槲皮素对非对称性二甲基精氨酸诱导损伤的脑血管内皮细胞的保护作用及其机制研究

Protective effect of quercetin on the injury of brain microvascular endothelial cells induced by asymmetric dimethylarginine and its mechanism

目的探讨槲皮素对非对称性二甲基精氨酸(ADMA)诱导下人脑血管内皮细胞(HBMECs)损伤的保护作用及其作用机制。方法采用ADMA(30μmol/L,作用24 h)诱导建立HBMECs损伤模型。实验分为对照组(不加任何干预因素)、ADMA组、ADMA+槲皮素处理组(加入终浓度分别为0. 1、1、10、100μmol/L的槲皮素预处理2 h后再加入终浓度为30μmol/L的ADMA作用24 h)、100μmol/L槲皮素处理组(100μmol/l的槲皮素预处理2 h后换常规培养液)、10μmol/l槲皮素处理组(10μmol/L的槲皮素预处理2 h后换常规培养液)、茴香霉素(ANISO)/P79350+ADMA+槲皮素处理组[加入终浓度为10μmol/L的槲皮素预处理2 h后再加入终浓度为30μmol/L的ADMA作用24 h,实验结束前1 h加入10μmol/L的ANISO或50μmol/L的P79350]以及ANISO/P79350处理组(常规培养液培养,实验结束前1 h加入10μmol/L的NAISO或50μmol/L的P79350),每组6个样本。不同浓度槲皮素预处理2 h后,噻唑蓝法测定细胞活性,酶联免疫吸附试验法测定细胞上清液中乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、活性氧、丙二醛、脑源性神经营养因子(BDNF)水平,Western blotting分析Bax、Bcl-2、c-Jun氨基末端激酶(JNK)、磷酸化JNK(p-JNK)、p38、磷酸化p38(p-p38)表达水平,逆转录-聚合酶链式反应检测内皮型一氧化氮合酶(e NOS) mRNA表达,caspase-3活性试剂盒检测caspase-3活性,应用JNK激动剂ANISO和p38激动剂P79350观察JNK/p38丝裂原活化蛋白激酶(p38 MAPK)信号通路在槲皮素介导的细胞损伤保护中的作用。结果与对照组比较,ADMA组HBMECs活性(52±8)明显降低,LDH水平(356±28)显著升高,BDNF浓度(51±8)明显下降,差异均有统计学意义(均P <0. 05);与ADMA组比较,ADMA+10μmol/L槲皮素处理组和ADMA+100μmol/L槲皮素处理组HBMECs细胞活性显著改善(分别为80±5、86±7),LDH水平明显下调(分别为162±20、141±17),BDNF浓度明显增加(分别为82±5、94±6),差异均有统计学意义(均P <0. 05)。应用10μmol/L的槲皮素处理进行后续实验,结果显示,与ADMA组比较,ADMA+槲皮素处理组Bcl-2 mRNA、e NOS mRNA和SOD水平均明显升高(分别为0. 75±0. 10、0. 81±0. 07、81±10),Bax表达水平和caspase-3活性、活性氧、丙二醛水平均明显减低(分别为1. 63±0. 12、1. 85±0. 16、169±16、159±13),差异均有统计学意义(均P <0. 05)。进一步机制分析表明,与对照组比较,ADMA组可显著提高HBMECs中JNK和p38的磷酸化水平(p-JNK/JNK和p-p38/p38分别为3. 46±0. 32、3. 66±0. 31);与ADMA组比较,ADMA+槲皮素处理组有效抑制了ADMA诱导下HBMECs的JNK和p38磷酸化水平(p-JNK/JNK和p-p38/p38分别为2. 60±0. 19、2. 72±0. 20),差异均有统计学意义(均P <0. 05);与ADMA+槲皮素处理组比较,ANISO+ADMA+槲皮素处理组p-JNK/JNK(4. 06±0. 30)和P79350+ADMA+槲皮素处理组p-p38/p38(3. 84±0. 32)明显增高,两组细胞活性和BDNF水平明显降低,LDH水平明显增加,差异均有统计学意义(均P <0. 05)。结论槲皮素可缓解ADMA诱导的HBMECs损伤,其机制可能与抑制JNK/p38 MAPK信号通路有关。

