芝麻素對自發(fā)性高血壓大鼠心肌氧化應(yīng)激損傷的保護作用

李偉,楊解人,李文星,張俊秀,唐麗娟,楊慧,熊鶯

(皖南醫(yī)學院藥理學教研室,安徽蕪湖241002)

【摘要】目的：觀察芝麻素對自發(fā)性高血壓大鼠(spontaneously hypertensive rat,SHR)心肌iNOS和c-fos蛋白表達來探討其可能保護機制。方法：SHR 35只，隨機分為SHR模型組、芝麻素[40、80、160 mg/(kg·d)]組、卡托普利[30 mg/(kg·d)]組及WKY陰性組，每天灌胃給藥1次，連續(xù)16周。末次給藥后，稱體質(zhì)量，HE染色觀察心肌病理變化；化學比色法測心肌組織中T-SOD的含量；免疫組化法檢測心肌誘導型一氧化氮合酶(inducible nitric oxide synthase，iNOS)和原癌基因(cellular oncogene fos，c-fos)陽性蛋白表達。結(jié)果：SHR組心肌細胞增生肥大且排列紊亂，組織中可見大量炎性細胞和脂肪組織浸潤，心肌T-SOD的含量明顯減少(P<0.01)，心肌iNOS和c-fos蛋白表達顯著增加(P<0.01)，芝麻素和卡托普利組上述病理變化則有不同程度的改善；心肌T-SOD明顯增加(P<0.01)，iNOS和c-fos蛋白表達明顯下調(diào)(P<0.01)。結(jié)論：芝麻素具有下調(diào)SHR心肌iNOS和c-fos蛋白表達，增強T-SOD能力，保護心肌損傷的作用。

【關(guān)鍵詞】芝麻素；自發(fā)性高血壓大鼠；心?。籘-SOD；iNOS；c-fos

【文獻標識碼】【中圖號】R 285.5A

DOI【】10.3969/j.issn.1002-0217.2015.02.001

文章編號：1002-0217(2015)02-0107-03

基金項目：安徽省大學生創(chuàng)新創(chuàng)業(yè)訓練計劃項目(AH201310368112)

收稿日期：2014-07-16

作者簡介：曹雨朦(1993-),女，2011級本科生，(電話)0553-3932464，(電子信箱)892466332@qq.com；

Effect of sesamin on the protein iNOS and c-fos expression in myocardia of spontaneouslyhypertensive ratsLIWei,YANGJieren,LIWenxing,ZHANGJunxiu,TANGLijuan,YANGHui,XIONGYin

Department of Pharmacology,Wannan Medical College,Wuhu 241002,China

Abstract【】Objective：To observe the effects of sesamin on the protein iNOS and c-fos expression in myocardia of spontaneously hypertensive rats(SHR),and explore the potential protective mechanisms.Methods:Thirty-five SHRs were randomly divided into group of SHR model,sesamin treatment in diverse dosage[40,80 and 160 mg/(kg·d)] and captopril intervention[30 mg/(kg·d)],and 7 Wistar-Kyoto rats(WKY) were included as negative controls.All rats were intragastrically administrated with the drugs once a day for consecutive 16 weeks.After the final intervention,the body weight (BW) was measured,and histopathological changes of the myocardia were observed by HE staining.Colorimetric analysis was performed to measure the contents of myocardial T-SOD,and immunohitochemical method was used to determine the expression of inducible nitric oxide synthase(iNOS) and c-fos.Results:SHR group demonstrated hypertrophied myocardia with disordered arrangement.Large quantity of inflammatory cells and adipose tissue infiltration were seen in the myocardial tissues,and the expression of T-SOD was significantly reduced (P<0.01),whereas the protein iNOS and c-fos expression in myocardia was significantly up-regulated(P<0.01).Myocardial pathological lesions were improved to a certain degree in the rats treated with sesamin and captopril,and T-SOD content was significantly increased (P<0.01),whereas the expression of iNOS and c-fos was notably reduced in dose-dependent manner(P<0.01).Conclusion:Sesamin could decrease the expression of iNOS and c-fos protein and enhance the ability of T-SOD,suggesting that it can be protective effect on myocardial injury in spontaneously hypertensive rats.

