吳秀琴,楊 威,尹玉嬌,何少棲,劉麗霞
茶多酚對(duì)一次性力竭運(yùn)動(dòng)大鼠氧化應(yīng)激和炎癥反應(yīng)的影響
吳秀琴,楊 威,尹玉嬌,何少棲,劉麗霞
目的:探討茶多酚對(duì)力竭運(yùn)動(dòng)大鼠氧化應(yīng)激和炎癥因子的影響,為減少運(yùn)動(dòng)損傷尋找有效的干預(yù)手段。方法:24只雄性SD大鼠按體重隨機(jī)分為對(duì)照組(C)、力竭組(E )和茶多酚力竭組(F,300 mg/kg/d茶多酚灌胃),4周后,進(jìn)行一次力竭游泳運(yùn)動(dòng),測(cè)定大鼠血清肌酸激酶(CK)、乳酸脫氫酶(LDH)、丙二醛(MDA)以及炎癥細(xì)胞因子白細(xì)胞介素(IL-1β,IL-10)和腫瘤壞死因子-α(TNF-α)含量。結(jié)果:與C組相比,E組大鼠力竭運(yùn)動(dòng)后血清CK、LDH活性和MDA含量顯著升高(P<0.05);促炎因子TNF-α和抗炎因子IL-10水平顯著增加(P<0.01和P<0.05)。與E組相比,F(xiàn)組大鼠血清CK、LDH活性和MDA含量顯著降低(P<0.05);促炎因子IL-1β和TNF-α水平顯著下降(P<0.05),而IL-10/TNF-α比值顯著升高(P<0.05)。相關(guān)性分析顯示,MDA與CK和TNF-α之間存在顯著正相關(guān)(P<0.05),與IL-10/TNF-α比值呈非常顯著負(fù)相關(guān)(P<0.01)。結(jié)論:茶多酚補(bǔ)充能提高機(jī)體抗氧化狀態(tài),減輕炎癥反應(yīng),對(duì)力竭運(yùn)動(dòng)的大鼠骨骼肌具有保護(hù)作用。
力竭運(yùn)動(dòng);茶多酚;炎癥因子;氧化應(yīng)激;肌肉損傷
規(guī)律的運(yùn)動(dòng)有利于健康,可以降低心血管病、癌癥和糖尿病的風(fēng)險(xiǎn)。然而,劇烈運(yùn)動(dòng)能引起攝氧量的顯著增加,隨著代謝活性的提高氧利用率的增加將引起活性氧(ROS)產(chǎn)生增多[24],當(dāng)ROS產(chǎn)生的數(shù)量超過(guò)內(nèi)源性抗氧化系統(tǒng)的防御能力時(shí),會(huì)出現(xiàn)氧化應(yīng)激。氧化應(yīng)激會(huì)導(dǎo)致組織和細(xì)胞大分子如脂類(lèi)、蛋白質(zhì)和核酸的破壞[15]。有證據(jù)表明,運(yùn)動(dòng)引起的氧化應(yīng)激可能與肌肉疲勞、肌肉損傷和運(yùn)動(dòng)能力下降有關(guān)[16,27,28]。運(yùn)動(dòng)后肌肉損傷或ROS增加促進(jìn)急性期局部炎癥反應(yīng),表現(xiàn)為炎癥因子從各類(lèi)型細(xì)胞中產(chǎn)生,刺激中性粒細(xì)胞和巨噬細(xì)胞向炎癥部位聚集以修復(fù)受損組織[19,29]。而浸潤(rùn)的中性粒細(xì)胞和巨噬細(xì)胞會(huì)產(chǎn)生額外的超氧陰離子,可能加劇肌肉損傷[11,25]。因此,各種能減少劇烈運(yùn)動(dòng)自由基產(chǎn)生和抗炎癥的補(bǔ)劑深受人們青睞。
茶多酚是茶葉中提取的多酚類(lèi)物質(zhì),是茶葉的主要有效成分。大量的流行病學(xué)研究、體外實(shí)驗(yàn)及動(dòng)物實(shí)驗(yàn)證實(shí),茶多酚生物活性廣泛,具有抗氧化,降血糖、抗炎、抗腫瘤和免疫調(diào)節(jié)作用[5]。以往的研究支持了茶多酚能減輕運(yùn)動(dòng)引起的氧化應(yīng)激[2,4]。然而,茶多酚對(duì)運(yùn)動(dòng)引起的炎癥的影響以及氧化應(yīng)激與炎癥之間的關(guān)系鮮有報(bào)道。本研究的目的是,探討補(bǔ)充茶多酚對(duì)力竭運(yùn)動(dòng)大鼠血清炎癥因子、氧化應(yīng)激和肌肉損傷的影響。
1.1 實(shí)驗(yàn)材料
