Pulsed electromagnetic fields inhibit atherosclerosis by regulating pyroptosis through membrane tension-mediated mechanosensitive channels

Abstract

Atherosclerosis serves as the core pathological basis of cardiovascular, cerebrovascular, and peripheral arterial diseases, posing a serious threat to human health. However, current mainstream treatments such as statin drugs and stent implantation are associated with significant side effects or limited efficacy, highlighting the urgent need for new therapeutic strategies. Pulsed electromagnetic fields (PEMFs), due to their noninvasive nature and anti-inflammatory properties, show potential in the treatment of atherosclerosis. This study utilized ApoE-/- mice, ApoE-/-NLRP3-/- knockout mice, human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), and human plasma samples for experiments, revealing significant endothelial cell (EC) inflammation and pyroptosis during the progression of atherosclerosis. PEMFs were found to effectively inhibit the activation of the NLRP3 inflammasome, reduce plaque formation, and delay the progression of atherosclerosis. Proteomic analysis of plasma from atherosclerosis patients further indicated elevated expression levels of proteins related to inflammation and pyroptosis, with particularly notable changes in membrane proteins. Mechanistic studies demonstrated that PEMFs improve mitochondrial dysfunction in ECs by regulating membrane tension and the mechanosensitive tension-mediated transient receptor potential vanilloid 4 (TRPV4) channels, thereby reducing pyroptosis. This discovery not only reveals a novel mechanobiological pathway but also provides a solid theoretical foundation for the development of PEMF-based therapies for atherosclerosis. Schematic diagram of the mechanism by which PEMFs treat atherosclerosis (created in BioRender). Wei, B. (2025) https://BioRender.com/undefined ).

Schematic diagram of the mechanism by which PEMFs treat atherosclerosis (created in BioRender). Wei, B. (2025) https://BioRender.com/undefined).

Fig. 1

PEMFs inhibit the formation of atherosclerotic plaques by attenuating pyroptosis and inflammation. a Oil red O staining was used to determine the lesion burden in the aorta in the ApoE-/- mouse groups: the normal chow diet (ND) group, high-fat diet (HFD, 15 weeks) group, and HFD+PEMFs (15 Hz, 1.5 mT, 1 h/day, 3 weeks) group; n = 5 mice per group. b Evans blue staining was used to assess aortic permeability under the same experimental conditions; n = 5 mice per group. Scale bar = 100 μm. c, d HE staining was used to visualize the plaque area (yellow arrows) in the aortic arch, and the results were quantified; n = 5 mice per group. Scale bar = 100 μm. e, f Serum levels of IL-1β and IL-18 were detected by ELISA; n = 5 mice per group. g Pyroptosis was assessed by flow cytometry via Hoechst 33342/PI staining in MVECs. h Western blot analysis of NLRP3, ASC, p20 caspase-1, and GSDMD-NT in aortic tissues; n = 5 mice per group. i Untargeted lipidomic analysis of aortic tissues from all experimental groups. KEGG pathway analysis of the top 10 differential lipids between the HFD + PEMF and HFD groups. Dots represent metabolites (blue: downregulated; red: upregulated). The pathway descriptions are described as follows: mmu00564: Glycerophospholipid metabolism; mmu00600: Sphingolipid metabolism; mmu04979: Cholesterol metabolism; mmu05231: Choline metabolism in cancer; mmu04975: Fat digestion and absorption; mmu04071: Sphingolipid signaling pathway; mmu04136: Autophagy–other; mmu04931: Insulin resistance; mmu00563: Glycosylphosphatidylinositol (GPI)−anchor biosynthesis; mmu04714: Thermogenesis; n = 5 mice per group. j Hierarchical clustering of the top 20 significantly altered lipids between the HFD + PEMF group and the HFD group, classified by LIPID MAPS: GL (glycerolipid), SP (sphingolipid), and GP (glycerophospholipid), n = 5 mice per group. k Double immunostaining of NLRP3 and CD31 in atherosclerotic lesions among different groups of mice; n = 5 mice per group. Scale bar = 100 μm. l Manders’ coefficient was used to evaluate the NLRP3-positive area within CD31-positive regions in the aortic arch plaques; n = 5 mice per group. m Quantitative comparison of NLRP3 colocalization with CD31 (ECs) and CD68 (macrophages) in the same mice; n = 5 mice per group. All the data represent biological replicates. The measured data are presented as the mean ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

