郭 文,羅學(xué)港,王 慧,陳 旦
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降鈣素基因相關(guān)肽對(duì)大鼠運(yùn)動(dòng)性疲勞的影響
郭 文1,羅學(xué)港2,王 慧2,陳 旦2
目的:探討降鈣素基因相關(guān)肽(CGRP)對(duì)大鼠運(yùn)動(dòng)性疲勞的影響。方法:成年健康雄性SD大鼠,依據(jù)不同的預(yù)處理措施隨機(jī)分成5組:空白對(duì)照組、磷酸鹽緩沖液(PBS)預(yù)處理組、CGRP預(yù)處理組、辣椒素(CAP)預(yù)處理組、CAP預(yù)處理+ CGRP預(yù)處理組,各組動(dòng)物在安靜狀態(tài)時(shí)、運(yùn)動(dòng)80 min時(shí)和運(yùn)動(dòng)至力竭后即刻處死。然后用免疫組化和放射免疫方法檢測(cè)上述條件下大鼠腓腸肌神經(jīng)肌肉接頭CGRP的表達(dá)變化;用蘇木精-伊紅(HE) 染色觀察上述條件下運(yùn)動(dòng)性疲勞大鼠骨骼肌形態(tài)結(jié)構(gòu)的變化;用化學(xué)比色的方法檢測(cè)上述條件下骨骼肌超氧化物歧化酶(SOD)、谷胱甘肽過氧化物酶(GSH-PX)、過氧化氫酶(CAT)及丙二醛(MDA)的變化。結(jié)果:1)給予CGRP預(yù)處理后,大鼠運(yùn)動(dòng)至力竭的時(shí)間明顯縮短(P<0.05),而給予CAP預(yù)處理后大鼠運(yùn)動(dòng)至力竭的時(shí)間明顯延長(zhǎng)(P<0.05);2)CGRP免疫組織化學(xué)和放射免疫結(jié)果顯示,CGRP的免疫陽(yáng)性產(chǎn)物在肌纖維縱切面上,其形狀為橢圓形或棒狀,主要分布于肌纖維膜的表面。給予CGRP預(yù)處理后,大鼠腓腸肌神經(jīng)肌肉接頭CGRP的表達(dá)明顯升高(P<0.05),而給予CAP預(yù)處理后,大鼠腓腸肌神經(jīng)肌肉接頭CGRP的表達(dá)明顯下降(P<0.05);3)HE染色顯示,安靜狀態(tài)下各組骨骼肌的形態(tài)結(jié)構(gòu)未見明顯改變;與安靜狀態(tài)相比,運(yùn)動(dòng)80min時(shí)除CGRP預(yù)處理組骨骼肌的細(xì)胞核明顯增多、增大外,其余各組均無明顯改變;力竭運(yùn)動(dòng)后即刻與安靜狀態(tài)下相比,除CAP預(yù)處理組無明顯改變外,其余各組骨骼肌的細(xì)胞核均明顯增多、增大;4)化學(xué)比色結(jié)果顯示,給予CGRP預(yù)處理后,大鼠骨骼肌中SOD、GSH-PX 和CAT的活性明顯下降(P<0.05),MDA的含量明顯上升(P<0.05),而給予CAP預(yù)處理后大鼠骨骼肌中SOD、GSH-PX 和CAT的活性明顯上升(P<0.05),MDA的含量明顯下降(P<0.05)。結(jié)論:CGRP 加劇了運(yùn)動(dòng)性疲勞的產(chǎn)生。
降鈣素基因相關(guān)肽;運(yùn)動(dòng)性疲勞;骨骼??;自由基
運(yùn)動(dòng)性疲勞是機(jī)體對(duì)運(yùn)動(dòng)訓(xùn)練刺激所產(chǎn)生的一系列綜合性復(fù)雜反應(yīng),易導(dǎo)致骨骼肌系統(tǒng)損傷,神經(jīng)內(nèi)分泌系統(tǒng)、心血管系統(tǒng)等功能障礙,機(jī)體的運(yùn)動(dòng)能力下降[7,19,28,32]。運(yùn)動(dòng)性疲勞的產(chǎn)生不僅與中樞神經(jīng)系統(tǒng)沒有足夠的運(yùn)動(dòng)神經(jīng)元驅(qū)動(dòng)有關(guān),而且與神經(jīng)肌肉接頭傳遞的失敗及周圍肌肉代謝水平的改變有關(guān)。因此,神經(jīng)肌肉接頭是運(yùn)動(dòng)性疲勞在外周產(chǎn)生的一個(gè)重要位點(diǎn)。目前,有關(guān)運(yùn)動(dòng)性疲勞與神經(jīng)肌肉接頭的研究主要集中于突觸前和突觸后[27,31]。在突觸前其可能存在的機(jī)制有:Ca2+內(nèi)流的下降;Ca2+對(duì)神經(jīng)末梢囊泡釋放敏感性的下降;可利用的囊泡數(shù)量下降。在突觸后其可能存在的機(jī)制有:乙酰膽堿受體(AChR)的脫敏;肌纖維膜興奮性的下降。
