腎臟缺血預(yù)適應(yīng)對(duì)核因子-κB活性及細(xì)胞凋亡的影響

1、腎臟缺血預(yù)適應(yīng)對(duì)核因子-B活性及細(xì)胞凋亡的影響 10-10-29 10:38:00 編輯：studa20作者：劉曉東， 曹長(zhǎng)春, 武煜, 陳宇【摘要】 目的 通過(guò)建立大鼠腎臟急性缺血/再灌注(I/R)及缺血預(yù)適應(yīng) (I/P+I/R)模型，初步研究不同的缺血方式后腎臟核因子-B（NF-B）活性及二聚體亞單位的構(gòu)成，探討其減輕腎臟腎小管細(xì)胞凋亡的機(jī)制。方法 30只雄性SD大鼠摘除右腎、分離出左腎動(dòng)脈后隨機(jī)均分為3組（n=10）：A組為假手術(shù)組（Sham組），只分離左腎動(dòng)脈，暴露術(shù)野60 min后直接縫合，24 h后取腎；B組為缺血/再灌注組(I/R組)，持續(xù)夾閉左腎動(dòng)脈45 min，恢復(fù)血供24

2、h后取腎；C組為缺血預(yù)適應(yīng)+缺血/再灌注組(I/P+I/R組)，左腎動(dòng)脈夾閉2 min，松開(kāi)5 min，重復(fù)3個(gè)循環(huán)，余同I/R組。分別用生化學(xué)方法檢測(cè)血肌酐值的變化，組織病理學(xué)評(píng)價(jià)腎小管損傷程度評(píng)分，凝膠電泳遲滯分析檢測(cè)腎組織NF-B/DNA 結(jié)合活性，超遲滯分析檢測(cè)NF-B亞單位的構(gòu)成，Tunel法檢測(cè)腎小管上皮細(xì)胞的凋亡。結(jié)果 血清學(xué)檢查及腎小管損傷評(píng)分提示，I/R組腎臟組織病理改變明顯，損傷程度重，與Sham組比較，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與I/R組比較，I/P+I/R組損傷程度則明顯減輕，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。凝膠電泳遲滯分析檢測(cè)腎組織NF-B/DNA結(jié)合活性I/P

3、+I/R組明顯高于Sham組，差異有統(tǒng)計(jì)學(xué)意義 (P0.05)；超遲滯分析檢測(cè)NF-B亞單位含有p65、p50；差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論 腎臟缺血預(yù)適應(yīng)可通過(guò)抑制NF-B轉(zhuǎn)錄活性，減輕腎小管上皮細(xì)胞凋亡，從而發(fā)揮損傷保護(hù)效應(yīng)。 【關(guān)鍵詞】 腎臟；缺血預(yù)適應(yīng)；核因子-B；細(xì)胞凋亡 Abstract: Objective To investigate the effect of different modes of renal ischemia on the activity of NF-B and the composition of dimeric subunit and to e

4、xplore the causative mechanism underlying the attenuated apoptosis of renal tubular cells with the establishment of animal models of acute renal ischemic/reperfusion (I/R) and ischemic preconditioning (I/P+I/R) in rats. Methods 30 male Sprague-Dawley rats were randomized into 3 groups (n=10 each) fo

5、llowing right nephrectomy and isolation of the left renal artery. Group A served as the sham-operated control, only with isolation of the left renal artery and suture following exposure for 60 min. Group B (I/R group) rats were subjected to ischemia for 45 min by clamping of the left renal artery an

6、d subsequent resumed blood supply. Group C (I/P+I/R) rats were pre-treated with 3 cycles of 2-minute ischemia and 5-minute reperfusion. All the rats were sacrificed at 24 h and nephrectomy was performed for analysis, including biochemical determination of the serum creatinine, histopathological eval

7、uation of the renal tubules, electrophoretic mobility shift assay (EMSA) of renal NF-B/DNA binding activity, super shift assay of the composition of NF-B complexes in renal tissues and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of the apoptosis of renal tubular epithe

