早期大劑量地塞米松對擠壓綜合征大鼠模型心肌特異性損傷的療效

於四軍，嚴(yán)　政，路　靖，金德奎，張　聰，劉惠亮

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早期大劑量地塞米松對擠壓綜合征大鼠模型心肌特異性損傷的療效

於四軍1，嚴(yán)政2，路靖1，金德奎1，張聰1，劉惠亮1

目的評估早期大劑量地塞米松對擠壓綜合征大鼠模型心肌特異性損傷的療效。方法將Wistar大鼠隨機(jī)分為4組?？瞻?Sham)組、對照(CS)組、生理鹽水(NS)組和地塞米松(DEX)組，每組20只。CS組造模后不做任何處理；DEX組和NS組造模后在解壓前即刻注射1 mg/kg地塞米松和等體積的生理鹽水；Sham組對正常Wistar大鼠無造模及藥物干預(yù)，行麻醉、取血和手術(shù)處理。各組收集不同時(shí)間點(diǎn)的血液和組織學(xué)樣本。結(jié)果心肌損傷觀察指標(biāo)：(1)血清cTnI OD值，解壓后3 h各組間差別無統(tǒng)計(jì)學(xué)意義。6 h時(shí)3個(gè)處理組血清cTnI均較Sham組顯著上升，CS組為(0.246+0.028)，NS組為(0.202+0.033)，DEX 組為(0.178+0.017)，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)；NS組和DEX組均低于CS組，NS組和DEX組之間差異無統(tǒng)計(jì)學(xué)意義。12 h時(shí)3個(gè)處理組均高于Sham組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)；各處理組間無統(tǒng)計(jì)學(xué)差異。24 h時(shí)CS組和NS組高于Sham組，P<0.05；DEX組與Sham組相比差異無統(tǒng)計(jì)學(xué)意義；(2)心肌細(xì)胞凋亡率CS組、NS組和DEX組各時(shí)間點(diǎn)均高于Sham組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。3 h時(shí)NS組和DEX組低于CS組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)，NS組和DEX組間差異無統(tǒng)計(jì)學(xué)意義12～24 h時(shí)間段，NS組和CS組差異無統(tǒng)計(jì)學(xué)意義，DEX顯著低于NS和CS組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)；(3) 心肌細(xì)胞HE染色 Sham組大鼠心肌組織正常。CS組中可見充血性改變，隨時(shí)間加重。NS組和DEX組充血水腫均有不同程度的減輕，DEX組減輕更明顯。結(jié)論擠壓綜合征大鼠存在心肌細(xì)胞的特異性損傷，大劑量的地塞米松和與之等體積的生理鹽水?dāng)U容均能改善這一現(xiàn)象，地塞米松明顯優(yōu)于生理鹽水單純擴(kuò)容。大劑量糖皮質(zhì)激素可能通過抑制全身炎性反應(yīng)從而減輕擠壓綜合征大鼠的心肌損傷。

擠壓綜合征；大鼠；炎性反應(yīng)；心肌特異性損傷；地塞米松

擠壓綜合征(crush syndrome，CS)是一類以創(chuàng)傷后循環(huán)休克及腎衰竭為特點(diǎn)的危重臨床疾病，在戰(zhàn)爭、交通事故、恐怖襲擊及地震等災(zāi)難事件中頗為常見。文獻(xiàn)[1,2]報(bào)道，地震中CS的發(fā)病率可達(dá)3%～20%，在高樓坍塌等恐怖事件中則可高達(dá)40%。臨床上對CS的認(rèn)識(shí)已有百年，CS的完整病理生理學(xué)過程目前仍存在爭議，目前，普遍認(rèn)為局部受壓導(dǎo)致的橫紋肌溶解后產(chǎn)生的細(xì)胞內(nèi)毒性物質(zhì)在血流恢復(fù)灌注之后，大量入血引起一系列全身性并發(fā)癥[3]。文獻(xiàn)[4]表明擠壓綜合征動(dòng)物模型存在心肌特異性損傷，其機(jī)制尚未清楚。另一項(xiàng)研究顯示，大劑量地塞米松通過PI3K-Akt-eNOS通道發(fā)揮抗炎作用以及降低缺血再灌注損傷，從而顯著提高了擠壓綜合征大鼠的早期生存率[4,5]。但大劑量糖皮質(zhì)激素對心肌特異性損傷的影響目前尚無研究報(bào)道。本研究通過觀察早期大劑量地塞米松對擠壓綜合征大鼠模型心肌特異性損傷的影響，初步探索心肌損傷的可能機(jī)制。