Objective To investigate the protective effect of quercetin on the injury of human brain microvascular endothelial cells(HBMECs)induced by asymmetric dimethylarginine(ADMA)and its mechanism.Methods An injury model of HBMECs was induced by ADMA(30μmol/L for 24 h).The experiment was divided into control group(without any intervention factors),ADMA group,ADMA+quercetin treatment group(pretreatment of quercetin with final concentration of 0.1,1,10 and 100μmol/L for 2 h followed by the addition of ADMA with final concentration of 30μmol/L for 24 h),100μmol/L quercetin treatment group(quercetin pretreatment with 100μmol/l for 2 h and then changing conventional culture medium),10μmol/L quercetin treatment group(quercetin pretreatment with 10μmol/L for 2 h and then changing conventional culture medium),ANISO/P79350+ADMA+quercetin treatment group(2 h after pretreatment with quercetin at the final concentration of 10μmol/L,adding ADMA with final concentration of 30μmol/L for 24 hours,and adding 10μmol/l anisomycin(ANISO)or 50μmol/L P79350 1 h before the end of the experiment),and the ANISO/P79350 treatment group(cultured with routine culture medium,and adding10μmol/L NAISO or 50μmol/L P79350 1 h before the end of the experiment)(n=6 samples in each group).Cell activity was measured by thiazole blue method after pretreatment with quercetin at different concentrations for 2 h.Lactate dehydrogenase(LDH),superoxide dismutase(SOD),reactive oxygen species,malondialdehyde,brain-derived neurotrophic factor(BDNF)levels in cell supernatants were detected by enzyme-linked immunosorbent assay.Bax,Bcl-2,c-Jun N-terminal kinase(JNK),phosphorylated JNK(p-JNK),p38,phosphorylated p38(p-p38)expression levels were analyzed with Western blotting.Reverse transcription-polymerase chain reaction was used to detect the expression of endothelial nitric oxide synthase(eNOS)mRNA.Caspase-3 activity kit was used to detect caspase-3 activity.The JNK agonist ANISO and p38 agonist P79350 were used to observe the role of the JNK/p38 mitogen-activated protein kinase(p38 MAPK)signaling pathway in the protection of quercetin-mediated cell injury.Results Compared with the control group,the activity of HBMECs(52±8)in the ADMA group was decreased significantly,the LDH level(356±28)was increased significantly,and the BDNF concentration(51±8)was decreased significantly.The differences were statistically significant(all P<0.05).Compared with the ADMA group,the activity of HBMECs in the ADMA+10μmol/L quercetin treatment group and ADMA+100μmol/L quercetin treatment group was significantly improved(80±5 and 86±7 respectively),the LDH level was significantly downregulated(162±20 and 141±17 respectively),and the concentration of BDNF was significantly increased(82±5 and 94±6 respectively).The differences were statistically significant(all P<0.05).The follow-up experiments were treated with 10 mol/L quercetin.The results showed that compared with the ADMA group,the levels of Bcl-2 mRNA,eNOS mRNA,and SOD in the ADMA quercetin treatment group were increased significantly(0.75±0.10,0.81±0.07,and 81±10,respectively),the expression level of Bax and the activity of caspase-3,reactive oxygen species,and malondialdehyde level were decreased significantly(1.63±0.12,1.85±0.16,169±16,and 159±13,respectively).There were significant differences(all P<0.05).Further mechanism analysis showed that compared with the control group,the ADMA group significantly increased the phosphorylation levels of JNK and p38 in HBMECs(p-JNK/JNK and p-p38/p38 3.46±0.32 and 3.66±0.31,respectively);Compared with the ADMA group,the ADMA+quercetin treatment group effectively inhibited the JNK and p38 phosphorylation levels of HBMCECs induced by ADMA(p-JNK/JNK and p-p38/p38 2.60±0.19 and 2.72±0.20 respectively).The differences were statistically significant(all P<0.05).Compared with the ADMA+quercetin treatment group,p-JNK/JNK(4.06±0.30)in ANISO+ADMA+quercetin treatment group,and p-p38/p38(3.84±0.32)in the P79350+ADMA+quercetin treatment group were significantly increased.The cell activity and the BDNF level were significantly decreased,while the LDH level was significantly increased in both groups.The differences were statistically significant(all P<0.05).Conclusion Quercetin attenuates ADMA-induced HBMEC injury,and its mechanism may be related to inhibition of JNK/p38 MAPK signaling pathway.