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.

Keywords: Mitochondrial dysfunction; Mycobacterium tuberculosis; NLRP3 inflammasome; Pyroptosis; Scutellarin; Warburg effect; host-directed therapy; hypoxia-inducible factor-1α.

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.

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.

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.

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.

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.

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.

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.