自從在腦、脊髓運(yùn)動(dòng)神經(jīng)元和運(yùn)動(dòng)神經(jīng)末梢檢測(cè)出降鈣素基因相關(guān)肽(calcitonin gene-related peptide, CGRP)后,CGRP在神經(jīng)肌肉接頭作為一種神經(jīng)調(diào)質(zhì)和/或營(yíng)養(yǎng)補(bǔ)劑就被廣泛研究[5,10,20,25]。CGRP被輸送到神經(jīng)肌肉接頭的神經(jīng)末梢后貯存在致密核心小泡(LDCV)[6,15],以Ca2+依賴方式伴隨神經(jīng)刺激而釋放[10,22,30],從而影響肌肉的代謝、結(jié)構(gòu)和功能特征。因此,CGRP的變化將導(dǎo)致該神經(jīng)所支配骨骼肌的代謝、結(jié)構(gòu)和功能特征的改變[24]。研究表明,CGRP有“雙重效應(yīng)”,正常生理?xiàng)l件下CGRP的釋放對(duì)靶組織起營(yíng)養(yǎng)作用,能夠擴(kuò)張周圍血管,促進(jìn)AChR的合成,并對(duì)睪丸下降、胃腸活動(dòng)、胃酸分泌等有良好的功效,并能抑制活性氧自由基的產(chǎn)生和緩減組織的氧化應(yīng)激,對(duì)組織起內(nèi)源性的保護(hù)作用,然而,在應(yīng)激或病理?xiàng)l件下,CGRP也能直接或間接的導(dǎo)致脊髓、骨骼肌興奮性的改變和炎癥的發(fā)生,對(duì)組織造成損傷[12,17]。有關(guān)CGRP與運(yùn)動(dòng)性疲勞的關(guān)系,Homonko等和Theriault(1997)等的研究表明,一次性大強(qiáng)度的運(yùn)動(dòng)可導(dǎo)致大鼠后肢運(yùn)動(dòng)神經(jīng)元CGRP顯著提高[13];另有研究表明,在運(yùn)動(dòng)過程中骨骼肌CGRP的改變與運(yùn)動(dòng)和/或骨骼肌的缺血缺氧有關(guān)[4],這提示,CGRP可能與運(yùn)動(dòng)性疲勞的產(chǎn)生有關(guān),但CGRP在運(yùn)動(dòng)性疲勞大鼠神經(jīng)肌肉接頭的生理和生物學(xué)角色目前仍不清楚。
本研究通過一次性運(yùn)動(dòng)至力竭疲勞動(dòng)物模型,運(yùn)用免疫組化、放射免疫、蘇木精-伊紅(hematoxylin-eosin,HE) 染色和化學(xué)比色的方法并結(jié)合補(bǔ)充外源性的CGRP和皮下注射CGRP耗竭劑的方法檢測(cè)CGRP對(duì)大鼠運(yùn)動(dòng)至力竭時(shí)間、骨骼肌的形態(tài)結(jié)構(gòu)、酶的活性和自由基代謝的影響,探索CGRP與運(yùn)動(dòng)性疲勞之間的潛在關(guān)系。
1.1 實(shí)驗(yàn)動(dòng)物與主要試劑
成年健康雄性SD大鼠150只,體重200~250 g,購(gòu)自湖南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)學(xué)院,所有動(dòng)物經(jīng)一般體查均無異常。所有大鼠都置于同一動(dòng)物房,分籠飼養(yǎng),室溫16~22℃,相對(duì)濕度45%~55%,正常晝夜節(jié)律,自由飲食。CGRP、辣椒素和多克隆兔抗CGRP抗體均購(gòu)于Sigma公司,骨骼肌超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽過氧化物酶(glutathione peroxidase,GSH-PX)、過氧化氫酶(catalase,CAT)及丙二醛(malondialdehyde,MDA)均購(gòu)于南京建成生物工程研究所,CGRP 放射免疫試劑盒購(gòu)于北京東亞放免技術(shù)研究所。
1.2 動(dòng)物模型制備
1.3 實(shí)驗(yàn)動(dòng)物分組
將150只大鼠依據(jù)不同的預(yù)處理措施隨機(jī)分成5組:空白對(duì)照組、PBS預(yù)處理組、CGRP預(yù)處理組、CAP預(yù)處理組、CAP預(yù)處理+CGRP預(yù)處理組,各組大鼠在安靜狀態(tài)時(shí)、運(yùn)動(dòng)80 min時(shí)和運(yùn)動(dòng)至力竭后即刻處死。
1.4 PBS、CGRP和CAP的預(yù)處理
大鼠皮下注射PBS、CAP、純化的大鼠CGRP。PBS預(yù)處理組、CGRP預(yù)處理組和CAP預(yù)處理組于力竭運(yùn)動(dòng)前4天兩次皮下給予PBS(每次300 μl)、CGRP(每次120 μg,300 μl PBS溶解,Sigma)和CAP (每次50 mg/kg,溶于10%無水酒精+10%吐溫-80+80%生理鹽水中)。CAP預(yù)處理+ CGRP預(yù)處理組于力竭運(yùn)動(dòng)前8天的前1~4天兩次皮下給予CAP (每次50 mg/kg,溶于10%無水酒精+10%吐溫-80+80%生理鹽水中),后5~8天兩次皮下給予CGRP(每次120 μg,300 μl PBS溶解,Sigma)。
1.5 組織制備
各組大鼠腹腔注射10%水合氯醛(4 ml/kg),待麻醉后快速取左側(cè)腓腸肌內(nèi)側(cè)頭,除去表面的凝血及結(jié)締組織,經(jīng)冰冷生理鹽水洗滌幾次,用濾紙吸干水分,用做放射免疫分析的標(biāo)本放入1 ml 0.1 M冰醋酸在手動(dòng)勻漿管中冰浴勻漿研磨,用做酶活性和自由基檢測(cè)的標(biāo)本按1 g組織中加入10 mL冰鹽水的比例作為勻漿介質(zhì)在手動(dòng)勻漿管中稀釋,并勻漿研磨,再經(jīng)4℃ 3 000 r/min離心10 min,取上清液貯存于-20℃冰箱保存待測(cè)。
