槲皮素预处理高糖环境培养的人脐静脉内皮细胞氧化应激、内质网应激反应观察

Effects of quercetin on oxidative stress and endoplasmic reticulum stress of human umbilical vein endo⁃thelial cells induced by high glucose

目的观察不同浓度槲皮素预处理后高糖培养条件培养的人脐静脉内皮细胞、氧化应激、内质网应激情况,并探讨其可能作用机制。方法体外培养内皮细胞,分为槲皮素组(Q20组、Q50组及Q100组)、高糖组(HG组)及正常对照组(Control组),Q20组预先加入20μmol/L的槲皮素预处理1 h,再加入10 mg/L的葡萄糖培养细胞,Q50组预先加入50μmol/L的槲皮素预处理1 h,再加入10 mg/L的葡萄糖培养细胞,Q100组预先加入100μmol/L的槲皮素预处理1 h,再加入10 mg/L的葡萄糖培养细胞。HG组在培养液中加入10 mg/L的HG培养细胞,Control组采用低糖DMEM培养基培养。干预24 h时采用CCK-8法观察各组细胞活性;培养24 h时采用流式细胞术测算内皮细胞凋亡率及细胞活性氧簇(ROS)含量,采用乳酸脱氢酶(LDH)试剂盒测定内皮细胞细胞培养液中LDH活性,采用WesternBlotting法检测各组内皮细胞内质网应激相关蛋白葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)和含半胱氨酸的天冬氨酸蛋白水解酶12(Caspase 12)。结果与Control组比较,培养24 h时HG组内皮细胞活性下降(P<0.05);与HG组比较,Q20组、Q50组及Q100组内皮细胞活性增加(P均<0.05),且随浓度增加,细胞活性上升(P均<0.05)。与Control组比较,培养24 h时HG组细胞凋亡率增加(P<0.05);与HG组比较,培养24 h时Q20组、Q50组及Q100组细胞凋亡率降低,且随槲皮素浓度增加,细胞凋亡率下降(P均<0.05)。与Control组比较,培养24 h时HG组细胞ROS含量升高(P<0.05);与HG组比较,Q20组、Q50组及Q100组细胞ROS含量降低,且随槲皮素浓度增加,ROS含量降低(P均<0.05)。与Control组比较,培养24 h时HG组细胞培养液中LDH活性升高(P<0.05);与HG组比较,Q50组及Q100组细胞培养液中LDH活性降低(P均<0.05);与Q50组比较,Q100组细胞培养液中LDH活性降低(P均<0.05)。与Control组比较,培养24 h时HG组内皮细胞GRP78、CHOP、Caspase 12表达升高(P均<0.01);与HG组比较,Q20组、Q50组及Q100组内皮细胞GRP78、CHOP、Caspase 12表达量降低(P均<0.01)。结论槲皮素预处理能提高高糖培养液中的内皮细胞活性,降低细胞培养液中LDH活性,抑制细胞凋亡,降低ROS含量,抑制细胞GRP78、CHOP及Caspase 12表达,且随着浓度的增加,其抑制作用增强。槲皮素可能通过抑制高糖条件下内皮细胞的氧化应激和内质网应激反应,抑制内皮细胞的凋亡,发挥内皮细胞保护作用。

Objective To investigate the effects of quercetin on oxidative stress and endoplasmic reticulum stress of human umbilical vein endothelial cells(HUVECs) induced by high glucose and their possible mechanism.Methods HUVECs were cultured in vitro,and the cells were divided into the control group(Control group),high glucose(HG) group,and quercetin groups with different concentrations(20 μmol/L,50 μmol/L,and 100 μmol/L,named as Q20,Q50,Q100 groups),respectively.HUVECs in the Q20 group were pretreated with 20 μmol/L quercetin for 1 h,and then were added with10 mg/L glucose for culture.HUVECs in the Q50 group were pretreated with 50 μmol/L quercetin for1 h,and then were added with glucose for culture.HUVECs in the Q100 group were pretreated with 100 μmol/L quercetin for 1 h,and then were added with 10 mg/L glucose for culture.In the HG group,10 mg/L HG was added to the culture medium,and the cells in the Control group were cultured in the low-glucose DMEM medium.After 24 hours of intervention,the cell viability was detected by cell counting Kit-8(CCK-8) method.The apoptosis rate and reactive oxygen species(ROS) level were detected by flow cytometry.The degree of cell membrane damage was evaluated by detecting lactate dehydrogenase(LDH) activity in the media.Western blotting was used to detect the expression levels of glucose-regulated protein 78(GRP78),CCAAT/enhancer-binding protein homologous protein(CHOP) and Caspasel2.Results Compared with the Control group,the activity of endothelial cells in the HG group decreased after 24 h(P<0.05).Compared with the HG group,the activity of endothelial cells in the Q20,Q50 and Q100 groups increased,and the cell viability increased with the increased concentrations of quercetin(all P<0.05).Compared with the Control group,the apoptosis rate of the HG group increased(P<0.05),while the apoptosis rates of the Q20,Q50 and Q100 groups decreased,and the apoptosis rate decreased with the increased concentrations of quercetin(all P<0.05).Compared with the Control group,the ROS in the HG group increased(P<0.05);compared with the HG group,the ROS in the Q20,Q50 and Q100 groups decreased,and the ROS decreased with the increased concentrations of quercetin(all P<0.05).Compared with the Control group,the LDH activity in the cell culture medium of the HG group increased(P<0.05).Compared with the HG group,the LDH activity in the cell culture medium of the Q50 group and Q100 group decreased(both P<0.05).Compared with the Q50 group,the LDH activity in the cell culture medium of the Q100 group decreased(P<0.05).Compared with the Control group,the expression levels of GRP78,CHOP,and Caspase 12 in the endothelial cells of the HG group increased(all P<0.01).Compared with the HG group,the expression levels of GRP78,CHOP,and Caspase 12 decreased in the endothelial cells of the Q20,Q50 and Q100 groups(all P<0.01).Conclusions Quercetin can increase endothelial cell activity,reduce LDH activity,inhibit the apoptosis,reduce ROS content,and inhibit the expression levels of GRP78,CHOP and Caspase 12 of endothelial cells in the high glucose solution,and its inhibitory effect is enhanced with the increased concentrations of quercetin.Quercetin may play a protective effect on endothelial cells by inhibiting endothelial cell oxidative stress,endoplasmic reticulum stress and reducing apoptosis under the condition of high glucose.