1　材料與方法

1.1造模和分組清潔級(jí)雄性Wistar大鼠140只，購于軍事醫(yī)學(xué)科學(xué)院，重320～380 g，飼養(yǎng)于武警總醫(yī)院中心實(shí)驗(yàn)室動(dòng)物房，保持室溫(23±3)℃，相對濕度55%±15%，日光燈照射，每12 h光暗交替。每5只大鼠飼養(yǎng)于1個(gè)籠中，自由進(jìn)食水。參考Isamu Murata等[6]造模方法。將大鼠稱重后腹腔注射0.4 ml/100g水合氯醛麻醉，仰臥放置于動(dòng)物恒溫毯，設(shè)置恒溫37 ℃。采用卡式止血帶作用于大鼠雙后肢，使用彈簧測力計(jì)確保壓力恒定為3 kg，連續(xù)擠壓5 h后解除壓迫正常飼養(yǎng)。

將Wistar大鼠按照每組保證每個(gè)時(shí)間點(diǎn)存活5只的原則，分為4組：空白(Sham)組(n=20)，對照(CS)組(n=47)，生理鹽水(NS)組(n=42)和地塞米松(DEX)組(n=25)，CS組造模后不做任何處理；DEX組和NS組分別在解壓前即刻注射1 mg/kg的地塞米松和等體積的生理鹽水；假手術(shù)組無造模及藥物干預(yù)，余處理相同。4組解壓后均飼養(yǎng)至特定時(shí)間點(diǎn)(解除壓迫后3、6、12、24 h)處死并立即獲取血樣和組織樣本。

1.2藥物和試劑地塞米松磷酸鈉注射液(瑞陽制藥有限公司)，水合氯醛溶液(北京軍區(qū)總醫(yī)院)，生理鹽水(辰欣制藥)，凋亡試劑盒由瑞士羅氏公司(In Situ Cell Death Detection Kit，POD)提供，大鼠血清肌鈣蛋白Elisa試劑盒(美國lifediagnosis公司，2010-2-HS)。4%多聚甲醛溶液、乙醇、二甲苯、固體石蠟、蘇木精染色等由武警總醫(yī)院眼眶病研究所病理實(shí)驗(yàn)室提供。

1.3血清cTnI的測定分離后肢靜脈取3 ml靜脈血2500 r/min,離心20 min取上清液，分裝后-80 ℃冷凍。擇期使用大鼠血清肌鈣蛋白Elisa試劑盒，嚴(yán)格按照說明書操作，待反應(yīng)完全于酶標(biāo)儀中測量其405 nm吸光度(OD)值。

1.4組織學(xué)檢查采血完成后，快速剪開胸部，在心臟仍在跳動(dòng)的情況下剪取心臟，用生理鹽水洗去血液，置入4%多聚甲醛中固定48 h。橫切取心尖部0.1 cm×0.1 cm×0.1 cm心室肌，脫水石蠟包埋切片。分別行HE染色以及TUNEL染色[7]，觀對比不同時(shí)間點(diǎn)各組的心肌細(xì)胞的形態(tài)學(xué)以及凋亡率情況。

2　結(jié)　　果

2.1納入實(shí)驗(yàn)情況實(shí)驗(yàn)共納入大鼠140只，其中麻醉意外死亡6只，未存活至特定時(shí)間點(diǎn)54只，最終每個(gè)時(shí)間點(diǎn)每組均為5只。除去麻醉意外死亡，Sham組共20只大鼠，未存活至特定時(shí)間點(diǎn)0只；CS組共47只，未存活至特定時(shí)間點(diǎn)27只；DEX組共25只，未存活至特定時(shí)間點(diǎn)5只；NS組共42只，未存活至特定時(shí)間點(diǎn)22只。每組3、6、12、24 h各入組5例大鼠。

