槲皮素通过调控PI3K/Akt信号通路对血管内皮祖细胞发挥保护作用
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摘要
目的探讨槲皮素(quercetin,Que)对氧化应激情况下血管内皮祖细胞(endothelial progenitor cells,EPCs)的保护作用及其作用机制。方法分离并诱导分化人脐血EPCs,随机分为对照组;过氧化氢(H_2O_2)组:加入500μmol·L~(-1)的H_2O_2,建立氧化应激损伤EPCs模型;Que干预组:先分别加入浓度为60、90μmol·L~(-1)的Que预处理30 min后,再加500μmol·L~(-1) H_2O_2共同培养,培养12、24、48 h后,WST-1法观察EPCs增殖情况;Western blot检测Akt、磷酸化Akt的表达水平;实时荧光RT-PCR法检测PI3K、Akt3 mRNA的表达水平。结果与空白对照组对比,经过H_2O_2处理的EPCs增殖能力明显降低,且EPCs的Akt蛋白表达明显下降,PI3K、AKT3 mRNA明显下降(P<0.01,P<0.05)。加入60、90μmol·L~(-1) Que处理12、24、48 h后,损伤的EPCs细胞增殖能力明显增加,与模型组比较差异具有统计学意义(P<0.05)。同时明显升高EPCs磷酸化Akt的蛋白表达(P<0.05),以及PI3K、AKT3 mRNA表达(P<0.01)。结论槲皮素对H_2O_2诱导血管EPCs氧化应激损伤起到修复作用,其作用机制可能是通过激活PI3K/Akt信号通路,减少氧化应激对脑血管病灶的影响,促进脑血管EPCs增殖和分化。
Aim To study the protective effect of quercetin(Que) on vascular endothelial progenitor cells(EPCs) under oxidative stress and the underlying mechanisms. Methods The human umbilical cord blood EPCs were separated,differentiated and randomized into two groups,namely,hydrogen peroxide(H_2O_2) group,in which the oxidative stress damage EPCs model was established by adding H_2O_2 of 500 μmol·L~(-1),and Que intervention group,in which Que pretreated with a concentration of 60 μmol·L~(-1) and 90 μmol·L~(-1) was added for 30 min respectively,and then 500 μmol·L~(-1) H_2O_2 was added for co-culture. WST-1 method was used to assess the proliferation of EPCs after 12,24 and 48 h culture. Western blot was used to detect the expression levels of Akt and phosphorylated Akt. Real-time fluorescence RT-PCR was employed to determine the expression levels of PI3 K and AKT3 mRNA. Results Compared with blank control group,the proliferation capacity of EPCs treated with hydrogen peroxide was significantly reduced. The expression of Akt protein in EPCs decreased significantly,and the relative expression of PI3 K and AKT3 mRNA decreased significantly(P<0.01,P<0.05). After 60,90 μmol·L~(-1) Que was added for 12,24 and 48 h,respectively,the proliferation capacity of damaged EPCs cells increased significantly,and the differencewas statistically significant compared with the model group( P < 0. 05). At the same time,the protein expression of p-Akt in EPCs significantly increased( P <0. 05),and mRNA expression of PI3 K and AKT3 increased( P < 0. 01). Conclusions Que can fix the EPCs oxidative stress induced by H2 O2,and its mechanism may be through the activation of PI3 K/Akt signa-ling pathway,reducing the influence of oxidative stress on cerebrovascular lesions,and promoting the proliferation and differentiation of cerebrovascular EPCs.
Aim To study the protective effect of quercetin(Que) on vascular endothelial progenitor cells(EPCs) under oxidative stress and the underlying mechanisms. Methods The human umbilical cord blood EPCs were separated,differentiated and randomized into two groups,namely,hydrogen peroxide(H_2O_2) group,in which the oxidative stress damage EPCs model was established by adding H_2O_2 of 500 μmol·L~(-1),and Que intervention group,in which Que pretreated with a concentration of 60 μmol·L~(-1) and 90 μmol·L~(-1) was added for 30 min respectively,and then 500 μmol·L~(-1) H_2O_2 was added for co-culture. WST-1 method was used to assess the proliferation of EPCs after 12,24 and 48 h culture. Western blot was used to detect the expression levels of Akt and phosphorylated Akt. Real-time fluorescence RT-PCR was employed to determine the expression levels of PI3 K and AKT3 mRNA. Results Compared with blank control group,the proliferation capacity of EPCs treated with hydrogen peroxide was significantly reduced. The expression of Akt protein in EPCs decreased significantly,and the relative expression of PI3 K and AKT3 mRNA decreased significantly(P<0.01,P<0.05). After 60,90 μmol·L~(-1) Que was added for 12,24 and 48 h,respectively,the proliferation capacity of damaged EPCs cells increased significantly,and the differencewas statistically significant compared with the model group( P < 0. 05). At the same time,the protein expression of p-Akt in EPCs significantly increased( P <0. 05),and mRNA expression of PI3 K and AKT3 increased( P < 0. 01). Conclusions Que can fix the EPCs oxidative stress induced by H2 O2,and its mechanism may be through the activation of PI3 K/Akt signa-ling pathway,reducing the influence of oxidative stress on cerebrovascular lesions,and promoting the proliferation and differentiation of cerebrovascular EPCs.
