。停訓(xùn)1周后,過度訓(xùn)練補(bǔ)充谷氨酰胺組巨噬細(xì)胞內(nèi)ROS恢復(fù)到安靜組水平。但另有研究報(bào)道,大鼠以85%˙VO2max強(qiáng)度進(jìn)行1 h跑臺(tái)運(yùn)動(dòng)對嗜中性粒細(xì)胞ROS生成無影響,而運(yùn)動(dòng)并補(bǔ)充谷氨酰胺時(shí) ROS生成增多[10]。這可能與運(yùn)動(dòng)強(qiáng)度、持續(xù)時(shí)間及細(xì)胞類型不同有關(guān)。

誘導(dǎo)型一氧化氮合酶(iNOS)催化產(chǎn)生的NO與 ROS的關(guān)系也是個(gè)存在許多爭論的問題?？v觀現(xiàn)有研究發(fā)現(xiàn),ROS與NO彼此消長關(guān)系存在于多種模式中,如糖尿病患者NADPH氧化酶激活引起過量 ROS生成,可導(dǎo)致eNOS解構(gòu),引起NO生成減少和內(nèi)皮依賴性血管舒張功能降低[24]。用一氧化氮合酶抑制劑(l-NAME)處理4周模擬ED患者,可使大鼠海綿體中亞硝酸鹽/硝酸鹽水平(NO含量)降低、介導(dǎo) ROS生成的 NADPH氧化酶催化亞基gp91phox表達(dá)增加近2倍,而運(yùn)動(dòng)可逆轉(zhuǎn) NO水平降低,使gp91phox亞基表達(dá)恢復(fù)至基礎(chǔ)水平[1]。此外,急性運(yùn)動(dòng)可通過NADPH氧化酶抑制機(jī)制來提高不?；顒?dòng)者內(nèi)皮祖細(xì)胞集落形成單位中NO水平,離體時(shí)NADPH氧化酶抑制劑夾竹桃麻素(apocynin)可抑制NADPH氧化酶活性,減少 ROS生成,使內(nèi)源性 NO生成增多[19]。但也有人不支持 NO與 ROS的這種相互消長關(guān)系。Zhao等[28]于2010年發(fā)表了一篇文章,他以小鼠 RAW264.7巨噬細(xì)胞為研究對象,用脂多糖LPS先活化iNOS,然后用 PMA刺激巨噬細(xì)胞 ROS生成,觀察到 ROS生成量及 NADPH氧化酶亞基p47phox(介導(dǎo) ROS生成的關(guān)鍵成分之一)磷酸化要強(qiáng)于不用LPS預(yù)激活組,從而得出了誘導(dǎo)iNOS表達(dá)可提高PMA刺激時(shí)ROS產(chǎn)量這一結(jié)論。但該實(shí)驗(yàn)設(shè)計(jì)存在不足,因?yàn)橛肔PS預(yù)處理,即可使巨噬細(xì)胞 NADPH氧化酶活化,ROS產(chǎn)量大幅增加[8],此時(shí)再用 PMA刺激巨噬細(xì)胞已無法確定哪種刺激劑在活化巨噬細(xì)胞時(shí)占主導(dǎo)地位。