2.2血清cTnI結(jié)果血清使用酶聯(lián)免疫法中405 nm下的吸光度值，與血清cTnI濃度呈正相關(guān)(表1)。解壓后3 h各組間差別無統(tǒng)計(jì)學(xué)意義。6 h時(shí)3個(gè)處理組血清cTnI均上升，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)；組間比較可見NS組和DEX組均低于CS組，P分別小于0.05和0.01，NS與DEX兩組之間無統(tǒng)計(jì)學(xué)差異。12 h時(shí)3個(gè)處理組均高于Sham組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。24 h時(shí)間點(diǎn)CS組和NS組仍高于Sham組，P分別小于0.01和0.05，DEX組則與Sham組無統(tǒng)計(jì)學(xué)差異；NS組和CS組、DEX組組別無統(tǒng)計(jì)學(xué)差異，而DEX組血清cTnI低于CS組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

表1各組擠壓綜合征大鼠血清cTnI吸光度(OD值)

組別cTnI吸光度3h6h12h24hSham組0.073±0.0180.052±0.031 0.067±0.0210.074±0.033CS組0.104±0.0240.246±0.028①0.162±0.029①0.147±0.039①NS組0.084±0.0380.202±0.033①③0.150±0.043①0.136±0.020②DEX組0.076±0.0100.178±0.017①④0.134±0.035①0.101±0.042③

注：與Sham組相比，①P<0.01,②P<0.05； 與CS組相比，③P<0.05；④P<0.01

2.3TUNEL染色結(jié)果見圖1。心肌細(xì)胞凋亡率采用隨機(jī)取80個(gè)400倍鏡下視野計(jì)數(shù)陽性細(xì)胞率取平均值。CS、NS和DEX組各時(shí)間點(diǎn)凋亡率均高于Sham組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。3組間3 h比較NS組和DEX組明顯低于CS組，但組間比較差異無統(tǒng)計(jì)學(xué)意義；6 h時(shí)NS組和DEX組仍低于CS組，同時(shí)DEX組低于NS組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；12～24 h時(shí)間段，NS組和CS組差異無統(tǒng)計(jì)學(xué)意義，但DEX組顯著低于NS組和CS組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見表2。

圖1　不同時(shí)間點(diǎn)擠壓綜合征大鼠TUNEL染色

表2　不同時(shí)間點(diǎn)擠壓綜合征大鼠心肌細(xì)胞凋亡率對比　(%)

注：與Sham組相比，①P<0.01,②P<0.05； 與CS組相比，③P<0.01,④P<0.05； 與NS組相比，⑤P<0.01,⑥P<0.05

2.4心肌細(xì)胞HE染色結(jié)果見圖2。Sham組大鼠心肌組織正常，微細(xì)血管、心肌間質(zhì)完好，細(xì)胞核呈卵圓形，位于細(xì)胞中央，核膜完整。CS組中可見充血性改變，肌間水腫，少許纖維斷裂，偶可見炎性細(xì)胞浸潤，并隨時(shí)間加重。NS組和DEX組充血水腫均有不同程度的減輕，DEX組減輕更明顯。

圖2　不同時(shí)間擠壓綜合征大鼠心臟組織HE染色

3　討　　論

近年來，擠壓綜合征的研究熱點(diǎn)逐漸從腎臟損傷向多器官衰竭等腎外臟器損傷轉(zhuǎn)移。多個(gè)研究相繼證實(shí)擠壓綜合征動(dòng)物模型存在心肌特異性損傷：血清中可見心肌酶上升，同時(shí)病理及免疫組織化學(xué)染色存在心肌細(xì)胞凋亡[4,8]。Shuiping等[5]研究顯示，大劑量地塞米松通過PI3K-Akt-eNOS通道發(fā)揮抗炎作用，從而顯著提高了擠壓綜合征大鼠的早期生存率。然而大劑量地塞米松對心肌細(xì)胞特異性損傷的研究至今未見報(bào)道。本研究觀察了大劑量地塞米松(DEX)與單純擴(kuò)容組(NS)、對照組(CS)及空白組(sham)之間心肌特異性損傷指標(biāo)的差異。結(jié)果顯示，在酶學(xué)指標(biāo)上DEX從解壓6 h開始即明顯低于對照組，且至24 h時(shí)間點(diǎn)即與正常大鼠無統(tǒng)計(jì)學(xué)差異，大劑量地塞米松使得心肌酶cTnI的酶峰提前，峰值下降。在組織病理學(xué)(HE染色)，變化更為明顯，DEX組各時(shí)間點(diǎn)心肌細(xì)胞的水腫及炎性浸潤均明顯較輕。在免疫組化(TUNEL染色)指標(biāo)上，80個(gè)40倍視野的DEX組平均凋亡率均明顯降低。同時(shí)本研究研究中觀察到，單純擴(kuò)容(NS組)亦能減輕心肌細(xì)胞損傷。

