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

Journal of Translational Medicine volume 23, Article number: 181 (2025) Cite this article

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 (UPRmt) 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.

Introduction

Sepsis results from an exaggerated immune response to an infection, leading to severe organ dysfunction and an increased risk of death. The lungs serve as a key defense mechanism, protecting the body from pathogens and foreign substances inhaled during respiration [1]. As such, acute lung injury (ALI) is the most common and severe manifestation of sepsis [2, 3].

Macrophages are considered critical cells in ALI and play essential roles in maintaining lung homeostasis. During sepsis, macrophages become activated and polarize into either M1 or M2 types. Excessive inflammatory activation of macrophages contributes to immune system imbalance in sepsis-induced ALI.

Accumulating evidence suggests that macrophages exhibit distinct metabolic profiles in response to their microenvironment. M1-type macrophage polarization is always accompanied by metabolic shifts from oxidative phosphorylation to glycolysis [4, 5], while M2-type polarization follows the opposite metabolic pattern. Recent findings indicate that macrophage functions are closely linked to glycolysis, and modulating glycolysis can improve macrophage immune function [6]. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) regulates glycolysis by controlling the rate-limiting enzyme PFK-1. Recent studies have shown that PFKFB3 plays a key role in modulating the inflammatory response and glycolysis in immune cells [7,8,9]. However, the exact function of PFKFB3 during the immune response remains unclear.

Oxidative stress is a common pathogenic mechanism in many inflammation-related diseases. Mitochondria are the center of cellular stress and the primary organelles responsible for reactive oxygen species (ROS) production. While moderate ROS levels help kill pathogens, excessive ROS, as often seen in sepsis, can damage lung tissue and exacerbate cellular inflammation. Patients treated with melatonin, whose therapeutic effect on sepsis is attributed to its antioxidant properties, have shown lower mortality risks. Therefore, regulating oxidative stress is an important therapeutic approach for ALI. Meanwhile, ROS can stabilize HIF-1α expression and promote downstream glycolytic pathways, but the relationship between ROS and glycolysis remains unclear.

Micheliolide (MCL) is a naturally occurring plant terpenoid from the Asteraceae family and can also be synthesized from parthenolide (PTL). MCL has demonstrated antitumor and immunomodulatory effects. Previous research has shown that MCL can suppress the growth of primary acute myeloid leukemia cells, as well as leukemia stem and progenitor cells [10]. MCL induces apoptosis via the endogenous apoptosis pathway, binding covalently with Sec at position 498 of TrxR, thereby inhibiting the biological function of TrxR and enhancing oxidative stress-induced apoptosis in hepatocellular carcinoma [11]. In inflammatory diseases, MCL reduces the inflammatory response by inhibiting the NF-κB and PI3K/AKT pathways [12, 13]. Additionally, MCL attenuates atherosclerosis by targeting the KEAP1/NRF2 interaction [14]. However, the specific targets and roles of MCL in macrophage polarization, glycolysis, and sepsis-induced ALI inflammation require further exploration.

Method

Materials

Micheliolide (MCL, CAS: 68,370-47-8) was obtained from YUANYE (China), and LPS, IFNγ, and N-acetylcysteine (NAC) were sourced from Sigma-Aldrich (USA). ELISA kits for mouse IL-1β, TNF-α, and IL-6 were purchased from Cusabio (China).

Animals

All animal experiments were conducted in accordance with National Institutes of Health guidelines and approved by the Southern Medical University Committee on Ethics of Animal Experiments. Male C57 mice, aged 6–8 weeks, were obtained from the Animal Center at Southern Medical University (application no.: IACUC-LAC-20230705-001). The mice were housed under standard conditions with a 12-h light/dark cycle and had free access to food and water.

The mice were randomly divided into four groups: sham, CLP, CLP + L-MCL (low-dose group, 20 mg/kg), and CLP + H-MCL (high-dose group, 40 mg/kg). The CLP model was established 1 h after MCL intraperitoneal injection. Similarly, the sham and CLP groups received equal volumes of saline. For the CLP model, mice were anesthetized with 2% pentobarbital, the abdominal cavity was opened, and the cecum was ligated with 4–0 silk thread and punctured with a 22G needle. The abdominal cavity and skin were then sutured. Finally, the mice were injected subcutaneously with 1 mL of saline solution. After 24 h, the mice were euthanized by an overdose of pentobarbital.