8、lial cells. Results Serological examinations and scores of renal tubular injury revealed evident histopatological changes and serious injury in renal tissues in group B. Compared with the sham group, the differences were statistically significant (P0.05); Compared with group B, group C had markedly

9、attenuated injury with significant statistical differences (P0.05); EMSA results showed that the renal NF-B/DNA binding activity was significantly higher in group C than in group A, with significant statistical differences (P0.05); and super shift assay of the composition of NF-B dimeric subunit con

10、firmed the presence of p65 and p50 in group C, with significant statistical differences (P0.05). Conclusion Renal ischemic preconditioning attenuates the apoptosis of renal tubular epithelial cells via the inhibition of the transcriptional activity of NF-B, and thus has the protective effect on rena

11、l tissues. Key words: kidney; ischemic preconditioning; nuclear factor-B; apoptosis缺血預(yù)適應(yīng)（ischemic preconditioning，IPC）是指臟器短暫缺血/再灌注（ischemia/reperfusion, I/R）后能耐受更長(zhǎng)時(shí)間的缺血，其內(nèi)在的分子生物學(xué)機(jī)制尚未明了1。我們的前期研究表明，大鼠腎臟也存在缺血預(yù)適應(yīng)的現(xiàn)象，具有一定的腎臟保護(hù)作用2。已有研究發(fā)現(xiàn)，細(xì)胞凋亡可能是缺血/再灌損傷的重要環(huán)節(jié)之一，而核因子-B（NF-B）是調(diào)控細(xì)胞凋亡的關(guān)鍵轉(zhuǎn)錄因子，可調(diào)控多種抗凋亡和促凋亡基因的轉(zhuǎn)錄3。

12、但對(duì)于腎臟缺血預(yù)適應(yīng)的抗凋亡作用及胞內(nèi)NF-B信號(hào)轉(zhuǎn)導(dǎo)通路的活化特點(diǎn)尚未闡明。為此，本實(shí)驗(yàn)通過(guò)觀察腎臟缺血預(yù)適應(yīng)對(duì)NF-B轉(zhuǎn)錄活性及細(xì)胞凋亡的影響，探討腎臟缺血預(yù)適應(yīng)的抗凋亡機(jī)制，為腎臟缺血/再灌注損傷的防治提供重要的理論依據(jù)。1 材料和方法1.1 實(shí)驗(yàn)動(dòng)物選擇與分組健康雄性SD大鼠30只，體重240250 g，由南京醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供。3%戊巴比妥鈉麻醉后，所有動(dòng)物均摘除右腎,穩(wěn)定10 min；隨機(jī)分成3組：A組為假手術(shù)組(Sham組，n=10)，只分離左腎動(dòng)脈，暴露術(shù)野60 min后直接縫合，24 h后取材;B組為缺血/再灌注組(I/R組，n=10)持續(xù)夾閉左腎動(dòng)脈45 min，恢復(fù)

13、血供24 h后取材；C組為缺血預(yù)適應(yīng)+缺血/再灌注組(I/P+I/R組，n=10)，左腎動(dòng)脈夾閉2 min，松開(kāi)5 min,重復(fù)3個(gè)循環(huán)，余同I/R組。1.2 血清學(xué)檢查大鼠腔靜脈采血1 ml,立即用離心機(jī)3 000 r/min離心5 min,取上清液進(jìn)行血肌酐值測(cè)定。1.3 組織學(xué)檢查常規(guī)石蠟切片進(jìn)行蘇木精-伊紅染色，光鏡下觀察其結(jié)構(gòu)變化，對(duì)腎小管壞死程度進(jìn)行評(píng)分。評(píng)分標(biāo)準(zhǔn)：每張切片在400倍鏡下取外髓質(zhì)部10個(gè)視野，依序觀察左上、右上、左下、右下、中間。正常為0分，輕微損傷（受損腎小管 75%）為4分；以此作半定量分析并計(jì)算其平均值，作為腎小管壞死的標(biāo)準(zhǔn)評(píng)分指數(shù)4。1.4 凝膠電泳遲滯分析