選取6周齡清潔級(jí)SD雄性大鼠24只,由福建醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供,質(zhì)量合格證號(hào):NO.0000999(許可證編號(hào):SCXK(閩)2012-001),體重157.47±7.65 g。采用國(guó)家級(jí)標(biāo)準(zhǔn)嚙齒類(lèi)動(dòng)物飼料(由北京華阜康生物科技股份有限公司提供,產(chǎn)品許可證號(hào):SCXK(京)2009-0008京飼審(2009)06170)分籠飼養(yǎng),每籠4只,自由飲食飲水。動(dòng)物房溫度控制在22±2℃之間,相對(duì)濕度45%-55%,自然光照。隔天用消毒液對(duì)動(dòng)物房進(jìn)行消毒。
1.2 實(shí)驗(yàn)分組和喂養(yǎng)方案
所有大鼠在適應(yīng)性喂養(yǎng)5天后,按體重隨機(jī)分為3組:對(duì)照組(C組),力竭組(E組),茶多酚力竭組(F組),每組各8只。F組大鼠按每天300 mg/kg體重的劑量灌胃茶多酚(茶多酚用蒸餾水溶解,含量為92%,由福建省寧德市仙洋洋食品科技有限公司提供),灌胃給藥體積為1 mL/100 g;C組和E組灌胃等劑量的蒸餾水;每周測(cè)量一次大鼠體重并調(diào)整給藥體積,補(bǔ)劑干預(yù)時(shí)間為上午9:00—10:00,每日灌服1次,持續(xù)4周。
1.3 運(yùn)動(dòng)方案
C組大鼠不進(jìn)行任何運(yùn)動(dòng)干預(yù);E組和F組大鼠在灌胃結(jié)束前4天開(kāi)始無(wú)負(fù)重適應(yīng)性游泳訓(xùn)練3天,每天20 min。游泳池的規(guī)格為直徑60 cm的塑料圓桶,水深60 cm,水溫32℃±1℃。E組和F組大鼠于最后一次灌胃結(jié)束后,次日上午9:00尾部負(fù)自身體重3%進(jìn)行一次性力竭游泳運(yùn)動(dòng)。判斷力竭標(biāo)準(zhǔn):大鼠連續(xù)在水面下運(yùn)動(dòng),沉入水中10 s不能回到水面,撈出后不能完成翻正反射。
1.4 樣本采集與保存
C組大鼠于灌胃結(jié)束后次日上午9:00,E組和F組于力竭運(yùn)動(dòng)結(jié)束后即刻,用20%的烏拉坦溶液麻醉,腹主動(dòng)脈取全血3 mL置于普通采血管內(nèi),室溫放置0.5 h待其自然凝固,4℃,3 000 rpm,離心10 min,留取上層血清,置于-80℃冰箱保存,用于測(cè)定CK、LDH、MDA以及炎癥因子IL-1β、TNF-α和IL-10。
1.5 指標(biāo)檢測(cè)
測(cè)定MDA采用硫代巴比妥酸法(TBA)法;測(cè)定CK采用乙酰半胱氨酸法;測(cè)定LDH采用苯肼顯色法;測(cè)定炎癥因子采用ELISA方法,試劑盒由南京建成生物工程研究所提供。
1.6 統(tǒng)計(jì)學(xué)分析
2.1 各組大鼠血清CK和LDH的變化
如表1所示,與C組相比,E組大鼠力竭運(yùn)動(dòng)后CK、LDH水平顯著升高(P<0.05);與E組相比,F(xiàn)組大鼠血清CK、LDH水平明顯下降(P<0.05)。
表 1 各組大鼠血清CK 、LDH和MDA水平一覽表
Table1 The Levels of Serum CK、LDH and MDA in Different Groups
CK(U/mL)LDH(U/L)MDA(nmol/mL)C組1.35±0.383377.89±1292.163.32±0.51E組1.86±0.35*5085.21±1340.39*4.01±0.34*F組1.41±0.47#3436.58±1299.85#3.53±0.46#
注:*P<0.05,代表與C組相比;#P<0.05,代表與E組相比,下同。
2.2 各組大鼠血清氧化應(yīng)激水平的變化
如表1所示,與C組比較,E組大鼠血清MDA含量顯著上升(P<0.05);與E組比較,F(xiàn)組大鼠血清MDA含量明顯下降(P<0.05)。
2.3 各組大鼠血清促/抗炎細(xì)胞因子的變化
如表2顯示,與C組相比,E組大鼠血清TNF-α和IL-10水平顯著升高(P<0.01和P<0.05),IL-1β水平升高但沒(méi)有顯著差異(P>0.05);F組大鼠IL-10水平顯著升高(P<0.05)。與E組相比,F(xiàn)組大鼠血清IL-1β、TNF-α水平顯著下降(P<0.05),IL-10有升高的趨勢(shì)(P>0.05),而IL-10/TNF-α比值顯著上升(P<0.05)。
2.4 CK、MDA與炎癥因子的相關(guān)性