【Key words】sesamin;spontaneously hypertensive rats;myocardium;T-SOD;iNOS;c-fos

高血壓病是一種危害人類健康的常見病，長期高血壓可導致重要靶器官損傷，尤其對心臟損傷更為嚴重，可引起心肌細胞肥大、間質(zhì)纖維化、細胞外基質(zhì)增加、心肌細胞凋亡、心臟擴大，最終導致心臟衰竭。故臨床上對高血壓的治療不僅需要降壓，同時要考慮保護靶器官。芝麻素(sesamin，Ses)是從芝麻中提取的活性有效成分，具有抗氧化、降血脂、降血壓等[1-3]作用。國外報道芝麻素不僅可以降低血壓，還能改善去氧皮質(zhì)酮-鹽敏感性高血壓及兩腎一夾腎性高血壓大鼠的心肌和血管的肥厚[4-5]，我們前期研究表明，芝麻素可以逆轉(zhuǎn)腎性高血壓、腎性高血壓伴高脂高糖飲食大鼠及SHR心肌肥厚的作用[6-8]。但芝麻素對SHR心肌保護作用機制是否與iNOS和c-fos表達有關(guān)未見報道，因此本研究以SHR為研究對象，通過檢測iNOS和c-fos蛋白表達來探討芝麻素對SHR心肌氧化應(yīng)激損傷的保護作用及可能機制。

1材料與方法

1.1實驗動物16周齡雄性SHR 35只和WKY 7只，體質(zhì)量(340±20)g，許可證號：SCXK(滬)2007-0005，購于上海斯萊克實驗動物有限責任公司。分籠飼養(yǎng)，每籠一只，保持溫度22～25 ℃，相對濕度60%～70%，自然光照，定時進食(飼料購于南京市江寧區(qū)青龍山動物繁殖場)和飲水。

1.2藥品和試劑芝麻素純度93.7%，分子式為C20H15O6；分子量354.34，北京興百祥科貿(mào)有限公司。卡托普利(captopril，Cap)，25 mg/片，批準文號：國藥準字H32023731，生產(chǎn)批號：09102411，常州制藥有限公司?？偝趸锲缁?total superoxide dismutase，T-SOD)試劑盒購于南京建成生物工程研究所。iNOS和c-fos一抗,SABC試劑盒和DAB顯色試劑盒均購于武漢博士德生物工程有限公司。

1.3分組及給藥SHR 35只隨機分成：SHR組[0.5%羧甲基纖維素鈉5 mL/(kg·d)]，Ses低、中、高組[40、80、160 mg/(kg·d)]，Cap陽性對照組[30 mg/(kg·d)]，另設(shè)WKY正常對照組[0.5%羧甲基纖維素鈉5 mL/(kg·d)]，每組7只，按上述設(shè)定劑量每日灌胃1次，連續(xù)給藥16周。

1.4心臟標本采集實驗結(jié)束，稱大鼠體質(zhì)量，3%戊巴比妥鈉(30 mg/kg)腹腔注射麻醉。取左心室心肌同一部位約2 g，按質(zhì)量體積比1∶9加冷生理鹽水，制成10%的心肌組織勻漿,3 000 r/min，4℃離心10 min，取上清液-20 ℃保存，用于生化測定。取心尖組織，PBS清洗，濾紙吸干，置于4%多聚甲醛中固定24 h，常規(guī)脫水、浸蠟、包埋。制成5 μm心肌切片用于HE染色及免疫組化。

1.5HE染色將各組大鼠心肌切成5 μm薄片，常規(guī)脫蠟復水，蘇木精染色5 min；水洗去多余染液，0.5%鹽酸酒精分化10 s；流水沖洗5 min；蒸餾水短洗；伊紅染色2 min；依次70%、85%、95%、100%酒精脫水；二甲苯透明，中性樹膠封片；光鏡下觀察心肌病理學變化。

