王一茗，張新昕，束彥頁

·論著·

·中醫(yī)·中西醫(yī)結(jié)合研究·

穴位埋線對(duì)缺血再灌注損傷高脂血癥合并2型糖尿病大鼠心肌保護(hù)作用的機(jī)制研究

王一茗1*，張新昕2，束彥頁1

目的探討穴位埋線對(duì)缺血再灌注損傷高脂血癥合并2型糖尿病(ZDF)大鼠心肌保護(hù)作用的機(jī)制，為臨床治療提供理論依據(jù)。方法2015年1月—2016年5月選取ZDF大鼠24只，隨機(jī)分為對(duì)照組、缺血再灌注組、缺血預(yù)處理組和穴位埋線組，每組6只。適應(yīng)性飼養(yǎng)1周后第7天，對(duì)照組直接開胸取材，其余3組均結(jié)扎冠狀動(dòng)脈左前降支，建立缺血再灌注損傷模型。缺血再灌注組缺血30 min，再灌注60 min；缺血預(yù)處理組缺血5 min、再灌注5 min，重復(fù)3次，之后操作同缺血再灌注組；穴位埋線組在適應(yīng)性飼養(yǎng)1周后第1天，“內(nèi)關(guān)”“膻中”“心俞”穴進(jìn)行穴位埋線，之后操作同缺血再灌注組。采用透射電鏡觀察各組ZDF大鼠心肌組織超微結(jié)構(gòu)及自噬水平，Western blotting法檢測(cè)心肌內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白葡萄糖調(diào)節(jié)蛋白78(GRP78)、自噬相關(guān)蛋白Beclin-1水平，酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)法檢測(cè)髓過氧化物酶(MPO)、丙二醛(MDA)水平。結(jié)果透射電鏡下對(duì)照組線粒體和心肌細(xì)胞形態(tài)結(jié)構(gòu)正常，未見明顯自噬體；缺血再灌注組線粒體和心肌細(xì)胞腫脹明顯，細(xì)胞膜破裂、內(nèi)容物泄漏，僅見空泡或空腔及少量自噬泡；缺血預(yù)處理組、穴位埋線組與缺血再灌注組相比，心肌細(xì)胞超微結(jié)構(gòu)損傷較輕，且自噬泡數(shù)量明顯增多；缺血再灌注組心肌細(xì)胞超微結(jié)構(gòu)破壞最為嚴(yán)重，且未見明顯自噬泡。缺血再灌注組、缺血預(yù)處理組、穴位埋線組GRP78、Beclin-1、MPO、MDA水平高于對(duì)照組(P<0.05)；缺血預(yù)處理組、穴位埋線組GRP78、Beclin-1水平高于缺血再灌注組，但MPO、MDA水平低于缺血再灌注組(P<0.05)。結(jié)論穴位埋線可能通過促進(jìn)GRP78、Beclin-1水平升高，進(jìn)而起到對(duì)再灌注損傷心肌的保護(hù)作用。

再灌注損傷；大鼠；內(nèi)質(zhì)網(wǎng)應(yīng)激；自噬；缺血預(yù)處理,心??；穴位埋線

王一茗，張新昕，束彥頁.穴位埋線對(duì)缺血再灌注損傷高脂血癥合并2型糖尿病大鼠心肌保護(hù)作用的機(jī)制研究[J].中國全科醫(yī)學(xué)，2017，20(30)：3776-3780.[www.chinagp.net]

WANG Y M,ZHANG X X,SHU Y Y.Mechanism of catgut implantation at acupoints for the protection of myocardium from ischemia-reperfusion injury in hyperlipidemia rats with type 2 diabetes mellitus[J].Chinese General Practice，2017，20(30)：3776-3780.

