摘要
目的在固有免疫细胞中明确18β-甘草次酸对I型干扰素通路的激动活性,并评价其在肝癌模型小鼠中的协同抑瘤作用。方法CCK-8法检测18β-甘草次酸对RAW264.7巨噬细胞增殖的影响;qRT-PCR法检测18β-甘草次酸对免疫细胞[RAW264.7细胞、小鼠骨髓来源巨噬细胞(bone marrow-derived macrophages,BMDM)、小鼠骨髓来源树突状细胞(bone marrow-dendritic cells,BMDC)]中干扰素β1(interferon beta 1,Ifnb1)、干扰素刺激基因15(interferon stimulated gene 15,Isg15)和干扰素诱导蛋白1(interferon-induced protein with tetratricopeptide repeats 1,Ifit1)mRNA表达的影响;qRT-PCR法检测18β-甘草次酸与干扰素α2(interferon alpha 2,IFNα2)联用对免疫细胞中Isg15和Ifit1 mRNA表达的影响;Western blotting法检测18β-甘草次酸对IFNα2诱导的RAW264.7细胞Janus激酶1(Janus kinase 1,JAK1)、酪氨酸激酶2(tyrosine kinase 2,TYK2)、信号传导及转录激活因子1(signal transducers and activators of transcription 1,STAT1)和STAT2磷酸化水平的影响。C57BL/6小鼠通过原位移植瘤手术建立肝癌模型,随机分为模型组、奥沙利铂组和奥沙利铂+18β-甘草次酸组,给予奥沙利铂和18β-甘草次酸治疗,评价小鼠体质量变化、肿瘤质量、脾质量、肝质量和肝脏系数。结果18β-甘草次酸显著升高了Ifnb1、Isg15和Ifit1的mRNA表达水平(P<0.05、0.01、0.001),协同增强了IFNα2诱导的Isg15和Ifit1的mRNA表达(P<0.05、0.01、0.001);18β-甘草次酸促I型干扰素的活性可能依赖于其对IFNα2诱导的下游信号转导关键蛋白JAK1、TYK2、STAT1、STAT2磷酸化的增强作用(P<0.05、0.01、0.001)。18β-甘草次酸与奥沙利铂联用显著抑制了小鼠肝细胞癌原位移植瘤的恶性生长(P<0.01),并改善了奥沙利铂毒性相关的体质量下降。结论18β-甘草次酸能够激活JAK-STAT信号并诱导I型干扰素下游ISGs的表达,同时能与奥沙利铂协同抑制肝癌生长,从而发挥激活肿瘤免疫的作用。
Objective To identify the regulative effects of 18β-glycyrrhetinic acid(18β-Ga) on type Ⅰ interferon(IFN-Ⅰ) responses in innate immune cells, and evaluate its antitumor effects in hepatocellular carcinoma(HCC) model mice. Methods CCK-8 assay was performed to detect the effect of 18β-Ga on proliferation of RAW264.7 cells. qRT-PCR was used to detect the effect of 18β-glycyrrhetinic acid on interferon beta 1(Ifnb1), interferon stimulated gene 15(Isg15) and interferon-induced protein 1(Ifit1) m RNA expressions in immune cells [RAW264.7 cells, mouse bone marrow-derived macrophages(BMDM), mouse bone marrow-derived dendritic cells(BMDC)]. q RT-PCR analysis was used to detect the m RNA levels of Isg15 and Ifit1 upon 18β-Ga and IFNα2 administration in immune cells. The phosphorylation levels of Janus kinase 1(JAK1), tyrosine kinase 2(TYK2), signal transducers and activators of transcription 1(STAT1) and STAT2 were detected by western blotting. Hepatocellular carcinoma model was established by orthotopic tumor transplantation, and model mice were randomly divided into model group, oxaliplatin group, oxaliplatin + 18β-Ga group. Mice were treated with oxaliplatin and 18β-Ga after modeling. Body weight, tumor weight, spleen weight, liver weight and liver coefficient were evaluated. Results 18β-Ga significantly increased the m RNA levels of Ifnb1, Isg15 and Ifit1 expression(P < 0.05, 0.01, 0.001) and facilidated IFNα2-induced Isg15 and Ifit1 expressions(P < 0.05, 0.01, 0.001). IFN-I-promoting activity of 18β-Ga may depend on the enhancement of JAK1, TYK, STAT1, and STAT2 phosphorylation(P < 0.05, 0.01, 0.001). The combination of 18β-Ga and oxaliplatin significantly inhibited the tumor growth of hepatocellular carcinoma orthotopic xenograft tumors in mice(P < 0.01), and improved the weight loss associated with oxaliplatin toxicity. Conclusion 18β-Ga has significant stimulatory effects on immune response by enhancing JAK-STAT signaling and inducing the ISGs expression in response to IFN-Ⅰ. Meanwhile, 18β-Ga c
作者
李硕
白金钊
刘闰平
LI Shuo;BAI Jin-zhao;LIU Run-ping(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《中草药》
CAS
CSCD
北大核心
2022年第16期5034-5043,共10页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(82004029)
北京市科技新星项目(Z201100006820025,Z211100002121167)
中华中医药学会青年人才托举工程(CACM-2020-QNRC2-04)。
关键词
18Β-甘草次酸
Ⅰ型干扰素
巨噬细胞
树突状细胞
肝癌
免疫调控
18β-glycyrrhetinic acid
type Ⅰ interferon
macrophage
dendritic cell
hepatocellular carcinoma
immunoregulation
