CytochalasinH对小鼠脾细胞增殖的抑制作用及其对Ca^(2+)/CaN/NFAT信号通路的影响

Inhibitory Effect of Cytochalasin H on the Proliferation of Mouse Spleen Cells and Its Effects on Ca(2+)/CaN/NFAT Signaling Pathway

【目的】研究红树林植物大红树内生真菌Phomopsis asparagi DHS-48得到的次级代谢产物cytochalasin H对小鼠(Mus musculus)脾细胞的免疫抑制活性及其作用机制。【方法】用不同浓度(3、6、12、25、30μmol/L)cytochalasin H处理小鼠脾细胞,通过CCK-8活力测定法检测其对刀豆蛋白A(Concanavalin A,ConA)诱导小鼠脾细胞增殖的抑制作用;流式细胞术检测cytochalasin H对小鼠脾细胞凋亡的影响;免疫荧光实验检测cytochalasin H对细胞中NFAT入核及钙离子内流的影响;酶联免疫吸附法(Enzyme-linked immune sorbent assay,ELISA)和免疫蛋白印迹法(Western blotting)测定cytochalasin H对脾细胞中NFAT信号通路的影响。【结果】CCK-8实验表明,cytochalasin H对于正常小鼠脾淋巴细胞的毒性比环孢菌素(Cyclosporin A,Cs A)弱,半抑制浓度IC_(50)=(35.07±1.16)μmol/L;流式细胞术显示,cytochalasin H对ConA刺激的脾淋巴细胞无促凋亡作用;cytochalasin H对ConA诱导增殖的小鼠脾淋巴细胞具有显著的抑制效果,IC_(50)=(14.81±0.81)μmol/L,且呈剂量依赖性;免疫荧光结果表明,cytochalasin H在浓度为15μmol/L时显著地抑制了钙离子内流;Western Blotting结果显示cytochalasin H对脾淋巴细胞中CaN、NFAT蛋白(nuclear factor of activated T-cells,NFAT)的表达具有显著的抑制作用;免疫荧光结果显示,cytochalasin H抑制细胞浆内的NFAT转移到细胞核;ELISA测试结果证明,cytochalasin H能显著抑制下游细胞因子白介素-2(IL-2)的分泌(α=0.05)。【结论】Cytochalasin H通过抑制Ca^(2+)/CaN/NFAT信号通路转导显著抑制T细胞增殖和活化。

【Objective】To study the immunosuppressive activity and mechanism of the secondary metabolite cytochalasin H obtained from the endophytic fungus Phomopsis asparagi DHS-48 of the mangrove plant Rhizophora mangle on mouse splenocytes.【Method】Mouse splenocytes were treated with different concentrations of cytochalasin H(3,6,12,25,30μmol/L),and its inhibitory effect on Concanavalin A(ConA)-induced proliferation of mouse splenocytes was detected by CCK-8 assay.The effect of cytochalasin H on the apoptosis of mouse splenocytes was detected by flow cytometry.The effect of cytochalasin H on the nuclear entry of NFAT and the influx of calcium ions in cells was detected by immunofluorescence experiment.Enzyme-linked immune sorbent assay(ELISA)and western blotting were used to determine the effect of cytochalasin H on NFAT signaling pathway in splenocytes.【Result】CCK-8 assay showed that cytochalasin H had a small toxic effect on lymphocytes,compared with Cyclosporin A(Cs A)(half maximal inhibitory concentration IC_(50)=35.07±1.16μmol/L).Flow cytometry showed that cytochalasin H did not promote apoptosis of spleen lymphocytes stimulated by ConA.Cell proliferation assays showed that cytochalasin H inhibited the proliferation of ConA-induced T murine splenic lymphocyte(IC_(50)=14.81±0.81μmol/L)in a dose dependent manner.Immunofluorescence showed that cytochalasin H significantly inhibited calcium influx at the concentration of 15μmol/L.Western Blotting showed that cytochalasin H significantly inhibited the expression of nuclear factor of activated T-cells(NFAT)in spleen lymphocytes.Immunofluorescence showed that cytochalasin H inhibited the transfer of NFAT from cytoplasm to nucleus.ELISA showed that cytochalasin H decreased the secretion of the downstream cytokine interleukin-2(IL-2)(α=0.05).【Conclusion】Cytochalasin H significantly inhibits T cell proliferation by inhibiting Ca^(2+)/CaN/NFAT signaling pathway transduction.