Protective effect of quercetin on the injury of brain microvascular endothelial cells induced by asymmetric dimethylarginine and its mechanism / 中国脑血管病杂志

ABSTRACT

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 (30 p.mol/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 lOOfimol/L for 2h followed by the addition of ADMA with final concentration of 30 pjnol/L for 24 h), 100 p.mol/L quercetin treatment group (quercetin pretreatment with 100 p.mol/1 for 2h and then changing conventional culture medium) , 10 pjnol/L quercetin treatment group (quercetin pretreatment with 10 n-mol/L for 2 h and then changing conventional culture medium) ,ANISO/P79350 + ADMA + quercetin treatment group (2h after pretreatment with quercetin at the final concentration of 10 yjnol/L, adding ADMA with final concentration of 30 jtmol/L for 24 hours,and adding 10 p.mol/1 anisomycin ( ANISO) or 50 jtmol/L P79350 1 h before the end of the experiment) ,and the ANI50/P79350 treatment group (cultured with routine culture medium,and addinglO jjlmol/L NAISO or 50 p,mol/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 supematants 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-polymcrase 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 ANISU and pjo agoiusi I'/yjDU were used to observe the role ot the JNK/p38 mitogen-activated protein kinase ( p38 MAPK) signaling pathway in the protection of quercetin-mediated cell injur)'. 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 + 10 pjnol/L quercetin treatment group and ADMA + 100 fjjnol/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). Hie 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 raRNA.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/p38160 ±0.19 and 172 ±0.20respectively). 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 tin? LDH level was significantly iiicreawil in both groups. Tlie 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 MARK signaling pathway.