如表3顯示,CK與MDA顯著正相關(guān)(P<0.05),CK、MDA與炎癥因子TNF-α非常顯著正相關(guān)(P<0.01),而MDA與IL-10/TNF-α比值呈非常顯著負(fù)相關(guān)(P<0.01)。
表 2 各組大鼠血清IL-1β、TNF-α、IL-10和IL-10/TNF-α水平一覽表
Table 2 The Levels of Serum IL-1β、TNF-α、IL-10 and L-10/TNF-α in Different Groups
IL-1β(ng/L)TNF-α(ng/L)IL-10(ng/L)IL-10/TNF-αC組36.11±1.46128.35±13.6073.32±3.890.57±0.03E組37.73±2.11160.37±7.03**83.57±12.19*0.52±0.07F組33.78±4.89#141.07±21.77#84.92±9.95*0.61±0.10#
注:**P<0.01,代表與C組相比。
表 3 CK、MDA和炎癥因子之間的相關(guān)性一覽表
Table 3 The Correlation between CK,MDA and Inflammation Cytokines
MDAIL-1βTNF-αIL-10IL-10/TNF-αCK0.59&0.380.65&&0.37-0.34MDA10.380.69&&0.12-0.59&&
注:&表示P<0.05,&&表示P<0.01。
許多文獻(xiàn)已證實(shí)力竭運(yùn)動(dòng)能引起氧化應(yīng)激、炎癥反應(yīng)和肌細(xì)胞結(jié)構(gòu)損傷,發(fā)現(xiàn)血漿中胞質(zhì)酶活性增加,即肌酸激酶(CK)和乳酸脫氫酶(LDH)活性增加[8,18,21]。血漿CK和LDH的活性是肌纖維損傷常見(jiàn)的標(biāo)志物。本研究結(jié)果顯示,力竭運(yùn)動(dòng)后血清CK、LDH水平顯著升高,與前人的研究結(jié)果相一致。運(yùn)動(dòng)引起血清CK水平增加表明了肌細(xì)胞膜通透性的增加,而CK的增加與脂質(zhì)過(guò)氧化產(chǎn)物丙二醛(MDA)的增加呈顯著正相關(guān)(r=0.59,P<0.05),反映氧化應(yīng)激參與了肌肉損傷過(guò)程。因此,可推測(cè)運(yùn)動(dòng)引起活性氧產(chǎn)生增加可能影響肌細(xì)胞膜的通透性,進(jìn)而使肌細(xì)胞中的酶進(jìn)入循環(huán)中[20]。與E組相比,F(xiàn)組運(yùn)動(dòng)后血清CK和LDH的水平顯著降低,表明茶多酚對(duì)力竭運(yùn)動(dòng)的大鼠骨骼肌具有保護(hù)作用。
在正常生理?xiàng)l件下,氧自由基的產(chǎn)生和抗氧化防御機(jī)制處于平衡狀態(tài),但劇烈運(yùn)動(dòng)時(shí),體內(nèi)的氧化與抗氧化作用將失衡并導(dǎo)致細(xì)胞損傷。MDA是氧自由基反復(fù)攻擊細(xì)胞膜的多不飽和脂肪酸而形成的脂質(zhì)過(guò)氧化產(chǎn)物,被廣泛地用做評(píng)價(jià)脂質(zhì)氧化損傷的指標(biāo),因此,MDA可作為反映氧化應(yīng)激的間接指標(biāo)[15]。力竭運(yùn)動(dòng)后血清MDA含量增加已得到證實(shí)[6,17,22]。研究認(rèn)為,力竭運(yùn)動(dòng)會(huì)使組織中抗氧化酶過(guò)度消耗,導(dǎo)致運(yùn)動(dòng)后即刻抗氧化酶活性下降,不足以平衡運(yùn)動(dòng)應(yīng)激情況下產(chǎn)生的自由基,多余的自由基會(huì)引起不飽和脂肪酸形成脂質(zhì)過(guò)氧化物,超過(guò)機(jī)體的保護(hù)機(jī)制,將導(dǎo)致氧化損傷[30]。本研究顯示,力竭運(yùn)動(dòng)后大鼠血清MDA含量顯著增加,表明急性力竭運(yùn)動(dòng)引起機(jī)體氧自由基脂質(zhì)過(guò)氧化反應(yīng)增強(qiáng),機(jī)體表現(xiàn)為氧化應(yīng)激狀態(tài)。茶多酚組大鼠血清MDA水平顯著下降,證實(shí)了茶多酚的抗氧化作用,與其他研究提出茶多酚能減輕氧化應(yīng)激的結(jié)果相一致[2,4]。茶多酚可能通過(guò)激活細(xì)胞內(nèi)抗氧化防御系統(tǒng)或者直接清除自由基[1],降低脂質(zhì)過(guò)氧化反應(yīng)產(chǎn)物來(lái)減輕急性力竭運(yùn)動(dòng)引起的氧化應(yīng)激,從而有助于推遲肌肉損傷的發(fā)生和發(fā)展。