H&E staining

Paraffin-embedded lung sections from the mice were stained with hematoxylin (Leagene, China) and eosin (Leagene, China), and images were captured using microscopy.

IHC staining

Mouse lung sections were incubated overnight at 4 °C with primary antibodies targeting SOD (Proteintech, China), PFKFB3 (Proteintech, China), and MPO (Proteintech, China). The sections were then treated with a secondary HRP-linked antibody (GK500710, Gene Tech) for 30 min and briefly exposed to DAB solution. The nuclei were counterstained with hematoxylin, and the sections were sealed with neutral gum.

Cell culture

RAW 264.7 cells, derived from mouse macrophages, were obtained from the Cell Bank of the Chinese Academy of Sciences (China). The cells were cultured in DMEM (GIBCO, USA) supplemented with 10% FBS (Excell, China) at 37 °C in a humidified atmosphere with 5% CO2. Bone marrow-derived macrophages (BMDMs) were isolated from the tibias and femurs of sacrificed mice and cultured for 6–7 days in DMEM containing 20% FBS, 1% penicillin/streptomycin (NSM, China), and 20 ng/mL M-CSF (MCE, China).

Assessment of lung moisture content ratio

After recording the original lung weight, the lungs were baked in a 65 °C oven for 48 h until the weight stabilized.

Cytokine measurements

Cytokine concentrations in cell supernatants, serum, and BALF were measured using ELISA kits for IL-1β, IL-6, and TNF-α (Cusabio, China) following the manufacturer’s instructions.

Measurement of GSH/GSSG, MDA production, SOD activity, lactate production, and ATP production

Kits for lactate production assay (A019-2–1) were purchased from Nanjing Jiancheng (Nanjing, China). Kits for GSH/GSSG (S0053), SOD (S0103), lipid peroxidation MDA (S0131S), and ATP production (S0027) were obtained from Beyotime (Nanjing, China). The assays were performed according to the manufacturer’s instructions.

Western blotting (WB)

Relative protein expression levels were determined by WB, as previously described [15]. The antibodies used in this study included PFKFB3 (13,763–1-AP, Proteintech), HK2 (22,029–1-AP, Proteintech), PKM2 (15,822–1-AP, Proteintech), β-actin (20,536–1-AP, Proteintech), Lonp1 (15,440–1-AP, Proteintech), ClpP (D163477, BBI), GAPDH (10,494–1-AP, Proteintech), iNOS (18,985–1-AP, Proteintech), IL-1β (41,610–1, Proteintech), IL-6 (bs-0782R, Bioss), and TNF-α (60,291–1-Ig, Proteintech).

qRT-PCR

RNA was extracted from samples using RNAiso (Takara, Japan), followed by reverse transcription using PrimeScript™ RT Master Mix (Takara, Japan). Quantitative PCR was conducted using TB Green™ Premix Ex Taq™ (RR420B, Takara) on the LightCycler® 480 system (Roche, Switzerland). The primers used in this study are presented in Table 1.

Table 1 The primers used in this study
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Mitochondrial superoxide detection

Mitochondrial superoxide levels were measured using MitoSOX™ Red (Invitrogen, USA) as a specific indicator for superoxide in mitochondria. Viable cells were incubated with MitoSOX™ Red (5 μM) for 15 min at 37 °C in the dark. After rinsing with warm PBS, the cells were observed using confocal microscopy (LSM880, Carl Zeiss).

Mitochondrial membrane potential detection

Viable cells were incubated with JC-1 (Beyotime, China) for 15 min at 37 °C in the dark. After rinsing with warm PBS, the cells were observed using confocal microscopy (LSM880, Carl Zeiss).

Statistical analysis

All data are presented as mean ± SD of at least three independent experiments. GraphPad Prism 9 was used for data analysis. ANOVA tests were applied to evaluate differences among multiple groups. p < 0.05 was considered statistically significant.

