黄芪多糖联合Smac shRNA对高糖诱导的血管内皮细胞凋亡的影响
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摘要
目的:探讨黄芪多糖联合Smac shRNA对高糖诱导的血管内皮细胞凋亡的影响。方法:以Smac shRNA慢病毒感染血管内皮细胞EVC-304,用Real-time PCR和Western blot法测定干扰效果。EVC-304细胞分为4组,对照组未处理,黄芪多糖组给予300 mg/L黄芪多糖,干扰组感染Smac shRNA慢病毒,黄芪多糖+干扰组用300 mg/L黄芪多糖培养感染Smac shRNA慢病毒的EVC-304细胞,随后4组均给予高糖处理。流式细胞术测定细胞凋亡,生化试剂盒测定培养液中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性及丙二醛(MDA)含量,Western blot法检测细胞激活型Caspase-3(C-Caspase-3)、C-Caspase-9蛋白的表达水平。结果:感染Smac shRNA慢病毒的EVC-304细胞Smac表达水平降低(P <0. 05)。Smac shRNA干扰或黄芪多糖处理均可降低高糖条件下EVC-304细胞凋亡率,提高SOD和CAT活性,减少MDA含量,抑制细胞中C-Caspase-3、C-Caspase-9蛋白表达,二者联用有协同作用(P <0. 05)。结论:黄芪多糖联合Smac干扰可抑制高糖诱导的血管内皮细胞凋亡,其作用机制可能与提高抗氧化酶活性、减少氧化损伤有关。
Aim: To investigate the effects of astragalus polysaccharide combined with Smac shRNA on apoptosis of vascular endothelial cells induced by high glucose. Methods: EVC-304 cells were infected with Smac shRNA lentivirus,and the interference effect was determined by Real-time PCR and Western blot. EVC-304 cells were divided into four groups,the control group was not treated,the astragalus polysaccharide group was treated with 300 mg/L astragalus polysaccharide,the interfering group was infected with Smac shRNA lentivirus,and the astragalus polysaccharide + interfering group was treated with 300 mg/L astragalus polysaccharide and infected with Smac shRNA,and then the four groups were cultured with high sugar. Results: Smac expression in EVC-304 cells infected with Smac shRNA lentivirus decreased( P <0. 05). Smac shRNA interference or astragalus polysaccharide treatment could reduce the apoptotic rate of EVC-304 cells,increase SOD and CAT activity,reduce MDA content,and inhibit the expressions of C-Caspase-3 and C-Caspase-9 protein in cells under high glucose conditions,and the combination of them had synergistic effects( P < 0. 05). Conclusion: Astragalus polysaccharide combined with Smac shRNA could inhibit the apoptosis of vascular endothelial cells induced by high glucose,and the mechanism may be related with enhancing antioxidant enzyme activity and reducing oxidative damage.
Aim: To investigate the effects of astragalus polysaccharide combined with Smac shRNA on apoptosis of vascular endothelial cells induced by high glucose. Methods: EVC-304 cells were infected with Smac shRNA lentivirus,and the interference effect was determined by Real-time PCR and Western blot. EVC-304 cells were divided into four groups,the control group was not treated,the astragalus polysaccharide group was treated with 300 mg/L astragalus polysaccharide,the interfering group was infected with Smac shRNA lentivirus,and the astragalus polysaccharide + interfering group was treated with 300 mg/L astragalus polysaccharide and infected with Smac shRNA,and then the four groups were cultured with high sugar. Results: Smac expression in EVC-304 cells infected with Smac shRNA lentivirus decreased( P <0. 05). Smac shRNA interference or astragalus polysaccharide treatment could reduce the apoptotic rate of EVC-304 cells,increase SOD and CAT activity,reduce MDA content,and inhibit the expressions of C-Caspase-3 and C-Caspase-9 protein in cells under high glucose conditions,and the combination of them had synergistic effects( P < 0. 05). Conclusion: Astragalus polysaccharide combined with Smac shRNA could inhibit the apoptosis of vascular endothelial cells induced by high glucose,and the mechanism may be related with enhancing antioxidant enzyme activity and reducing oxidative damage.