心肌梗死為全球范圍內(nèi)患者的主要致死原因之一，心肌急性且持續(xù)性的缺血缺氧，會(huì)嚴(yán)重?fù)p傷心肌。心肌梗死面積的大小關(guān)系患者的預(yù)后，及時(shí)有效的再灌注、恢復(fù)心肌血供、縮小梗死面積，是心肌梗死的治療目標(biāo)。然而，再灌注過程十分復(fù)雜，常伴隨一些適應(yīng)性調(diào)控機(jī)制的作用，如內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)及自噬等，是學(xué)者們一直關(guān)注的焦點(diǎn)[1]。

ERS是自噬的重要調(diào)控通路[2-3]。ERS與自噬作為細(xì)胞對(duì)損傷刺激的適應(yīng)性反應(yīng)，維持著細(xì)胞內(nèi)環(huán)境穩(wěn)態(tài)。ERS時(shí)未折疊蛋白反應(yīng)激活自噬，而自噬則通過降解錯(cuò)誤折疊或未折疊的蛋白來減輕內(nèi)質(zhì)網(wǎng)負(fù)荷，從而抑制ERS的過度激活[1]。

針灸預(yù)處理可發(fā)揮內(nèi)源性保護(hù)作用，從而減輕因缺血再灌注對(duì)心肌造成的損傷[4-5]。自噬可保護(hù)再灌注損傷心肌[6]。ERS與自噬關(guān)系密切，存在交互效應(yīng)。本研究通過制備心肌缺血再灌注損傷模型，觀察ERS通路相關(guān)蛋白-葡萄糖調(diào)節(jié)蛋白78(GRP78)及自噬相關(guān)蛋白Beclin-1的表達(dá)情況，探討穴位埋線對(duì)缺血再灌注損傷心肌的保護(hù)作用。

1 材料與方法

1.1 主要試劑 Beclin-1、GRP78(Abcam公司)；髓過氧化物酶(MPO)、丙二醛 (MDA)試劑盒(DECQ上?；鄯f生物科技公司)，其余試劑(如Tris-base、Tween-20、Western專用脫脂奶粉、過硫酸銨粉、甘氨酸G7126、SDS粉末、甲醇、甲叉雙丙烯酰胺、TEMED、Western bloting及缺血預(yù)處理裂解液、PMSF、20XTBS、PVDF膜等)購自Sigma公司，實(shí)驗(yàn)動(dòng)物由北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司提供〔動(dòng)物許可證編號(hào)SCXK(京)2012-0001〕。

1.2 實(shí)驗(yàn)分組及模型制備 研究時(shí)間：2015年1月—2016年5月。

SPF級(jí)雄性高脂血癥合并2型糖尿病(ZDF)大鼠24只，8周齡，體質(zhì)量220～230 g，在SPF級(jí)實(shí)驗(yàn)環(huán)境適應(yīng)性飼養(yǎng)1周，使用符合國家標(biāo)準(zhǔn)及有生產(chǎn)許可的誘導(dǎo)飼料Purina#5008，飲用無菌過濾水，空氣凈化度10 000級(jí)，氨濃度<14 mg/m2，相對(duì)濕度40%，溫度22～25 ℃，噪聲低于50 dB。本實(shí)驗(yàn)符合《實(shí)驗(yàn)動(dòng)物管理?xiàng)l例》。將ZDF大鼠隨機(jī)分為對(duì)照組、缺血再灌注組、缺血預(yù)處理組和穴位埋線組，每組6只。

本文創(chuàng)新點(diǎn)：

(1)首次深入觀察穴位埋線法對(duì)高脂血癥合并2型糖尿病(ZDF)大鼠心肌缺血再灌注損傷的影響及內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)-自噬機(jī)制；(2)選用ZDF大鼠作為實(shí)驗(yàn)動(dòng)物更接近臨床實(shí)際。