Results

MCL attenuated sepsis-induced ALI in vivo

Firstly, we established a CLP mouse model to examine the protective effects of MCL on sepsis-induced ALI. CLP caused changes such as edema, thickening of the alveolar space, and infiltration of inflammatory cells, while HE results showed that both high and low doses of MCL improved these changes (Fig. 1A, B). To evaluate lung injury and inflammatory responses, we calculated the wet-to-dry weight ratio and measured the concentrations of inflammatory cytokines in blood, BALF, and lung tissue after MCL treatment. Compared with the control group, we observed a high wet-to-dry weight ratio in lung tissues, and the proinflammatory mediators (IL-1β, IL-6, and TNF-α) in blood, bronchoalveolar lavage fluid( BALF), and lung tissue were significantly increased in the CLP group. These markers were reduced by MCL treatment, with the therapeutic effect being better in the high-concentration group than in the low-concentration group (Fig. 1C–F). These results indicated that MCL successfully suppresses inflammation in the sepsis-ALI mice model.

Fig. 1
figure 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

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MCL ameliorates M1-type macrophage polarization and the inflammatory response induced by LPS and IFN-γ

The impact of MCL on the survival and growth of RAW264.7 cells and bone marrow-derived macrophages (BMDMs) was assessed using the CCK-8 assay. The results showed that MCL exhibited minimal toxicity towards RAW264.7 cells and BMDMs at concentrations of 10 μM and 5 μM, respectively (Fig. 2B, C). We pretreated RAW264.7 cells and BMDMs with MCL for 1 h, followed by exposure to lipopolysaccharides (LPS; 100 ng/mL) and interferon-gamma (IFN-γ; 20 ng/mL) for 12 h. As shown in Fig. 2D, E, RAW264.7 cells and BMDMs expressed higher levels of inflammatory factors (pro-IL-1β, IL-6, and TNF-α) after LPS and IFN-γ stimulation. In contrast, MCL treatment reduced the expression of these inflammatory factors. ELISA results were consistent with the WB findings (Fig. 2F, G). Additionally, we used WB to assess the expression of iNOS, a marker for M1 macrophage polarization. WB results revealed that MCL reduced the expression of the M1-type polarization marker iNOS (Fig. 2H, I). In conclusion, MCL inhibits M1-type macrophage polarization and reduces the inflammatory response induced by LPS and IFN-γ.

Fig. 2
figure 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)

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MCL attenuates mitochondrial oxidative stress in macrophages

Transcriptome sequencing was performed on MCL-treated and untreated BMDMs. Volcano plots demonstrated the significance of differentially expressed genes in relation to fold changes. In the MCL-pretreated group, 1097 genes were upregulated, while 1721 genes were downregulated compared with the LPS + IFN-γ group (Fig. 3A). GO enrichment analysis indicated that MCL's effects were focused on cellular defense and inflammation, including cytokine-stimulated responses and the regulation of cytokine production processes (Fig. 3B). We highlighted changes in the downregulation of inflammation-associated genes (Fig. 3C). KEGG pathway analysis showed enrichment primarily in the PI3K-Akt signaling and Ras signaling pathways (Fig. 3D). Among these pathways, we observed that MCL also regulated the glutathione pathway, with upregulation of glutathione-related genes (Fig. 3D–E). Glutathione is a key antioxidant involved in the inflammatory response to oxygen free radicals, reducing cell damage.

Fig. 3
figure 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

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To further investigate MCL's effect on inflammation-related oxidative stress, we examined LPS and IFN-γ-induced oxidative stress in macrophages, which included increased MDA levels, decreased SOD activity, and depletion of the GSH/GSSG ratio. MCL treatment significantly restored these parameters (Fig. 4A–C). Mitochondria play an essential role in cellular inflammation and are the primary organelles for combating oxidative stress. Thus, we examined whether MCL affects mitochondrial function. Our results showed that MCL enhances mitochondrial membrane potential (Fig. 4D, E). MitoSOX Red staining revealed that mitochondrial ROS, which were elevated by LPS and IFN-γ stimulation, were inhibited by MCL (Fig. 4F, G). These findings indicate that MCL improves mitochondrial dysfunction caused by an inflammatory environment.

