红树林植物红茄苳内生烟曲霉代谢产物对小鼠脾细胞增殖的影响

Effects of Metabolites of Aspergillus fumigatus from Mangrove Plant Rhizophora mucronata on the Proliferation of Mouse Spleen Cells

为了研究红树林植物红茄苳内生真菌烟曲霉次级代谢产物Af-8[16-O-deacetylhelvolic acid 21,16-lactone]对小鼠脾细胞中免疫抑制活性的影响,采用CCK-8细胞活力测定方法、体外钙调蛋白磷酸酶(calcineurin,CN)活性抑制方法、酶联免疫吸附测定法(enzyme-linked immuno sorbent assay,ELISA)和免疫蛋白印迹法(Western blot)研究Af-8对脾细胞中NFAT信号通路的影响。Af-8对于正常小鼠脾细胞显示较弱的细胞毒性,IC_(50)为(168.96±1.35)μmol/L;Af-8对钙调蛋白磷酸酶有显著的抑制作用,抑制率达到88.81%;随着Af-8浓度的增加,Af-8对ConA诱导脾细胞增殖也显示出明显的抑制效果,IC_(50)为(12.11±0.11)μmol/L;ELISA测试结果表明Af-8对细胞白介素-2(IL-2)的表达也起到显著抑制作用;Western Blot显示Af-8对NFAT1蛋白(nuclear factor of activated T-cells1,NFAT1)的去磷酸化也表现出显著的抑制作用。Af-8通过对CN酶的抑制进而影响NFAT信号通路,从而发挥免疫抑制效果。

To study the effect of the secondary metabolite Af-8[16-O-deacetylhelvolic acid 21,16-lactone]isolated from the endophytic Aspergillus fumigatus of the mangrove plant Rhizophora mucronata on the immunosuppressive activity in mouse spleen cells,CCK-8 cell viability determination method,calcineurin activity inhibition method in vitro,enzyme-linked immunosorbent assay(ELISA)and Western blot were used to study the effect of Af-8 on the NFAT signaling pathway in spleen cells.At concentrations resulting in relatively low cytotoxicity(IC_(50)=(168.96±1.35)μmol/L)Af-8 significantly inhibited calcineurin activity with its inhibition rate reaches 88.81%and ConA-induced splenocytes proliferation in a dose-dependent manner with IC_(50)value of(12.11±0.11)μmol/L.The secretion of interleukin-2(IL-2)were down-regulated by Af-8 in cell supernatant as determined by ELISA.Western blotting revealed that Af-8 had a significant inhibitory effect on the dephosphorylation of NFAT1 protein in mouse T lymphocyte.The results showed that Af-8 affects the NFAT signaling pathway by inhibiting CN enzyme,thereby exerting an immunosuppressive effect.