在本研究中,腹膜巨噬細(xì)胞內(nèi)iNOS與 ROS也存在這種消長關(guān)系。對各組巨噬細(xì)胞內(nèi) ROS與iNOS mRNA表達(dá)量進(jìn)行相關(guān)分析,結(jié)果顯示,二者呈直線負(fù)相關(guān),相關(guān)系數(shù) r=-0.45(P=0.004)。以各組 ROS和iNOS表達(dá)量相對C組的倍比關(guān)系作圖(圖5),發(fā)現(xiàn) ROS生成量較高時(shí),iNOS mRNA表達(dá)處于較低水平(E2組);而隨著iNOS mRNA表達(dá)水平增加,ROS生成量呈現(xiàn)降低趨勢,且這種趨勢在ED1組最為明顯,ED1組iNOS mRNA表達(dá)量是C組的6.3倍,而 ROS生成量顯著降低(P＜0.01),只有 C組的28.6%。雖然有人報(bào)道離體時(shí)DPI對某些細(xì)胞NOS活性起一定的抑制作用[14],但DPI的這種抑制作用可被NADPH、NADP+和 2’5’-ADP 阻斷[2]。此外 ,運(yùn)動(dòng)狀態(tài)下機(jī)體生理功能發(fā)生深刻變化,釋放多種激素以適應(yīng)運(yùn)動(dòng)的需要,因此,運(yùn)動(dòng)時(shí)補(bǔ)充DPI的效應(yīng)與離體時(shí)不一定完全一致(但抑制ROS生成效應(yīng)與離體時(shí)相似,圖3)。本研究結(jié)果表明,iNOS表達(dá)增強(qiáng),可促進(jìn) NO產(chǎn)生,從而在降低ROS生成中發(fā)揮一定作用。有較多證據(jù)支持這一推測,如大鼠支氣管肺泡灌洗液細(xì)胞(主要是巨噬細(xì)胞)進(jìn)行NO暴露(十萬分之一NO濃度),在用酵母多糖刺激時(shí),NO暴露過的細(xì)胞超氧自由基釋放能力顯著受損[20];培養(yǎng)的人微血管內(nèi)皮細(xì)胞用NO供體(DETA-NONOate)處理0.5～24 h,DETA-NONOate(10～300μmol/L)可顯著抑制內(nèi)皮細(xì)胞中過氧化氫的生成,呈濃度和時(shí)間依賴性,且NO抑制過氧化氫生成效應(yīng)不依賴于降低NADPH氧化酶亞基表達(dá),也不依賴于NADPH氧化酶亞基的解離,而是通過使p47phox亞基S-亞硝基化來實(shí)現(xiàn)[25]。除外源性 NO可抑制巨噬細(xì)胞 ROS生成外,內(nèi)源性一氧化氮合酶活性增加也可降低ROS生成:如LPS刺激巨噬細(xì)胞時(shí),類胡蘿卜素和葉黃素可通過提高iNOS等酶的活性來減少 H2O2積聚[9];運(yùn)動(dòng)可誘導(dǎo)糖尿病大鼠二聚體型eNOS增加,使NO生成增多,ROS生成減少,從而減少糖尿病相關(guān)的氧化應(yīng)激,使糖尿病相關(guān)并發(fā)癥減少[4];規(guī)律性有氧運(yùn)動(dòng)可使老年大鼠血管中eNOS表達(dá)和活性增高,減少NADPH氧化酶的表達(dá)和活化,從而降低氧化損傷[3]。此外,用一氧化氮合酶抑制劑抑制iNOS活性使NO產(chǎn)生減少,可增加LPS刺激時(shí)O-2生成量[16]。雖然iNOS表達(dá)受 ROS-NF-κb等信號途徑的調(diào)控[14],但iNOS催化產(chǎn)生的 NO也可作用于ROS生成,二者相互作用已被大量實(shí)驗(yàn)所證明。因此,本實(shí)驗(yàn)中過度訓(xùn)練及補(bǔ)充DPI或谷氨酰胺影響巨噬細(xì)胞ROS產(chǎn)生過程中,iNOS催化產(chǎn)生的NO可能起一定的調(diào)控作用。

4 結(jié)論

過度訓(xùn)練使腹膜巨噬細(xì)胞ROS生成量顯著低于生理水平,引起運(yùn)動(dòng)性免疫抑制;補(bǔ)充抗氧化劑DPI可使過度訓(xùn)練時(shí)巨噬細(xì)胞內(nèi)ROS生成量進(jìn)一步降低,補(bǔ)充谷氨酰胺對過度訓(xùn)練引起的巨噬細(xì)胞ROS生成量降低沒有改善作用;過度訓(xùn)練及補(bǔ)充DPI或谷氨酰胺可影響巨噬細(xì)胞內(nèi)ROS生成,這在一定程度上受iNOS催化產(chǎn)生的NO調(diào)控。

[1]CLAUDINO M A,FRANCO-PENTEADO C F,PRIVIERO F B,et al.Upregulation of gp91phox subunit of NAD(P)H oxidase contributes to erectile dysfunction caused by long-term nitric oxide inhibition in rats:reversion by regular physical training[J].Urology,2010,75(4):961-967.

[2]DENNIS J S,OLUFUNMILAYO A F,NYOUN S K,et al.Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs[J].J FASEB,1991,5:98-103.

[3]DOUGLAS R S,ASHLEY E W,GARY L P,et al.Habitual exercise and vascular ageing[J].J Physiol,2009,587:5541-5549.

[4]GRIJALVA J,HICKS S,ZHAO X,et al.Exercise training enhanced myocardial endothelial nitric oxide synthase(eNOS)function in diabetic Goto-Kakizaki(GK)rats.Cardiovasc Diabetol,2008,7:34.

