目的：观察穿心莲内酯（Andrographolide， AD）对肺癌细胞基质金属蛋白酶9（matrix metal oproteinase-9，MMP-9）表达的影响及其分子机制。方法体外培养肺癌细胞系H3255，分别用1.0，3.0和5.0μmol/L AD处理细胞24 h，未处理组为空白对照。伤口愈合实验和侵袭实验分别检测AD对H3255细胞迁移与侵袭的影响。RT-PCR检测MMP-9 mRNA的表达；Western blot检测MMP-9蛋白表达和Akt磷酸化。荧光素酶报告基因分析NF-κB和MMP-9的活性。结果伤口愈合实验和Transwel 小室实验结果显示，AD能在不影响细胞活性的条件下显著抑制H3255细胞侵袭和迁移。同时，Western blot和RT-PCR结果证实AD能抑制MMP-9蛋白及mRNA表达。此外，AD能抑制Akt磷酸化以及NF-κB的转录活性。PI3 K抑制剂LY 294002处理后，NF-κB-luc活性以及MMP-9蛋白表达显著降低。此外，不同浓度AD处理能显著抑制MMP-9的启动子活性。结论 AD是一种潜在的抗肺癌细胞转移药物，它可能通过影响PI3 K/NF-κB通路抑制MMP-9表达而发挥作用。%Objective To investigate the effect and molecular mechanism of Andrographolide (AD) on matrix metalloproteinase-9 (MMP-9) expression in human colon cancer H 3255 cells. Methods Human lung cancer H3255 cell line weve cultured in vitro, and treated with 1.0, 3.0, 5.0μmol/L AD for 24 h, untreated cells was used as blank control. Cell viability, cell migration and cell invasion were analyzed by MTT assay, scratch healing assay and transwell membrane assay, respectably. Expression of MMP-9 mRNA was analyzed by RT-PCR. Protein expression and phosphorylation of Akt were detected by Western blot. Activity of NF-κB and MMP-9 were analyzed by luciferase reporter assay. Results AD could significantly reduced H 3255 cells invasion and migration without affecting the viability of cells, as demonstrated by scratch healing and transwell membrane assay. Furthermore, Western blot and RT-PCR results showed that AD could markedly inhibited MMP-9 activity and its expression in both protein and mRNA levels. AD could attenuated Akt’s phosphorylation and the activity of NF-κB. Moreover, LY 294002, an inhibitor of PI3 K, could significantly inhibited NF-κB transcriptional activity and MMP-9 expression. In addition, different concentrations of AD could inhibit the promote activity of MMP-9. Conclusion AD was a potential anti-invasive agent by inhibiting MMP-9 involved in PI3 K/NF-κB pathways.
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