曹慶雷,趙海濤,李小蘭
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熱預(yù)適應(yīng)對(duì)大鼠中樞疲勞后體溫及腦神經(jīng)遞質(zhì)5-HT分泌的影響研究
曹慶雷,趙海濤,李小蘭
北京科技大學(xué)體育部,北京,100083。
目的:觀察熱預(yù)適應(yīng)通過調(diào)節(jié)中樞神經(jīng)遞質(zhì)對(duì)大鼠運(yùn)動(dòng)誘導(dǎo)的中樞疲勞的作用。方法:12只成年雄性SD大鼠,隨機(jī)分為2組,6只為預(yù)熱組,6只為對(duì)照組。運(yùn)動(dòng)疲勞模型通過力竭跑步模型建立,熱預(yù)處理組是42℃熱處理15min,每天3次,連續(xù)5天,對(duì)照組不做任何處理。記錄最后運(yùn)動(dòng)力竭時(shí)間及運(yùn)動(dòng)前后體內(nèi)核心溫度,同時(shí)用液相色譜測量腦組織中5羥色胺(5-HT)的水平。結(jié)果:(1)熱預(yù)處理組的平均力竭時(shí)間為178.24±42.43 min,顯著高于對(duì)照組318.49±37.25min,熱預(yù)適應(yīng)顯著增加了運(yùn)動(dòng)的耐受時(shí)間(P<0.05);(2)熱預(yù)處理組運(yùn)動(dòng)前后肛溫差0.382±0.396℃顯著低于對(duì)照組(1.954±0.603)℃,熱預(yù)適應(yīng)顯著降低了運(yùn)動(dòng)后體溫(P<0.05);結(jié)論:熱預(yù)適應(yīng)可以有效的降低運(yùn)動(dòng)前后體溫差,并且可能是通過減少5-HT含量來起到延緩中樞運(yùn)動(dòng)疲勞的作用。
熱預(yù)適應(yīng);神經(jīng)遞質(zhì);運(yùn)動(dòng)疲勞
運(yùn)動(dòng)性疲勞是因?yàn)檫\(yùn)動(dòng)引起的肌肉運(yùn)動(dòng)及做功能力下降的現(xiàn)象,其中長時(shí)間中等強(qiáng)度的運(yùn)動(dòng)所產(chǎn)生的疲勞主要由中樞神經(jīng)系統(tǒng)發(fā)出的抑制信號(hào)而引起的,稱之為運(yùn)動(dòng)中樞性疲勞[1-3],是目前運(yùn)動(dòng)疲勞機(jī)制研究的熱點(diǎn)之一。緩解疲勞對(duì)提高運(yùn)動(dòng)能力有重要作用,是運(yùn)動(dòng)領(lǐng)域重要的研究課題。運(yùn)動(dòng)性中樞疲勞機(jī)制研究提出多種假說,涉及中樞神經(jīng)遞質(zhì)失衡,內(nèi)穩(wěn)態(tài)失調(diào),氨基酸、離子代謝紊亂,自由基增多,能源衰竭,神經(jīng)內(nèi)分泌、免疫系統(tǒng)的平衡打破等諸多變化[4]。隨著神經(jīng)生物學(xué)相關(guān)技術(shù)方法在運(yùn)動(dòng)醫(yī)學(xué)研究中應(yīng)用,運(yùn)動(dòng)性中樞疲勞機(jī)制的研究取得了一些進(jìn)展,長時(shí)間運(yùn)動(dòng)時(shí)中樞神經(jīng)遞質(zhì)的變化是導(dǎo)致疲勞產(chǎn)生的重要原因[1,3]。
有關(guān)神經(jīng)遞質(zhì)分泌失衡導(dǎo)致中樞性疲勞的研究發(fā)現(xiàn),運(yùn)動(dòng)過程中體溫升高可能是誘導(dǎo)神經(jīng)遞質(zhì)分泌的主要原因之一。熱應(yīng)激條件下,體溫升高的熱敏信號(hào),傳遞至下丘腦視前區(qū)等腦區(qū)域,腦區(qū)域內(nèi)熱敏神經(jīng)元接受并整合形成反饋信號(hào)作用于下丘腦、延髓中縫區(qū)等不同腦區(qū),影響腦內(nèi)神經(jīng)遞質(zhì)的釋放和相應(yīng)受體的表達(dá),從而產(chǎn)生一系列反應(yīng),調(diào)整各種機(jī)能以適應(yīng)環(huán)境溫度的改變,并可影響機(jī)體長時(shí)間的運(yùn)動(dòng)能力,并從而在運(yùn)動(dòng)性中樞疲勞的產(chǎn)生和恢復(fù)中發(fā)揮特定的作用[5-7]。