中国被毛孢醇提物通过MAPK/NF-κB通路双向调控巨噬细胞的免疫功能研究

Hirsutella sinensis alcohol extract regulates immune function of macrophages through MAPK/NF-κB pathway

目的利用巨噬细胞和小鼠模型,探究中国被毛孢醇提物(Hirsutella sinensis alcohol extract,HSAE)的免疫调控功能和机制。方法CCK-8法检测HSAE对RAW264.7巨噬细胞活力的影响;通过血清溶血素实验明确HSAE对小鼠免疫功能的影响;利用qRT-PCR技术检测炎症相关因子表达,探究HSAE对RAW264.7细胞静息状态下的免疫促进功能和脂多糖(lipopolysaccharide,LPS)刺激的炎症激活状态下的炎症抑制作用;通过Western blotting检测HSAE对丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和核因子-κB(nuclear factor-κB,NF-κB)信号通路的调控作用。结果HSAE能够显著增加巨噬细胞的吞噬功能和小鼠血清溶血素水平(P<0.01、0.001);HSAE能够激活静息状态下的巨噬细胞肿瘤坏死因子(tumor necrosis factor,Tnf)、干扰素β1(interferon beta 1,Ifnb1)、CXC趋化因子配体10(CXC chemokine ligand 10,Cxcl10)的m RNA表达(P<0.01、0.001),而在LPS诱导的炎症激活的巨噬细胞中,HSAE可显著抑制上述炎症因子的m RNA表达水平(P<0.05、0.01、0.001);此外,HSAE显著增强静息状态下巨噬细胞中MAPK信号通路关键蛋白细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、p38和NF-κB信号通路p65的磷酸化水平(P<0.05、0.01、0.001),并在炎症激活后抑制相关蛋白的磷酸化(P<0.01、0.001)。结论HSAE对小鼠巨噬细胞具有双向免疫调节作用,即静息状态下的免疫促进作用和炎症激活状态下的免疫抑制作用,该双重调控作用可能与其调控MAPK和NF-κB免疫信号通路相关。

Objective To explore the immunomodulatory function and molecular mechanism of Hirsutella sinensis alcohol extract(HSAE)using macrophages and mice model.Methods The cytotoxicity of HSAE against RAW264.7 cells was measured by CCK-8 assay,and immune-regulating functions of HSAE was determined by serum hemolysin test in vivo.To explore the immune-promoting function of HSAE on RAW264.7 cells in resting state and inflammatory inhibitory effect of HSAE on RAW264.7 cells in the presence of LPS stimulation,the expressions of inflammatory related cytokines were detect by qRT-PCR.Furthermore,regulatory effects of HSAE on mitogen-activated protein kinase(MAPK)and nuclear factor-κB(NF-κB)pathway were detected by Western blotting.Results HSAE significantly increased the phagocytosis of macrophages and level of serum hemolysin in mice(P<0.01,0.001).HSAE activated Tnf,Ifnb1 and Cxcl10 mRNA expressions in resting macrophages(P<0.01,0.001),while in macrophages activated by LPS-induced inflammation,HSAE significantly inhibited the mRNA expression levels of above inflammatory factors(P<0.05,0.01,0.001).In addition,HSAE significantly enhanced the phosphorylation levels of extracellular regulated protein kinase(ERK),c-Jun N-terminal kinase(JNK),p38 and p65 in resting macrophages(P<0.05,0.01,0.001),which play essential roles in MAPK or NF-κB signaling pathway.However,phosphorylation levels of these proteins were inhibited by HSAE in macrophages after LPS stimulation(P<0.01,0.001).Conclusion HSAE has a two-way immunomodulatory effect on mouse macrophages,that is,immune promotion at rest and immune suppression at inflammatory activation,which may be related to its regulation of MAPK and NF-κB immune signaling pathways.