1.6心肌T-SOD的測定比色法測定心肌勻漿中T-SOD含量(U/mgprot)，具體步驟按試劑盒說明書操作。用DNM-9602酶標分析儀(北京普朗新技術(shù)有限公司)測定，波長550 nm。組織勻漿總蛋白含量測定采用BCA法。

1.7免疫組化法表達iNOS和c-fos陽性蛋白各組心肌切成5 μm薄片，常規(guī)脫蠟至水，免疫組化步驟如下：0.01 mol/L枸櫞酸鹽微波抗原修復，室溫冷卻，PBS洗滌3次；滴加3%過氧化氫室溫孵育15 min滅活內(nèi)源性過氧化物酶，PBS洗3次；滴加5% BSA封閉液37 ℃,30 min，甩去多余液體，不洗；滴加iNOS、c-fos一抗(1∶200)，4 ℃冰箱過夜，37 ℃復溫45 min，PBS洗滌3次；滴加生物素化山羊抗兔的二抗，置37 ℃,30 min，PBS漂洗3次；滴加SABC，37 ℃,30 min，PBS漂洗4次；DAB顯色5 min，蒸餾水洗滌；蘇木素復染2 min，自來水沖洗，分色液分色6～7 s，自來水沖洗；無水酒精脫水，二甲苯透明，中性樹脂封片。

2結(jié)果

2.1芝麻素對SHR心肌HE染色的影響光鏡下觀察到WKY組心肌細胞間隙、組織形態(tài)正常，呈長梭形，心肌纖維結(jié)構(gòu)清晰排列整齊。SHR組心肌細胞增生肥大，心肌纖維增粗且排列紊亂，細胞核增大，組織中可見炎性細胞和脂肪組織浸潤，提示心肌細胞出現(xiàn)病理損傷。當給予不同劑量的Ses和Cap后，心肌細胞的形態(tài)及增生均有不同程度的改善，心肌纖維排列較為整齊，炎性細胞和脂肪組織浸潤均有所減少，說明心肌細胞的病理損傷均有不同程度的好轉(zhuǎn)。提示Ses和Cap對SHR心肌細胞損傷具有一定的保護作用(圖1)。

2.2芝麻素對SHR心肌T-SOD的影響與WKY比較，SHR心肌T-SOD含量顯著減少，具有顯著統(tǒng)計學意義(P<0.01)；與SHR比較，Ses各劑量組和Cap組心肌T-SOD不同程度的增加，具有顯著統(tǒng)計學意義(P<0.01)，且芝麻素各劑量組呈明顯的劑量依賴關(guān)系(r=0.963 7，R2=0.928 7)。顯示芝麻素對SHR有一定的抗氧化能力(表1)。

2.3芝麻素對SHR心肌iNOS和c-fos蛋白表達的影響與WKY比較，SHR心肌iNOS和c-fos蛋白表達顯著增多，具有顯著統(tǒng)計學意義(P<0.01)；與SHR比較，Ses 160、80 mg/kg劑量組和Cap組心肌iNOS和c-fos表達明顯減少，具有統(tǒng)計學意義(P<0.01或P<0.05)，說明芝麻素可下調(diào)SHR心肌iNOS和c-fos蛋白的表達(表2、圖2、3)。

A.WKY,B.SHR,C.Ses160 mg/kg,D.Ses80 mg/kg,E.Ses40 mg/kg,F.Cap30 mg/kg

圖1芝麻素對SHR心肌HE染色的影響(200×)
Fig 1Effect of sesamin on the myocardium of SHR (HE,200×)

表1芝麻素對自發(fā)性高血壓大鼠心肌T-SOD的影響

Groupdose(mg/kg)nT-SOD(U/mgprot)WKY-795.21±2.73SHR-757.07±6.72**Sesamin160791.81±7.62##Sesamin80783.62±6.25**##Sesamin40760.3±7.44**Captopril30792.34±4.84##