引文
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[15]SENA CM,PEREIRA AM,SEICA R. Endothelial dysfunction:a major mediator of diabetic vascular disease[J]. Biochim Biophys Acta,2013,1832(12):2216
[16]LANG M,ZHOU Z,SHI L,et al. Influence of zoledronic acid on proliferation,migration,and apoptosis of vascular endothelial cells[J]. Br J Oral Maxillofac Surg,2016,54(8):889
[17]WANG D,WANG Q,YAN G,et al. High glucose and interleukin 1β-induced apoptosis in human umbilical vein endothelial cells involves in down-regulation of monocarboxylate transporter 4[J]. Biochem Biophys Res Commun,2015,466(4):607
[18]NAKAO T,MORITA H,MAEMURA K,et al. Melatonin ameliorates angiotensinⅡ-induced vascular endothelial damage via its antioxidative properties[J]. J Pineal Res,2013,55(3):287
[19]PATEL SN,PANDYA K,CLARK GJ,et al. Comparison of taurine and pantoyltaurine as antioxidants in vitro and in the central nervous system of diabetic rats[J]. Exp Toxicol Pathol,2016,68(2/3):103
[20]梁薇,谭年花,潘子怡,等.益智仁-乌药醚提取物对糖尿病肾病氧化应激Mn-SOD、MDA影响实验研究[J].山西中医学院学报,2016,17(3):20
[2] WANG J,JIANG H,WANG J,et al. Propofol attenuates high glucose-induced superoxide anion accumulation in human umbilical vein endothelial cells[J]. Fundam Clin Pharmacol,2016,30(6):511
[3]李眺,陈利国,李权,等.川芎嗪配伍黄芪多糖对血管内皮细胞的保护作用[J].中华中医药杂志,2011,26(11):2672
[4]张玲,宋杰,孙燕玲,等.黄芪多糖通过激活AMPK减轻同型半胱氨酸对人血管内皮细胞的损伤[J].中国病理生理杂志,2018,34(7):1195
[5] PHILCHENKOV A,MIURA K. The IAP protein family,SMAC mimetics and cancer treatment[J]. Crit Rev Oncog,2016,21(3/4):185
[6]孔宪如,方春钱,陈魁,等.褪黑素对糖尿病大鼠氧化抗氧化系统及Smac表达的影响[J].江苏医药,2010,36(17):2066
[7]杨志霞,王琦.黄芪多糖免疫作用的基础与临床研究进展[J].世界中医药,2013,8(7):833
[8]刘蓓,朱海燕,高永红,等.黄芪多糖对TNF-α诱导心脏微血管内皮细胞黏附分子基因转录及p38MAPK信号通路的影响[J].世界中医药,2011,6(3):263
[9] ZHU YP,SHEN T,LIN YJ,et al. Astragalus polysaccharides suppress ICAM-1 and VCAM-1 expression in TNF-α-treated human vascular endothelial cells by blocking NF-κB activation[J]. Acta Pharmacol Sin,2013,34(8):1036
[10]HAN R,TANG F,LU M,et al. Astragalus polysaccharide ameliorates H2O2-induced human umbilical vein endothelial cell injury[J]. Mol Med Rep,2017,15(6):4027
[11]HUANG WM,LIANG YQ,TANG LJ,et al. Antioxidant and anti-inflammatory effects of Astragalus polysaccharide on EA. hy926 cells[J]. Exp Ther Med,2013,6(1):199
[12]HOU L,CHEN K,HAO Y,et al. Effect of Smac in combination with cisplatin on esophageal cancer cell line ECA109[J]. Int J Clin Exp Med,2015,8(10):18506
[13]LUECK SC,RUSS AC,BOTZENHARDT U,et al. Smac mimetic induces cell death in a large proportion of primary acute myeloid leukemia samples,which correlates with defined molecular markers[J]. Oncotarget,2016,7(31):49539
[14]LI J,WANG Y,DU L,et al. Radiation-induced cytochrome c release and the neuroprotective effects of the pan-caspase inhibitor z-VAD-fmk in the hypoglossal nucleus[J]. Exp Ther Med,2014,7(2):383
[15]SENA CM,PEREIRA AM,SEICA R. Endothelial dysfunction:a major mediator of diabetic vascular disease[J]. Biochim Biophys Acta,2013,1832(12):2216
[16]LANG M,ZHOU Z,SHI L,et al. Influence of zoledronic acid on proliferation,migration,and apoptosis of vascular endothelial cells[J]. Br J Oral Maxillofac Surg,2016,54(8):889
[17]WANG D,WANG Q,YAN G,et al. High glucose and interleukin 1β-induced apoptosis in human umbilical vein endothelial cells involves in down-regulation of monocarboxylate transporter 4[J]. Biochem Biophys Res Commun,2015,466(4):607
[18]NAKAO T,MORITA H,MAEMURA K,et al. Melatonin ameliorates angiotensinⅡ-induced vascular endothelial damage via its antioxidative properties[J]. J Pineal Res,2013,55(3):287
[19]PATEL SN,PANDYA K,CLARK GJ,et al. Comparison of taurine and pantoyltaurine as antioxidants in vitro and in the central nervous system of diabetic rats[J]. Exp Toxicol Pathol,2016,68(2/3):103
[20]梁薇,谭年花,潘子怡,等.益智仁-乌药醚提取物对糖尿病肾病氧化应激Mn-SOD、MDA影响实验研究[J].山西中医学院学报,2016,17(3):20