用作免疫組織化學(xué)染色和HE染色的組織,在上述大鼠麻醉快速取下左側(cè)腓腸肌內(nèi)側(cè)頭后,開胸經(jīng)左心室插管入升主動(dòng)脈,先灌以生理鹽水(150 ml),繼之以4%多聚甲醛磷酸緩沖液(500 ml,pH7.4,4℃)灌注固定,先快后慢。取右側(cè)腓腸肌內(nèi)側(cè)頭,剔除筋膜,入相同的灌注液4℃后固定過夜,0.01 M PBS充分洗滌后,OCT包埋于-80℃保存?zhèn)溆谩?/p>
1.6 CGRP免疫組織化學(xué)染色
標(biāo)本于-80℃冰箱取出后,-20℃三頓恒低溫切片機(jī)切片,片厚200 μm,進(jìn)行CGRP免疫組織化學(xué)染色,采用漂浮法,主要過程如下:1)切片用0.01 M PBS(pH 7.4)振動(dòng)漂洗3次,每次10 min;2)入含0.3%的甲醇雙氧水,20 min;3)0.01 M PBS(pH 7.4)振動(dòng)漂洗3次,每次10 min;4)入5 %正常小牛血清白蛋白(BSA)室溫封閉2 h;5)入1∶1 000的多克隆兔抗CGRP抗體(Sigma)4℃孵育過3夜;6)0.01 M PBS(pH 7.4)振動(dòng)漂洗3次,每次10 min;7)入1∶200生物素化山羊抗兔IgG(Vector)37℃孵育2 h;8)0.01 M PBS(pH 7.4)振動(dòng)漂洗3次,每次10 min;9)入1∶200 ABC 復(fù)合物(Vector,預(yù)先 30 min配制),37℃ 2 h;10)入0.05% DAB + 0.03%雙氧水顯色,0.01 M PBS終止反應(yīng)、貼片、常規(guī)脫水、透明、封片。
陰性對(duì)照采用正常小牛血清代替一抗,其余步驟與上述相同,結(jié)果為陰性。
1.7 骨骼肌CGRP濃度的測(cè)定
測(cè)定時(shí)用0.1 M的PBS 5倍稀釋。按要求依次加樣后,充分混勻,室溫放置20 min后,4℃ 3 000 rpm離心25 min,取上清液,在γ計(jì)數(shù)器上測(cè)定CPM數(shù)。通過預(yù)先編制的程序,直接給出CGRP濃度。
1.8 HE染色
標(biāo)本于-80℃冰箱取出后,0.01 M PBS充分洗滌后,梯度乙醇脫水,二甲苯透明,石蠟包埋,切片厚4 μm。HE染色按下列步驟進(jìn)行:常規(guī)脫蠟,梯度乙醇脫水后行HE染色。蘇木精液染色6 min,流水沖洗,1%鹽酸處理3 s,稍水洗,1%氨水返藍(lán)5 s,流水沖洗,0.5%伊紅液染色3 min,稍水洗,80%、90%、100%乙醇脫水,二甲苯透明,中性樹膠封固。
1.9 SOD、GSH-PX、CAT活性測(cè)定及MDA含量測(cè)定
SOD、GSH-PX、CAT的活性及MDA的含量測(cè)定采用比色法,組織蛋白含量的測(cè)定采用考馬斯亮藍(lán)法。具體操作如下:1)取樣品加入潔凈試管中,依次加入各試劑,混勻,95℃或37℃水浴,加入中止液;2)蒸餾水調(diào)零,測(cè)定吸光度OD值;3)空白管用蒸餾水代替樣品,標(biāo)準(zhǔn)管用標(biāo)準(zhǔn)液代替樣品,其他步驟相同;4)根據(jù)樣品、空白管、標(biāo)準(zhǔn)管在分光光度計(jì)上的吸光度值,按公式計(jì)算MDA的含量和SOD、GSH-PX、CAT的活性。
1.10 數(shù)據(jù)收集處理
切片在Motic顯微鏡下觀察,CGRP的免疫組織化學(xué)染色和HE染色于每組每只動(dòng)物各取3張切片,每張切片采用Nikon Coolpix 950數(shù)碼相機(jī)在Motic顯微鏡下隨機(jī)取8個(gè)部位攝片。
2.1 平均力竭時(shí)間
各組大鼠平均運(yùn)動(dòng)至力竭的時(shí)間如表1所示:空白對(duì)照組和PBS預(yù)處理組相比,兩組大鼠平均運(yùn)動(dòng)至力竭的時(shí)間無顯著性差異;CAP預(yù)處理組大鼠運(yùn)動(dòng)至力竭的時(shí)間明顯延長(zhǎng),與空白對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。CGRP預(yù)處理組大鼠運(yùn)動(dòng)至力竭的時(shí)間明顯縮短,與空白對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義(P<0.05);CAP預(yù)處理+CGRP預(yù)處理組與空白對(duì)照組相比,大鼠運(yùn)動(dòng)至力竭的時(shí)間有所下降,但差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。
