Scutellarin suppresses Mycobacterium tuberculosis-induced pyroptosis in macrophages by inhibiting the HIF-1α-mediated Warburg effect

ABSTRACT

Background:

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains a major global health threat due to prolonged treatment and drug-resistant strains. Host-directed therapy (HDT), which modulates host-pathogen interactions, offers potential to shorten treatment and limit resistance. This study investigates the effects of Scutellarin (SCU), a flavonoid from Scutellaria baicalensis, on Mtb-infected macrophages within the HDT framework.

Methods:

Anti-pyroptotic and anti-inflammatory effects of SCU were assessed in Mtb-infected THP-1 and J774A.1 macrophages, and in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. Mitochondrial function was evaluated by oxygen consumption rate(OCR), membrane potential, and superoxide levels; glycolytic activity was measured by proton efflux rate (GlycoPER). Expression of inflammasome-related markers was analyzed by Western blot, qPCR, ELISA, immunofluorescence, and flow cytometry. The role of hypoxia-inducible factor 1-alpha (HIF-1α) was examined via siRNA knockdown.

Results:

SCU inhibited NLRP3 inflammasome activation, reduced IL-1β and IL-18 secretion, and attenuating pyroptosis. It restored mitochondrial integrity by regulating p-DRP1, MFN2, and Cytochrome C expression, and suppressed HIF-1α-mediated glycolytic reprogramming. Silencing of HIF-1α confirmed its role in SCU’s mechanism. In vivo, SCU reduced pulmonary inflammation and cytokine release in LPS-induced ALI.

Conclusion:

SCU alleviates Mtb-induced pyroptosis and inflammation in macrophages by inhibiting the HIF-1α-mediated Warburg effect.

Introduction

Despite the availability of effective treatments, the rapid spread of Tuberculosis(TB) and the growing issue of drug resistance continue to pose significant public health challenges, particularly in low-income countries and regions where prevention and control efforts face substantial barriers[1]. In recent years, despite continuous improvements in prevention and control measures, the incidence and mortality rates of TB have remained high. Notably, the COVID-19 pandemic has hindered progress in TB prevention and control[2]. Currently, major challenges in TB treatment include the prolonged treatment duration and the frequent emergence of drug-resistant strains. Therefore, optimizing treatment strategies to enhance efficacy and reduce the risk of drug resistance has become a key research focus.

HDT is increasingly recognized for its ability to modulate the host immune response and counteract the adaptive advantages of pathogens[3, 4]. Studies have identified host immune metabolism as a crucial link between metabolic regulation and immune function, playing a pivotal role in TB pathogenesis[5]. Among these metabolic changes, the reprogramming of immune cells, particularly the Warburg effect, is especially significant in Mtb infection. The Warburg effect refers to the phenomenon in which cells maintain high levels of glycolysis even under aerobic conditions. While commonly observed in tumor cells, this metabolic shift is also widespread in immune cells during infection[6–8].

Macrophages, the primary host cells of Mtb, undergo metabolic changes that directly impact the intensity of immune responses. Mtb infection can drive macrophages toward aerobic glycolysis, enabling rapid energy production for immediate immune defense. However, prolonged activation may lead to excessive pro-inflammatory cytokine release, contributing to chronic inflammation and tissue damage[9, 10]. Specifically, Mtb infection promotes the expression of crucial enzymes involved in glycolysis in macrophages while reducing the expression of enzymes associated with oxidative phosphorylation. Moreover, Mtb stimulates macrophages to secrete IL-1β, which, through the IL-1R1 signaling pathway, promotes the accumulation of lactate, further exacerbating macrophage activation[11]. In this process, HIF-1α plays a crucial role. HIF-1α not only enhances pro-inflammatory immune responses by promoting glycolysis but also regulates the inflammatory metabolic balance, making it critical for the host’s anti-infection immunity[12]. Therefore, HIF-1α could serve as a potential therapeutic target, providing new intervention strategies for tuberculosis treatment. It should be noted, however, that studies have found that different types of macrophages may exhibit distinct metabolic responses during Mtb infection, leading to variations in their immune responses[13].

Mitochondria play a critical role in maintaining cellular energy metabolism and regulating cell survival[14]. Research has shown that Mtb infection disrupts mitochondrial homeostasis in macrophages, leading to a decrease in mitochondrial membrane potential, increased mitochondrial fission, and excessive production of reactive oxygen species (ROS)[15]. Mitochondrial homeostasis relies on a dynamic balance between fission and fusion. The mitochondrial fission protein dynamin-related protein 1 (Drp1) is a key regulator of fission, promoting mitochondrial division by aggregating on the outer mitochondrial membrane to form fission complexes[16, 17]. Under oxidative stress or pathological conditions, abnormal Drp1 activation exacerbates mitochondrial damage and induces apoptosis. Conversely, the mitochondrial fusion protein MFN2 is essential for maintaining mitochondrial morphology and preserving metabolic homeostasis[18, 19]. MFN2 deficiency not only intensifies oxidative stress in macrophages but also promotes their polarization toward the M1 pro-inflammatory phenotype, further amplifying inflammation[20]. An imbalance between mitochondrial fission and fusion, coupled with ROS accumulation, creates a vicious cycle. Excessive ROS production can activate the NLRP3 inflammasome and exacerbate the inflammatory response[21, 22]. Additionally, ROS can stabilize the HIF-1α protein, enhancing IL-1β and glycolysis-related gene expression, thereby perpetuating a sustained pro-inflammatory positive feedback loop[23–25]. This mechanism sustains high glycolytic activity and worsens the inflammatory environment, ultimately driving chronic inflammation and immune dysfunction.

Although HDT has shown great promise in TB treatment, research on regulating host metabolic reprogramming and cellular processes through natural products remains limited. Scutellarin (SCU), a flavonoid compound derived from Scutellaria baicalensis and other Chinese medicinal herbs, has been shown to possess anti-inflammatory, antioxidant, and anti-apoptotic properties [26, 27]. However, no studies have yet explored the effects of SCU on Mtb infection and its underlying mechanisms.

This study, using an in vitro model of Mtb infection, evaluates for the first time the role of SCU in regulating the Warburg effect and maintaining macrophage mitochondrial homeostasis. The findings suggest that Mtb infection induces aerobic glycolytic metabolic reprogramming in macrophages, and SCU may prevent the overactivation of glycolysis by inhibiting HIF-1α. By suppressing aerobic glycolysis, SCU also modulates mitochondrial dynamics, restores the homeostatic expression of MFN2 and p-DRP1, stabilizes mitochondrial membrane potential, and reduces excessive ROS production, thereby mitigating the release of pro-inflammatory cytokines. The research demonstrates that SCU plays a crucial role in suppressing immune hyperactivation and alleviating pyroptosis, offering novel therapeutic targets and strategies for HDT. This study not only provides new insights into the application of SCU in tuberculosis immunotherapy but also further broadens its potential value in regulating host immunometabolism.

