穿琥宁抗炎作用的血红素加氧酶-1的信号转导机制

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。

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

OBJECTIVE To explore the anti-inflammatory mechanism of Chuanhuning. METHODS Cell survival was measured with WST-8 kit. NF-κBP65 and mitogen activated protein kinase APk2(MAPK-APk2) nuclear translocation was studied in CHO cells with NF-κBP65-enhanced green fluorescent 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-κBP65 nuclear translocation or MAPK-APk2 nuclear translocation induced by interlukin-1β (IL-1β) or lipopolysaccharides(LPS). Chuanhuning 3-100 μmol·L^-1 weakly inhibited NF-κBP65 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 Chuanhuning 3, 10, 30 and 100 μmol·L^-1 for 4 h. Chuanhuning 100 μmol·L^-1 for 6 h inhibited COX-2 expression and PGE₂ production, and for 12 h inhibited iNOS expression and NO production induced by IL-1β 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-κB signaling system remains unclear.