引文
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[3] 展恩欣,范昊昌,陈晓凤,等. 载脂蛋白A-I模拟肽L-4F减轻过氧化氢诱导的小鼠骨髓内皮祖细胞损伤[J]. 中国病理生理杂志,2016,32(1):1-7.
[3] Zhan E X,Fan H C,Chen X F,et al. Apolipoprotein A-I mimic peptide L-4F to reduce the injury of mouse bone marrow endothelial progenitor cells induced by hydrogen peroxide[J]. Chin J Pathophysiol,2016,32(1):1-7.
[4] 张薇,何龙,刘潇忆,等. 槲皮素通过影响吲哚胺-2,3-双加氧酶活性抑制HeLa细胞增殖的研究[J]. 中国药理学通报,2018,34(2):219-24.
[4] Zhang W,He L,Liu X Y,et al. Quercetin inhibits the proliferation of HeLa cells by affecting the activity of indole amine-2,3-dioxygenase[J]. Chin Pharmacol Bull,2018,34(2):219-24.
[5] 夏鑫,吴明华. 血管相关内皮祖细胞标志物研究[J]. 脑与神经疾病杂志,2018,26(1):65-7.
[5] Xia X,Wu M H. Study on vascular endothelial progenitor cell markers[J]. J Brain Neurol Dis,2018,26(1):65-7.
[6] 戴备军,翁剑枫,胡志斌. 基质金属蛋白酶-9和内皮祖细胞在心脏移植术患者中表达的相关性及临床意义[J]. 中国临床药理学与治疗学,2018,23(3):296-300.
[6] Dai B J,Weng J F,Hu Z B. Correlation and clinical significance of matrix metalloproteinase-9 and endothelial progenitor cells expression in patients with heart transplantation[J]. Chin Clin Pharmacol Ther,2018,23(3):296-300.
[7] Wang H J,Zhang D,Tan Y Z,et al. Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions[ J]. Am J Physiol Cell Physiol,2013,304(7): 617-26.
[8] Wiltrout C,Lang B,Yan Y,et al. Repairing brain after stroke: a review on post-ischemic neurogenesis[J]. Neurochem Int,2007,50(7-8):1028-41.
[9] 张伟,刘晓丹,李菲,等. 血府逐瘀汤调控内皮祖细胞修复损伤血管内皮的研究[J]. 中国药理学通报,2016,32(3):427-33.
[9] Zhang W,Liu X D,Li F,et al. Study on the effect of Xuefu Zhuyu decoction on vascular endothelium injury induced by endothelial progenitor cells[J]. Chin Pharmacol Bull,2016,32(3):427-33.
[10] 刘暖,杨雷,毛秉豫,等. 蛋白激酶D1在大鼠骨髓源性内皮祖细胞中的促血管新生作用[J]. 中国药理学通报,2015,31(9):1259-64.
[10] Liu N,Yang L,Mao B Y,et al. Angiogenesis of protein kinase D1 in bone marrow derived endothelial progenitor cells in rats[J]. Chin Pharmacol Bull,2015,31(9):1259-64.
[11] 王艳,胥光热,彭永权,等.氧自由基对内皮祖细胞的损伤及槲皮素的保护作用[J].四川医学,2012,33(3):386-8.
[11] Wang Y,Xu G R,Peng Y Q,et al. The damage of oxygen free radical to endothelial progenitor cells and the protective effect of quercetin[J]. Sichuan Med,2012,33(3):386-8.
[12] Davis J M,Murphy E A,Carmichael M D. Effects of the dietary flavonoid quercetin upon performance and health[J]. Curr Sports Med Rep,2009,8(4): 206-13.