目前尚無研究表明，大劑量糖皮質(zhì)激素為何能夠減輕CS大鼠心肌細(xì)胞特異性損傷。本研究認(rèn)為，全身炎性反應(yīng)及低血容量休克是兩個(gè)可能原因。炎性反應(yīng)在擠壓綜合征病理生理中扮演著重要角色，主要在以下三個(gè)方面：(1)擠壓及其所致的缺血狀態(tài)均可導(dǎo)致中性粒細(xì)胞趨化因子含量上升，誘導(dǎo)中性粒細(xì)胞聚集產(chǎn)生炎性反應(yīng)，從而進(jìn)一步加劇肌細(xì)胞的不穩(wěn)定性；(2)解壓后的再灌注過程產(chǎn)生大量氧自由基及活化的中性粒細(xì)胞，活性氧通過激活轉(zhuǎn)錄因子NF-κB誘導(dǎo)促炎細(xì)胞因子基因的表達(dá)，而促炎因子TNF、IL-1等通過NADPH氧化酶再次誘導(dǎo)活性氧的生成，從而形成正反饋[9,10]，氧自由基再次誘導(dǎo)細(xì)胞溶解的同時(shí)，大量的炎性反應(yīng)介質(zhì)使毛細(xì)血管擴(kuò)張、通透性增高，加劇血管的損傷以及血流進(jìn)入肌肉的第三間隙，進(jìn)一步惡化低血容量狀態(tài)；(3)局部的毒性物質(zhì)、炎性反應(yīng)因子和氧自由基等進(jìn)入循環(huán)產(chǎn)生全身炎性反應(yīng)，導(dǎo)致遠(yuǎn)端臟器的損傷，最終可致全身多器官衰竭[11]。文獻(xiàn)[5]報(bào)道其他疾病導(dǎo)致的全身炎性反應(yīng)綜合征中也存在心肌細(xì)胞損傷。傳統(tǒng)的糖皮質(zhì)激素在藥理劑量下具有強(qiáng)大的抗炎作用，Murata 等[4]已證實(shí)大劑量地塞米松通過PI3K-Akt-eNOS通道降低缺血再灌注損傷以及局部和全身的炎性反應(yīng)等，從而顯著提高了擠壓綜合征動(dòng)物模型中的生存率。Rie等[11]報(bào)道活性氧與擠壓綜合征的遠(yuǎn)端臟器衰竭相關(guān)，抗炎作用抑制中性粒細(xì)胞的聚集活化，同時(shí)減少了進(jìn)入循環(huán)的氧自由基和炎性反應(yīng)介質(zhì)的含量，降低遠(yuǎn)端臟器的損傷。本研究中大劑量地塞米松能夠顯著改善心肌細(xì)胞特異性損傷也是從治療效果這個(gè)側(cè)面證明，CS大鼠心肌特異性損傷的主要由全身炎性反應(yīng)引起，并能被強(qiáng)效抗菌藥改善。同時(shí)本研究注意到單純擴(kuò)容的生理鹽水同樣能抑制心肌細(xì)胞的凋亡，且在某些時(shí)間點(diǎn)地塞米松和生理鹽水對凋亡指標(biāo)的影響無統(tǒng)計(jì)學(xué)意義；加之正常大鼠在抽取大量血液后可見心肌細(xì)胞凋亡上升，本研究推測低血容量同樣是心臟特異性損傷的原因之一。