Materials and methods

Reagents

Scutellarin (purity 98%, PubChem CID: 185617) was obtained from Tauto Biotech (E-0554,Shanghai, China). Dimethyl sulfoxide (DMSO,D2650), lipopolysaccharide (LPS, L2630), and fetal bovine serum (F0193-500ML) were sourced from Sigma-Aldrich (Darmstadt, Germany). Dexamethasone (HY-14648) was purchased from MedChemExpress (New Jersey, USA). Radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B), Western and IP cell lysis buffer (P0013)were provided by Beyotime Biotechnology (Shanghai, China). An IL-1β ELISA kit (4,336,726) was obtained from R&D Systems (Minnesota, USA). Antibodies against PKM2 (4053), NLRP3 (15,101), Gasdermin D(39754),HMGB1 (3935), Cleaved Caspase-1 (89,332),HRP-conjugated anti-rabbit IgG (7074),and F4/80 (70,076) were purchased from Cell Signaling Technology (Massachusetts, USA). Anti-human caspase-1 (p20) (AG-20B-0048-C100) was sourced from Adipogen Life Sciences (California, USA). Antibodies against HIF-1α (DF12004) and ASC (sc-514414) were obtained from Santa Cruz Biotechnology (California, USA). Phospho-DRP1 (S616) antibody (HA72776) was a product of HUABIO (Hangzhou, China). Cytochrome C(A4912), HRP-conjugated anti-mouse IgG (AS003), phospho-DRP1-S616 (A1353), MFN2 (A19678), MFN2 (A13606), and HRP-conjugated β-actin (AC028) were purchased from ABclonal (Wuhan, China).

Cell culture

The murine macrophage cell line J774A.1 (ATCC) was cultured in DMEM (HyClone, USA) with 10% FBS, 100 U/mL penicillin G, and 0.1 mg/mL streptomycin (P1400,Solarbio, China) at 37°C in 5% CO₂. Human THP-1 cells (ETHEPHON, China) were cultured in RPMI-1640 (HyClone, USA) with 10% FBS, 100 U/mL penicillin G, and 0.1 mg/mL streptomycin, and differentiated into macrophages by 100 nM PMA treatment for 24 hours.

MTT assay

The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was used to assess cell proliferation. Briefly, cells were seeded into 96-well plates at a density of 2 × 10⁴ cells per well. After grouping, the cells were treated accordingly. Following 24 or 48 hours of treatment, the culture medium was aspirated, and 100 μL of MTT solution (5 mg/mL) was added to each well for 4 hours of incubation at 37°C. After carefully removing the solution, 150 μL of DMSO was added to each well, and the plate was gently shaken for 5 minutes at 37°C. The absorbance was measured at 490 nm using a microplate reader (Tecan, Switzerland).

Macrophage infection and treatment

On the day of infection, a suspension of Mtb H37Ra was added to the cells at a multiplicity of infection (MOI) of 10:1. Adherent THP-1-derived macrophages or J774A.1 cells were exposed to a Mycobacterium-containing medium at 37°C for 4 hours. Afterward, the cells were washed three times with sterile phosphate-buffered saline (PBS) and cultured in fresh medium, with or without different concentrations of SCU (20, 40, and 80 µM), for varying durations (6, 12, and 24 hours).

siRNA knockdown in THP-1 cells

After differentiating THP-1 cells into macrophages, HIF-1α silencing was achieved by transfecting the cells with HIF-1α siRNA (20 µM) and a corresponding negative control using the RiboFECT CP transfection kit (C10511,RiboBio, China), according to the manufacturer’s instructions. The sequences of HIF-1α siRNA were as follows: forward, 5′-CCUCAGUGUGGGUAUAAGA-3′; reverse, 5′-UCUUAUACCCACACUGAGG-3′. The siRNA was chemically synthesized by GenePharma (Shanghai, China), and the negative control siRNA was also obtained from GenePharma. Cells were incubated in transfection medium for 48 hours before proceeding with subsequent experiments.

Animal handling

Specific-pathogen-free (SPF) male C57BL/6 mice (6 weeks old, weighing 20–22 g) were purchased from Jihui Company (Shanghai, China) and housed at the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The mice were maintained under controlled environmental conditions with free access to food and water and were used after a two-week isolation and acclimatization period. All animal procedures were approved by the Animal Ethics Committee of the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The ALI model was induced in mice by intraperitoneal injection of LPS (10 mg/kg). Scutellarin (SCU) was dissolved in a solution of 1% DMSO and 99% saline. For treatment, mice in the SCU group received an intraperitoneal injection of SCU (25 or 50 mg/kg) one hour after LPS injection. The dexamethasone (DEX) group received an intraperitoneal injection of DEX (5 mg/kg) one hour post-LPS injection. The control group received an equivalent volume of saline. Twelve hours after LPS administration, all mice were euthanized for sample collection and analysis. Euthanasia was conducted by gradual-fill carbon dioxide (CO₂) inhalation in accordance with ethical guidelines.

A total of thirty mice were used in this study, and they were randomly assigned to groups(n = 6 per group) using a random number table. Every effort was made to minimize the number of mice used and to reduce their suffering. The study was approved by the Ethics Committee of Shanghai University of Traditional Chinese Medicine(PZSHUTCM210820006), and all experiments were conducted in accordance with the regulations of the National Science and Technology Commission of China regarding the use of experimental animals.

LDH assay

THP-1 or J774A.1 cells were seeded in 24-well plates and cultured overnight at 37°C. The cells were then treated with H37Ra and SCU for 24, 48, and 72 hours. Cell culture supernatants were collected, and LDH levels were measured using an LDH cytotoxicity detection kit (C0016,Beyotime, China) according to the manufacturer's protocol. The absorbance was measured at 490 nm using a microplate reader (Tecan, Switzerland).

Enzyme-Linked immunosorbent assay (ELISA)

Cell culture supernatants and mouse serum were collected, and the levels of the inflammatory cytokine IL-1β were measured using an ELISA kit (DY401-05/DY201-05,R&D Systems, USA) according to the manufacturer’s instructions. The levels of IL-18 were also measured using an ELISA kit (EK118/EK218, MULTI SCIENCES, China), following the manufacturer’s instructions.

