Micheliolide attenuates sepsis-induced acute lung injury by suppressing mitochondrial oxidative stress and PFKFB3-driven glycolysis

Abstract

Background: Sepsis is a potentially fatal condition with a significant risk of death. Acute lung injury (ALI) is a life-threatening complication of sepsis, and the inflammatory response plays a critical role in sepsis-induced ALI. The protective effects of micheliolide (MCL) against renal fibrosis and leukemia have been demonstrated, but the precise underlying mechanisms remain unclear.

Methods: In vitro, lipopolysaccharides (LPS) and interferon-gamma (IFN-γ) were used to stimulate RAW264.7 cells and bone marrow-derived macrophages (BMDMs) to investigate the protective effect of MCL on sepsis-induced ALI. Cecal ligation and puncture (CLP) models were constructed in mice to induce ALI in vivo. The expression of inflammatory factors, macrophage polarization markers, and the glycolysis-related enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) were measured in vivo. Mitochondrial function, oxidative stress, and mitochondrial-related proteins were evaluated in vitro.

Results: MCL inhibited CLP-induced ALI, as evidenced by improvements in proinflammatory factor levels, lung wet/dry ratios, and histopathological findings. In vitro, MCL treatment significantly suppressed LPS + IFN-γ-induced M1-type polarization of RAW264.7 cells and BMDMs, as well as the production of inflammatory factors and oxidative stress. Mechanistic experiments revealed that MCL suppresses PFKFB3-driven glycolysis to reduce inflammation and activates the mitochondrial unfolded protein response (UPR^mt) to alleviate mitochondrial stress. However, the therapeutic effect of MCL was diminished when PFKFB3 was overexpressed in cells.

Conclusion: This study is the first to demonstrate that MCL attenuates sepsis-induced ALI by reducing M1-type macrophage polarization. Its therapeutic effect is closely related to the suppression of oxidative stress and PFKFB3-driven glycolysis.

Keywords: Acute lung injury; Glycolysis; Macrophage polarization; Oxidative stress; PFKFB3.

Fig. 1

MCL attenuated sepsis-induced ALI in vivo. A Lung tissue was analyzed using H&E staining (upper panel: 40 × , scale bar = 500 μm; bottom panel: 400 × , scale bar = 50 μm). B The lung injury score of lung tissues. C Measurement of the W/D ratio in the lungs of mice. n = 5. D Inflammatory cytokines IL-1β, IL-6, and TNF-α production in blood. n = 5. E Inflammatory cytokines IL-1β, IL-6, and TNF-α production in BALF. n = 5. F Expression of IL-1β, IL-6, and TNF-α in lung tissue detected using WB. n = 3. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant, compared with the CLP group

Fig. 2

MCL ameliorates M1-type macrophage polarization and the inflammatory response induced by LPS and IFN-γ. A MCL structure. B, C RAW264.7 cells and BMDMs were exposed to MCL for 24 h, and cell viability was determined by CCK8. D Protein expression of IL-1β, IL-6, and TNF-α in RAW264.7 cells. E Protein expression of IL-1β, IL-6, and TNF-α in BMDMs. F Levels of IL-1β, IL-6, and TNF-α in the supernatants of RAW264.7 cells (MCL: 10 μM). G Levels of IL-1β, IL-6, and TNF-α in the supernatants of BMDMs (MCL: 5 μM). H Protein expression of iNOS in RAW264.7 cells. I Protein expression of iNOS in BMDMs. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant, compared with the IFNγ + LPS group (n = 3)

Fig. 3

Targets of MCL acting on BMDMs. A Volcano plot of upregulated and downregulated genes (I_L_M means IFNγ + LPS + MCL group, I_L means IFNγ + LPS group). B GO enrichment analysis for IFNγ + LPS + MCL versus IFNγ + LPS. C Heatmap of genes related to inflammation. D Top 10 KEGG pathways enriched in IFNγ + LPS + MCL versus IFNγ + LPS. E Heatmap of genes in the glutathione pathway

Fig. 4

MCL attenuates mitochondrial oxidative stress in macrophages. A, C The GSH:GSSG ratio, SOD activity, and MDA production were measured in BMDMs. D, E Mitochondrial ROS production was measured by MitoSOX™ Red in RAW264.7 cells and BMDMs (scale bar = 10 μm). F, G Mitochondrial membrane potential was estimated using JC-1 in both RAW264.7 cells and BMDMs (scale bar = 10 μm). H Protein levels of LONP1, CLpP, Drp1, and MFN1 were estimated in BMDMs using WB. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001, compared with the IFNγ + LPS group (n = 3)

Fig. 5

MCL regulates M1-type macrophage polarization by reducing mitochondrial oxidative stress. (A-D) BMDMs treated with H₂O₂ (100 μM). A The GSH:GSSG ratio, SOD activity, and MDA production. B Mitochondrial membrane potential was estimated using JC-1 (scale bar = 10 μm). C Mitochondrial ROS production was measured by MitoSOX™ Red (scale bar = 10 μm). D iNOS protein level was detected using WB. E–H Pretreated BMDMs with NAC (2 mM) and MCL (5 μM). E The GSH:GSSG ratio, SOD activity, and MDA production. F Mitochondrial membrane potential was estimated using the JC-1 kit (scale bar = 10 μm). G Mitochondrial ROS production was measured by MitoSOX™ Red (scale bar = 10 μm). H iNOS protein level was analyzed using WB. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ns = not significant (n = 3)

Fig. 6

MCL regulates PFKFB3-driven glycolysis. A Volcano plot showing PFKFB3 downregulation after MCL pretreatment (LPS + IFNγ + MCL group vs LPS + IFNγ group). B Relative mRNA expression of PFKFB3. C WB was used to quantify the protein levels of partial glycolysis-related proteins. D Levels of partial respiratory chain proteins were measured using WB. E Lactic acid levels in BMDM supernatants. F ATP levels. G iNOS and IL-1β expression after PFKFB3 overexpression. H IL-1β, IL-6, and TNF-α mRNA expression following PFKFB3 overexpression. I Mitochondrial membrane potential was estimated using the JC-1 kit (scale bar = 10 μm). J Mitochondrial ROS production was measured by MitoSOX™ Red (scale bar = 10 μm). K Change in the NADPH/NADP⁺ ratio after PFKFB3 overexpression. L PFKFB3 expression level after NAC pretreatment. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3)

Fig. 7

MCL protects mice from sepsis-induced ALI. A WB to assess iNOS and PFKFB3 levels in lung homogenates. B Expression of PFKFB3 was detected using immunohistochemistry (scale bar = 100 μm). C Lactic acid levels in mouse blood. D Expression of MPO and SOD was detected using immunohistochemistry (scale bar = 100 μm). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3)

Fig. 8

Micheliolide attenuates sepsis-induced ALI by suppressing oxidative stress and PFKFB3-driven glycolysis