葡萄籽提取物對(duì)缺血再灌注導(dǎo)致衰老大鼠腎小管上皮細(xì)胞凋亡的保護(hù)作用

1、葡萄籽提取物對(duì)缺血再灌注導(dǎo)致衰老大鼠腎小管上皮細(xì)胞凋亡的保護(hù)作用 10-09-14 10:58:00 編輯：studa20作者：洪權(quán) 肖博晗 魏日胞 汪楊 劉曉巒 馮哲 陳香美【摘要】 目的 探討葡萄籽提取物（Grape seed extract proanthocyanidin，GSPE）對(duì)缺血再灌注（I/R）導(dǎo)致衰老大鼠腎小管上皮細(xì)胞凋亡的保護(hù)作用。方法 分離培養(yǎng)24月齡Wistar雄性大鼠的原代腎小管上皮細(xì)胞，第3代后，將細(xì)胞分為對(duì)照組、10 mol/L Antimycin A處理組、GSPE干預(yù)組以及Antimysin A+GSPE 組。Anexin V/PI染色，流式細(xì)胞儀檢測(cè)腎小管

2、上皮細(xì)胞凋亡情況，定量PCR和Western印跡法分別檢測(cè)Caspase3mRNA和蛋白表達(dá)水平，化學(xué)發(fā)光法檢測(cè)細(xì)胞Capsase3活性及過(guò)氧化物丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。激光共聚焦檢測(cè)細(xì)胞內(nèi)活性氧(ROS)的濃度。結(jié)果 （1）衰老大鼠腎小管細(xì)胞經(jīng)Antimycin A處理后，凋亡率增高，Caspase3，MDA，ROS明顯增加，SOD下降；（2）經(jīng)GSPE干預(yù)后，其凋亡情況明顯好轉(zhuǎn)，caspase3、MDA、ROS明顯降低。SOD含量部分恢復(fù)（P0.05）。結(jié)論 老年大鼠腎小管細(xì)胞在I/R損傷時(shí)，ROS 堆積，線(xiàn)粒體損傷導(dǎo)致腎小管上皮細(xì)胞凋亡。GSPE可清除ROS從

3、而達(dá)到抑制腎小管上皮細(xì)胞凋亡、減輕I/R損傷的作用。 【關(guān)鍵詞】 衰老；活性氧；腎小管上皮細(xì)胞；缺血/再灌注損傷；凋亡【Abstract】 Objective To explore the protective effects of Grape seed extract proanthocyanidin(GSPE）to the renal tubular epithelial cells of aging rats induced by ischemia and reperfusion. Methods The 24 month Wistar rats renal tubular epithe

4、lial cells were Isolated and cultured. After the third passage, the cells were divided into control, and 10 mol/L Antimysin A, GSPE, Antimysin A + GSPE treatment groups. The apoptosis of the renal tubular epithelial cells was determined by flow cytometry, which was dyed by Anexin V/PI. PCR and Weste

5、rn blot were used to detect the expressions of mRNA and protein of caspase3 respectively. Chemoluminescence technique was used to detect the activity of caspase3, the content of MDA and the activity of SOD. Laser confocal microscopy was used to test the concentration of the ROS in the cells. Results

6、 (1)The rates of apoptosis and the levels of caspase3，MDA，ROS were increased obviously,and the level of SOD was decreased in aging rats renal tubular epithelial cells by Antimysin A; (2)After the treatment of GSPE, the apoptosis was improved, the levels of caspase3, MDA and ROS were decreased signif

7、icantly. The content of SOD was partial recovered（P0.05）. Conclusions At the I/R injury of aging rats kidney tubular cells, the accumulation of ROS and the damage of mitochondria lead to the apoptosis of renal tubular epithelial cell. GSPE can remove ROS and prevent the apoptosis of kidney tubular e

8、pithelial cells, lessen the impaired effects of I/R. 【Key words】 Aged; ROS; Renal tubular epithelial cell; Ischemia/reperfusion; Apoptosis急性腎臟缺血再灌注(I/R)損傷是休克、嚴(yán)重創(chuàng)傷及腎移植過(guò)程中一種常見(jiàn)的臨床病理生理現(xiàn)象，可以導(dǎo)致急性腎衰竭（ARF）和移植腎功能喪失，在這一過(guò)程中最先受到損傷的細(xì)胞是腎小管上皮細(xì)胞，老年人由于容易出現(xiàn)腎臟低灌注狀態(tài)，如心肌梗死、脫水、利尿劑的使用以及老年人常見(jiàn)的腎動(dòng)脈硬化因此更容易出現(xiàn)ARF。近年來(lái)ARF中活性氧（rea

9、ctive oxygen species ，ROS）激增導(dǎo)致的腎小管上皮細(xì)胞凋亡引起了人們的關(guān)注1。很多的證據(jù)顯示ROS能增加線(xiàn)粒體通透性轉(zhuǎn)變孔道（PTP）的通透性，導(dǎo)致細(xì)胞色素C釋放，引起凋亡。既往研究認(rèn)為葡萄籽提取物(GSPE)具有抗氧化功能，可減少動(dòng)脈硬化，能影響內(nèi)皮細(xì)胞的凋亡增殖過(guò)程。本研究模擬并觀察老年大鼠腎小管上皮細(xì)胞在缺血再灌注（I/R）損傷情況下，ROS在腎小管上皮細(xì)胞凋亡中起到的作用。使用抗氧化劑葡萄籽提取物（GSPE）給與實(shí)驗(yàn)性治療，觀察GSPE抑制ROS產(chǎn)生后，是否對(duì)腎小管上皮細(xì)胞有保護(hù)作用及對(duì)老年高ROS負(fù)荷狀態(tài)的治療效果。1 材料與方法1.1 實(shí)驗(yàn)動(dòng)物與器材 雄性Wistar大鼠6只，27月齡（700750 g），清潔動(dòng)物，購(gòu)自中國(guó)醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物研究所。GSPE（天津尖峰公司）、胎牛血清、DMEM培養(yǎng)基、Trizol試劑、MMLV反轉(zhuǎn)錄試劑盒（美國(guó)Invitrogen公司），Antimycin A（抗霉素A，AA），Annexin V/PI凋亡檢測(cè)試劑盒（美國(guó)Biodesign公司），兔來(lái)源抗caspase3多克隆抗體（美國(guó)Santa Cruze公司），CaspACET