Seahorse metabolic assay

THP-1 monocytes were differentiated into macrophages by treatment with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 h. J774A.1 macrophages were directly seeded and used at ∼90% confluence. Seahorse experiments were conducted using the XFe24 analyzer from Agilent, following the manufacturer’s instructions. Metabolic studies were conducted using the XF Cell Mito Stress Test Kit and the XF Glycolytic Rate Assay Kit. Cells were treated with Mtb and SCU for 12 h as previously described. After treatment, the cells were washed three times with PBS and pre-incubated with Seahorse XF base medium supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. The corresponding compounds were added to the drug wells of the probe plates. For the glycolysis rate measurement, the final concentrations in the cell wells were: Rot/AA at 0.5 µM and 2-DG at 50 mM. For the XF cellular mitochondrial stress test, the final concentrations in the cell wells were: oligomycin at 2 µM, FCCP at 2 µM, and Rot/AA at 0.5 µM. After the experiment, Seahorse medium was discarded, and the cells were lysed. The protein content in each well was quantified, and data analysis was performed using the XF Wave software.

Western blotting assay

Total proteins were extracted from cells and tissues using RIPA lysis buffer, and protein concentration was determined using the BCA protein assay kit (WB6501,NCM Biotech, China). Protein samples were first separated by 10% SDS-PAGE gel electrophoresis, followed by transfer of the protein bands to a nitrocellulose (NC) membrane (66485,Pall, USA). The membrane was then blocked with 5% non-fat dry milk. Membranes were incubated at 4°C overnight with primary antibodies diluted at 1:1000. After incubation, the membrane was washed three times with TBST and then treated with HRP-conjugated secondary antibody at a dilution of 1:2000 at room temperature for 2 hours. After three washes with TBST, the protein bands were visualized using a Tanon Chemi Doc Ultra chemiluminescence imaging system (Tanon, China), and the images were analyzed using ImageJ software.

To determine the oligomerization of PKM2 and ASC, cells were treated as described previously, washed with PBS, resuspended in Triton buffer (PBS containing 0.2% Triton X-100), and crosslinked with 4 mM DSS for 30 minutes at room temperature. After centrifugation, the pellet was resuspended in 1× SDS buffer, boiled, and the proteins were separated by SDS-PAGE.

Co-Immunoprecipitation (Co-IP)

Cell lysates with equal protein concentrations were prepared as described. Lysates were incubated overnight at 4°C with anti-PKM2 antibody (1 mg/mL). To perform immunoprecipitation, 40 µL of protein A/G agarose beads were added, and the mixture was incubated for an additional 3 hours at 4°C. Beads were washed five times with IP lysis buffer. The beads were then resuspended in 1×SDS-PAGE loading buffer and heated at 100°C for 5 minutes. Finally, SDS-PAGE was performed for protein immunoblotting analysis.

Quantitative PCR (qPCR)

RNA was extracted using an RNA extraction kit (B0004DP,EZBioscience, China) and reverse-transcribed into cDNA using PrimeScript reverse transcription reagents (RR037A,TaKaRa, Japan), following the manufacturer's protocol. Quantitative PCR was conducted using the TB Green qPCR kit (RR420A,TaKaRa, Japan). mRNA levels were normalized to β-actin expression, and the relative expression was calculated using the 2^-ΔΔCT method. The sequences of the qRT-PCR primers are as follows:HK1 (human, targets NM_000188.3) forward, 5′-CACATGGAGTCCGAGGTTTATG-3′;HK1 (human, targets NM_000188.3) reverse, 5′-CGTGAATCCCACAGGTAACTTC-3′;HK2(human, targets NM_000189.5) forward, 5′-TTGACCAGGAGATTGACATGGG-3′;HK2(human, targets NM_000189.5) reverse, 5′-CAACCGCATCAGGACCTCA-3′;SLC2A1 (human, targets NM_006516.4)forward, 5′-ATTGGCTCCGGTATCGTCAAC-3′;SLC2A1 (human, targets NM_006516.4)reverse, 5′-GCTCAGATAGGACATCCAGGGTA-3′;PFKFB3(human, targets NM_004566.4)forward, 5′-ATTGCGGTTTTCGATGCCAC-3′;PFKFB3(human, targets NM_004566.4)reverse, 5′-GCCACAACTGTAGGGTCGT-3′;PGAM1(human, targets NM_002629.4)forward, 5′-GTGCAGAAGAGAGCGATCCG-3′;PGAM1(human, targets NM_002629.4)forward, 5′-CGGTTAGACCCCCATAGTGC-3′;HIF-1α(human, targets NM_001530.4)forward, 5′-ATCCATGTGACCATGAGGAAATG-3′;HIF-1α(human, targets NM_001530.4) reverse, 5′-TCGGCTAGTTAGGGTACACTTC-3′;PKM2(human, targets NM_002654.6)forward, 5′-ATAACGCCTACATGGAAAAGTGT-3′;PKM2(human, targets NM_002654.6) reverse, 5′-TAAGCCCATCATCCACGTAGA-3′;ALDOB(human, targets NM_000035.4)forward, 5′-CCACCGTAACAGCTCTCCAC-3′;ALDOB(human, targets NM_000035.4) reverse, 5′-CACTCATGCCACCAGACAAAA-3′。

Confocal microscope detection

Mito-Tracker staining was performed by incubating the cells with 400 nM Mito-Tracker Red CM-H2XRos (C1035,Beyotime, China) at 37°C for 30 minutes, followed by observation of mitochondrial morphology using a laser confocal microscope. Mitochondrial membrane potential (ΔΨm) was assessed using the JC-1 fluorescence kit (C2006,Beyotime, China). Cells were incubated with JC-1 staining solution at 37°C for 30 minutes, and fluorescence intensity was analyzed using a laser confocal microscope(Zeiss, Germany).

Flow cytometry

For the mitochondrial total ROS assay, after treating the cells for 12 hours as described previously, the cells were washed three times with sterile PBS and incubated with MitoSOX (2.5 µM) (M36006,Thermo, MA, USA) for 30 minutes at 37°C. The cells were then analyzed using flow cytometry (Beckman Coulter, USA).

Histopathology

For tissue fixation, mouse lung tissues were fixed in 4% paraformaldehyde. After fixation, the tissues were embedded in paraffin and sectioned. The tissue sections were then stained using hematoxylin and eosin (H&E) for histological analysis[28].

Immunohistochemistry (IHC)

Mouse lung tissue paraffin sections were dehydrated through an ethanol gradient, followed by antigen retrieval and blocking of endogenous peroxidase activity. After blocking, the sections were incubated with the primary antibody, washed, and then incubated with the secondary antibody. Staining was performed using DAB (DA1010,Solarbio, China) and hematoxylin, and the sections were finally mounted with neutral resin.

Lactate detection

Mouse lung tissues were processed according to the manufacturer's instructions, and lactate production was measured using a lactate assay kit (A019-2-1,Jiancheng, China).

Statistical analysis

Data are presented as mean ± SD (for in vitro) or median and interquartile range (IQR) (for in vivo). Statistical analysis was performed using GraphPad Prism 8.0. For two-group comparisons, a two-tailed Student’s t-test was used. For multiple groups, one-way ANOVA was performed, followed by Dunnett’s post-hoc test for comparisons to a single control. A p-value of <0.05 was considered significant.