[5]HOHL R,FERRARESSO R L,DE OLIVEIRA R B,et al.Development and Characterization of an Overtraining Animal Model[J].Med Sci Sport Exe.2009,41(5):1155-1163.

[6]KAMATA H,HONDA S,MAEDA S,et al.Reactive oxygen species promote TNFα-induced death and sustained JNK activation by Inhibiting MAP Kinase phosphatases[J].Cell,2005,120(5):649-661.

[7]KAWANISHI N,YANO H,YOKOGAWA Y,et al.Exercise training inhibits inflammation in adipose tissue via both suppression of macrophage infiltration and acceleration of phenotypic switching from M1 to M2 macrophages in high-fat-diet-induced obese mice[J].Exe Immunol Rev,2010,16:105-118.

[8]KIM H G,YOON D H,LEE W H,et al.Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-kappaB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophag e[J].J Ethnopharmacol,2007,114(3):307-315.

[9]KIM J H,NA H J,KIM C K,et al.The non-provitamin A carotenoid,lutein,inhibits NF-kappaB-dependentgene expression through redox-based regulation of the phosphatidylinositol 3-kinase/PTEN/Akt and NF-kappaB-inducing kinase pathways:role of H2O2in NF-kappaB activation[J].Free Radic Biol Med,2008,45(6):885-896.

[10]LAGRANHA C J,DE LIMA T M,SENNA S M,et al.The effect of glutamine supplementation on the function of neutrophils from exercised rats.Cell Biochem Funct,2005,23(2):101-107.

[11]LI Q X,XIONG Z Y,HU B P,et al.Aging-associated insulin resistance predisposes to hypertension and its reversal by exercise:the role of vascular vasorelaxation to insulin[J].Basic Res Cardiol,2009,104(3):269-284.

[12]MARCELO MACEDO ROGERO,JULIO TIRAPEGUI,MARCO AURELIO RAMIREZ VINOLO,et al.Dietary Glutamine Supplementation Increases the Activity of Peritoneal Macrophages and Hemopoiesis in Early-Weaned Mice Inoculated with Mycobacteriumbovis Bacillus Calmette-Guérin[J].J Nutrition,2008,138(7):1343-1348.

[13]MAXIME PELLEGRIN,CAROLE MIGUET-ALFONSI,KARIMA BOUZOURENE,et al.Long-term Exercise Stabilizes Atherosclerotic Plaque in ApoE Knockout Mice[J].Med Sci Sport Exe,2009,41(12):2128-2135.

[14]MENDES A F,CARVAL HO A P,CARAMONA M M,et al.Diphenyleneiodonium inhibits NF-kappaB activation and iNOS expression induced by IL-1beta:involvement of reactive oxygen species[J].Mediators Inflamm,2001,10(4):209-215.

[15]MINAKAMI R,SUMIMOTOA H.Phagocytosis-coupled activation of the superoxide-producing phagocyte oxidase,a member of the NADPH oxidase(Nox)family[J].Int J Hematol,2006,84:193-198.

[16]MUZAFFAR S,J EREMYJ Y,ANGELINI GD,et al.Role of the endothelium and nitric oxide synthases in modulating superoxide formation induced by endotoxin and cytokines in porcine pulmonary arteries[J].Thorax,2003,58:598-604.

[17]NA KYUNGLEE,YOUNG GEUM CHOI,JI YOUN BAIK,et al.A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation[J].Blood,2005,106(3):852-859.

[18]NATALIE HISCOCK,BENTE KLARLUND PEDERSEN.Exercise-induced immunodepression-plasma glutamine is not the link[J].J Appl Physiol,2002,93:813-822.

[19]NATHAN T J,SARAH W,ESPEN E S,et al.Effects of acute and chronic endurance exercise on intracellular nitric oxide in putative endothelial progenitor cells:role of NAPDH oxidase[J].Am JPhysiolHeart Circ Physiol,2009,297:H1798-H1805.

[20]OL KER C,SIESE A,STUMPF S,et al.Impaired superoxide radical production by bronchoalveolar lavage cells from NO(2)-exposed rats[J].Free Radic Biol Med,2004,37(7):977-987.

[21]SAKURAI T,IZAWA T,KIZAKI T,et al.Exercise training decreases expression of inflammation-related adipokines through reduction of oxidative stress in rat white adipose tissue[J].Biochem Biophys Res Commun,2009,379(2):605-609.