神經(jīng)網(wǎng)絡(luò)調(diào)節(jié)是多重而復(fù)雜的過程,遞質(zhì)的分泌受內(nèi)外環(huán)境影響,并且可因環(huán)境的改變而進(jìn)行適應(yīng)性調(diào)節(jié),保持身體穩(wěn)態(tài)。那么過量運(yùn)動(dòng)引起的遞質(zhì)變化,也可能通過全身性調(diào)節(jié)引起自身適應(yīng)性改變,從而使失衡的神經(jīng)遞質(zhì)分泌趨于正常。那么,預(yù)適應(yīng)后達(dá)到的新的遞質(zhì)平衡就有可能適應(yīng)機(jī)體的應(yīng)激狀態(tài),從而在過量運(yùn)動(dòng)過程中推遲疲勞出現(xiàn)的時(shí)間、延長運(yùn)動(dòng)時(shí)間,減輕疲勞程度。在自身適應(yīng)調(diào)節(jié)機(jī)制研究中,預(yù)適應(yīng)是目前常用且安全有效的方法之一,常用于減輕機(jī)體損傷,如缺血預(yù)適應(yīng)保護(hù)腦組織、肌肉組織等[8-9]。熱預(yù)適應(yīng)是指通過反復(fù)暴露在高熱的條件下,使得體內(nèi)和皮膚溫度升高并且大量出汗[10-13]。熱預(yù)適應(yīng)的研究也應(yīng)用于諸多領(lǐng)域,如熱預(yù)適應(yīng)對(duì)保護(hù)和改善心肌細(xì)胞的損傷,對(duì)神經(jīng)系統(tǒng)及小鼠的學(xué)習(xí)行為也有積極的影響[14-15]。已有研究顯示,在熱應(yīng)激條件下,可通過提高DA、NE的活性抑制機(jī)體溫度升高導(dǎo)致的中樞疲勞,改善熱應(yīng)激下運(yùn)動(dòng)能力[16-17]。同時(shí)也有研究顯示,DA能提高神經(jīng)元活性也能使受試者在高熱環(huán)境中運(yùn)動(dòng)時(shí)有更高的耐受性[18]。由此可見,人體對(duì)熱應(yīng)激的調(diào)節(jié)反應(yīng)與神經(jīng)遞質(zhì)分泌有密切的關(guān)系。調(diào)節(jié)神經(jīng)遞質(zhì)的活性,達(dá)到自身在高溫環(huán)境中新的平衡,有利于維持機(jī)體的穩(wěn)態(tài)。
因此,我們的研究擬通過熱預(yù)適應(yīng)調(diào)節(jié)中樞神經(jīng)遞質(zhì)的平衡,研究并闡明運(yùn)動(dòng)時(shí)重要神經(jīng)遞質(zhì)的變化規(guī)律,明確其與運(yùn)動(dòng)性中樞疲勞的關(guān)系,并且尋找中樞性疲勞的有效的預(yù)防與恢復(fù)方法。
成年雄性SD大鼠(8周齡)12只,體重230g—260g之間(購自北京維通利華實(shí)驗(yàn)動(dòng)物公司)。大鼠飼養(yǎng)在首都醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心大鼠飼養(yǎng)房。大鼠正常分組(3只/籠子),自由飲水,飲食,溫度為18—24℃,空氣相對(duì)濕度40%—60%,正常晝夜節(jié)律。
1.2.1 高溫預(yù)熱處理 預(yù)熱組動(dòng)物用6%水合氯醛0.6ml/100g腹腔注射麻醉,置于60℃恒溫水浴箱,保持大鼠呼吸通暢,直至肛溫升至42℃,維持肛溫42℃ 15min,再置于常溫環(huán)境下恢復(fù)體溫,1小時(shí)后,再升高體溫42℃ 15min,反復(fù)3次,連續(xù)5天。對(duì)照組不做任何處理。
1.2.2 急性力竭疲勞模型建立 采用Bedford[19]建立的一次性平板跑臺(tái)力竭跑步模型,兩組動(dòng)物測試前進(jìn)行適應(yīng)性跑臺(tái)訓(xùn)練一次/天(共3天),低于力竭程度。恢復(fù)2天后進(jìn)行一次性力竭跑步實(shí)驗(yàn),采用treadmill平板跑臺(tái)法,測試由15cm/s開始,持續(xù)15min,增加至25cm/s,持續(xù)15min,最后保持在35 cm/s跑步直至力竭,記錄每只動(dòng)物三階段跑步時(shí)間。
1.2.3 核心體溫測定 采用體溫計(jì)測量肛溫表示核心體溫,跑步前測定初始肛溫,力竭跑步完成后立即測肛溫,用運(yùn)動(dòng)前后核心體溫的變化觀察體溫變化的差異。
