1、姜黃素與依那普利對(duì)單側(cè)輸尿管梗阻大鼠模型干預(yù)的對(duì)比研究         11-05-15 09:35:00     編輯：studa20                 作者：趙愛青,張曉明,侯恒,李榮山,馬存根【摘要】  目的 觀察姜黃素對(duì)單側(cè)輸尿管梗阻(UUO)大鼠腎間質(zhì)纖維化的影響,并與依那普利進(jìn)行比較

2、。方法 采用UUO致腎間質(zhì)纖維化大鼠模型,術(shù)后第4周處死各組大鼠,用HE、Masson染色評(píng)定腎小管間質(zhì)纖維化程度；用免疫組化方法檢測(cè)腎組織內(nèi)轉(zhuǎn)化生長(zhǎng)因子1(TGF-1)、結(jié)締組織生長(zhǎng)因子(CTGF)和基質(zhì)金屬蛋白酶抑制劑1(TIMP-1)的表達(dá)部位及蛋白表達(dá)水平。結(jié)果 與假手術(shù)組相比,模型組大鼠腎間質(zhì)纖維組織面積顯著擴(kuò)大(P<0.01),腎組織內(nèi)TGF-1、CTGF和TIMP-1蛋白質(zhì)表達(dá)均顯著上調(diào)(P<0.01)。姜黃素或依那普利干預(yù)后,上述上調(diào)指標(biāo)都被顯著抑制(P<0.05或P<0.01)。兩種藥物作用比較無(wú)顯著差異(P>0.05)。結(jié)論 姜黃素或依那普利對(duì)

3、UUO大鼠的干預(yù)效果相似,此可能與下調(diào)腎組織內(nèi)TGF-1和CTGF,進(jìn)而部分抑制TIMP-1的表達(dá)有關(guān)。 【關(guān)鍵詞】  姜黃素；依那普利；轉(zhuǎn)化生長(zhǎng)因子1；結(jié)締組織生長(zhǎng)因子；基質(zhì)金屬蛋白酶抑制劑-1；腎間質(zhì)纖維化Abstract：Objective To investigate the effect of curcumin on renal interstitial fibrosis following unilateral ureteral obstruction (UUO), and compare the effects of Curcumin with that of Enal

4、april. Method UUO rat model was used. Male Wistar rats were randomly divided into four groups with 8 rats each：shame-operated group (0.9% saline solution), model group (dimethyl sulphoxide, DMSO), Curcumin treatment group curcumin 100 mg/(kg·d), and Enalapril treatment group enalapril 10 mg/(kg

5、·d). All rats were killed 4 w after surgery. The degree of tubulointerstitial fibrosis was scored by HE and Masson staining. The sites and levels of protein expression of TGF-1, CTGF and TIMP-1 were detected by immunohistochemistry staining. Result Compared with those of shame-operated group, t

6、he blood urea nitrogen level was significantly increased (P<0.01), and the relative area of interstitial fibrosis was also significantly enlarged, the expression of TGF-1, CTGF and TIMP-1 protein was significantly up-regulated in kidney tissue of model group (P<0.01). After intervention with C

7、urcumin or Enalapril, the upregulation of the above-mentioned parameters were all significantly inhibited (P<0.05 or P<0.01). There was no significant difference of intervention effects between Curcumin and Enalapril (P>0.05). Conclusion Pharmacological effects of Curcumin or Enalapril on u

8、nilateral ureteral obstruction rats are quite similar. These effects may result from the down-regulation of TGF-1, CTGF and TIMP-1 expression in kidney tissue.Key words：Curcumin；Enalapril；TGF-1；CTGF；TIMP-1；renal interstitial fibrosis姜黃素(Curcumin)是從姜科植物姜黃中提取的一種植物多酚,也是姜黃發(fā)揮藥理作用最重要的活性成分。近年研究表明,姜黃素對(duì)多種原因所