[22]SEBASTIAN SIXT,SEBASTIAN BEER,MATTHIAS BLüHER,et al.Long-but not short-term multifactorial intervention with focus on exercise training improves coronary endothelial dysfunction in diabetes mellitus type 2 and coronary artery disease[J].Eur Heart J,2010,31(1):112-119.

[23]SHATROV V A,BRUNE B.Induced expression of manganese superoxide dismutase by non-toxic concentrations of oxidized low-density lipoprotein(oxLDL)protects against oxLDL-mediated cytotoxicity[J].Biochem J,2003;374(2):505-511.

[24]SHEN G X.Oxidative stress and diabetic cardiovascular disorders:roles of mitochondria and NADPH oxidase[J].Can J Physiol Pharmacol[J].2010,88(3):241-248.

[25]STAVROS SELEMIDIS,GREGORY J DUSTING,HITESH PESHAVARIYA,et al.Nitric oxide suppresses NADPH oxidase-dependent superoxide production by S-nitrosylation in human endothelial cells[J].Cardiovascular Res,2007,75:349-358.

[26]WANG YIJIE,MANDY MZ,GREGORY KL,et al.The Role of the NADPH Oxidase Complex,p38 MAPK,and Akt in Regulating Human Monocyte/Macrophage Survival[J].Am J Res Cell Molecular Biology,2007,36:68-77.

[27]YASUHARU MATSUMOTO,VOL KER ADAMS,SASKIA JACOB,et al.Regular Exercise Training Prevents Aortic Valve Disease in Low-Density Lipoprotein-Receptor-Deficient Mice[J].Circulation,2010,121:759-767.

[28]ZHAO K,HUANG Z,LU H,et al.Induction of inducible nitric oxide synthase increases the production of reactive oxygen species in RAW264.7 macrophages[J].Biosci Rep,2010,30(4):233-241.

Effects of Overtraining and Supplement with DPI or Glutamine on the Production of ROS and the Expression of iNOS in Pritoneal Macrophages

XIAO Wei-hua1,2,CHEN Pei-jie1,WANG Ru1,DON G Jing-mei1

目的:觀察過度訓(xùn)練及補(bǔ)充二聯(lián)甲苯(DPI)或谷氨酰胺(Gln)對大鼠腹膜巨噬細(xì)胞活性氧(ROS)生成能力的影響,探討誘導(dǎo)型一氧化氮合酶(iNOS)在此過程中發(fā)揮的作用。方法:8周齡健康雄性wistar大鼠56只,隨機(jī)分為安靜對照組(C)、過度訓(xùn)練組(E)、過度訓(xùn)練補(bǔ)充DPI組(ED)、過度訓(xùn)練補(bǔ)充 Gln組(EG)。后3組根據(jù)取材時(shí)間不同各分為2組,運(yùn)動(dòng)后36 h取材組(E1、ED1、EG1),運(yùn)動(dòng)后 7天取材組(E2、ED2、EG2)。總計(jì) 7組,每組 8只,除C組外,其他6組都進(jìn)行11周遞增負(fù)荷跑臺(tái)訓(xùn)練。斷頭處死大鼠并分離純化腹膜巨噬細(xì)胞,流式細(xì)胞術(shù)測定ROS生成量,熒光定量PCR技術(shù)測定iNOS基因表達(dá)。結(jié)果:ROS生成:E1組顯著低于C組;ED1組顯著低于 E1組(P＜0.05),與C組相比極顯著降低(P＜0.01);EG1組與 E1組相比沒有顯著差異,與C組相比極顯著降低(P＜0.01)。E2組、ED2組、EG2組分別與 E1組、ED1組、EG1相比均顯著增高;E2組、ED2組、EG2組、C組4組之間比較無顯著差異。iNOS mRNA表達(dá):ED1組相對 E1組顯著增加(P＜0.05),相對C組極顯著增加(P＜0.01);ED2組相對 ED1組顯著降低;其他相關(guān)組間比較無顯著差異。對各組中ROS與iNOS表達(dá)量進(jìn)行相關(guān)分析,顯示二者呈直線負(fù)相關(guān),相關(guān)系數(shù) r=-0.45(P=0.004)。以各組 ROS和iNOS表達(dá)量相對,C組的倍比關(guān)系作圖,發(fā)現(xiàn) ROS生成量較高時(shí),iNOS mRNA表達(dá)處于較低水平(E2組),隨著iNOS mRNA表達(dá)水平增加,ROS生成量呈現(xiàn)降低趨勢。結(jié)論:過度訓(xùn)練使腹膜巨噬細(xì)胞ROS生成量顯著低于生理水平,引起運(yùn)動(dòng)性免疫抑制;補(bǔ)充抗氧化劑DPI可使過度訓(xùn)練時(shí)巨噬細(xì)胞ROS生成量進(jìn)一步降低,補(bǔ)充谷氨酰胺對過度訓(xùn)練引起的巨噬細(xì)胞ROS生成量降低沒有改善作用;過度訓(xùn)練及補(bǔ)充DPI或谷氨酰胺可影響巨噬細(xì)胞內(nèi)ROS生成,這在一定程度上受iNOS催化產(chǎn)生的NO調(diào)控。