1.2.4 腦內(nèi)神經(jīng)遞質(zhì)檢測 兩組力竭跑步實(shí)驗(yàn)結(jié)束后,采用斷頭法獲取腦組織,置于冰上并迅速分離皮質(zhì)、下丘腦、海馬、紋狀體。高氯酸提取法充分勻漿后,4 000g/min離心5min,離心取上清液,-80℃凍存。采用高效液相色譜法分析各部腦組織內(nèi)5-HT含量。
所有結(jié)果采用均數(shù)±標(biāo)準(zhǔn)差表示,組間差異采用t檢驗(yàn)分析,< 0.05為顯著性差異,P < 0.01 為極顯著性差異。
兩組動(dòng)物經(jīng)水平跑臺(tái)三階段力竭跑步后,測定各階段跑步時(shí)間,結(jié)果顯示預(yù)熱組動(dòng)物較對(duì)照組跑步持續(xù)時(shí)間明顯延長,疲勞出現(xiàn)時(shí)間延后。其中一、二階段跑步時(shí)間和動(dòng)物體能狀態(tài)無差別,兩組動(dòng)物均能按跑臺(tái)速度勻速跑步,無疲勞表現(xiàn);而在第三階段后,尤其進(jìn)入跑步后期,兩組動(dòng)物出現(xiàn)疲勞的時(shí)間、體能表現(xiàn)差異明顯,對(duì)照組動(dòng)物首先出現(xiàn)疲勞癥狀,如,速度下降,跑步暫停等,跑步至力竭平均時(shí)長為178.24±42.43min,而預(yù)熱組動(dòng)物在第三階段,特別是跑步后期,耐力明顯提高,能長時(shí)間保持勻速跑步,延遲疲勞出現(xiàn),力竭時(shí)間較對(duì)照組平均時(shí)長顯著延長,力竭時(shí)間為318.49±37.25min(P<0.05)。(如圖1)
圖1 第三階段力竭時(shí)間
隨著運(yùn)動(dòng)過程的進(jìn)行,體溫會(huì)逐步升高,溫度升高時(shí)是中樞疲勞的誘因之一。兩組動(dòng)物力竭跑步后均即刻檢測肛溫,結(jié)果顯示對(duì)照組動(dòng)物平均肛溫,升高幅度,預(yù)熱組動(dòng)物平均肛溫較跑步前升高0.382±0.396℃(p<0.05),較對(duì)照組(1.954±0.603℃)肛溫升高幅度較小。(如圖2)
圖2 力竭前后肛溫差
5-HT色氨是重要的中樞疲勞的標(biāo)志物之一,在熱適應(yīng)組中5-HT色氨的含量顯著低于對(duì)照組(p<0.01),熱預(yù)適應(yīng)處理后5-HT下降了接近50%。(見圖3)
圖3 神經(jīng)遞質(zhì)5-HT的變化
熱預(yù)適應(yīng)在運(yùn)動(dòng)損傷以及腦保護(hù)研究中已經(jīng)有較為深入的研究。實(shí)驗(yàn)發(fā)現(xiàn)熱預(yù)適應(yīng)可提高動(dòng)物熱休克蛋白(Hsp)和抗氧化劑表達(dá)水平,對(duì)大鼠骨骼肌運(yùn)動(dòng)損傷具有保護(hù)作用[20]。另外,熱預(yù)適應(yīng)產(chǎn)生熱休克蛋白,在多種中樞性疾病或損傷中被發(fā)現(xiàn)具有有效的保護(hù)作用[21-23]。此外,在缺氧及低能量條件下培養(yǎng)的大腦海馬細(xì)胞,其損害程度也可因熱休克蛋白而減輕[24]。這些研究表明,熱預(yù)適應(yīng)對(duì)運(yùn)動(dòng)中機(jī)體和腦有保護(hù)作用。我們測量了熱預(yù)適應(yīng)組跟對(duì)照組大鼠跑臺(tái)的平均力竭時(shí)間。結(jié)果表明,熱預(yù)適應(yīng)后,大鼠的平均力竭時(shí)間為320min 而對(duì)照組的平均力竭時(shí)間約為180min,說明經(jīng)過熱預(yù)適應(yīng)后大鼠的力竭時(shí)間提高了約75%。說明了熱預(yù)適應(yīng)可以延緩運(yùn)動(dòng)性疲勞的出現(xiàn)時(shí)間。
機(jī)體在運(yùn)動(dòng)后,體溫會(huì)有升高的現(xiàn)象,與正常的體溫相比,機(jī)體在高溫下的運(yùn)動(dòng)能力會(huì)有所下降。熱預(yù)適應(yīng)是經(jīng)常被作為減少這種損傷的措施之一。熱預(yù)適應(yīng)可以誘導(dǎo)機(jī)體生理上對(duì)高溫的適應(yīng),從而可以用來提高運(yùn)動(dòng)成績。人們已經(jīng)對(duì)熱適應(yīng)進(jìn)行了大量的研究,例如,Patterson等人研究表明健康機(jī)體在經(jīng)過8天和22天的熱預(yù)適應(yīng)后體內(nèi)核心溫度分別下降了0.20℃與0.32℃[25]。