Results

Effect of SCU on the viability of THP-1 and J774A.1 cells

The chemical structure of SCU is illustrated in Figure 1a. Cell viability was assessed using the MTT assay, evaluating the viability of SCU-treated THP-1 (Figure 1b) and J774A.1 cells (Figure 1c). The results indicated that SCU (10, 20, 40, and 80 μM) had no significant effect on cell viability within 24 hours. Due to SCU's cytotoxicity at higher concentrations, we limited its administration to 24 hours in subsequent experiments, using concentrations of 20, 40, and 80 μM.

Figure 1.

Effect of SCU on Macrophage Activity.a The chemical structure of SCU is, (2S,3S,4S,5R,6S)-6-[5,6-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-7-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid (Molecular formula: C21H18O12; Molecular weight: 462.36; CAS Registry Number: 27740-01-8; PubChem CID: 185617). b THP-1 cell viability after exposure to SCU (10–80 μM) for 24, 48, and 72 h, measured using the MTT assay (n = 4).c J774A.1 cell viability after exposure to SCU (10–80 μM) for 24, 48, and 72 h, measured using the MTT assay (n = 4).*p < 0.05, **p < 0.01, and ***p < 0.001.

SCU inhibits the warburg effect in Mtb-Infected THP-1 and J774A.1 cells

A hallmark of pro-inflammatory immune cells is the reprogramming of their metabolism, which involves a shift from mitochondrial oxidative phosphorylation to glycolysis, a process known as the Warburg effect[29]. To investigate the effect of SCU on aerobic glycolysis in Mtb-infected THP-1 and J774A.1 cells, we measured the glycolytic proton efflux rate (glycoPER) and oxygen consumption rate (OCR) using the Seahorse Extracellular Flux Analyzer. The main sources of extracellular protons are lactic acid produced by aerobic glycolysis and CO₂ generated by mitochondrial oxidative phosphorylation (OXPHOS). GlycoPER was estimated by subtracting the CO₂-dependent acidification from the total proton efflux rate (PER), providing a measure of aerobic glycolysis and extracellular lactate production. Our results showed that Mtb infection increased glycolytic flux in both THP-1 and J774A.1 cells (Figure 2a,b). The energy map revealed that Mtb infection induced both cell types to shift toward aerobic glycolysis, an effect that was inhibited by SCU(Figure 2c,d). Mtb infection upregulated basal glycolysis, compensatory glycolysis, basal proton efflux rate, and post-2-DG acidification in both THP-1 and J774A.1 cells, whereas SCU inhibited these changes (Figure 2e-l). These results suggest that SCU regulates cellular energy supply primarily by modulating mitochondrial aerobic glycolysis. In the Cell Mito StressTest (Figure 2m,n), we observed that Mtb infection impaired the basal oxygen consumption and mitochondrial oxidative phosphorylation of ATP production in both cell lines, which was reversed by SCU (Figure 2o-r). These results indicate that SCU maintains mitochondrial homeostasis and respiration in vitro by inhibiting the Warburg effect.

Figure 2.

Evaluation of real-time glycoPER and OCR in Mtb-infected THP-1 and J774A.1 cells after SCU treatment.a-b Glycolysis rate test plots for THP-1 and J774A.1 cells(n = 3).c-d Energy map at the first time point of the glycolysis rate test for THP-1 and J774A.1 cells(n = 3).e-f Basal glycolysis for THP-1 and J774A.1 cells(n = 3).g-h Compensatory glycolysis for THP-1 and J774A.1 cells (n = 3).i-j Basal proton efflux rate for THP-1 and J774A.1 cells (n = 3).k-l Post-2-DG acidification for THP-1 and J774A.1 cells (n = 3).m-n Mitochondrial stress test plots for THP-1 and J774A.1 cells(n = 3).o-p Basal respiration for THP-1 and J774A.1 cells (n = 3).q-r ATP production from mitochondrial oxidative phosphorylation for THP-1 and J774A.1 cells (n = 3).ns: not statistically significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

SCU mitigates mitochondrial damage in macrophages infected with Mtb

Mitochondrial shape and bioenergetics are closely interconnected[30]. Studies have shown that in pro-inflammatory macrophages, mitochondrial morphology undergoes significant alterations, leading to the formation of smaller, fragmented mitochondria that are dispersed throughout the cytoplasm[31]. Confocal microscopy of Mitotracker-labeled macrophages, which stains mitochondria independently of membrane potential, revealed that macrophages in the Mtb group exhibited punctate and numerous mitochondria(Figure 3a, white arrow). In contrast, mitochondria in the SCU-treated group appeared more tubular and were fewer in number, resembling the control cells (Figure 3a). JC-1 is a probe for assessing mitochondrial membrane potential (MMP) in living cells. In healthy mitochondria with intact MMP, JC-1 forms J-aggregates that emit red fluorescence. In contrast, in mitochondria with compromised MMP, JC-1 remains in its monomeric form, emitting green fluorescence. JC-1 staining revealed that mitochondria in the control and SCU-treated (40 μM) groups predominantly emitted red fluorescence (Figure 3b, white arrows), indicating preserved MMP. In Mtb-infected cells, mitochondria primarily showed green fluorescence (Figure 3b, white arrow), reflecting a decreased red-to-green fluorescence ratio and mitochondrial dysfunction. SCU treatment (40 μM) effectively mitigated Mtb-induced mitochondrial dysfunction by restoring red fluorescence, indicative of preserved MMP (Figure 3b).

Figure 3.

SCU inhibits Mtb damage to mitochondrial metabolism in THP-1 and J774A.1 cells.a Changes in mitochondrial morphology of THP-1 cells treated with different concentrations of SCU were observed by confocal microscopy using Mitotracker staining(n = 3). The white arrow points to the mitochondria.b Mitochondrial membrane potential changes in THP-1 cells were assessed using JC-1 staining and observed by confocal microscopy(n = 3). Bar graphs show the statistical results of mitochondrial membrane potential intensity for each group. The white arrow points to the mitochondria.c-d The expression levels of p-DRP1, MFN2, and Cytochrome C proteins in THP-1 and J774A.1 cells treated with 20, 40, and 80 μM SCU were detected by western blotting(n = 3). Bar graphs show the quantification of grey scale values for the protein levels in each group.e-f The expression levels of p-DRP1, MFN2, and Cytochrome C proteins in THP-1 and J774A.1 cells after 40 μM SCU treatment for different time points (6, 12, and 24 h) were detected by western blotting(n = 3). Bar graphs show the quantification of grey scale values for the protein levels in each group.g-h Mitochondrial ROS levels in THP-1 and J774A.1 cells were detected by flow cytometry using the mitochondrial superoxide indicator MitoSOX(n = 3). Bar graphs show the statistical results of mitochondrial ROS mean fluorescence intensity (MFI) for each group.ns: not statistically significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

