青蒿双琥酯通过保护线粒体抑制cGAS-STING通路减轻LPS诱导的巨噬细胞炎性反应OA

Objective Endotoxemia is a common high-risk subtype of sepsis,primarily caused by lipopolysaccharide(LPS)released from Gram-negative bacteria.Macrophages,essential components of the innate immune system,represent one of the most critical defense cells within the immune system once LPS breaches physical barriers;LPS can activate macrophages and induce a severe inflammatory response.Mitochondria,vital organelles regulating cellular metabolism and inflammatory responses,undergo structural and functional damage that further exacerbates the inflammatory activation of macrophages.This study aims to investigate the inhibitory effects of a novel artemisinin derivative,disuccinate artemisinin(DA),on macrophage mitochondrial damage and inflammatory responses induced by low-concentration LPS,as well as the interrelationships between these effects and the preliminary mechanisms of action.Methods Low-concentration LPS(20 ng/mL)was used to induce mitochondrial damage and inflammatory cytokine expression in the mouse monocyte/macrophage cell line RAW264.7.Cell toxicity of DA at concentrations of 0,1,2,4,8,16,32,and 64 µg/mL was assessed using CCK-8 assay(n=6);qPCR was employed to monitor the dynamic changes in mRNA levels of TNF-α,IL-6 and IL-1β in RAW264.7 cells at 0,1/12,1/6,1/3,0.5,1,2,4,8,24,and 48 h after stimulation with 20 ng/mL LPS(n=3 or 4).qPCR was further performed to assess the effects of 5,10,and 20 µg/mL DA on the expression of the aforementioned genes in RAW264.7 cells stimulated with 20 ng/mL LPS(n=3 or 4).RAW264.7 cells were divided into the control group(medium),20 µg/mL DA group,20 ng/mL LPS group,LPS+DA(5 µg/mL)group,LPS+DA(10 µg/mL)group,and LPS+DA(20 µg/mL)group.Western blotting was applied to detect the expression of nuclear factor kappa B p65(NF-κB p65),NF-κB p50,and NF-κB inhibitor alpha(IκB-α)in cytoplasmic and nuclear proteins(n=3).RAW264.7 cells were divided into a control group(medium),a 10 µg/mL DA group,a 20 ng/mL LPS group,and an LPS+DA(10 µg/mL)group.Western blotting was applied to detect the expression of Toll-like receptor 4(TLR4)pathway proteins in total cell lysates(n=3).RAW264.7 cells were divided into a control group(medium),a 20 ng/mL LPS group,and an LPS+DA(10 µg/mL)group.Western blotting,fluorescence imaging,and qPCR were utilized to detect the expression of p-NF-κB p65(Ser536),inhibitor of κB kinase α(IKKα),inhibitor of κB kinase β(IKKβ),p-IKKα/β(Ser176/180)and cGAS-STING pathway proteins in total cell lysates(n=3).The mitochondrial morphology,membrane potential(n=5),and reactive oxygen species(ROS)(n=4)were observed.Free mitochondrial DNA and chromosomal DNA in the cytoplasm were also detected(n=4).Results The IC10 value of DA was 13.2 µg/mL for the proliferation of RAW264.7 cells.Peak TNF-α expression occurred at 1 h after LPS stimulation(P<0.05),that of IL-6 at 24 h(P<0.05),and that of IL-1β at 4 h(P<0.05).DA sustainably downregulated the expression of these LPS-induced genes.Compared with 20 ng/mL LPS,DA also downregulated the expression of TNF-α,IL-6,and IL-1β genes in a concentration-dependent manner(P<0.05),with an optimal concentration of 10 µg/mL(<IC10).Western blotting showed that,compared with 20 ng/mL LPS,DA concentration-dependently inhibited LPS-induced IκB-α degradation,reduced the nuclear translocation of NF-κB p50 and NF-κB p65,and decreased p-NF-κB p65(Ser536)levels,thereby inhibiting the activation of NF-κB(P<0.05).DA exerted no significant effect on the TLR4 pathway(P>0.05),but it did inhibit the activation of p-IKKα/β(Ser176/180)(P<0.05).Mitochondrial damage assays demonstrated that,compared to 20 ng/mL LPS,DA inhibited LPS-induced mitochondrial fragmentation,membrane potential decline,ROS production,and mitochondrial DNA release(P<0.05),thereby downregulating cGAS,p-STING(Ser366),p-TBK1(Ser172),and p-IRF3(Ser396)(P<0.05),thereby inhibiting the activation of the cGAS-STING pathway.Conclusion DA mitigates mitochondrial damage induced by low-concentration LPS in macrophages,thereby inhibiting the activation of cGAS-STING pathway to alleviate mitochondria-related inflammatory responses.DA is considered an anti-inflammatory candidate drug with mitochondrial protective properties.