熱休克反應(yīng)通常是經(jīng)過改善的耐熱性的標(biāo)志之一,有研究表明,熱預(yù)適應(yīng)誘導(dǎo)的細(xì)胞內(nèi)熱休克蛋白(Hsp72)濃度增加[26]。這些數(shù)據(jù)表明,細(xì)胞內(nèi)的熱休克蛋白可能對(duì)熱預(yù)適應(yīng)更為敏感。通過測量實(shí)驗(yàn)組跟對(duì)照組力竭運(yùn)動(dòng)前后體溫表明,熱預(yù)處理組的運(yùn)動(dòng)前后的溫差變化顯著小于對(duì)照組。先前研究表明,運(yùn)動(dòng)過程中體溫的升高可能是誘發(fā)神經(jīng)遞質(zhì)分泌失衡的主要原因之一。運(yùn)動(dòng)過程中會(huì)引起體溫升高,運(yùn)動(dòng)員機(jī)體核心溫度隨時(shí)間與運(yùn)動(dòng)負(fù)荷的增加而平穩(wěn)的、緩慢的升高[27]。已經(jīng)有證據(jù)表明,正是過量運(yùn)動(dòng)時(shí)體溫的升高引起了多種遞質(zhì)分泌,例如,體溫升高后腦內(nèi)視前區(qū)和下丘腦前部區(qū)域中5-HT含量上升[28];DA、NE伴隨大腦和機(jī)體核心溫度的升高而增加,該類遞質(zhì)參與調(diào)節(jié)降溫過程[29-30]。
研究證實(shí)多種神經(jīng)遞質(zhì)與中樞性運(yùn)動(dòng)疲勞產(chǎn)生有密切關(guān)系并起到重要的調(diào)節(jié)作用,因而成為疲勞產(chǎn)生機(jī)制、緩解疲勞方法研究中的重要內(nèi)容。目前已經(jīng)發(fā)現(xiàn)多種神經(jīng)遞質(zhì)參與了運(yùn)動(dòng)疲勞的發(fā)生,主要包括多巴胺、γ-氨基丁酸(GABA)、5-HT和NE等;也有興奮性遞質(zhì)谷氨酸(Glu)和天冬氨酸(Asp)等及其他類型神經(jīng)遞質(zhì)如乙酰膽堿等參與[4]。過量運(yùn)動(dòng)可引起多種神經(jīng)遞質(zhì)分泌失衡,并導(dǎo)致遞質(zhì)之間相互作用的平衡失調(diào),因而引起腦內(nèi)代謝紊亂,引起中樞性疲勞,這可能是運(yùn)動(dòng)疲勞發(fā)生的重要原因。
5-HT是研究最多的的中樞疲勞的標(biāo)志物之一[31-33]。研究發(fā)現(xiàn),長時(shí)間大強(qiáng)度運(yùn)動(dòng)使血漿游離的氨基酸濃度上升,氨基酸和色氨酸爭奪結(jié)合位點(diǎn)從而增加了血漿中游離色氨酸的濃度和色氨酸/支鏈氨基酸的比值,促使更多的色氨酸等遞質(zhì)穿過血腦屏障,使腦內(nèi)特定區(qū)域抑制性遞質(zhì)5-HT的合成增加,同時(shí)會(huì)引起多種酶活性下降,ATP再合成下降,繼而使肌肉運(yùn)動(dòng)能力下降[34]。5-HT是最明顯的中樞疲勞的衡量物。我們測量了熱預(yù)適應(yīng)組和對(duì)照組的5-HT的含量(圖3),結(jié)果發(fā)現(xiàn),熱預(yù)處理組5-HT在腦中的含量顯著低于對(duì)照組,這表明,熱預(yù)適應(yīng)抑制了由運(yùn)動(dòng)疲勞導(dǎo)致的5-HT濃度的上升。
綜上所述,本研究觀察到熱預(yù)處理干預(yù)后導(dǎo)致大鼠運(yùn)動(dòng)至力竭的時(shí)間縮短,運(yùn)動(dòng)前后體溫差減小,中樞神經(jīng)遞質(zhì)5-HT的含量下降,我們推測通過熱應(yīng)激預(yù)適應(yīng),誘導(dǎo)并激活神經(jīng)遞質(zhì)網(wǎng)絡(luò)的活動(dòng),適應(yīng)溫度變化,使腦內(nèi)神經(jīng)遞質(zhì)分泌預(yù)先進(jìn)入調(diào)節(jié)機(jī)制,尤其是提高抑制性遞質(zhì)的活性,使神經(jīng)遞質(zhì)分泌與身體機(jī)能趨于平衡,達(dá)到高溫環(huán)境中的穩(wěn)態(tài),調(diào)節(jié)機(jī)體適應(yīng)高溫環(huán)境,將有效延緩因神經(jīng)遞質(zhì)失調(diào)造成代謝失衡、中樞性疲勞的出現(xiàn)。研究中,我們通過熱預(yù)處理使得機(jī)體適應(yīng)了5-HT的下降,從而在運(yùn)動(dòng)力竭實(shí)驗(yàn)中,大鼠的5-HT的水平相應(yīng)的下降。這無疑對(duì)改善鍛煉機(jī)能,提高疲勞運(yùn)動(dòng)耐受力有重要意義。
