血管内皮生长因子对乙醇诱发大鼠肝窦内皮细胞凋亡的作用

Effects of vascular endothelial growth factor on apoptosis of sinusoidal endothelial cells caused by ethanol:experiment with rats

目的研究乙醇引起肝窦内皮细胞(SEC)死亡的类型和血管内皮生长因子(VEGF)对这种死亡的作用,以及涉及 Ets-1和半胱氨酸蛋白水解酶(Caspase)-8的作用机制。方法按照 Braet等的方法并作改进,从 Wistar 雄性大鼠分离和培养 SEC。然后,将 SEC 与乙醇(25～100 mmol/L)和VEGF(20～30 ng/ml)共同孵育6 h,采用缺口末端标记(TUNEL)技术检测 SEC 凋亡。以 Western 印迹法测定 Ets-1蛋白表达和 FLICE/Caspase-8比色蛋白酶检测试剂盒测定 Caspase-8活性的变化。结果相差显微镜下,培养3 d 的SEC 呈纺锤状并基本融合。而与乙醇孵育的过程中 SEC 趋于皱缩和死亡。荧光显微镜下,对照 SEC 绝大多数呈 TUNEL 染色阴性;添加乙醇(100 mmol/L)2 h 后TUNEL 染色阳性细胞开始增加,6 h 后约75%细胞呈 TUNEL 染色阳性;并且,SEC 与乙醇(25～100 mmol/L)共同孵育6 h,TUNEL 染色阳性的 SEC 增加呈明显剂量依赖性关系(均 P<0.05)。VEGF(20～30 ng/ml)明显抑制100 mmol/L 的乙醇引起 SEC 凋亡,呈明显剂量依赖性关系(P<0.05);加入 VEGF(30 ng/ml)以后引起的 TUNEL 染色阳性细胞数只是单纯与乙醇孵育的71%(P<0.05)。加入100 mmol/L 乙醇培养6 h 后,SEC 的 Ets-1蛋白表达明显减少,VEGF(30 ng/ml)防止乙醇(100 mmol/L)引起 SEC 表达 Ets-1蛋白减少。SEC 与乙醇(100 mmol/L)孵育2 h 时 Caspase-8活性明显增加至44.9±14.3;VEGF(20和30 ng/ml)防止乙醇(100 mmol/L)引起 SEC 表达 Caspase-8活性增加(分别为30.4±2.0和25.2±2.2,均 P<0.05)。结论 VEGF 防止乙醇诱导的 SEC 凋亡,至少部分可能是通过抑制乙醇下调 SEC 表达 Ets-1蛋白和乙醇上调 Caspase-8活性表达。

Objective To investigate the cell death pattern of sinusoidal endothelial cells （SECs） caused by ethanol and the effects of vascular endothelial growth factor （VEGF） on this cell death, as well as the underlying mechanism involving Ets-1 and Caspase-8. Methods SECs were isolated from male Wister rats and cultured in medium containing ethanol（25 -100 mmol/L）. VEGF（20 -30 ng/ml）was added into the medium to be co-incubated for up to 6 h. Apoptosis was detected by terminal deoxynucleotidyl transferase （TdT）-mediated d-uridine triphosphate （dUTP）-biotin nick end labeling （TUNEL） technique. The protein expression of Ets-1, prototype of anti-apoptotic Ets family, was determined by Western blotting and the Caspase-8 was measured by FLICE/caspase-8 colorimetric protease assay kit. Results Three days after culture, the SECs showed spindle-like shapes and nearly confluent, however, the cells tended to shrink and die during the time course of ethanol incubation under phase contrast microscope. The control SECs contained only a few percent of TUNEL-positive cells; however, the TUNEL-positive cells started to increase 2 hours after the addition of ethanol （ 100 mmol/L）, and about 75% of the cells were TUNEL-positive 6 hours after ethanol incubation （ P 〈 0. 05 ） under fluorescent microscope. Again, TUNEL-positive cells increased 6 hours after ethanol （25 - 100 mmol/L） incubation in a dose dependent manner （P 〈 0. 05 ）. Six hours after VEGF （20 -30 ng/ml） was added into the medium with ethanol （100 mmol/L） the percentage of TUNEL positive SECs decreased in a dose dependent manner （ P 〈 0. 05 ）, the level of ethanol-induced apoptotic cells in the presence of VEGF （30 ng/ml） being around 71% 6 hours after ethanol incubation alone. The Ets-1 protein level of the SECs decreased 6 hours after ethanol （ 100 mmol/L） incubation, which was prevented almost completely by VEGF （30 ng/ml ）. The Caspase-8 activity level was significantly increased to 44. 9±14. 3 2 hours after ethanol （ 100 mmol/L） incubation, and decrease to 30.4±2.0 and 25.2±2.2 respectively after the addition of VEGF（20-30ng/ml） （both P〈0.05）. Conclusion VEGF prevents the apoptosis of primary cultured SECs induced by ethanol, through at least in part, inhibition of ethanol-induced down-regulation of Ets-1 protein expression and ethanol-induced up-regulation of Casepase-8 activity in SECs.