穿琥宁抗炎作用的血红素加氧酶-1的信号转导机制  

Heme oxygenase-1 signaling mechanism of Chuanhuning anti-inflammatory effect

作　　者：张波[1] 龙隆[2] 王莉莉[2] 刘克良[2] 宫泽辉[1] 

机构地区：[1]军事医学科学院毒物药物研究所新药评定研究室,北京100850 [2]军事医学科学院毒物药物研究所药物化学研究室,北京100850

出　　处：《中国药理学与毒理学杂志》2013年第1期84-90,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：国家十一五科技重大专项综合性新药研究开发技术平台(2009ZX09301-002)~~

摘　　要：目的初步探讨穿琥宁抗炎作用的信号转导机制。方法用WST-8细胞计数试剂盒检测穿琥宁的细胞毒性;用IN Cell Analyzer 2000活细胞成像系统及组成型增强表达绿色荧光蛋白偶联NF-κBP65(EGFP-NF-κBP65)融合蛋白的CHO细胞和EGFP偶联促分裂原活化蛋白激酶APk2(EGFP-MAPK-APk2)融合蛋白的BHK细胞观察穿琥宁对白细胞介素1β(IL-1β)或肿瘤坏死因子α(TNF-α)诱导NF-κBP65核转位及脂多糖(LPS)诱导的P38MAPK下游分子MAPK-APk2核转位的影响;Western印迹法检测穿琥宁和血红素加氧酶-1(HO-1)特异性抑制剂锌原卟啉(ZnPP)对RAW264.7细胞HO-1、诱导型一氧化氮合酶(iNOS)和环氧化酶2(COX-2)表达的影响;Griess反应和ELISA法测定穿琥宁对RAW264.7细胞一氧化氮(NO)和前列腺素E2(PGE2)生成的影响。结果穿琥宁3～100μmol·L-1作用24 h对RAW264.7细胞无细胞毒性。穿琥宁不能抑制由IL-1β诱导的NF-κBP65核转位和LPS诱导的MAPK-APk2核转位,对TNF-α诱导的NF-κBP65核转位有较弱的抑制作用,抑制率为20%,无明显浓度效应关系。穿琥宁3,10,30和100μmol·L-1作用4 h能诱导RAW264.7细胞HO-1表达,穿琥宁100μmol· L-1作用6 h显著抑制IL-1β诱导的COX-2表达和PGE2产生,作用12 h抑制iNOS表达和NO产生。HO-1活性抑制剂ZnPP能部分逆转穿琥宁的上述作用。结论穿琥宁的抗炎作用可能主要通过HO-1信号转导,继而抑制iNOS和COX-2的表达及NO和PGE2的产生。对NF-κB信号传导系统的调节作用尚不清楚。OBJECTIVE To explore the anti-inflammatory mechanism of Chuanhuning. METHODS Cell survival was measured with WST-8 kit. NF-KBP65 and mitogen activated protein kinase APk2 （MAPK-APk2） nuclear translocation was studied in CHO cells with NF-KBP65-enhanced green fluores- cent protein （EGFP） fusion protein and BHK cells with EGFP-MAPK-APk2 fusion protein. The protein expression of heme oxygenase-1 （HO-1）, inducible nitric oxide synthase （iNOS） and cyclooxygenase 2 （COX-2） of RAW264.7 cells was detected by Western blotting. The concentration of nitric oxide （NO） and prostaglanddins E2 （PGE2） of RAW264.7 cells was measured by Griess reaction and ELISA. RESULTS Chuanhuning 3 -100 μmol·L-1 for 24 h had no cytotoxicity for RAW264.7 cells. Chuanhuning 3-100 μmol·L-1 could not inhibit NF-KBP65 nuclear translocation or MAPK-APk2 nuclear translocation in- duced by interlukin-113 （IL-115） or lipopolYSaccharides（LPS）. Chuanhuning3 -100μmol·L-1 weakly inhibi- ted NF-KBP65 nuclear translocation induced by tumor necrosis factor-α （TNF-α）, and the inhibitory rate was 20% and had no concentration-effect relationship. The protein expression of HO-1 in RAW264.7 cells was induced by Chuanhuning3, 10, 30 and 100 μmol·L-1for 4 h. Chuanhuning 100 μmol·L-1for 6 h in- hibited COX-2 expression and PGE2 production, and for 12 h inhibited iNOS expression and NO production induced by IL-115 of RAW264. 7 cells. Zinc protoporphyria partially reversed the inhibitory effect of Chuanhuning on iNOS and COX-2 expression and NO and PGE2 production. CONCLUSION The anti- inflammatory mechanism of Chuanhuning may be related to its inhibition of iNOS and COX-2 expression and NO and PGE2 production via up-regulation of HO-1 pathway. The regulatory role of Chuanhuning for the NF-KB signaling system remains unclear.

关 键 词：穿琥宁 抗炎药(中药) 血红素加氧酶-1 一氧化氮合酶 环氧化酶2 

分 类 号：R285[医药卫生—中药学]