適應(yīng)性飼養(yǎng)1周后第7天，對(duì)照組不做任何處置，直接開胸取材；其余3組制備缺血再灌注損傷模型：10%水合氯醛(0.4 ml/100 g)腹腔注射，仰臥位固定，剪毛后氣管插管，連接小動(dòng)物呼吸機(jī)，開胸并暴露心臟，剪開心包，冠狀動(dòng)脈左前降支位于心耳下緣約0.15 cm，繞左前降支穿線，線兩端同時(shí)穿過聚乙烯小管形成閉環(huán)，監(jiān)測(cè)導(dǎo)聯(lián)變化，S-T段發(fā)生顯著(超過0.1 mv為顯著)抬高為缺血成功，S-T段下降1/2以上為再灌注成功。缺血再灌注組：缺血30 min，再灌注60 min。缺血預(yù)處理組：缺血5 min、再灌注5 min，反復(fù)3次，之后操作同缺血再灌注組。穴位埋線組：適應(yīng)性飼養(yǎng)1周后第1天對(duì)“內(nèi)關(guān)”“膻中”及“心俞”進(jìn)行穴位埋線，之后操作同缺血再灌注組。各組造模成功后取心肌組織-80 ℃保存?zhèn)溆谩?/p>

1.3 實(shí)驗(yàn)方法

1.3.1 透射電鏡觀察心肌組織超微結(jié)構(gòu)及自噬水平 沿肌纖維走向取左心室前壁心肌，將標(biāo)本切成(1×1×1)mm3大小。經(jīng)預(yù)固定、再固定、逐級(jí)脫水、包埋、切片等步驟，透射電鏡下觀察心肌組織超微結(jié)構(gòu)及自噬水平。

1.3.2 Western blotting法檢測(cè)GRP78、Beclin-1水平 分別于各組心肌組織滴入其體積3倍量的組織裂解液，0 ℃研磨成組織勻漿，靜置10 min；經(jīng)低溫(4 ℃)15 000 r/min離心10 min(離心半徑10 cm)；滴入蛋白上樣緩沖液后，置于沸水中5 min。用BCA法定量蛋白濃度。滴入相同劑量2×SDS上樣緩沖液，95 ℃靜置5 min?？装迕靠准?0 μg胞質(zhì)蛋白，用12%十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分離，低溫恒壓轉(zhuǎn)膜，用5%脫脂奶粉25 ℃下封膜后靜置2 h，加入兔抗鼠抗(1∶500)或內(nèi)參GAPDH(1∶800)一抗，4 ℃靜置12 h后洗膜，用辣根過氧化酶標(biāo)記的羊抗兔Ig G (1∶5 000) 25 ℃靜置1 h；經(jīng)漂洗后電化學(xué)發(fā)光法(ECL)常規(guī)顯影。用Gelpro4.0凝膠光密度分析軟件進(jìn)行分析，以目的條帶灰度/內(nèi)參條帶灰度比值表示GRP78、Beclin-1水平。

1.3.3 酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)法檢測(cè)MPO、MDA水平 各組ZDF大鼠再灌注60 min后，用含有EDTA抗凝劑采血管采血2 ml，3 000 r/min離心10 min(離心半徑10 cm)，收集分離血清后靜置于-20 ℃冰箱，測(cè)定ZDF大鼠MPO、MDA水平。

2 結(jié)果

2.1 心肌組織超微結(jié)構(gòu) 透射電鏡下對(duì)照組線粒體和心肌細(xì)胞形態(tài)結(jié)構(gòu)正常，未見明顯自噬體(見圖1A)；缺血再灌注組線粒體腫脹明顯，嵴突紊亂、斷裂甚至消失，心肌細(xì)胞明顯水腫，肌絲斷裂、溶解，肌小節(jié)明暗帶模糊不清，細(xì)胞器結(jié)構(gòu)破壞，胞質(zhì)內(nèi)可見許多大小不等的空泡或空腔及少量自噬泡，細(xì)胞膜破裂，細(xì)胞內(nèi)容物泄漏(見圖1B)；缺血預(yù)處理組、穴位埋線組與缺血再灌注組相比，心肌細(xì)胞超微結(jié)構(gòu)損傷較輕，且自噬泡數(shù)量明顯增多；缺血再灌注組心肌細(xì)胞超微結(jié)構(gòu)破壞較缺血預(yù)處理組嚴(yán)重，且未見明顯自噬泡(圖1C、D)。