Mitochondria are highly dynamic organelles that regulate their morphology through fission and fusion processes. Excessive mitochondrial fission results in fragmentation, disrupts the electron transport chain, and causes abnormal accumulation of mitochondrial ROS[32, 33]. p-DRP1 is a crucial protein that facilitates mitochondrial fission and promotes fragmentation, whereas MFN2 plays an essential role in mediating mitochondrial fusion. To evaluate the effect of SCU on mitochondrial fission and fusion proteins in Mtb-infected THP-1 and J774A.1 cells, western blotting was performed. The results showed that mitochondrial fission was increased in Mtb-infected cells compared to controls (Figure 3c-f), and SCU inhibited the phosphorylation of DRP1. Moreover, we observed a reduction in MFN2 expression during Mtb infection, which SCU treatment reversed (Figure 3c-f). Additionally, SCU suppressed the expression of Cytochrome C, a key indicator of mitochondrial damage (Figure 3c-f). These findings suggest that SCU mitigates Mtb-induced mitochondrial dysfunction by modulating the balance between mitochondrial fission and fusion in macrophages.

The level of mitochondrial ROS is a critical factor in oxidative stress, and studies have shown that mitochondrial ROS can activate the NLRP3 inflammasome[34]. To examine changes in mitochondrial ROS levels following Mtb stimulation, we used the mitochondrial superoxide indicator MitoSOX. The results demonstrated a marked increase in mitochondrial ROS in macrophages from the Mtb group compared to the control, while SCU treatment notably decreased ROS levels in comparison to the Mtb group (Figure 3g,h).

The results suggest that SCU exerts a protective effect by preserving mitochondrial function following Mtb stimulation. Overall, SCU treatment reduces excessive mitochondrial division, improves mitochondrial morphology by inhibiting the reduction of MFN2 and the phosphorylation of DRP1 at Ser616, and mitigates mitochondrial ROS production, thereby alleviating mitochondrial dysfunction.

SCU inhibits inflammation and pyroptosis in Mtb-Infected THP-1 and J774A.1 cells

Our previous study demonstrated that Mtb infection in macrophages promotes the activation of NLRP3 inflammasomes, leading to pyroptosis, bacterial dissemination, and severe inflammatory responses[35, 36]. In this study, we observed that SCU significantly reduced the expression of NLRP3 in a manner dependent on both time and concentration.(Figure 4a-d). Additionally, SCU suppressed the formation of apoptosis-associated speck-like proteins (ASC), which contain polymerized caspase recruitment domains (Figure 4e,f). SCU also effectively inhibited caspase-1 activity and blocked the N-terminal fragment of GSDMD(Figure 4a-d), preventing the onset of pyroptosis. During pyroptosis, lactate dehydrogenase (LDH) is released from cells, and its activity serves as an indicator of cell membrane integrity. Mtb infection increased LDH release, while SCU (40μM) reversed this effect at all time points (Figure 4g, h). In Mtb-infected macrophages, pyroptosis leads to IL-1β production, which is released through the cell membrane and triggers inflammation. After treating Mtb-infected macrophages with different concentrations of SCU (20, 40, 80μM) for 12 hours, the results indicated that SCU at 40 and 80μM inhibited IL-1β expression (Figure 4i, j). Similarly, IL-18 levels were elevated in Mtb-infected macrophages and SCU (20, 40, 80μM) treatment significantly reduced IL-18 release (Figure 4k, l). Additionally, SCU inhibited the expression of the inflammation-related protein high-mobility group box 1 (HMGB1) (Figure 4a-d).

Figure 4.

SCU Inhibits Inflammation and Pyroptosis in Mtb-Infected THP-1 and J774A.1 Cells.a The expression levels of NLRP3, GSDMD-N, Cleaved-Caspase-1, and HMGB1 proteins in THP-1 cells treated with 20, 40, and 80 μM of SCU were detected by western blotting(n = 3). Bar graphs represent the quantification of protein levels from grey scale values.b The levels of NLRP3, GSDMD-N, cleaved caspase-1, and HMGB1 proteins in THP-1 cells treated with 40 μM SCU for different durations (6, 12, and 24 hours) were detected by western blotting(n = 3). Bar graphs present the statistics of protein levels from grey scale values.c The expression levels of NLRP3, GSDMD-N, cleaved caspase-1, and HMGB1 proteins in J774A.1 cells treated with 20, 40, and 80 μM of SCU were detected by western blotting(n = 3). Bar graphs represent the quantification of protein levels from grey scale values.d The levels of NLRP3, GSDMD-N, cleaved caspase-1, and HMGB1 proteins in J774A.1 cells treated with 40 μM SCU for different durations (6, 12, and 24 hours) were detected by western blotting(n = 3). Bar graphs present the statistics of protein levels from grey scale values.e-f The expression of ASC proteins cross-linked with DSS in THP-1 and J774A.1 cells treated with different concentrations of SCU was measured by western blotting(n = 3).g-h The levels of LDH released into the supernatants of THP-1 and J774A.1 cells were measured using the LDH Cytotoxicity Assay Kit(n = 3). SCU was treated at a concentration of 40 μM.i-j The inhibitory effect of different concentrations of SCU on Mtb-induced IL-1β release from THP-1 and J774A.1 cells was assessed by ELISA(n = 4).k-l The inhibitory effect of different concentrations of SCU on Mtb-induced IL-18 release from THP-1 and J774A.1 cells was assessed by ELISA (n = 4). ns: not statistically significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

Collectively, the findings suggest that SCU provides protection by attenuating Mtb-induced macrophage inflammation and inhibiting pyroptosis.