Fig. 4
figure 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)

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Mitochondrial quality control (MQC) helps maintain relatively low and stable levels of ROS [16]. MQC eliminates damaged mitochondrial components through processes such as mitochondrial proteases, the mitochondrial unfolded protein response (UPRmt), mitochondrial dynamics, and mitochondrial autophagy/mitophagy [17] Mitochondrial damage activates UPRmt [18], 19, which promotes the refolding and degradation of misfolded proteins in mitochondria. In our study, MCL at a high dose induced the expression of LONP1 and ClpP, suggesting that UPRmt was activated by MCL (Fig. 4H). Given that the mitochondrial fission/fusion mechanism also responds to oxidative stress to maintain mitochondrial homeostasis [20], we investigated whether MCL influences these processes. To test this, we examined the protein levels of Drp1 and Mfn1, which are key players in the mitochondrial fission/fusion process (Fig. 4H). Surprisingly, MCL did not alter Drp1 or Mfn1 levels, suggesting that MCL might not affect the mitochondrial fission/fusion process. Differences in Drp1 and Mfn1 functional diversity and expression modifications may be associated with the disease model and tissue cell differences. Our data suggest that MCL alleviates mitochondrial oxidative stress and dysfunction by modulating UPRmt.

MCL regulates M1-type macrophage polarization by reducing mitochondrial oxidative stress

Mitochondrial oxidative stress is associated with macrophage polarization. To investigate the contribution of mitochondrial oxidative stress to macrophage polarization and verify the pharmacologic action of MCL, we stimulated macrophages with the ROS generator H₂O₂ (20 μM) and ROS scavenger NAC (2 mM) for 12 h. As shown in Fig. 5A, SOD activity and the GSH/GSSG ratio decreased, while MDA production increased in cells treated with H₂O₂. These effects were accompanied by an increase in JC-1 monomers and mitochondrial superoxide (Fig. 5B, C). Additionally, H₂O₂ treatment increased the expression of M1-type macrophage-related proteins (Fig. 5D). These results suggest that M1-type macrophage polarization is driven by elevated intracellular ROS levels. Conversely, MCL reduced oxidative stress induced by H₂O₂ and restored macrophage polarization. In an inflammatory environment, both NAC and MCL reversed the changes in intracellular SOD, GSH/GSSG, and MDA levels in LPS- and IFN-γ-treated cells (Fig. 5E). Moreover, mitochondrial transmembrane potential and mitochondrial ROS levels were reduced following NAC or MCL treatment (Fig. 5F, G). Alleviating mitochondrial stress also helped reduce M1-type macrophage polarization (Fig. 5H). Therefore, we conclude that MCL exerts an antioxidant effect on mitochondrial stress, thereby inhibiting M1-type macrophage polarization.

Fig. 5
figure 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)

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MCL regulates PFKFB3-driven glycolysis

Metabolic reprogramming is a key characteristic of macrophage polarization. M1 macrophages shift from oxidative phosphorylation to glycolysis. Transcriptome analysis revealed that the glycolysis-related enzyme PFKFB3 underwent significant changes after MCL intervention (Fig. 6A). Following LPS + IFN-γ induction, PFKFB3 mRNA levels increased significantly, while pre-incubation with MCL reduced the mRNA levels induced by LPS + IFN-γ (Fig. 6B). We also assessed whether MCL affects the expression of other key enzymes in glycolysis and oxidative phosphorylation-related complexes. The results showed a decreasing trend in the protein expression levels of glycolysis enzymes (HK2, PKM2, LDHA) after MCL treatment, although the changes were not significant compared with the LPS + IFN-γ group. Among these, PFKFB3 was the most significantly regulated glycolysis gene by MCL pretreatment (Fig. 6C). Further analysis revealed that MCL did not significantly affect the levels of proteins involved in the mitochondrial respiratory chain (SDHA, UQCRC1, NDUFB8, and ATP5a1), suggesting that MCL specifically targets the glycolytic process, with relatively limited effects on mitochondrial respiratory chain complexes (Fig. 6D). Lactic acid, the endproduct of glycolysis, serves as an indicator of glycolysis levels. We found that MCL reduced lactic acid secretion (Fig. 6E), while ATP levels increased after MCL treatment (Fig. 6F). These results indicate that MCL suppresses LPS + IFN-γ-induced glycolytic metabolism.

Fig. 6
figure 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)

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Previous studies have shown that PFKFB3 influences inflammatory responses through various mechanisms. To further explore the role of PFKFB3 in macrophage polarization, we overexpressed PFKFB3 in BMDMs. Our results demonstrated that increased PFKFB3 levels led to higher expression of IL-1β and iNOS proteins after LPS + IFN-γ stimulation (Fig. 6G), as well as elevated mRNA levels of IL-1β, TNF-α, and IL-6 (Fig. 6H). However, MCL reversed the proinflammatory effects of PFKFB3 overexpression. Thus, our findings revealed that MCL inhibits the inflammatory response by regulating the key glycolysis enzyme PFKFB3.

