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.

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).

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.

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.