SCU inhibits HIF-1α-mediated enhancement of the warburg effect

Metabolic reprogramming in macrophages is primarily driven by HIF-1α, which upregulates the expression of glycolytic enzymes and pro-inflammatory cytokines (such as IL-1β) while inhibiting OXPHOS activity[37]. The increased aerobic glycolysis observed in macrophages in the previous experiments led us to investigate whether HIF-1α levels were abnormally elevated in these cells. qRT-PCR analysis showed that SCU significantly reduced the expression of key glycolytic enzymes and HIF-1α-related genes, including HK1, HK2, SLC2A1, PFKFB3, PGAM1, HIF-1α, PKM2, and ALDOB in Mtb-infected THP-1 cells (Figure 5a). Western blotting analysis further demonstrated that HIF-1α protein levels were markedly elevated in the Mtb group compared to control cells, while SCU effectively inhibited this increase (Figure 5b, c). Studies suggest that HIF-1α is a key regulator of host defense during Mtb infection, enhancing glycolysis and promoting the pro-inflammatory immune response[38]. In addition, Severe hypoxia is present in human TB lesions, indicating that hypoxia-driven HIF-1α activation promotes lung tissue destruction, which may be a crucial mechanism underlying TB transmission and disease progression[39]. Previous studies have shown that PKM2, as a coactivator of HIF-1α, interacts with HIF-1α to promote metabolic reprogramming[40]. Co-immunoprecipitation (Co-IP) results showed that the interaction between HIF-1α and PKM2 was significantly enhanced in Mtb-infected cells. SCU effectively inhibited this interaction compared to the Mtb group (Figure 5d, e). PKM2 dimers can translocate to the nucleus, where they directly interact with HIF-1α to regulate the expression of glycolytic enzymes[41]. There is a dynamic equilibrium between PKM2 monomers or dimers and PKM2 tetramers. PKM2 dimers can stabilize HIF-1α, thereby influencing the expression of HIF-1α target genes, including IL-1β and those involved in glycolytic processes. In this study, we assessed PKM2 protein expression and observed that DSS cross-linking of THP-1 and J774A.1 cell lysates revealed significantly lower levels of the tetrameric form of PKM2 (240 kDa) in the Mtb group compared to control and SCU-treated macrophages (Figure 5f, g). Additionally, the dimeric form of PKM2, which is accessible to the nucleus, was more prevalent in Mtb-infected cells. Subcellular localization analysis revealed elevated levels of HIF-1α and PKM2 in the nucleus of cells treated with Mtb (Figure 5h, i). These results suggest that Mtb infection induces a shift towards HIF-1α-mediated aerobic glycolysis in macrophages, a process that is inhibited by SCU.

Figure 5.

SCU inhibits HIF-1α-induced aerobic glycolysis.a The mRNA expression levels of HK1, HK2, SLC2A1, PFKFB3, PGAM1, HIF-1α, PKM2, and ALDOB were assessed by qRT-PCR in THP-1 cells(n = 3).b-c The expression levels of HIF-1α proteins were detected by western blotting in THP-1 and J774A.1 cells treated with 20, 40, and 80 μM SCU(n = 3). Bar graphs show the quantification of the grey scale values of protein levels for each group.d-e Co-IP assay was performed to detect the interaction between HIF-1α and PKM2 in THP-1 and J774A.1 cells(n = 3). F-g Western blotting was used to detect the expression of PKM2 protein cross-linked using DSS in THP-1 and J774A.1 cells treated with different concentrations of SCU(n = 3).h-i The levels of HIF-1α and PKM2 proteins in the nuclei of THP-1 and J774A.1 cells after nucleoplasmic separation were detected by western blotting at 20, 40, and 80 μM SCU(n = 3). Bar graphs show the quantification of the grey scale values of protein levels for each group. ns: not statistically significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

SCU maintains mitochondrial homeostasis and inhibits pyroptosis by regulating HIF-1α

To explore the role of HIF-1α in SCU’s effects on macrophages, we transfected THP-1 cells with HIF-1α siRNA. Our findings showed that transfection with HIF-1α siRNA reduced the levels of pyroptosis-related proteins, including NLRP3, GSDMD-N, Cleaved-Caspase-1, and HMGB1, in Mtb-infected THP-1 cells (Figure 6a). This suggests that interference with HIF-1α protein can inhibit Mtb-induced pyroptosis. Furthermore, after HIF-1α siRNA transfection, the mitochondrial fission protein p-DRP1 was decreased, while the mitochondrial fusion protein MFN2 was increased in Mtb-infected THP-1 cells, indicating that HIF-1α interference can block mitochondrial division in macrophages (Figure 6b). Similarly, we assessed the subcellular localization of HIF-1α and PKM2. As shown in Figure 6c, interference with HIF-1α inhibited the Mtb-induced upregulation of both HIF-1α and PKM2 proteins in the nucleus.

Figure 6.

SCU protects mitochondrial metabolism in macrophages by inhibiting HIF-1α.a After transfection of small interfering RNA (siRNA) targeting HIF-1α in THP-1 cells, the expression levels of NLRP3, GSDMD-N, Cleaved-Caspase-1, and HMGB1 proteins were detected by western blotting(n = 3). Bar graphs show the quantification of grey scale values for the protein levels in each group.b After transfection of siRNA targeting HIF-1α in THP-1 cells, the levels of p-DRP1, MFN2, and Cytochrome C proteins were detected by western blotting(n = 3). Bar graphs show the quantification of grey scale values for the protein levels in each group.c After transfection of siRNA targeting HIF-1α in THP-1 cells, the expression levels of HIF-1α and PKM2 proteins in the nuclei of the cells after nucleoplasmic separation were detected by western blotting(n = 3). Bar graphs show the quantification of grey scale values for the protein levels in each group. ns: not statistically significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

SCU protects against LPS-Induced acute lung injury in mice

To determine whether SCU plays a role in an in vivo inflammation model, we investigated its effect on an LPS-induced ALI model in mice. First, we measured its ability to reduce serum IL-1β levels in the presence of ALI. The results showed that intraperitoneal injection of SCU 1 hour after LPS induction attenuated the elevation of serum IL-1β levels in the acute lung injury model (Figure 7a).

Figure 7.

SCU attenuates LPS-induced acute lung injury and inflammatory cytokine expression in mice by in vivo inhibition of HIF-1α.a Serum levels of IL-1β in mice (n = 5) were detected by ELISA.b Lactate levels in mouse lung tissue homogenates were detected using a lactate assay kit (n = 6).c Lung tissues from mice were stained with H&E (20×), with dexamethasone (DEX) used as a positive control(n = 3). Bar graphs show the quantification of grey scale values for protein levels in each group. Scale bar = 150 μm.d Immunohistochemical images of F4/80 expression in mouse lung tissue (20×), and the positive area was quantified (n = 3). Bar graphs show the quantification of grey scale values for protein levels in each group. Scale bar = 150 μm.e-g Immunohistochemical images of HIF-1α, p-DRP1, and MFN2 expression in mouse lung tissue (20×), with the positive area quantified (n = 3). Bar graphs show the quantification of grey scale values for protein levels in each group. Scale bar = 150 μm.h-i The expression levels of NLRP3, GSDMD-N, HMGB1, HIF-1α, p-DRP1, MFN2, and Cytochrome C proteins in mouse lung tissues were detected by western blotting (n = 4). Bar graphs show the quantification of grey scale values for protein levels in each group. ns: not statistically significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

In the LPS-induced ALI model, lung tissue lactate levels were significantly elevated, indicating abnormal activation of glycolytic metabolism. SCU treatment significantly reversed LPS-induced lactate accumulation (Figure 7b). Immunohistochemical analysis revealed that LPS may drive the glycolytic process by upregulating the protein expression of HIF-1α, whereas SCU suppressed the abnormal elevation of HIF-1α (Figure 7e, 7i), suggesting that it alleviates metabolic imbalance by regulating the HIF-1α-glycolysis axis. Additionally, LPS induced an increase in p-DRP1 protein expression in lung tissues (Figure 7f, 7i) while reducing MFN2 protein expression (Figure 7G, 7I). Similarly, SCU inhibited these changes, indicating that it maintains mitochondrial dynamic homeostasis by regulating the DRP1/MFN2 balance. This effect may be achieved by inhibiting HIF-1α-mediated mitochondrial dysfunction.