表1 本研究大鼠運(yùn)動(dòng)至力竭的時(shí)間一覽表Table 1 The Time of the Rat Treadmill
2.2 不同干預(yù)措施條件下運(yùn)動(dòng)性疲勞大鼠腓腸肌神經(jīng)肌肉接頭CGRP的表達(dá)變化
CGRP的免疫陽(yáng)性產(chǎn)物在肌纖維縱切面上,其形狀為橢圓形或棒狀,主要分布于肌纖維膜的表面。
1.在安靜狀態(tài)下??瞻讓?duì)照組(圖1A)、PBS預(yù)處理組(圖1B)和CAP預(yù)處理+CGRP預(yù)處理組(圖1E)的免疫陽(yáng)性產(chǎn)物表達(dá)較少,呈色較淺;CGRP預(yù)處理組(圖1C)的免疫陽(yáng)性產(chǎn)物較空白對(duì)照組明顯增多,反應(yīng)增強(qiáng),呈色較深;而CAP預(yù)處理組大鼠(圖1D)的免疫陽(yáng)性產(chǎn)物較空白對(duì)照組有所減少,呈色變淺。
圖1 運(yùn)動(dòng)性疲勞大鼠腓腸肌CGRP免疫組織化學(xué)圖Figure1.CGRP Immunohistochemistry of Gastrocnemius Muscle after Exercise-induced Fatigue(Bar=50 μm)
放射免疫結(jié)果顯示(表2),空白對(duì)照組、PBS預(yù)處理組和CAP預(yù)處理+CGRP預(yù)處理組相比,大鼠神經(jīng)肌肉接頭CGRP的表達(dá)量無明顯差別(P>0.05);但CGRP預(yù)處理組與空白對(duì)照組相比,大鼠神經(jīng)肌肉接頭CGRP的表達(dá)量明顯升高(P<0.05),而CAP預(yù)處理組大鼠與空白對(duì)照組相比,大鼠神經(jīng)肌肉接頭CGRP的表達(dá)量明顯下降(P<0.05)。
表2 運(yùn)動(dòng)性疲勞大鼠腓腸肌CGRP 濃度一覽表Table 2 The CGRP Concentration of Gastrocnemius Muscle after Exercise-induced Fatigue(pg/mg wet weight)
2.運(yùn)動(dòng)80 min時(shí)。與安靜狀態(tài)時(shí)相比,除CGRP預(yù)處理組免疫陽(yáng)性產(chǎn)物明顯增多,反應(yīng)增強(qiáng),呈色較深外,其余各組的免疫陽(yáng)性產(chǎn)物均有所下降,反應(yīng)減弱,呈色變淺(圖1);CGRP預(yù)處理組(圖1H)的免疫陽(yáng)性產(chǎn)物較空白對(duì)照組增多,反應(yīng)增強(qiáng),呈色較深; 而CAP預(yù)處理組大鼠(圖1I)的免疫陽(yáng)性產(chǎn)物較空白對(duì)照組有所減少,呈色變淺。
放射免疫結(jié)果顯示(表2),與安靜狀態(tài)時(shí)相比,各組運(yùn)動(dòng)80 min時(shí),大鼠神經(jīng)肌肉接頭CGRP的表達(dá)量除CGRP預(yù)處理組顯著上升外(P<0.05),其余各組CGRP的表達(dá)量均有所下降(P<0.05);CGRP預(yù)處理組與空白對(duì)照組相比,大鼠神經(jīng)肌肉接頭CGRP的表達(dá)量明顯升高(P<0.05),而CAP預(yù)處理組大鼠與空白對(duì)照組相比,大鼠神經(jīng)肌肉接頭CGRP的表達(dá)量明顯下降。
3.力竭運(yùn)動(dòng)后即刻。各組的免疫陽(yáng)性產(chǎn)物與安靜狀態(tài)下各對(duì)應(yīng)組相比明顯增多,反應(yīng)增強(qiáng),呈色變深(圖1);CGRP預(yù)處理組(圖1M)的免疫陽(yáng)性產(chǎn)物較空白對(duì)照組明顯增多,反應(yīng)增強(qiáng),呈色變深; 而CAP預(yù)處理組大鼠(圖1N)的免疫陽(yáng)性產(chǎn)物較空白對(duì)照組有所減少,呈色變淺。
放射免疫結(jié)果顯示(表2),與安靜狀態(tài)時(shí)相比,各組力竭運(yùn)動(dòng)后即刻大鼠神經(jīng)肌肉接頭CGRP的表達(dá)量顯著上升(P<0.05);CGRP預(yù)處理組與空白對(duì)照組相比,大鼠神經(jīng)肌肉接頭CGRP的表達(dá)量明顯升高(P<0.05),而CAP預(yù)處理組大鼠與空白對(duì)照組相比,大鼠神經(jīng)肌肉接頭CGRP的表達(dá)量明顯下降(P<0.05)。
2.3 HE染色
HE染色結(jié)果顯示(圖2),安靜狀態(tài)下各組骨骼肌的形態(tài)結(jié)構(gòu)未見明顯改變;運(yùn)動(dòng)80 min時(shí)與安靜狀態(tài)下相比,除CGRP預(yù)處理組骨骼肌的細(xì)胞核明顯增多、增大外,其余各組均無明顯改變;力竭運(yùn)動(dòng)后即刻與安靜狀態(tài)下相比除CAP預(yù)處理組無明顯改變外,其余各組骨骼肌的細(xì)胞核均明顯增多、增大。2.4 骨骼肌MDA含量及SOD、GSH-PX、CAT活性的檢測(cè)