全身炎性反應(yīng)究竟如何導(dǎo)致CS大鼠心肌特異性損傷？本研究結(jié)合擠壓綜合征病理生理過程，推測其心臟損傷的原因可能是，擠壓和再灌注期間產(chǎn)生的大量炎性反應(yīng)介質(zhì)及氧自由基通過循環(huán)到達(dá)心臟，使心肌細(xì)胞細(xì)胞膜處于不穩(wěn)定狀態(tài)，膜通透性上升，代謝毒性物質(zhì)更易進(jìn)入心肌細(xì)胞；全身炎性反應(yīng)時(shí)，機(jī)體處于應(yīng)激狀態(tài)，交感系統(tǒng)興奮，能量消耗加大，部分炎性反應(yīng)介質(zhì)促使冠狀動(dòng)脈痙攣，冠脈血流減少，同時(shí)全身的低血容量休克狀態(tài)使心肌氧供進(jìn)一步惡化，從而產(chǎn)生心肌細(xì)胞的凋亡。

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(2015-08-11收稿2015-12-11修回)

(責(zé)任編輯郭青)

Effect of early stage high-dose dexamethasone on cardiac-specific injury in crush-syndrome rats

YU Sijun1, YAN Zheng2,LU Jing1, JIN Dekui1, ZHANG Cong1, and LIU Huiliang1.

1.Department of Cardiology, General Hospital of Chinese People’s Armed Police Force,100039 Beijing,China;2.Department of Cardiology, Shouyi District of the 3rd Hospital of Wuhan, 430536 Wuhan, China

ObjectiveTo evaluate the effect of the early stage high-dose dexamethasone on cardiac-specific injury in the crush-syndrome rats.MethodsCS rats were divided into three groups:CS group, NS group and DEX group (n=20 each). DEX group and NS group were intravenously administered 5mg/kg of dexamethasone and an equal volume of nomal saline respectively immediately before reperfusion while CS group received no treatment.A sham group serving as a blank control underwent the same procedures as all the three CS groups except for the compression and decompression and drug intervention. ResultsMyocardial specific injuries: (1) In serum cTnI OD, there were no statistically significant differences between all groups at 3 h after decompression. At 6h, the serum levels of cTnI increased in all three groups (compared with sham group),P<0.01. In NS group and DEX group were significantly lower than in CS group, and thePvalue was smaller than 0.05 and 0.01 respectively. There was no significant difference between DEX and NS groups. At 12 h in all three treatment groups were higher than in sham group (P<0.01).The differences between the three treatment groups were not statistically significant. At 24 h in group CS group and NS group were significantly higher than in sham group,P<0.01 andP<0.05 respectively.There was no significant difference between DEX group and sham group. In DEX group was significantly lower than in CS group,P<0.05. There were no significant differences between NS and DEX group, NS and CS group. (2) Apoptosis rate in myocardium: the apoptosis rate in CS group, NS group and DEX group at each time point were higher than that in sham group,P<0.01. At 3 h in NS group and DEX group were lower than in CS group,P<0.05 andP<0.01, respectively. There was no differences between NS and DEX group. At 6 h in NS group and DEX group were still lower than in CS group,P<0.05 andP<0.01, respectively. In DEX group were lower than in NS group,P<0.05.At 12 h and 24 h, there was no difference between NS and CS group. In DEX group were significantly lower than in NS group,P<0.01. (3) HE staining of myocardium: in sham group was normal. In the CS group, the congestive changes were observed and aggravated along with increasing time. NS group and DEX group had different degree of congestion and edema relief, and the relief in DEX group was more obvious.ConclusionsThere is cardiac specific injury in CS rat model, which may be threated by high-dose dexamethasone and normal saline. High-dose dexamethasone is more effective than an equal volume of normal saline. High dose of glucocorticoid may reduce the myocardial injury in crush syndrome rats through its suppression of systemic inflammatory response.

crush syndrome; rats; inflammation; cardiac-specific injury; dexamethasone

於四軍，博士，主治醫(yī)師。

1.100039北京，武警總醫(yī)院心內(nèi)科，2.430536，武漢市第三醫(yī)院心內(nèi)科

劉惠亮，E-mail:lhl518@vip.sina.com

R514