摘要
目的研究硫氧还蛋白结合蛋白(TXNIP)介导的NLRP3炎症小体激活在心肌微血管内皮细胞(CMECs)缺氧/复氧(H/R)损伤中的作用。方法分离培养C57BL/6J小鼠CMECs,随机分为对照组、H/R损伤组、H/R+Scrambled siRNA组和H/R+TXNIP siRNA组。各组处理后使用ELISA试剂盒检测细胞IL-1β水平,采用免疫共沉淀的方法检测TXNIP和NLRP3的相互作用,Western blot检测TXNIP和NLRP3的表达水平,乳酸脱氢酶试剂盒检测细胞培养上清LDH释放情况和细胞Caspase-3活性。结果与对照组相比,H/R后CMECs的NLRP3表达水平明显升高(P<0.05),NLRP3炎症小体激活(IL-1β水平升高,P<0.01),且TXNIP和NLRP3的结合效果明显增强(P<0.01)。运用TXNIP siRNA抑制TXNIP表达后行H/R处理,与H/R+Scrambled siRNA组相比,NLRP3炎症小体激活水平明显下降(IL-1β水平降低),细胞Caspase-3活力和LDH释放水平均显著降低(均P<0.05)。结论在CMECs发生H/R损伤时,NLRP3炎症小体激活,TXNIP和NLRP3的相互作用增强;抑制TXNIP表达后,NLRP3炎症小体激活水平降低,内皮损伤减轻。说明TXNIP介导的NLRP3炎症小体激活参与CMECs的H/R损伤,可能成为CMECs的H/R损伤的新机制。
AIM To determine the effect of thioredoxin-interacting protein (TXNIP) mediated NLRP3 inflammasome activation on cardiac microvascular endothelial cells (CMECs) during hypoxia/reoxygenation (H/R) injury. METHODS CMECs isolated from the hearts of adult mice were randomized into control group, H/R group, H/R + scrambled siRNA group and H/R + TXNIP siRNA group. Levels of IL-IL were detected by ELISA kits. The interaction between TXNIP and NLRP3 was determined by immunoprecipitation. Expressions of TXNIP and NLRP3 were analyzed by Western blot, and caspase-3 activity and LDH release were assessed with test kits. RESULTS H/R increased NLRP3 expression ( P 〈 0. 05 ), IL-1 [3 level ( P 〈 0. 01 ) and the interaction between TXNIP and NLRP3 ( P 〈 0. 01 ) in CMECs compared with control group. Blocking TXNIP signaling with siRNA significantly inhibited NLRP3 activation (P 〈 0. 05 ) and alleviated endothelial injury evidenced by increased caspase-3 activity LDH release (P 〈 0.05 ). CONCLUSION H/R injury promotes NLRP3 activation and increases the interaction between TXNIP and NLRP3. Blocking TXNIP signaling reduces NLRP3 activation and endothelial injury. TXNIP mediated NLRP3 activation may be a novel mechanism in I-L/R injury of CMECs.
出处
《心脏杂志》
CAS
2015年第5期510-513,519,共5页
Chinese Heart Journal
基金
国家自然科学基金项目资助(81170186)
关键词
NLRP3
TXNIP
心肌微血管内皮细胞
缺氧/复氧损伤
NLRP3
thioredoxin-interacting protein
cardiac mierovascular endothelial cells
hypoxia/ reoxygenation injury