表3表明,在安靜狀態(tài)時(shí),各組之間MDA含量的變化差異無顯著性(P>0.05)。運(yùn)動(dòng)80 min時(shí),空白對(duì)照組、PBS預(yù)處理組和CAP預(yù)處理+CGRP預(yù)處理組相比,MDA的含量也無明顯差別;但CGRP預(yù)處理組與空白對(duì)照組相比,MDA的含量顯著上升(P<0.05);而CAP預(yù)處理組與空白對(duì)照組相比,MDA的含量略有上升,但差異無顯著性(P>0.05)。力竭運(yùn)動(dòng)后即刻,空白對(duì)照組、PBS預(yù)處理組和CAP預(yù)處理+CGRP預(yù)處理組相比,MDA的含量也無明顯差別;但CGRP預(yù)處理組與空白對(duì)照組相比,MDA的含量顯著上升(P<0.05);CAP預(yù)處理組與空白對(duì)照組相比,MDA的含量也有所上升,但差異無顯著性(P>0.05)。運(yùn)動(dòng)80 min時(shí)、力竭運(yùn)動(dòng)后即刻各組與安靜狀態(tài)下各對(duì)應(yīng)組相比較,MDA的含量均顯著上升(P<0.05)。
表4、表5、表6表明,在安靜狀態(tài)時(shí),各組之間SOD、GSH-PX和CAT的活性變化差異無顯著性(P>0.05)。運(yùn)動(dòng)80 min時(shí),空白對(duì)照組、PBS預(yù)處理組和CAP預(yù)處理+CGRP預(yù)處理組相比,SOD、GSH-PX和CAT的活性差異也無顯著性(P>0.05);但CGRP預(yù)處理組與空白對(duì)照組相比,SOD、GSH-PX和CAT的活性顯著下降;而CAP預(yù)處理組大鼠與空白對(duì)照組相比,SOD、GSH-PX和CAT的活性顯著上升(P<0.05)。力竭運(yùn)動(dòng)后即刻,空白對(duì)照組、PBS預(yù)處理組和CAP預(yù)處理+CGRP預(yù)處理組相比,SOD、GSH-PX和CAT的活性也無明顯差別;但CGRP預(yù)處理組與空白對(duì)照組相比,SOD、GSH-PX和CAT的活性顯著下降(P<0.05);而CAP預(yù)處理組大鼠與空白對(duì)照組相比,SOD、GSH-PX和CAT的活性顯著上升(P<0.05)。運(yùn)動(dòng)80 min時(shí)、力竭運(yùn)動(dòng)后即刻各組與安靜狀態(tài)下各對(duì)應(yīng)組相比較,骨骼肌SOD和GSH-PX的活性均顯著上升(P<0.05);CAT的活性在運(yùn)動(dòng)80 min時(shí)、力竭運(yùn)動(dòng)后即刻各組與安靜狀態(tài)下各對(duì)應(yīng)組相比較,除CGRP預(yù)處理組差異無顯著性外,其余各組均顯著上升(P<0.05)。
本研究運(yùn)用一次性運(yùn)動(dòng)至力竭疲勞動(dòng)物模型,通過給大鼠皮下注射CGRP和用CAP耗竭CGRP的方法從行為學(xué)、形態(tài)學(xué)和生理機(jī)能的角度檢測(cè)CGRP對(duì)大鼠運(yùn)動(dòng)性疲勞的影響,探索CGRP與運(yùn)動(dòng)性疲勞之間的潛在關(guān)系。
本研究首先測(cè)量了不同干預(yù)措施條件下大鼠運(yùn)動(dòng)至力竭的平均時(shí)間。研究結(jié)果表明,給大鼠皮下注射CGRP后,在其他組運(yùn)動(dòng)至80 min時(shí)該組已達(dá)力竭狀態(tài),大鼠運(yùn)動(dòng)至力竭的時(shí)間縮短大約37%;而用CAP耗竭內(nèi)源性CGRP后大鼠運(yùn)動(dòng)至力竭的時(shí)間延長(zhǎng)大約32%。這提示,CGRP可能加速了大鼠運(yùn)動(dòng)性疲勞的產(chǎn)生。
圖2 運(yùn)動(dòng)性疲勞大鼠腓腸肌HE染色圖Figure 2. HE Stain of Gastrocnemius Muscle after Exercise-induced Fatigue(Bar=50 μm)
表3 運(yùn)動(dòng)性疲勞大鼠腓腸肌MDA 含量一覽表Table 3 The MDA Content of Gastrocnemius Muscle after Exercise-induced Fatigue(nmol/mg prot)
表4 運(yùn)動(dòng)性疲勞大鼠腓腸肌SOD 活性一覽表Table 4 The SOD Activity of Gastrocnemius Muscle after Exercise-induced Fatigue(U/mg)
表5 運(yùn)動(dòng)性疲勞大鼠腓腸肌GSH-PX的活性一覽表Table 5 The GSH-PX Activity of Gastrocnemius Muscle after Exercise-induced Fatigue(U/mg)
