| Background and Objective:Licorice,a widely used traditional Chinese medicine,exhibits various pharmacological activities,including potent anti-inflammatory effects.Licorice exerts its anti-inflammatory effects through multiple mechanisms and has shown significant efficacy in the treatment of various inflammatory diseases.Recent research has highlighted the crucial role of the c GAS-STING signaling pathway in regulating inflammatory responses.The c GAS-STING pathway is an intracellular immune pathway responsible for detecting DNA damage and viral infections,triggering immune responses.Abnormal activation of the c GAS-STING pathway is closely associated with the development of inflammatory diseases.Targeted inhibition of this pathway has emerged as a novel approach for treating inflammatory conditions.Licorice’s anti-inflammatory effects may be attributed to its modulation of the c GAS-STING signaling pathway.This study aims to investigate the novel mechanism by which licorice exerts its anti-inflammatory effects by targeting the c GAS-STING pathway.By doing so,it seeks to enhance our understanding of the scientific basis for licorice’s "clearing heat and detoxification" properties and provide insights for the development of c GAS-STING inhibitors in the future.Method:In this study,we aimed to explore the active components of Licorice that exhibit anti-inflammatory effects through the c GAS-STING signaling pathway,building on previous studies from our research group.We used a Methionine and choline deficient diet(MCD)to induce Non-alcoholic steatohepatitis in mice,as well as a Trex1 gene knockout mouse model,to assess the anti-inflammatory effects of these active components and the impact of Licorice on the c GAS-STING signaling pathway.1.Mouse bone marrow-derived macrophages(BMDMs)and human THP-1 cells were treated with Licorice extract.The c GAS-STING signaling pathway was activated using HT-DNA,2’3’-c GAMP,or other STING agonists.Real-time quantitative PCR(RT-qPCR)was used to analyze the expression of related target genes,while western blot and immunofluorescence assay were used to analyze the expression of phosphorylated IRF3 protein and the nuclear shift of IRF3,respectively.These experiments aimed to investigate whether Licorice extract affects the activation of c GAS-STING signaling pathway.To evaluate the anti-inflammatory effect of Licorice extract in a mouse NASH model induced by MCD diet,the STING inhibitor C-176 was used as the control.Licorice extract(500 mg/kg)and/or intraperitoneal injection of STING inhibitor C-176 were administered every two days.After six weeks of treatment,pathological analysis of liver tissues was conducted,and plasma biochemical indices and m RNA expressions of α-SMA,Col1a1,IL-6,and TNF-a were measured to evaluate the anti-inflammatory effect of Licorice extract in the mouse NASH model.2.BMDMs and THP-1 cells were treated with Licochalcone B,followed by induction of the c GAS-STING signaling pathway with HT-DNA,2’3’-c GAMP or other STING agonists.The expression of related target genes was analyzed by RT-qPCR,and the expression of phosphorylated IRF3 protein was analyzed by western blotting.Immunofluorescence assay was used to analyze the nuclear shift of IRF3 to investigate the impact of Licochalcone B on the c GAS-STING signaling pathway.The mechanism of Licochalcone B on the c GAS-STING signaling pathway was explored through co-immunoprecipitation and pull-down experiments.In mice,Licochalcone B was administered intraperitoneally(20 mg/kg or 40 mg/kg)for one hour,followed by intraperitoneal injection of the STING agonist CMA to induce the production of type I interferon and pro-inflammatory factors.The expression levels of type I interferon and inflammatory factors were determined in mice intraperitoneal lavage solution and serum 4 hours later to evaluate the in vivo effects of Licochalcone B.Furthermore,in Trex1 knockout mice,Licochalcone B was injected intraperitoneally(40 mg/kg)for 15 consecutive days.Pathological analysis and RT-qPCR were performed on core,liver,intestine,tongue,and other tissues to observe the anti-inflammatory effect of Licochalcone B in the Trex1 knockout mouse model.Results:1.Licorice extract was found to inhibit the activation of the c GAS-STING signaling pathway in BMDMs and THP-1 cells induced by HT-DNA,as well as the activation of the pathway induced by various STING agonists.Mechanistically,the Licorice extract inhibited the activation of the downstream signaling pathway by preventing STING oligomerization.In vivo experiments showed that licorice extract was effective in reducing inflammation and fibrosis in MCD diet-induced NASH model mice.Interestingly,combined treatment with licorice extract and C-176 did not result in any additional benefits over individual treatment with licorice extract or the STING inhibitor C-176 alone in the NASH model mice.These findings suggest that licorice extract inhibits inflammation and liver fibrosis in NASH model mice by suppressing the c GAS-STING signaling pathway.2.Licochalcone B was found to have the ability to inhibit the activation of the c GAS-STING signaling pathway both in vitro and in vivo.Specifically,it was able to inhibit the activation of the downstream signaling pathways by targeting the STING-TBK1-IRF3 signaling axis.In vivo studies showed that Licochalcone B was able to effectively inhibit the elevated levels of type I interferon and inflammatory cytokines induced by STING agonist CMA in mice.Furthermore,in Trex1 knockout mice,Licochalcone B was found to significantly improve the pathological changes associated with Trex1 knockout and inhibit the expression of inflammatory factors.These results suggest that Licochalcone B has strong anti-inflammatory properties and may be a promising therapeutic agent for the treatment of inflammation and autoimmune diseases.Conclusion: In summary,Licorice extract and Licochalcone B have been identified as inhibitors of the c GAS-STING signaling pathway,indicating their potential as candidate compounds for the development of c GAS-STING signaling pathway inhibitors. |