Next, we investigated whether PFKFB3 influences cellular oxidative stress. To clarify the relationship between PFKFB3 and oxidative stress, we examined mitochondrial ROS levels and mitochondrial membrane potential in BMDMs. The results showed that PFKFB3 overexpression increased mitochondrial superoxide production and elevated the mitochondrial membrane potential, indicating that PFKFB3 exacerbates mitochondrial stress (Fig. 6I, J). Previous research suggests that PFKFB3 redirects glucose flux from the pentose phosphate pathway (PPP) to glycolysis [21]. PFKFB3 overexpression reduced the NADPH/NADP + ratio, which indicated that PFKFB3 helped maintain the oxidative stress state, whereas MCL treatment increased the NADPH/NADP⁺ ratio (Fig. 6K). Finally, reversing mitochondrial oxidative stress with NAC reduced the PFKFB3 level elevation caused by inflammation (Fig. 6L). These results suggest that PFKFB3 promotes mitochondrial stress and that intracellular oxidative stress also enhances PFKFB3’s effects.

MCL protects mice from sepsis-induced ALI

The expression of PFKFB3 and iNOS in the lung tissue of mice subjected to the CLP model showed an increase in septic mice (Fig. 7A). Treatment with high doses of MCL decreased the CLP-induced levels of PFKFB3 and iNOS. Immunohistochemical analysis validated the WB results, indicating that MCL reduced pulmonary PFKFB3 expression in septic mice (Fig. 7B). Additionally, MCL lowered blood lactate levels in septic mice (Fig. 7C). MCL treatment also increased lung SOD levels and reduced MPO in septic mice (Fig. 7D).

Fig. 7
figure 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)

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Discussion

This study demonstrated that MCL could regulateregulates PFKFB3-mediated glycolysis and intracellular oxidative stress, reducing M1 macrophage polarization and mitigating septic lung damage. The key findings are as follows: (i) MCL alleviates lung damage in septic mice; (ii) MCL modulates mitochondrial oxidative stress, which drives macrophage polarization toward the M1 phenotype; and (iii) MCL limits PFKFB3-mediated glycolysis to reduce the inflammatory response.

The inflammatory response often coincides with elevated intracellular free radicals, diminished antioxidant capacity, and an imbalance between oxidants and antioxidants. This imbalance leads to ROS accumulation, causing cellular damage and exacerbating inflammation [22]. ROS play a key role in promoting and sustaining the M1 phenotype in macrophages. Therefore, controlling oxidative stress is crucial for reducing inflammation. Our study found that increased mitochondrial oxidative stress contributes to M1-type macrophage polarization, while MCL helps reduce this polarization. MCL demonstrated similar effectiveness to the antioxidant NAC in diminishing M1-type macrophage polarization by managing mitochondrial oxidative stress. However, ROS influence both proinflammatory and anti-inflammatory responses [23, 24], and the variance may depend on the context, with factors like cell differentiation stage and ROS levels influencing M1 or M2 polarization. Further research is needed to clarify the effects of MCL on the M2 macrophage phenotype and the factors that determine whether ROS production triggers M1 or M2 signaling pathway activation.

In our study, MCL also activated UPRmt, a pathway that targets misfolded proteins in the mitochondrial matrix and responds to stress from unfolded or misfolded mitochondrial proteins. In senescent cells, UPRmt reduces mitochondrial damage and enhances mitochondrial function [25]. Unfortunately, this study did not explore the precise molecular mechanisms through which MCL influences UPRmt or how UPRmt specifically manages mitochondrial stress.

Multiple studies have shown that glycolysis-related genes and pathways are significantly upregulated in sepsis [26, 27]. Previous research indicated that MCL targets PKM2 to inhibit leukemia cell growth [28], but the mechanism of MCL in modulating glycolysis during inflammatory responses remains unclear. Our previous study demonstrated that PFKFB3 promotes LPS-induced lung injury [29]. In this study, we found that MCL significantly counteracted the proinflammatory effects of PFKFB3 overexpression.