We further investigated the effect of SCU on inflammation-related proteins in the lung tissue of mice with acute lung injury. Immunoblotting assays showed that SCU administration reduced the expression of NLRP3, GSDMD-N, and HMGB1 proteins in LPS-induced mouse lung tissues (Figure 7h). Meanwhile, we performed immunohistochemical analysis of F4/80 in the lung tissues of mice with acute lung injury to investigate the effects of SCU on LPS-induced macrophages in the lung. The results showed that, compared to the control group, the LPS group exhibited elevated expression of the macrophage surface marker F4/80, while the SCU treatment group significantly reduced its expression (Figure 7d).

Additionally, pathological damage to mouse organs was assessed in H&E-stained tissue sections. LPS-treated mice exhibited inflammatory cell infiltration, alveolar wall thickening, interstitial edema, and vascular congestion in the lungs. However, SCU treatment significantly alleviated these pathological changes (Figure 7c).

Collectively, hese findings indicate that SCU exerts anti-inflammatory effects through the glycolysis-mitochondria-pyroptosis regulatory network, effectively attenuates LPS-induced acute lung injury in mice, and holds therapeutic potential for inflammatory lung injury.

Discussion

Tuberculosis (TB) remains one of the most significant global health threats, with Mycobacterium tuberculosis (Mtb), the causative agent of TB, estimated to infect a quarter of the world's population. In 2023, the number of newly diagnosed and reported TB cases reached a record high of 8.2 million[42, 43]. Effective control of inflammation is critical for improving TB prognosis[44]. Scutellaria baicalensis-derived SCU, a glyoxylated flavonoid, has demonstrated broad pharmacological activities, particularly in the treatment of cardiovascular and cerebrovascular diseases, and has been clinically used for these conditions[45]. This study examined the effects of SCU on Mtb-induced inflammation and pyroptosis in macrophages using a HDT approach.

Macrophage energy metabolism is critical for controlling inflammatory processes and maintaining tissue homeostasis, especially in the context of diseases associated with inflammation. It has been found that pro-inflammatory stimuli lead to a Warburg-like upregulation of macrophage glycolysis, similar to what is observed in tumors[46]. The shift from oxidative phosphorylation to aerobic glycolysis is crucial for the balance between inflammatory and regulatory immune phenotypes in macrophages. However, the exact mechanism of the Warburg effect in tuberculosis (TB) remains unclear. Recent evidence indicates that the miR-26a/SIRT6/HIF-1α axis plays a pivotal role in regulating glycolysis and inflammatory responses during Mtb infection, further underscoring the complexity of metabolic reprogramming in infected macrophages[47]. In this study, we assessed real-time glycoPER and OCR in live cells. The data demonstrated that SCU treatment inhibited the Mtb-induced increase in the rate of aerobic glycolysis. Moreover, SCU reversed the decrease in cellular basal oxygen consumption and mitochondrial oxidative phosphorylation of ATP production observed in the Mtb group. These findings suggest that SCU can regulate the switch from aerobic glycolysis to oxidative phosphorylation in macrophages.

Glycolysis is a metabolic process that converts glucose to pyruvate and subsequently to lactate, and it is regulated by various genes, including SLC2A1, which is involved in glucose uptake, and enzymes such as HK1, HK-2, PGAM1, PKM2, which directly catalyze glycolysis, as well as PFKFB3 and ALDOB, which are indirectly involved[48]. In this research, we employed qPCR assays to analyze gene expression and found that these genes were upregulated in Mtb-infected cells, with HIF-1α serving as an important transcription factor regulating their expression. Mtb-infected cells also exhibited elevated expression of HIF-1α and PKM2, and SCU reduced the expression of these genes, along with lowering the HIF-1α protein level. SCU further inhibited the interaction between HIF-1α and PKM2, potentially by inhibiting their nuclear translocation. Additionally, a previous study found that SCU promotes HIF-1α degradation through direct interaction with HSP90[49]. The results indicate that SCU might mediate its therapeutic actions via various molecular targets, although the precise pathways involved are not yet completely understood. Based on these results, we propose that SCU may prevent inflammatory changes and pyroptosis in macrophages by inhibiting HIF-1α-induced enhancement of aerobic glycolysis.

Mitochondria are essential organelles in eukaryotic cells, regulating various biological processes, including ROS production, energy metabolism, stress responses, and cell fate decisions[50]. Moreover, mitochondria are essential for the activation of the NLRP3 inflammasome. The majority of ROS are produced by mitochondria, and several studies have shown that mitochondria-derived ROS (mtROS) can activate the NLRP3 inflammasome[51–53]. Additionally, the dynamic properties of mitochondria, such as fusion, fission, and degradation, are vital for their role in energy production[54]. Excessive mitochondrial fission leads to fragmentation, while increased fusion promotes the integration of neighboring mitochondria. Dynamin-related protein 1 (Drp1), a crucial cytoplasmic GTPase, is required for mitochondrial fission. Phosphorylation of Drp1 at the S616 site drives its translocation to the mitochondria, thereby facilitating fission[55]. Mfn2, a mitochondrial fusion protein located on the outer membrane, is essential for outer membrane fusion and plays a role in mitochondrial quality control by helping to remove damaged mitochondria[56]. In our study, Mtb-infected macrophages exhibited increased p-Drp1 expression and decreased Mfn2 expression, leading to altered mitochondrial morphology, which was inhibited by SCU. Furthermore, Cytochrome C, a small protein normally located in the inner mitochondrial membrane, participates in electron transfer and energy conversion. It has been suggested that p-Drp1 promotes mitochondrial outer membrane permeabilization, leading to Cytochrome C release, which is considered a marker of mitochondrial dysfunction[57, 58]. In contrast, SCU treatment inhibited the elevated Cytochrome C expression in Mtb-infected macrophages. Beyond protein expression, we also assessed several mitochondrial parameters. The results demonstrated that SCU preserved mitochondrial morphology after Mtb infection and significantly reduced mitochondrial ROS levels. Mtb infection caused damage to mitochondrial structure and reduced mitochondrial membrane potential, whereas SCU treatment restored the membrane potential, ultimately maintaining mitochondrial homeostasis.