*P<0.05,**P<0.01vsWKY;#P<0.05,##P<0.01vsSHR

表2芝麻素對SHR心肌iNOS和c-fos蛋白表達的影響

Groupdose(mg/kg)niNOS(AIOD)c-fos(AIOD)WKY-70.43±0.080.42±0.10SHR-70.96±0.16**0.79±0.15**Sesamin16070.51±0.12##0.47±0.07##Sesamin8070.69±0.12**##0.64±0.10**#Sesamin4070.92±0.15**0.74±0.10**Captopril3070.46±0.10##0.44±0.09##

*P<0.05,**P<0.01vsWKY;#P<0.05,##P<0.01vsSHR

3討論

形態(tài)學實驗結(jié)果顯示，SHR心肌細胞增生肥厚，形態(tài)異常，心肌纖維增粗及排列紊亂，出現(xiàn)大量炎性細胞和脂肪組織浸潤，證明SHR的心肌細胞出現(xiàn)病理性損傷。給予芝麻素治療16周后，心肌細胞及纖維有不同程度的改善，炎性浸潤有所減輕，說明芝麻素對心肌細胞的損傷有一定的保護作用。但通過何種途徑發(fā)揮保護作用值得進行探討和研究。

A.WKY,B.SHR,C.Ses160 mg/kg,D.Ses80 mg/kg,E.Ses40 mg/kg,F.Cap30 mg/kg

圖2芝麻素對SHR心肌iNOS蛋白表達的影響(HE,400×)
Fig 2Effect of sesamin on iNOS expression in myocardium of SHR(HE,400×)

A.WKY,B.SHR,C.Ses160 mg/kg,D.Ses80 mg/kg,E.Ses40 mg/kg,F.Cap30 mg/kg

圖3芝麻素對SHR心肌c-fos表達的影響(HE,400×)
Fig 3Effect of sesamin on c-fos expression in myocardium of SHR(HE,400×)

SHR心肌出現(xiàn)氧化應(yīng)激，活性氧誘導原癌基因c-fos的表達增強，心肌細胞蛋白由胎兒型向合成型轉(zhuǎn)換，蛋白合成增加，觸發(fā)和調(diào)節(jié)心肌肥厚的發(fā)展，被眾多學者認為是AngⅡ心肌肥厚的標志物[17-19]。前期研究表明[8]，SHR心肌全心濕質(zhì)量，左心濕質(zhì)量及左心濕質(zhì)量指數(shù)明顯增加，而心肌纖維和膠原也有不同程度增加，細胞超微結(jié)構(gòu)顯示心肌細胞濁腫，空泡樣變化，膠原纖維明顯增生，線粒體腫脹，基質(zhì)密度降低，部分空泡變性，而芝麻素各組有不同的好轉(zhuǎn)，說明SHR心肌出現(xiàn)肥厚及損傷，但對于標志物c-fos并未檢測。因此本研究采用免疫組化法，觀察SHR心肌中c-fos表達是否有改變，結(jié)果證明SHR心肌組織中c-fos蛋白表達明顯增強，與文獻報道相一致[12,20]，說明SHR心肌損傷可能與心肌c-fos表達上調(diào)有關(guān)。而SHR心肌損傷大鼠經(jīng)芝麻素治療后，心肌組織中c-fos蛋白表達不同程度減少，說明芝麻素改善SHR心肌細胞損傷的作用機制可能與下調(diào)c-fos蛋白表達有關(guān)。

綜上所述，芝麻素具有保護SHR心肌細胞損傷的作用，其機制可能與提高心肌細胞中T-SOD能力，下調(diào)iNOS和c-fos的蛋白表達有關(guān)，其確切機制有待進一步研究。

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·基礎(chǔ)醫(yī)學·

鄭書國，男，副教授，碩士生導師，(電話)13955300871，(電子信箱)zhengsg2000@163.com，通訊作者.