红树林内生真菌来源dicerandrol A及其对肝癌HepG2细胞的增殖抑制作用和机制的初步研究

摘要：目的 研究红树林植物大红树内生真菌Phomopsis asparagi DHS-48发酵提取物中的化学成分及其对肝癌HepG2细胞的抑制作用，并初步探讨其作用机制。方法 通过反复硅胶柱色谱、Sephadex LH-20柱色谱等方法进行分离纯化，根据理化性质、波谱数据并结合参考文献鉴定化合物DD-38的结构；用不同浓度DD-38处理HepG2细胞，采用MTT比色法检测其对HepG2细胞增殖的抑制作用；平板克隆形成实验检测HepG2细胞克隆形成能力；细胞划痕实验观察HepG2细胞细胞的体外迁移能力；Annexin V/PI双染流式细胞术检测细胞凋亡；Western blot检测β-catenin的表达水平。结果 通过1H NMR、13C NMR及MS等波谱学方法，结合相关文献数据，鉴定化合物结构为：dicerandrol A(DD-38)；MTT实验显示，DD-38对HepG2细胞的增殖具有明显抑制作用，存在时间和剂量依赖性(P<0.05，P<0.01)；根据细胞增殖率得出24 h时HepG2细胞的IC50为(4.8273±0.2216) μmol/L；平板克隆形成实验显示，DD-38明显降低了HepG2细胞的克隆形成能力，具有剂量依赖性(P<0.01)；细胞划痕实验表明DD-38可以显著降低HepG2细胞的迁移能力(P<0.01)，加药24 h后，细胞迁移率从77.09%±2.16%降低到12.14%±5.32%；流式细胞术检测实验显示，DD-38可促进HepG2细胞凋亡，凋亡率存在剂量依赖性；Western blot实验显示，DD-38可抑制β-catenin蛋白在细胞质和细胞核中的表达(P<0.05和P<0.01)。结论 DD-38可抑制HepG2细胞的增殖、迁移和克隆形成，促进细胞凋亡，其机制可能与抑制Wnt/β-catenin信号通路活化有关。

Mangrove endophytic fungi-derived dicerandrol A and its inhibitory effects and preliminary mechanism on HepG2 cells

Abstract Objective To study the chemical constituents from Chinese mangrove plant Rhizophora mangleendophytic fungus Phomopsis asparagi DHS-48, and investigate the inhibitory effect of isolated metabolites onHepG2 cells, and to preliminarily explore its mechanism in vitro. Methods Crude extracts of P. asparagi DHS-48were isolated and purified by the chromatography on silica gel and Sephadex LH-20. The isolated metabolite DD-38 wasidentified as dicerandrol A on the basis of comprehensive spectroscopic data analyses and comparison with literaturedata. HepG2 cells were treated with different concentrations of DD-38, and MTT colorimetry was used to detect itsinhibitory e?ect on the proliferation of HepG2 cells. The clone formation ability of HepG2 cells was detected usingplate clone formation assay. Cell scratch assay was used to observe the migration ability of HepG2 cells. Apoptosiswas detected by Annexin V/PI double staining flow cytometry. The expression of β-catenin was detected by Westernblot. Results MTT assay showed that DD-38 significantly inhibited the proliferation of HepG2 cells in a time anddose dependent manner (P<0.05, P<0.01). The IC50 value of DD-38 for HepG2 cells at 24 h was (4.8273±0.2216)μmol/L. Colony formation assay showed that DD-38 signi?cantly inhibited the colony forming ability of HepG2 cellsin a dose-dependent manner (P<0.01). Cell scratch assay showed that DD-38 signi?cantly inhibited the migration ofHepG2 and Hela cells (P<0.01) and migration rate was decreased from 77.09%±2.16% to 12.14%±5.32% after DD-38treated for 24 h. Flow cytometry showed that DD-38 promoted the HepG2 cells apoptosis in a dose-dependent mannerafter the treatment with DD-38. Western blot showed that DD-38 suppressed the expression of β-catenin proteinin cytoplasm and nucleus (P<0.05, P<0.01). Conclusion We showed that DD-38 can inhibit the proliferation,migration and colony formation of HepG2 cells and induced cell apoptosis, and its mechanism may be related to theinhibition of activation of the Wnt/β-catenin signaling pathway.