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-β at 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.
Tetrandrine enhances the host antiviral response through cGAMP-mediated cGAS-STING signaling pathway
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-β at 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.
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