劇烈運(yùn)動(dòng)引起肌肉損傷,機(jī)體對(duì)運(yùn)動(dòng)后組織損傷的反應(yīng)與感染引起的炎癥免疫應(yīng)答相似[26]。白細(xì)胞被動(dòng)員和激活,誘導(dǎo)急性期反應(yīng),促炎細(xì)胞因子產(chǎn)生增加,細(xì)胞浸潤(rùn)和組織損傷[10]。事實(shí)上,肌肉損傷引起促炎細(xì)胞因子如白細(xì)胞介素IL-1β,IL-6和TNF-α的釋放,緊接著激活抗炎細(xì)胞因子IL-10及IL-1受體拮抗劑(IL-1ra)的產(chǎn)生??寡准?xì)胞因子通過(guò)限制炎癥細(xì)胞因子產(chǎn)生,上調(diào)其可溶性拮抗劑結(jié)合蛋白,抑制炎癥細(xì)胞活動(dòng)來(lái)減輕炎癥[7]。近年來(lái),IL-10/TNF-α比值被認(rèn)為是衡量炎癥反應(yīng)程度的重要指標(biāo),高比值被稱為抗炎比值,低比值則說(shuō)明存在炎癥反應(yīng)[3]。本研究發(fā)現(xiàn),力竭運(yùn)動(dòng)后大鼠血清TNF-α含量明顯增加,IL-1β水平有上升的趨勢(shì)(P>0.05),同時(shí)IL-10濃度也顯著升高,與運(yùn)動(dòng)時(shí)促炎細(xì)胞因子和抗炎細(xì)胞因子之間的平衡假設(shè)一致[23,31]。炎癥細(xì)胞因子有助于調(diào)節(jié)中性粒細(xì)胞的快速遷移,進(jìn)而巨噬細(xì)胞進(jìn)入損傷的肌細(xì)胞部位啟動(dòng)修復(fù)過(guò)程[9],通過(guò)促炎細(xì)胞因子如TNF-α的釋放,有助于受損組織降解。促炎細(xì)胞因子如TNF-α和IL-1β的存在可以刺激IL-6和生長(zhǎng)因子的產(chǎn)生,從而啟動(dòng)衛(wèi)星細(xì)胞的增值和肌纖維的再生[12]。因此,劇烈運(yùn)動(dòng)后血漿TNF-α的升高可能是吞噬作用和肌肉重建的標(biāo)志[32]。用茶多酚干預(yù)后,發(fā)現(xiàn)力竭組大鼠血清TNF-α、IL-1β水平顯著下降,IL-10/TNF-α比值明顯升高,表明茶多酚在力竭運(yùn)動(dòng)中可能通過(guò)降低促炎因子水平發(fā)揮抗炎作用。
細(xì)胞因子與氧化損傷標(biāo)志物之間存在聯(lián)系,通過(guò)相關(guān)性分析發(fā)現(xiàn),MDA與TNF-α呈顯著正相關(guān)、與IL-10/TNF-α比值呈顯著負(fù)相關(guān),證實(shí)了活性氧參與TNF-α從肌肉和免疫細(xì)胞的釋放,并且,TNF-α也可能通過(guò)激活NADPH氧化酶引起活性氧的合成[13],使肌肉恢復(fù)過(guò)程中損傷進(jìn)一步得到發(fā)展。此外,TNF-α也與CK呈顯著正相關(guān)的關(guān)系,表明由于運(yùn)動(dòng)和ROS產(chǎn)生增加引起的肌細(xì)胞膜完整性的變化與促炎性免疫反應(yīng)相一致。因此,茶多酚可以通過(guò)降低脂質(zhì)過(guò)氧化反應(yīng)產(chǎn)物和促炎細(xì)胞因子的水平,顯著提高力竭運(yùn)動(dòng)大鼠的抗氧化狀態(tài)并減輕炎癥反應(yīng)。
4周茶多酚干預(yù)能提高機(jī)體抗氧化狀態(tài),減輕炎癥反應(yīng),對(duì)力竭運(yùn)動(dòng)的大鼠骨骼肌具有保護(hù)作用。
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The Effects of Tea Polyphenols Supplemneton Oxidative Stress and InflammatoryResponse in Rats after Exhaustive Exercise
WU Xiu-qin,YANG Wei,YIN Yu-jiao,HE Shao-xi,LIU Li-xia