表6 運(yùn)動(dòng)性疲勞大鼠腓腸肌CAT的活性一覽表Table 6 The CAT Activity of Gastrocnemius Muscle after Exercise-induced Fatigue(U/mg)
通過免疫組織化學(xué)和放射免疫的方法,測(cè)試不同干預(yù)措施條件下大鼠安靜狀態(tài)、運(yùn)動(dòng)至80 min時(shí)、力竭運(yùn)動(dòng)后即刻腓腸肌神經(jīng)肌肉接頭CGRP的變化,發(fā)現(xiàn)力竭運(yùn)動(dòng)后即刻,大鼠腓腸肌神經(jīng)肌肉接頭CGRP的表達(dá)量與安靜狀態(tài)時(shí)相比明顯上升。以往研究表明,一次大強(qiáng)度的下坡跑后,大鼠后肢運(yùn)動(dòng)神經(jīng)元的CGRP顯著提高,并持續(xù)2周左右,到第四周恢復(fù)到安靜水平[13],這與本研究結(jié)果基本一致。運(yùn)動(dòng)神經(jīng)元CGRP表達(dá)的增加可能是由于運(yùn)動(dòng)時(shí)腓腸肌的收縮提高了相關(guān)運(yùn)動(dòng)神經(jīng)元的發(fā)放活動(dòng),從而引起運(yùn)動(dòng)神經(jīng)元CGRP的表達(dá)均增加。運(yùn)動(dòng)80 min時(shí)各組與安靜狀態(tài)下各對(duì)應(yīng)組相比較,除CGRP預(yù)處理組外,其余各組CGRP的表達(dá)量有所下降,這可能是由于大鼠經(jīng)過一段時(shí)間的運(yùn)動(dòng),機(jī)體機(jī)能狀態(tài)未達(dá)到力竭水平出現(xiàn)的一種保護(hù)性現(xiàn)象。給大鼠皮下注射CGRP后,無論安靜狀態(tài)、運(yùn)動(dòng)80 min時(shí),還是力竭運(yùn)動(dòng)后即刻,大鼠腓腸肌神經(jīng)肌肉接頭CGRP的表達(dá)均明顯升高,而補(bǔ)充CAP后,無論安靜狀態(tài)、運(yùn)動(dòng)80 min時(shí),還是力竭運(yùn)動(dòng)后即刻大鼠腓腸肌神經(jīng)肌肉接頭CGRP的表達(dá)均明顯下降。這說明,本研究的干預(yù)是有效的。
為了進(jìn)一步了解CGRP對(duì)運(yùn)動(dòng)性疲勞的影響,本研究檢測(cè)了不同干預(yù)措施條件下CGRP對(duì)大鼠骨骼肌形態(tài)結(jié)構(gòu)及骨骼肌酶的活性和自由基含量的影響。1990年,F(xiàn)isher等通過建立大鼠鈍挫傷模型,發(fā)現(xiàn)傷后骨骼肌出現(xiàn)了許多肌細(xì)胞核,形態(tài)類似衛(wèi)星細(xì)胞,說明,骨骼肌細(xì)胞核的變化可初步反映骨骼肌受損傷的程度。本研究發(fā)現(xiàn),給大鼠皮下注射CGRP,運(yùn)動(dòng)至80 min時(shí)(力竭運(yùn)動(dòng)后即刻)大鼠骨骼肌的細(xì)胞核明顯增多、增大,而用CAP耗竭CGRP后大鼠骨骼肌細(xì)胞核增多、增大的現(xiàn)象不明顯。提示,CGRP干預(yù)導(dǎo)致了顯著的肌細(xì)胞形態(tài)學(xué)改變,因而,CGRP可能加速了骨骼肌的損傷。骨骼肌細(xì)胞核的增多、增大可能與以下原因有關(guān):1)骨骼肌損傷時(shí),肌衛(wèi)星細(xì)胞分裂,變成紡錘形的成肌細(xì)胞,然后多個(gè)成肌細(xì)胞融合成管狀的多核細(xì)胞(稱為肌管),最后變成骨骼肌纖維;2)破碎骨骼肌纖維的細(xì)胞核及其周圍的肌漿可變?yōu)槌杉〖?xì)胞;3)結(jié)締組織中的某種細(xì)胞也可分化為成肌細(xì)胞[1]。
研究表明,通過測(cè)量大鼠骨骼肌中MDA的含量及SOD、GSH-PX 和CAT的活性能反映大鼠的運(yùn)動(dòng)能力及運(yùn)動(dòng)性疲勞的產(chǎn)生[8,9,33]。MDA是細(xì)胞脂質(zhì)過氧化的一種主要產(chǎn)物,生物膜脂質(zhì)的不飽和脂肪酸最易受到自由基的攻擊而發(fā)生過氧化,所以,組織MDA的含量可反映機(jī)體脂質(zhì)過氧化水平,間接反映機(jī)體細(xì)胞受自由基攻擊損傷的嚴(yán)重程度[16,18]。在自由基的連鎖反應(yīng)中,SOD、GSH-PX 和CAT都是機(jī)體內(nèi)的抗過氧化物酶。SOD是體內(nèi)主要的自由基清除酶,能夠催化超氧陰離子歧化為O2和H2O2,從而保護(hù)細(xì)胞膜和線粒體等免受氧化損傷,而H2O2可以被GSH-PX 和CAT催化分解為O2和H2O[2,14]。CAT 主要集中在過氧化物酶體中,而GSH-PX 主要在細(xì)胞質(zhì)中。