2.2 4組ZDF大鼠GRP78、Beclin-1水平比較 4組ZDF大鼠GRP78、Beclin-1水平比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。缺血再灌注組、缺血預(yù)處理組、穴位埋線組GRP78、Beclin-1水平高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；缺血預(yù)處理組、穴位埋線組 GRP78、Beclin-1水平高于缺血再灌注組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表1、圖2)。

Table1 Comparison of the levels of GRP78 and Beclin-1 among the 4 groups of ZDF rats

組別只數(shù)GRP78Beclin-1對(duì)照組60.47±0.020.77±0.04缺血再灌注組60.74±0.04a0.97±0.02a缺血預(yù)處理組60.73±0.01ab1.28±0.02ab穴位埋線組61.74±0.05ab1.29±0.05abF值816.709150.053P值<0.001<0.001

注：GRP78=葡萄糖調(diào)節(jié)蛋白78；與對(duì)照組比較，aP<0.05；與缺血再灌注組比較，bP<0.05

注：GRP78=葡萄糖調(diào)節(jié)蛋白78

圖2 4組ZDF大鼠GRP78、Beclin-1表達(dá)

Figure2 Expression levels of GRP78 and Beclin-1 among 4 groups of ZDF rats

2.3 4組ZDF大鼠MPO、MDA水平比較 4組ZDF大鼠MPO、MDA水平比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。對(duì)照組MPO、MDA水平低于缺血再灌注組、缺血預(yù)處理組、穴位埋線組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；缺血預(yù)處理組、穴位埋線組 MPO、MDA水平低于缺血再灌注組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表2)。

注：圖A為對(duì)照組，圖B為缺血再灌注組，圖C為缺血預(yù)處理組，圖D為穴位埋線組

圖1 各組ZDF大鼠心肌組織透射電鏡顯示結(jié)果(×8 200)

Figure1 Results of examination of myocardium in the 4 groups of ZDF rats by transmission electron microscope

Table2 Comparison of levels of MPO and MDA among the 4 groups of ZDF rats

組別只數(shù)MPO(U/g)MDA(μmol/g)對(duì)照組62.34±0.111.98±0.26缺血再灌注組618.35±0.38a4.34±0.46a缺血預(yù)處理組65.64±0.15ab2.89±0.34ab穴位埋線組66.97±0.35ab3.23±0.88abF值1905.7338.520P值<0.0010.007

注：MPO=髓過氧化物酶，MDA=丙二醛；與對(duì)照組比較，aP<0.05；與缺血再灌注組比較，bP<0.05

3 討論

ERS是應(yīng)激發(fā)生時(shí)在細(xì)胞中的最初反應(yīng)[7]，是某種原因所致的細(xì)胞內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)失衡，并引起生理功能紊亂的一種亞細(xì)胞器上的病理過程[8]。目前研究已證實(shí)，ERS參與多種心血管疾病的病理生理過程，并發(fā)揮著非常重要的作用，其在細(xì)胞內(nèi)介導(dǎo)保護(hù)性反應(yīng)和凋亡反應(yīng)[9],而GRP78是其介導(dǎo)這兩種反應(yīng)的關(guān)鍵信號(hào)因子。在一定程度上，GRP78水平反應(yīng)了細(xì)胞ERS保護(hù)性反應(yīng)水平[10]。Beclin-1作為細(xì)胞自噬的關(guān)鍵調(diào)控因子，其是形成自噬體不可或缺的因素，通過與配體結(jié)合來調(diào)節(jié)細(xì)胞內(nèi)自噬活性[11]。

ERS可通過多條信號(hào)通路調(diào)節(jié)自噬，與維持內(nèi)質(zhì)網(wǎng)正常功能關(guān)系密切[12-14]。ERS誘導(dǎo)細(xì)胞發(fā)生自噬[15-21]，而自噬可減輕內(nèi)質(zhì)網(wǎng)負(fù)擔(dān)，這可能與細(xì)胞抗凋亡的代償機(jī)制有關(guān)[22]。ERS時(shí)未折疊蛋白反應(yīng)激活自噬，而自噬通過降解錯(cuò)誤折疊或未折疊蛋白，減輕內(nèi)質(zhì)網(wǎng)負(fù)荷，抑制過度激活的ERS；但過度激活的ERS-自噬又會(huì)造成細(xì)胞損傷的加重，甚至導(dǎo)致細(xì)胞死亡。