Objectives:To study the effects of tea polyphenols supplemnet on oxidative stress and inflammatory response in rats after exhaustive exercise,find out effective interventions for reducing the sports injuries.Methods:Twenty-four male SD rats were randomly divided into three groups according to weight:control group(C),exhaustive exercise group (E),exhaustive exercise with Tea polyphenols (F,dose:300mg/kg/d).4 weeks later,the rats performed once exhaustive exercise,and the serum creatine kinase (CK),lactate dehydrogenase (LDH),malondialdehyde(MDA),the level of inflammatory cytokine:interleukin (IL-1β,IL-10) and the tumor necrosis factor alpha (TNF -a) were examined.Results:Compared with C group,the serum CK and LDH activity and MDA content of E group rats were significantly increased after exhaustive exercise (P<0.05);also,the levels of pro-inflammatory cytokine TNF-a and anti-inflammatory cytokine IL-10 were significantly increased (P<0.01 andP<0.01).Compared with E group,the serum CK and LDH activity and MDA content of F group rats significantly decreased (P<0.05); the levels of pro-inflammatory Cytokine TNF-a and IL-1β were all significantly decreased (P<0.05),whereas the ratio of IL-10/TNF-α was significantly improved(P<0.05).Correlation analysis showed that it appeared positive correlation MDA with CK and TNF-a (P<0.05),but with ratio of IL - 10 / TNF-a,they appeared very significantly negative correlation (P<0.01).Conclusion:The supplement of tea polyphenols can improve the body's antioxidant status,reduce inflammation response and play a protective role to skeletal muscle in rat performing exhaustive exercise.
exhaustiveexercise;teapolyphenols;inflammationcytokines;oxidativestress;skeletalmuscleinjury
1002-9826(2016)01-0092-04
10.16470/j.csst.201601013
2015-05-19;
2015-08-12
吳秀琴(1964-),女,福建霞浦人,副教授,碩士,碩士研究生導(dǎo)師,主要研究方向?yàn)檫\(yùn)動(dòng)人體科學(xué),E-mail:xiuqin415@163.com。
福建師范大學(xué) 體育科學(xué)學(xué)院,福建 福州 350108 Fujian Normal University,F(xiàn)uzhou 350108,China.
G804.23
A