摘要
目的研究粉防己碱是否可以联合cGAMP作为cGAMP的激动剂增强cGAS-STING信号通路,从而探究粉防己碱的抗病毒功能。方法采用不同浓度的粉防己碱联合cGAMP分别处理THP1-Lucia-ISRE及RAW-Lucia-ISRE细胞,荧光素酶报告试验探究IRF3免疫应答水平;Western blot检测cGAS-STING信号通路激活情况;实时荧光定量PCR检测IFN-β、CXCL10及CCL5的mRNA表达水平;ELISA检测IFN-β表达情况。构建稳定表达STING-GFP基因的HeLa细胞(HeLa-STNG-GFP),粉防己碱联合cGAMP处理该细胞后,观察STING-GFP点聚集现象。Ⅰ型疱疹病毒(herpes simplex virus type 1,HSV-1)感染THP1-Lucia-ISG细胞,粉防己碱联合cGAMP处理细胞,Western blot及荧光强度探究粉防己碱的抗病毒能力。结果粉防己碱能够增强cGAMP介导的IRF3免疫应答,与cGAMP联用可激活cGAS-STING信号通路。粉防己碱联合cGAMP处理HeLa-STNG-GFP细胞后,观察到明显的STING-GFP点聚集现象。对感染HSV-1的THP1-Lucia-ISG细胞进行粉防己碱联合cGAMP孵育,HSV-1病毒滴度、HSV-糖蛋白D/UL30的表达及HSV-GFP荧光强度均显著下降。结论粉防己碱联合cGAMP可激活cGAS-STING信号通路从而增强宿主的抗病毒反应。
Objective To investigate whether tetrandrine could be used as an agonist of cGAMP to enhance the activation of cGAS-STING signaling pathway and analyze the antiviral function of tetrandrine.Methods THP1-Lucia-ISRE and RAW-Lucia-ISRE cells were incubated with different doses of tetrandrine in combination with cGAMP,respectively.IRF3 reporter activity was analyzed by luciferase reporter assay.Western blot was used to detect the activation of cGAS-STING signaling pathway.The expression of IFN-β,CXCL10 and CCL5 at mRNA level was quantified by real-time quantitative PCR.The expression of IFN-βat protein level was assessed by ELISA.HeLa cells stably expressing STING-GFP gene(HeLa-STNG-GFP cells)were constructed and stimulated with tetrandrine and cGAMP,then puncta-like structures were imaged by ZEISS LSM780.THP1-Lucia-ISRE cells were infected with herpes simplex virus type 1(HSV-1)in the presence or absence of tetrandrine or cGAMP.The antiviral function of tetrandrine was analyzed by Western blot and fluorescence intensity assay.Results Tetrandrine enhanced cGAMP-mediated IRF3 responses and activated cGAS-STING signaling pathway in combination with cGAMP.Tetrandrine combined with cGAMP triggered STING translocation and the formation of puncta-like structures in HeLa-STNG-GFP cells.The titer of HSV-1,the expression of HSV-glycoprotein D/UL30 and the fluorescence intensity of HSV-GFP were all decline after treating HSV-1-infected THP1-Lucia-ISRE cells with tetrandrine and cGAMP.Conclusions Tetrandrine combined with cGAMP activates cGAS-STING signaling pathway,thus enhancing the host antiviral response.
作者
王志文
张亚明
解智慧
Wang Zhiwen;Zhang Yaming;Xie Zhihui(Department of Laboratory Medicine,Huaihe Hospital of Henan University,Kaifeng 475000,China;Department of Otorhinolaryngology,Huaihe Hospital of Henan University,Kaifeng 475000,China;Department of Oncology,Huaihe Hospital of Henan University,Kaifeng 475000,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2024年第1期17-26,共10页
Chinese Journal of Microbiology and Immunology