臨床將透射電鏡觀察自噬體囊泡作為判定自噬的金標(biāo)準(zhǔn)[23-25]。本研究制備ZDF大鼠心肌缺血再灌注損傷模型，透射電鏡觀察示：除對(duì)照組外，其余3組ZDF大鼠心肌組織均表現(xiàn)出不同程度的梗死以及梗死后的缺血壞死，提示心肌缺血再灌注損傷模型成功建立；與缺血再灌注組相比，缺血預(yù)處理 組及穴位埋線組的心肌超微結(jié)構(gòu)損傷較輕，且自噬泡數(shù)量增多；穴位埋線組與缺血預(yù)處理組的損傷相對(duì)較輕，提示穴位埋線與缺血預(yù)處理具有近似的心肌保護(hù)作用。

心肌缺血再灌注期間，缺血預(yù)處理組及穴位埋線組GRP78、Beclin-1水平明顯高于缺血再灌注組，提示缺血預(yù)處理組和穴位埋線組可通過調(diào)控ERS-自噬通路對(duì)細(xì)胞凋亡發(fā)揮抑制作用，從而對(duì)損傷心肌起到保護(hù)作用。

MPO、MDA是反映機(jī)體氧化應(yīng)激情況的重要指標(biāo)[26-27]。本實(shí)驗(yàn)結(jié)果顯示，缺血再灌注組MPO、MDA水平明顯升高，說明缺血區(qū)發(fā)生炎性反應(yīng)，自由基增多，進(jìn)而誘導(dǎo)心肌組織損傷加劇。而缺血預(yù)處理組及穴位埋線組的MPO、MDA水平雖高于對(duì)照組，但較缺血再灌注組明顯降低，說明兩種預(yù)處理手段均可減輕缺血再灌注損傷。

本研究采用經(jīng)典的中醫(yī)外治法——穴位埋線，通過可吸收縫合線的持久性刺激與穴位功效相結(jié)合，具有實(shí)用性及易操作等優(yōu)點(diǎn)。俞募配穴是治療心臟疾病的常用配穴，可調(diào)暢氣機(jī)、寬胸理氣。膻中穴是心包的募穴，八會(huì)穴之一；心俞穴是心經(jīng)背俞穴；內(nèi)關(guān)穴是心包經(jīng)的絡(luò)穴，也是八脈交會(huì)穴之一，與心經(jīng)相通，因而也常配合應(yīng)用。無菌操作下，將可吸收縫合線(聚乙醇酸PGA縫線)剪成1 cm左右，放入0.7×30 TWLB一次性無菌注射針針頭中。確定刺入上述穴位前，用愛爾碘局部皮膚常規(guī)消毒，一次性無菌注射針刺入穴位后用特制平頭針將縫合線推入穴位中，縫合線埋入穴位后迅速出針并用無菌棉球按壓止血。該法既簡便易行，又彌補(bǔ)針灸治療結(jié)束后療效衰減的不足，在治療慢性疾病方面優(yōu)勢(shì)突出。

本實(shí)驗(yàn)通過多水平、多角度對(duì)缺血再灌注損傷心肌ERS-自噬通路機(jī)制進(jìn)行研究發(fā)現(xiàn)，穴位埋線與缺血預(yù)處理作用近似，可能是其可發(fā)揮內(nèi)源性保護(hù)。對(duì)ERS-自噬通路的激活，可能是穴位埋線通過促進(jìn)GRP78、Beclin-1水平升高，維持內(nèi)質(zhì)網(wǎng)正常功能并減輕其負(fù)擔(dān)，以及適度調(diào)節(jié)自噬活性、抗凋亡及抑制過度ERS，從而起到對(duì)再灌注損傷心肌的保護(hù)作用。對(duì)于穴位埋線治療心肌缺血再灌注損傷的作用機(jī)制，仍需深入研究。