GSH-PX能特異地催化GSH對(duì)H2O2的還原反應(yīng),起到保護(hù)細(xì)胞膜結(jié)構(gòu)和功能完整性的作用[23]。本研究表明,給大鼠皮下注射CGRP后,與空白對(duì)照組相比,大鼠骨骼肌中MDA含量顯著升高,SOD、GSH-PX 和CAT的活性顯著下降,而用CAP耗竭CGRP后,與空白對(duì)照組相比,大鼠骨骼肌中MDA含量顯著下降,SOD、GSH-PX 和CAT的活性顯著升高。這提示,CGRP在一定程度上增加了大鼠力竭運(yùn)動(dòng)后體內(nèi)自由基的生成量,降低了體內(nèi)自由基的清除,增加了自由基對(duì)細(xì)胞脂質(zhì)成分的氧化損傷作用,加速了運(yùn)動(dòng)性疲勞的產(chǎn)生。研究報(bào)道,CGRP有預(yù)防小鼠自身免疫性糖尿病發(fā)病及抗氧化應(yīng)激的作用,增加SOD和CAT的生成,降低MDA的含量[26]。這一報(bào)道與本研究結(jié)果正好相反,這可能與實(shí)驗(yàn)?zāi)P突駽GRP劑量有關(guān)。在脊椎動(dòng)物骨骼肌,CGRP通過作用于CGRP受體,誘發(fā)肌肉的收縮[29],在成年嚙齒類動(dòng)物膈肌,CGRP縮短了肌肉收縮的相對(duì)不應(yīng)期[11],上調(diào)了骨骼肌纖維cAMP的水平[21]。結(jié)合本研究實(shí)驗(yàn)結(jié)果,認(rèn)為,CGRP對(duì)大鼠運(yùn)動(dòng)性疲勞的影響可能是由于CGRP對(duì)骨骼肌長(zhǎng)時(shí)間的作用導(dǎo)致骨骼肌長(zhǎng)時(shí)間收縮,從而引起大鼠運(yùn)動(dòng)性疲勞的提前發(fā)生。
綜上所述,本研究觀察到CGRP干預(yù)可導(dǎo)致大鼠運(yùn)動(dòng)至力竭的時(shí)間縮短,形態(tài)學(xué)上骨骼肌損傷加劇,骨骼肌自由基生成增多,提示,CGRP加速了運(yùn)動(dòng)性疲勞的產(chǎn)生。但是,CGRP加速運(yùn)動(dòng)性疲勞產(chǎn)生的具體機(jī)制,尤其是其下游的作用靶點(diǎn),需要更進(jìn)一步的研究。
CGRP干預(yù)后,大鼠運(yùn)動(dòng)至力竭的時(shí)間明顯下降,骨骼肌的細(xì)胞核明顯增多、增大,MDA的含量上升,SOD、GSH-PX和CAT的活性下降,提示,CGRP可能介導(dǎo)了運(yùn)動(dòng)性疲勞的產(chǎn)生。
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Effects of Calcitonin Gene-related Peptide on the Exercise-induced Fatigue of Rats
GUO Wen1,LUO Xue-gang2,WANG Hui2,CHEN Dan2
The purpose of this paper is to study the effects of CGRP on the exercise-induced fatigue of rats.Methods:Healthy adult male Sprague-Dawley rats were randomly divided into five groups according to the different pretreatment:the blank control group,the PBS pretreatment group,the CGRP pretreatment group,the CAP pretreatment group,the CAP pretreatment +CGRP pretreatment group.The rats were sacrificed at the resting state,the exercise to 80 min and the exercise to exhaustion,Then the immunohistochemistry and radioimmunoassay was used to detecting the expression of CGRP at the condition of the different pretreatment,the HE staining was used to detecting the change of morphous formation of skeletal muscle at the condition of the different pretreatment,the chemistry colorimetric method was used to detecting the change of superoxide dismutase,glutathione peroxidase,catalase,and malondialdehyde at the conditions