We also investigated the relationship between increased PFKFB3 and oxidative stress. Overproduction of ROS has been shown to significantly affect glycolysis in acute myeloid leukemia [30]. PFKFB3 overexpression reduces the NADPH/NADP + ratio and increases mitochondrial stress, indicating that PFKFB3 exacerbates cellular oxidative stress and impairs mitochondrial function. Treatment with antioxidants such as NAC and MCL reduced PFKFB3 expression, suggesting that alleviating ROS reduces inflammatory glycolysis. Our results reveal that ROS and glycolysis are not in an upstream–downstream relationship but rather mutually influence each other.

In conclusion, our data demonstrate, for the first time, the effect of MCL in regulating mitochondrial function, macrophage polarization, and glycolysis in septic lung injury. MCL reverses inflammation-compromised glycolysis and mitochondrial dysfunction driven by PFKFB3. These findings suggest that MCL may be a potential therapeutic agent for treating septic lung injury.

Conclusion

Our study demonstrates that MCL regulates macrophage proinflammatory polarization and reduces the production of inflammatory factors by mitigating intracellular oxidative stress. Furthermore, MCL controls the production of PFKFB3, a key enzyme in glycolysis, leading to decreased cellular glycolysis and, ultimately, reduced inflammation. Glycolysis and ROS mutually reinforce each other to amplify the inflammatory response. Therefore, MCL could be a promising therapeutic option for treating lung injury caused by sepsis (Fig. 8).

Fig. 8
figure 8

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

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Availability of data and materials

The datasets generated are available from the corresponding author on reasonable request.

Abbreviations

ALI:

Acute lung injury

BMDM:

Bone marrow-derived macrophages

CCK-8:

Cell counting kit-8

CLP:

Cecal ligation and puncture

IFN-γ:

Interferon-gamma

IHC:

Immunohistochemical

IL-1β:

Interleukin-1β

IL-6:

Interleukin-6

LPS:

Lipopolysaccharides

MCL:

Micheliolide

MDA:

Malondialdehyde

NAC:

N-acetylcysteine

SOD:

Superoxide dismutase

PFKFB3:

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3

PTL:

Parthenolide

ROS:

Reactive oxygen species

PCR:

Polymerase chain reaction

TNF-α:

Tumor necrosis factor-α

UPRmt :

Mitochondrial unfolded protein response

WB:

Western blotting

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Acknowledgements

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Funding

This work was supported by the National Natural Science Foundation of China (82270089, 81870068) and National College Students' Innovation and Entrepreneurship Training Program (202312121006).

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Author notes

  1. Wenhan Li, Yuhan Li and Linjie Xiao contribution equally to this work.

Authors and Affiliations

  1. Department of Respiratory and Critical Care Medicine, Nanfang Hospital, Southern Medical University, 1838 Guangzhou North Road, Guangzhou, 510515, China

    Wenhan Li, Linjie Xiao, Zhanzhan Xie, Jun Peng & Ying Meng

  2. The Second School of Clinical Medicine, Southern Medical University, Guangzhou, China

    Yuhan Li

  3. Department of Respiratory and Critical Care Medicine, Guangdong Provincial Key Laboratory of Major Obstetric Diseases, Guangdong Provincial Clinical Research Center for Obstetrics and Gynecology, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510000, China

    Wenhui Huang

  4. Department of Emergency Medicine, Nanfang Hospital, Southern Medical University, 1838 Guangzhou North Road, Guangzhou, 510515, China

    Xu Li

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  1. Wenhan Li
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Contributions

Wenhan Li: Conceptualization, Data curation, Investigation, Visualization, Writing–original draft. Yuhan Li:Linjie Xiao: Data curation, Investigation, Visualization. Zhanzhan Xie: Jun Peng: Data curation, Investigation. Wenhui Huang: Conceptualization, Writing–original draft. Xu Li: Conceptualization, Funding acquisition, Supervision. Ying Meng: Conceptualization, Funding acquisition, Supervision.

Corresponding authors

Correspondence to Xu Li or Ying Meng.

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Ethics approval and consent to participate

All mice experiments were approved by the Southern Medical University Committee on Ethics of Animal Experiments (application NO: IACUC-LAC-20230705–001).

Competing interests

The authors declare that they have no competing interests.

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Li, W., Li, Y., Xiao, L. et al. Micheliolide attenuates sepsis-induced acute lung injury by suppressing mitochondrial oxidative stress and PFKFB3-driven glycolysis. J Transl Med 23, 181 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05906-0

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Journal of Translational Medicine

ISSN: 1479-5876

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