作者貢獻(xiàn)：王一茗進(jìn)行研究設(shè)計(jì)與實(shí)施、資料收集整理、撰寫論文并對(duì)文章負(fù)責(zé)；張新昕、束彥頁進(jìn)行研究實(shí)施、資料收集；王一茗進(jìn)行質(zhì)量控制及審校。

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MechanismofCatgutImplantationatAcupointsfortheProtectionofMyocardiumfromIschemia-reperfusionInjuryinHyperlipidemiaRatswithType2DiabetesMellitus

WANGYi-ming1*，ZHANGXin-xin2，SHUYan-ye1

1.DepartmentofAcupunctureandMoxibustion,AffiliatedHospitalofJiangsuUniversity，Zhenjiang212001,China2.JiangsuUniversityHospitalWorkers，Zhenjiang212013，China

ObjectiveTo explore the mechanism of catgut implantation at acupoints for the protection of myocardium from ischemia-reperfusion injury in hyperlipidemia zucker diabetic fatty (ZDF)rats,models of type 2 diabetes mellitus,in order to provide a theoretical basis for clinical treatment.MethodsThis study was conducted during January 2015 to May 2016.Twenty-four ZDF rats were selected and randomly divided into control group,ischemia reperfusion (IR) group,ischemic pretreatment (IP) group and catgut implantation (CI) group，with 6 in each.On the 7th day after one week of adaptive feeding,myocardial tissues of the control group were taken out via thoracotomy,the other three groups were used for establishing IR models after ligation of the left anterior descending coronary artery.IR group first

myocardial ischemia treatment for 30 min,then received reperfusion for 60 min.IP group received 3 rounds of 5-min myocardial ischemia treatment and 5-min reperfusion,then received 30-min myocardial ischemia treatment and 60-min reperfusion.CI group received the same treatment given to IR group except on the first day after one week of adaptive feeding,they received catgut implantation at Neiguan,Danzhong,and Xinshu acupoints.The ultrastructure and level of autophagy of myocardium of ZDF rats were observed by transmission electron microscope.Western blotting was used to detect the levels of myocardial endoplasmic reticulum stress-related protein glucose-regulated protein 78 (GRP78) and autophagy-related protein Beclin-1.The levels of myeloperoxidase (MPO) and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay (ELISA).ResultsTransmission electron microscope observation showed that,the control group demonstrated normal mitochondria and cardiomyocytes without many autophagysomes;IR group presented significantly swollen mitochondria and cardiomyocytes with ruptured cell membrane and leaked contents,leaving vacuoles or cavities and a small amount of autophagic vacuoles.Compared with IR group,the myocardial cell ultrastructural damage in IP and CI groups was lighter and the number of autophagic vacuoles was significantly increased.The myocardial cell ultrastructural damage in IR group was the most serious,even there were no many autophagic vacuoles.The levels of GRP78,Beclin-1,MPO and MDA were significantly lower in the control group than in the other three groups (P<0.05).IR group had lower levels of GRP78 and Beclin-1 but higher levels of MPO and MDA compared with IP and CI groups (P<0.05).ConclusionCatgut implantation at Neiguan,Danzhong,and Xinshu acupoints may play a role in the protection of myocardium from ischemia-reperfusion injury by increasing the levels of GRP78 and Beclin-1.

Reperfusion injury;Rats;Endoplasmic reticulum stress;Autophagy;Ischemic pretreatment,myocardium;Catgut implantation at acupoints

R 619.9

A

10.3969/j.issn.1007-9572.2017.00.074

2017-03-15；

2017-08-12)

(本文編輯：崔莎)

國家自然科學(xué)基金青年科學(xué)基金項(xiàng)目(81303037)

1.212001江蘇省鎮(zhèn)江市，江蘇大學(xué)附屬醫(yī)院針灸科 2.212013江蘇省鎮(zhèn)江市，江蘇大學(xué)職工醫(yī)院

*通信作者：王一茗，主治中醫(yī)師；E-mail:kingonetea@163.com

*Correspondingauthor:WANGYi-ming，AttendingTCMdoctor；E-mail:kingonetea@163.com