摘要
目的研究补骨脂酚(BAK)对血管内皮细胞模拟缺血再灌注损伤(SIR)的保护作用及其分子机制。方法用不同浓度BAK(2,5,10μmol/L)预处理体外培养的人脐静脉内皮细胞(HUVECs)24 h,接受模拟缺血再灌注(SIR)损伤(缺血45min,再灌注4 h),用CCK-8法和TUNEL法分别检测细胞活力和凋亡率,酶活性测定法检测培养液中乳酸脱氢酶(LDH)活性,细胞中丙二醛(MDA)的含量及超氧化物歧化酶(SOD)的活性,DHE荧光探针检测细胞中活性氧(ROS)的含量,Western blot法检测细胞膜PKCε、细胞质PKCε、Bcl-2、Bax、cleaved caspase 3和gp91phox蛋白的表达水平。使用PKCε特异性阻断剂εV1-2阻断该信号通路,进一步探究PKCε信号通路在其中的作用。结果 BAK预处理呈剂量依赖性地改善内皮细胞SIR损伤,且10μmol/L剂量效果最佳。BAK预处理可明显提高缺血再灌注损伤后内皮细胞活力,减少LDH释放,增强细胞中SOD活性,降低MDA含量及gp91phox蛋白表达量,减少ROS的产生;促进抗凋亡蛋白Bcl-2的表达,抑制凋亡蛋白cleaved caspase 3,Bax的表达,降低再灌注损伤后细胞凋亡率;并且显著促进PKCε膜转位,而特异性阻断PKCε信号通路可逆转BAK的上述保护作用(均P<0.05)。结论 BAK通过激活PKCε信号通路,进而增强细胞抗氧化应激和抗凋亡能力,最终缓解内皮细胞缺血再灌注损伤。
Objective To explore the protective effects of bakuchiol（ BAK） against simulated ischemia reperfusion（ SIR）-induced injury in human umbilical vein endothelial cells（ HUVECs） and its underlying mechanisms. Methods The cultured HUVECs were pretreated with BAK（2,5,10 μmol/L） for 24 h and then subjected to SIR（45 min ischemia,4 h reperfusion）. Cell viability and apoptotic index were detected by CCK-8 and TUNEL methods,and the lactate dehydrogenase（ LDH） releasing,intracellular malondialdehyde（ MDA） content,superoxide dismutase（ SOD） were all assessed by specialized kits. The translocation of protein kinase C epsilon（PKCε） and the expression levels of Bcl-2,Bax,cleaved caspase 3 and oxidative stress marker gp91 phoxwere detected by Western blot. Dihydroethidium（ DHE）,a fluorescence probe,was used to detect the presence of reactive oxygen species（ ROS）. The εV1-2,a specific inhibitor of PKCε signaling,was used to further study the role of PKCε in this process. Results BAK pretreatment protected HUVECs against SIR injury in a dose-dependent manner,especially at the concentration of 10 μmol/L. BAK pretreatment significantly improved the cell viability,reduced LDH releasing,up-regulated the SOD activity and down-regulated the MDA content of SIR-treated HUVECs（ P 〈 0. 05）. In addition,BAK supplementation significantly reduced the apoptotic index,increased PKCε translocation and Bcl-2 expression,and decreased the expression levels of cleaved caspase 3,Bax and gp91 phox（ P 〈 0. 05）. However,the protective effects of BAK mentioned above were significantly attenuated by εV1-2（ P 〈 0. 05）. Conclusion BAK pretreatment can protect against SIR-induced oxidative stress and apoptosis on HUVECs via PKCε signaling pathway.
出处
《山西医科大学学报》
CAS
2017年第10期975-981,共7页
Journal of Shanxi Medical University
基金
北京市自然科学基金资助项目(7122178)
总参军事医学和老年病科研基金资助项目(ZCWS14C12)