of the different pretreatment.Results:1) The time of the treadmill exercise to exhaustion was significantly decurtated after CGRP pretreatment (P<0.05),and the time of the treadmill exercise to exhaustion was significantly prolonged after CAP pretreatment (P<0.05).2) Immunohistochemistry and radioimmunoassay showed that CGRP immunopositive products were elliptical or rod-shape at longitudinal section of muscle fiber.The expression of CGRP was significantly increased after the CGRP pretreatment (P<0.05),and the expression of CGRP was significantly decreased after the CAP pretreatment (P<0.05).3) HE staining showed that there were no obvious differences in amorphous constitution of skeletal muscle in all groups at the resting state.There were also no obvious changes in morphous constitution of skeletal muscle at the other groups at the exercise to 80 min than at the resting state,excluding cell nucleus of skeletal muscle were significantly increased and augmented at the CGRP pretreatment group,Cell nucleus of skeletal muscle were significantly increased and augmented in all other groups,while there were no obvious changes in morphous constitution of skeletal muscle at the CAP pretreatment group at exercise to exhaustion than at the resting state.4) The chemistry colorimetric showed that the content of MDA was significantly increased (P<0.05),and the activity of SOD,GSH-PX,CAT was significantly decreased after the CGRP pretreatment (P<0.05),While the content of MDA was significantly decreased (P<0.05),and the activity of SOD,GSH-PX,CAT was significantly increased after the CAP pretreatment (P<0.05).Conclusions:The CGRP intensified the generation of exercise-induced fatigue.
calcitoningene-relatedpeptide;exercise-inducedfatigue;skeletalmuscles;freeradical
2015-12-08;
2016-06-23
湖南省教育科學(xué)規(guī)劃基金項(xiàng)目(XJK011QTM002)。
郭文(1978-),女,湖南益陽(yáng)人,副教授,博士,主要研究方向?yàn)檫\(yùn)動(dòng)與神經(jīng)系統(tǒng)的損傷修復(fù),E-mail:gw7808200@163.com。
1.湖南師范大學(xué) 體育學(xué)院,湖南 長(zhǎng)沙 410012;2.中南大學(xué) 湘雅醫(yī)學(xué)院,湖南 長(zhǎng)沙 410013 1.Hunan Normal University,Changsha 410012,China;2.Central South University,Changsha 410013,China.
G804.7
A
10.16469/j.css.201607008