經(jīng)過熱預(yù)適應(yīng)處理后,大鼠運(yùn)動(dòng)力竭時(shí)間明顯延長,肛溫的運(yùn)動(dòng)前后溫差顯著變小,5-HT的含量明顯下降,說明熱預(yù)處理通過對(duì)神經(jīng)遞質(zhì)的調(diào)控從而改善了運(yùn)動(dòng)中樞疲勞的發(fā)生。
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Heat Preconditioning Reduce Exercise Induced Central Fatigue by Neurotransmitters DA, 5-HT, NE Manipulation
CAO Qinglei, ZHAO Haitao, LI Xiaolan
P.E. Department of University of Science and Technology Beijing, 100083, China.
objective: To observe the effect of heat preconditioning on exercise induced central fatigue by regulate theneurotransmitters manipulation. Methods: Twelve male adult SD rats were randomly divided into 2 control group(n=6), heat preconditioning model group(n=6). Sports fatigue model was established by treadmill running. Heat preconditioning was applied by 42℃, per times for 15min, three times per day for 5 days. The exhausted exercise time and body core temperature were recorded. The 5 hydroxytryptamine(5-HT)levels in the brain tissue were measured by HPLC. Results: 1)The duration of exhausted exercise in heat preconditioning group and control group were(56.00 ± 12.27)min and(70.88 ± 13.74)min respectively, significant increase in exercise tolerance(P<0.05); 2)The duration of exhausted exercise in heat preconditioning group and control group were(0.382 ± 0.396)℃ and(1.954 ± 0.603)℃respectively, significant increase in exercise tolerance(P<0.05); Conclusions: Heat preconditioning can effectively reduce the temperature difference before and after exercise, and may be through the reduction of 5-HT content to play a role in delaying the movement of fatigue.
Heat preconditioning; Neurotransmitters; Exercise fatigue
1007―6891(2017)06―0024―04
10.13932/j.cnki.sctykx.2017.06.06
G804.7
A
2017-06-14
2017-07-29
中央高?;究蒲袠I(yè)務(wù)費(fèi)資助項(xiàng)目,項(xiàng)目編號(hào):FRF-BR -16-003B。