過度訓(xùn)練;巨噬細(xì)胞;活性氧;NADPH氧化酶;誘導(dǎo)型一氧化氮合酶;鼠;動(dòng)物實(shí)驗(yàn)

Objective:To investigate the effect of overtraining and supplement with DPI(diphenylene iodonium)or glutamine on the production of ROS(reactive oxygen species)in pritoneal macrophages and the function of iNOS(inducible nitric oxide synthase)in this process.Method:56 male wistar rats were randomly divided into 7 groups:sedentary group(C,n=8),overtraining group(E),overtraining supplement with DPI group(ED),overtraining supplement with Gln group(EG).E,ED and EG group were respectively divided into two groups which sacrificed at 36h(E1,ED1,EG1,n=8)and 7 days(E2,ED2,EG2,n=8)after the last training.All groups except C were training in standard treadmill with an increasing load for 11 weeks.Peritoneal macrophages were isolated and purified after all rats were sacrificed by decapitation.The production of ROS was detected by FACS,and real time-PCR was used to detect the mRNA expression of iNOS.Result:Overtraining group showed a significant decline in production of ROS when compared with pritoneal macrophage from the sedentary group,and ED1 group significant lower than E1 group(P＜0.05)and sedentary group(P＜0.01).The production of ROS in EG1 group had no significant difference when compared with overtraining group,however,it significant lower than the sedentary group(P＜0.01).E2,ED2 and EG2 group showed a significant increase when compared with E1,ED1 and EG1 group respectively.There were no significant differences in E2,ED2,EG2 and C group.The expression of iNOS in ED1 group significant higher than overtraining group(P＜0.05) ,sedentary group(P＜0.01)and ED2 group(P＜0.05).The iNOS mRNA levels had no significant differencies in E2,ED2,EG2 and C group.The correlation analysis showed that ROS and iNOS were nega-tive correlation and the pearson correlation was-0.45(P=0.004).When the production of ROS was highness,the expression of iNOS was the lowest(E2 group).When the iNOS mRNA levels increase,the production of ROS showed a tendency of decline.Conclusion:Overtraining made the production of ROS in pritoneal macrophage lower than physiological level significantly,which was the performance of exercise-induced immunosuppression.DPI supplement made the production of ROS in overtraining group decline more seriously.And the decline of ROS in overtraining group could not be ameliorated by Gln supplement.Overtraining and supplement with DPI or Gln certainly had an influence on ROS production in pritoneal macrophages,and it was regulated by NO which mediate by iNOS in a certain extent.

overtraining;macrophage;reactive oxygen species;NA D P H oxidase;inducible nitric oxide synthase

G804.7

A

1000-677X(2011)02-0049-06

2010-12-12;

2011-01-10

國家自然科學(xué)基金項(xiàng)目(30971422);上海市重點(diǎn)學(xué)科建設(shè)資助項(xiàng)目(S30802)。

肖衛(wèi)華(1981-),男,湖南安化人,講師,在讀博士研究生,研究方向?yàn)檫\(yùn)動(dòng)免疫學(xué)與青少年體質(zhì),Tel:(021)51253242,E-mail:xiaoweihua1115@163.com;陳佩杰(1962-),男,浙江舟山人,教授,博士,博士研究生導(dǎo)師,研究方向?yàn)檫\(yùn)動(dòng)免疫學(xué)與青少年體質(zhì),Tel:(021)51253626,E-mail:chengpjk@online.sh.cn。

1.Shanghai University of Sport,Shanghai 200438,China;2.Xiangnan University,Chenzhou 423000,China.