14、(EMSA)測(cè)定NF-B/DNA結(jié)合活性提取腎組織核蛋白，以微量考馬斯亮藍(lán)法測(cè)定核蛋白濃度，調(diào)至2 g/L，并于-70 保存。按試劑盒(Promega 公司)說(shuō)明進(jìn)行：在T4激酶作用下，將-32P-ATP (北京亞輝公司) 標(biāo)記到NF-B位點(diǎn)ODN(探針)上后純化。將核蛋白抽提物與標(biāo)記的NF-B 探針?lè)磻?yīng)，同時(shí)做特異性競(jìng)爭(zhēng)結(jié)合試驗(yàn)。超遲滯分析（super shift assay）部分：將2 l抗NF-B亞單位抗體p65、p50、Rel-B、p52分別在加入探針前加入反應(yīng)體系，室溫孵育30 min，反應(yīng)產(chǎn)物經(jīng)4%非變性聚丙烯酰胺凝膠電泳完畢后，- 70 放射性自顯影，X線膠片上的滯后帶用凝膠成像

15、分析系統(tǒng)進(jìn)行分析。以相對(duì)密度單位(RDU) 表示NF-B/DNA 結(jié)合活性。1.5 原位末端脫氧核苷酸轉(zhuǎn)移酶標(biāo)記法(TUNEL)測(cè)定腎小管上皮細(xì)胞凋亡試劑盒由南京凱基生物公司提供。切片厚5 m,常規(guī)脫蠟入水，經(jīng)3過(guò)氧化氫室溫處理10 min以消除內(nèi)源性過(guò)氧化物酶，PBS緩沖液沖洗3次，蛋白酶K消化30 min，然后用PBS緩沖液沖洗3次，加入TUNEL的反應(yīng)液37孵育60 min，用PBS緩沖液沖洗3次后加親和素辣根過(guò)氧化物酶，37濕盒孵育30 min，PBS緩沖液沖洗3次，加DAB試劑顯色，蘇木精染色，脫水、透明、封片。光鏡下觀察凋亡細(xì)胞，細(xì)胞核呈棕黃色者為陽(yáng)性細(xì)胞，于凋亡細(xì)胞分布區(qū)域，隨機(jī)

16、選取5個(gè)視野，用400倍光鏡計(jì)數(shù)凋亡細(xì)胞，以腎小管上皮細(xì)胞凋亡陽(yáng)性細(xì)胞數(shù)占總腎小管上皮細(xì)胞數(shù)的百分比作為腎小管上皮細(xì)胞凋亡指數(shù)(AI)。1.6 統(tǒng)計(jì)學(xué)處理所有數(shù)據(jù)均以s表示，各組間檢驗(yàn)用方差分析(F檢驗(yàn)和q檢驗(yàn))。所有數(shù)據(jù)使用SPSS 11.0 統(tǒng)計(jì)軟件進(jìn)行分析。P0.05被認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義。2 結(jié)果2.1 各組大鼠血肌酐的變化I/R組和I/P+I/R組大鼠血肌酐值與Sham組比較都有明顯升高，差異具有統(tǒng)計(jì)學(xué)意義（P0.01），但是I/P+I/R組血肌酐值明顯低于I/R組，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。見(jiàn)表1。表1 各組大鼠血肌酐、腎小管損傷積分及AI的比較（略）2.2 各組大鼠腎小管損傷積分的變化I/R組腎臟組織病理改變明顯，受損最嚴(yán)重的部位是皮髓交界處、外髓內(nèi)帶的近端小管，主要表現(xiàn)為小管上皮細(xì)胞脫落、刷狀緣消失和腎小管阻塞。與I/R組比較，I/P+I/R組損傷程度則明顯減