高迁移率族蛋白1对小鼠脑微血管内皮细胞 P-糖蛋白表达的影响

摘要

[Abstract ] Objective High-mobility group box-1 (HMGB1) is abundantly released in the epileptogenic brain tissue , but few reports are seen about the effect of HMGB 1 on the expression of P-glycoprotein ( P-gp) in the vascular endothelial cells of the epi-leptogenic tissue .This study is to explore whether HMGB 1 can regulate P-gp expression in the brain microvascular endothelial cells of the mouse in vitro . Methods Immortalized brain microvascular endothelial bEnd .3 cells of the mouse were cultured in vitro and al-located to different concentration groups ( treated with culture medium containing 10 , 100 , 500 , and 1000 ng/mL HMGB1 for 8 hours), treatment duration groups (treated with culture medium containing 100 ng/mL HMGB1 for 4, 8, 16, 24, and 32 hours), and a control group ( treated with culture medium without HMGB 1 ) .The mRNA expression of P-gp-encoding gene-multidrug resistance gene 1a (mdr1a) was detected by real-time qPCR, and its protein expression determined by Western blot and immunocytochemistry . Results The results of qPCR manifested that the expressions of mdr 1a mRNA were 1.646 ±0.176, 1.777 ±0.135, 1.617 ±0.043, and 1.398 ±0.182 in the 10, 100, 500, and 1000 ng/mL HMGB1 groups, respectively, significantly higher than 1.030 ±0.284 in the control group (P<0.05), and so were those in the 4, 8, 16, 24 h, and 32 h groups (2.655 ±0.112, 2.168 ±0.212, 1.823 ± 0.232, 1.418 ±0.376, and 1.445 ±0.123) than in the control (1.010 ±0.164) (P <0.05).Western blot showed a significant increase in the P-gp protein expression in all the concentration groups (P<0.05) as well as in the 8 h and 16 h treatment duration groups as compared with the control group (P<0.05).Immunocytochemis-try also revealed a higher P-gp expression in the HMGB1-treated than in the control cells (P<0.01). Conclusion HMGB1 can upregu-late the expressions of mdr1a mRNA and P-gp protein in the brain microvascular endothelial cells of the mouse , which may associated with drug resistance of central nervous system diseases , especially that of epilepsy .%目的：癫痫脑内可大量释放高迁移率族蛋白1（high mobility group box protein 1， HMGB1），但针对HMGB1与癫痫脑内血管内皮细胞过表达的耐药蛋白P-糖蛋白（P-glycoprotein， P-gp）关系的研究甚少。文中探讨HMGB1对体外培养的小鼠脑微血管内皮细胞P-gp表达的影响。方法体外培养永生化小鼠脑微血管内皮细胞株bEnd．3，分为不同浓度HMGB1组（10、100、500、1000 ng／mL HMGB1处理bEnd．3细胞8 h）；不同时间HMGB1处理组（以100 ng／mL HMGB1处理bEnd．3细胞4、8、16、24、32 h）；对照组（给予等量正常培养基）。采用实时定量PCR检测bEnd．3细胞中P-gp编码基因多药耐药基因1a （mdr1a）mRNA表达水平，免疫印迹法、免疫细胞化学法检测P-gp蛋白表达水平。结果 qPCR结果显示：10、100、500、1000 ng／mL HMGB1组mdr1a mRNA表达量分别为1．646±0．176、1．777±0．135、1．617±0．043和1．398±0．182，较对照组（1．030±0．284）明显升高（P＜0．05）。 HMGB1处理4、8、16、24、32 h组mdr1a mRNA表达量分别为2．655±0．112、2．168± 0．212、1．823±0．232、1．418±0．376和1．445±0．123，较对照组（1．010±0．164）明显升高（P＜0．05）。免疫印迹法结果显示：与对照组比较，各浓度组P-gp蛋白表达水平均增高（P＜0．05），HMGB1处理8、16 h组P-gp蛋白表达增高（P＜0．05）。免疫细胞化学染色结果显示：100 ng／mL HMGB1处理16 h后P-gp蛋白表达灰度值（110．843±4．036）较对照组（160．303±2．193）明显减少（P＜0．01）。结论 HMGB1能够上调小鼠脑微血管内皮细胞耐药蛋白P-gp和其编码基因mdr1a表达，可能与中枢神经系统疾病尤其是癫痫的耐药相关。