Mitochondrial dysfunction induced by Mycobacterium tuberculosis (Mtb) infection, as well as its specific role in Mtb infection, has not been adequately explored[59]. In particular, the relationship between mitochondrial morphological disruption caused by Mtb infection and the pro-inflammatory phenotype of macrophages remains under-investigated[60, 61]. To determine whether these mitochondrial changes were linked to aerobic glycolysis, we transfected THP-1 cells with siHIF-1α for 24 hours, followed by Mtb infection and SCU (40 μM) treatment for 12 hours. The results showed that HIF-1α knockdown significantly reduced Mtb-induced inflammatory alterations and pyroptosis, decreased the expression of the mitochondrial fission protein p-Drp1, and increased the expression of the mitochondrial fusion protein MFN2. These effects were comparable to those observed with SCU treatment. These observations propose that SCU protects Mtb-infected macrophages from pyroptosis by inhibiting HIF-1α-mediated aerobic glycolysis.

It has been shown that an enhanced inflammatory response does not improve Mtb clearance [62], but instead exacerbates host damage[63]. Macrophages, as key phagocytic cells of the innate immune system, play a crucial role in maintaining tissue homeostasis and responding to pathogenic challenges. Pyroptosis is a form of programmed cell death that aids in pathogen defense, but when excessive, it can cause sustained inflammation and contribute to inflammatory diseases[64]. Previous studies from our team have demonstrated that Mtb infection induces pyroptosis in macrophages[65, 66]. In this study, we found that SCU treatment inhibited the activation of the NLRP3 inflammasome, the cleavage of caspase-1 and GSDMD, and the release of IL-1β and IL-18 in Mtb-infected macrophages. Additionally, SCU treatment also reduced the expression of the inflammatory protein HMGB1. The evidence points to that SCU effectively inhibits the inflammatory changes and pyroptosis in Mtb-infected macrophages.

Finally, we explored the effects of SCU in an LPS-induced acute lung injury model in mice. The results demonstrated that SCU reduced LPS-induced lactate secretion in lung tissue, indicating its inhibitory effect on the abnormal activation of glycolysis. Additionally, SCU suppressed the expression of HIF-1α and p-DRP1 proteins while upregulating MFN2 protein expression in lung tissue, suggesting its role in maintaining mitochondrial homeostasis. Further analysis revealed that SCU decreased serum levels of the pro-inflammatory factor IL-1β, reduced macrophage infiltration in lung tissue, and reversed the expression of inflammation – and pyroptosis-related proteins. Therefore, we propose that the protective effects of SCU in this mouse model may be attributed to its inhibition of HIF-1α-mediated aerobic glycolysis, thereby preserving mitochondrial function and mitigating inflammatory and pyroptotic responses.

However, the current study has several limitations. First, this study utilized the avirulent Mycobacterium tuberculosis H37Ra strain as an in vitro infection model, which adheres to international standards. Compared to H37Rv, which is derived from the same parent strain, M. tuberculosis H37, H37Ra exhibits only 272 identified sequence differences – significantly fewer than the differences observed between the attenuated BCG Pasteur strain and M. bovis AF2122/97 [67]. Research has shown that H37Rv infection induces more pronounced pyroptosis and stronger inflammatory responses in macrophages compared to H37Ra infection[68, 69]. Furthermore, it has been reported that H37Rv infection reduces glycolysis and mitochondrial metabolism in macrophages, differing from the effects observed with H37Ra[59]. Conversely, some studies have shown that macrophages infected with H37Rv exhibit significant upregulation of genes involved in glycolytic processes and pyruvate metabolism[70]. Secondly, although the Mtb viable infection model more closely resembles the clinical symptoms of tuberculosis, its application is constrained by biosafety restrictions and the limitations of animal experimental centers. Therefore, to evaluate the anti-inflammatory effects of drugs within the HDT strategy, we established an ALI mouse model via intraperitoneal injection of LPS to simulate lung inflammation triggered by Mtb infection[71], rather than directly using Mycobacterium tuberculosis. Additionally, previous studies have demonstrated that LPS-stimulated macrophages undergo a metabolic shift from mitochondrial OXPHOS to glycolysis under normoxic conditions[72], further justifying the use of the LPS model to investigate inflammation-related metabolic changes. We acknowledge the limitation of using this model and suggest that future studies could incorporate a true Mtb-infected mouse model for more direct relevance to tuberculosis infection.

In conclusion, we found that the expression of HIF-1α was upregulated in Mtb-infected macrophages, thereby enhancing macrophage aerobic glycolysis and subsequently promoting mitochondrial fission and dysfunction. These dysfunctional mitochondria significantly contribute to mtROS production, and the excessive accumulation of ROS induces redox stress, which subsequently upregulates the expression of HIF-1α and its target genes. The large-scale production of mtROS triggers the activation of the NLRP3 inflammasome, leading to the release of IL-1β via the caspase-1/GSDMD-N pathway. These factors contribute to the inflammatory microenvironment in lung tissue, exacerbating lung tissue injury. Our findings provide evidence that SCU, or the development of synthetic analogues, could yield more potent and effective molecules that may serve as valuable drug candidates for treating Mtb infections.

Conclusions

In summary, our findings demonstrate that Scutellarin (SCU) effectively mitigates pyroptosis and inflammatory impairments in both Mtb-infected macrophages and inflammatory lung injury models. SCU enhances mitochondrial function, maintains cellular energy homeostasis, and inhibits HIF-1α-dependent metabolic reprogramming induced by Mtb infection. Moreover, SCU significantly reduces NLRP3/GSDMD-mediated pyroptosis, thereby attenuating inflammation and cellular damage. These results suggest that SCU could be a promising therapeutic strategy for addressing inflammation and cellular dysfunction in tuberculosis (TB) and related inflammatory disorders.

Author contribution statement

Jianchao Wu: Conceptualization, Methodology, Data Curation, Writing – Original Draft. Fanglin Liu: Conceptualization, Methodology, Data Curation, Writing – Original Draft. Jingjing Shen: Investigation, Data Curation. Hemin Zhang and Yaqi Liu: Investigation, Data Curation. Jinxia Sun: Data Curation, Writing – Review & Editing. Guizhen Yang: Project administration. Yuejuan Zheng: Supervision, Funding Acquisition, Writing – Review & Editing. Xin Jiang: Supervision, Funding Acquisition, Writing – Review & Editing.Equal Contribution: Jianchao Wu and Fanglin Liu contributed equally to this work.

Funding Statement

This work was supported by the Ministry of Science and Technology of the People's Republic of China, the National Natural Science Foundation of China (No. 81873069), and the Shanghai Municipal Science and Technology Major Project (No. ZD2021CY001).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data supporting the findings of this study are available from the corresponding author upon request. Clinical trial number: Not applicable.

Supplemental Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/13510002.2025.2565861.