9、致的腎損害、糖尿病大鼠腎臟都具有保護(hù)作用1。在大鼠動(dòng)物模型中,可明顯減輕肺纖維化、肝纖維化程度2。但姜黃素能否明顯減輕單側(cè)輸尿管梗阻模型(UUO)大鼠腎間質(zhì)纖維化？目前國(guó)內(nèi)外鮮見報(bào)道。為此,筆者通過(guò)一定劑量的姜黃素作用于UUO大鼠,觀察其抗腎纖維化作用及對(duì)轉(zhuǎn)化生長(zhǎng)因子1(TGF-1)、結(jié)締組織生長(zhǎng)因子(CTGF)和基質(zhì)金屬蛋白酶抑制劑-1(TIMP-1)表達(dá)的影響,并與依那普利進(jìn)行比較。1  實(shí)驗(yàn)材料1.1  動(dòng)物    68周齡清潔級(jí)雄性Wistar大鼠32只,體重180200 g,山西醫(yī)科大學(xué)動(dòng)物實(shí)驗(yàn)中心提供。1.2  主要試劑兔

10、抗大鼠TGF-1多克隆抗體(150)、兔抗大鼠CTGF多克隆抗體(1100)、山羊抗大鼠TIMP-1多克隆抗體(1100)、生物素化山羊抗兔IgG,鏈酶親和素-生物素-過(guò)氧化物酶復(fù)合物工作液,二甲基鄰苯二胺(DAB)顯色試劑均購(gòu)自武漢博士德公司；多聚賴氨酸粘片劑購(gòu)自北京中杉金橋生物技術(shù)有限公司；姜黃素購(gòu)自美國(guó)Sigma公司。2  實(shí)驗(yàn)方法2.1  分組和造模動(dòng)物隨機(jī)分為4組：假手術(shù)組(A組,僅予生理鹽水)、模型組(B組,僅予二甲基亞砜)、姜黃素組C組,100 mg/(kg·d)、依那普利組D組,10 mg/(kg·d),每組8只。各組大鼠于術(shù)后第2天開始

11、腹腔注射給藥。UUO致腎小管間質(zhì)纖維化模型參考文獻(xiàn)3方法。2.2  血生化指標(biāo)檢測(cè)假手術(shù)組打開腹腔后分離左側(cè)輸尿管,并不結(jié)扎即關(guān)閉腹腔。術(shù)后第4周,右心耳心臟穿刺抽血約6 mL,用自動(dòng)生化儀測(cè)定尿素氮(BUN)。之后處死各組大鼠,左側(cè)腎臟行原位灌流去除血細(xì)胞后,部分腎組織用10%中性福爾馬林液固定,其余腎組織放入液氮中保存?zhèn)溆谩?.3  腎組織病理學(xué)檢查    動(dòng)物麻醉后取雙側(cè)腎臟觀察其大體外觀變化,取部分梗阻腎經(jīng)固定、脫水、透明、浸蠟、包埋,切4 m厚的切片,行HE染色及Masson染色,光學(xué)顯微鏡(×200)采集Masson染色圖

12、像(每只大鼠隨機(jī)采集15個(gè)視野),以多功能彩色病理圖像分析系統(tǒng)(山西醫(yī)科大學(xué))進(jìn)行自動(dòng)測(cè)量分析,計(jì)算腎間質(zhì)纖維組織相對(duì)面積(腎間質(zhì)綠染區(qū)面積/視野總面積×100%)。2.4  免疫組化染色采用SABC法進(jìn)行免疫組化檢測(cè)。切片常規(guī)處理后,用微波處理10 min(TGF-1、CTGF),或用0.075%胰蛋白酶(TIMP-1) 37 消化20 min修復(fù)抗原。分別滴加相應(yīng)第一抗體,4 冰箱孵育過(guò)夜。然后加入相應(yīng)生物素化第二抗體,37,20 min,滴加鏈酶親和素-生物素-過(guò)氧化物酶,37 ,20 min,最后加入DAB顯色液,顯微鏡下控制顯色。每次染色用PBS緩沖液代替一抗設(shè)立陰性對(duì)照。免疫組化半定量分析借助多媒體病理彩色分析系統(tǒng)完成。在200倍光鏡下采集免疫組化圖像,每張切片隨機(jī)采集10個(gè)不重疊視野(避開腎小球及動(dòng)脈血管),計(jì)算腎小管間質(zhì)相對(duì)陽(yáng)性面積(腎小管間質(zhì)陽(yáng)性面積/視野