PEMFs reduce the Ox-LDL-induced inflammatory response and pyroptosis and improve EC survival in an inflammatory environment. a CCK8 was used to detect the viability of HUVECs in the following groups: the normal cell (NC) group, the Ox-LDL (100 μg/mL, 24 h) group, and the Ox-LDL+PEMFs (15 Hz, 1.5 mT, 1 h/day, 1 d) group; n = 5 independent experiments per group. b LDH release in the supernatant of HUVECs was detected with an LDH kit; n = 5 independent experiments per group. c–g Levels of IL-1β, IL-18, IL-6, MCP-1, and TNF-α in the supernatants of HUVECs or HAECs were detected via ELISA; n = 5 independent experiments per group. h, i Western blot analysis of NLRP3, ASC, p20 caspase-1, and GSDMD-NT in HUVECs or HAECs; n = 5 independent experiments per group. j, k Pyroptosis was assessed by flow cytometry via Hoechst 33342/PI staining in HUVECs; n = 5 independent experiments per group. l, m Pyroptosis was assessed by flow cytometry via Hoechst 33342/PI staining in HAECs; n = 5 independent experiments per group. All the data represent biological replicates. The measured data are presented as the mean ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

The bidirectional regulation of NLRP3 downregulation has the same effect on PEMFs, whereas excessive upregulation counteracts the therapeutic effect of PEMFs. a Oil red O staining was used to determine the lesion burden in the aorta in the ApoE-/- (AKO) and ApoE-/-NLRP3-/- (DKO) mouse groups: the HFD AKO group, HFD DKO group, HFD AKO+PEMFs group, HFD AKO+PEMFs+MCC950 group, and HFD AKO+PEMFs+Nigericin group, n = 5 mice per group. b HE staining was used to visualize the plaque area in the aortic arch; n = 5 mice per group. Scale bar = 100 μm. c Western blot analysis of NLRP3, ASC, p20 caspase-1, and GSDMD-NT in aortic tissues; n = 5 mice per group. d, e Serum levels of IL-1β and IL-18 were detected via ELISA; n = 5 mice per group. f Oil red O staining was used to determine the lesion burden in the aorta in the following groups of AAV-injected ApoE-/- mice: the HFD + AAV NC group, HFD + AAV NLRP3 KD group, HFD+PEMFs+AAV NC group, and HFD+PEMFs+AAV NLRP3 KD group, n = 5 mice per group. g Statistical results of oil red O staining in mice in each group; n = 5 mice per group. h HE staining was used to visualize the plaque area in the aortic arch in AAV-injected ApoE-/- mice; n = 5 mice per group. Scale bar = 100 μm. i Statistical results of HE staining in mice in each group; n = 5 mice per group. j The mean fluorescence intensity (MFI) of NLRP3 was measured via flow cytometry in mouse vascular endothelial cells (MVECs) isolated from heart and aortic tissues; n = 5 mice per group. k The MFI of GSDMD-NT in MVECs isolated from heart and aortic tissues (n = 5 mice per group) was measured via flow cytometry. All the data represent biological replicates. The measured data are presented as the mean ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Downregulation of NLRP3 can protect cells, and upregulation of NLRP3 can aggravate EC damage. a CCK8 was used to detect cell viability in the following HUVEC groups: the Ox-LDL group, the Ox-LDL + MCC950 group, the Ox-LDL+PEMFs group, the Ox-LDL+ PEMFs+MCC950 group, and the Ox-LDL+PEMFs+Nigericin group; n = 5 independent experiments per group. b LDH release in the supernatant of HUVECs was detected with an LDH kit; n = 5 independent experiments per group. c–g Levels of IL-1β, IL-18, IL-6, MCP-1, and TNF-α in the supernatants of HUVECs or HAECs were detected via ELISA; n = 5 independent experiments per group. h Pyroptosis was assessed by flow cytometry via Hoechst 33342/PI staining in HUVECs; n = 5 independent experiments per group. i Pyroptosis was assessed by flow cytometry via Hoechst 33342/PI staining in HAECs; n = 5 independent experiments per group. j, k Western blot analysis of NLRP3, ASC, p20 caspase-1, and GSDMD-NT in HUVECs or HAECs; n = 5 independent experiments per group. All the data represent biological replicates. The measured data are presented as the mean ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Mitochondrial dysfunction is a bridge between inflammation and atherosclerosis. a Subcellular localization analysis of differentially expressed proteins in the CHD group versus the non-CHD group. The count indicates the number of differentially expressed proteins. n = 20 participants per group. b Mitochondrial permeability transition pore (mPTP) opening was detected with an mPTP detection kit in the following HUVEC groups: the NC group, the Ox-LDL group, and the Ox-LDL+PEMFs group; n = 5 independent experiments per group. c Mitochondrial membrane potential was detected by JC-1 staining; n = 5 independent experiments per group. Scale bar = 100 μm. d OCRs of HUVECs subjected to sequential injections of oligomycin (1 mM), FCCP (0.5 mM), and rotenone/antimycin A (0.5 mM). The values were normalized to the cell count. e–h Bar graphs quantifying basal respiration, maximal respiration, ATP production and spare respiration capacity; n = 5 independent experiments per group. i OCRs of MVECs subjected to sequential injections of oligomycin (1 mM), FCCP (0.5 mM), and rotenone/antimycin A (0.5 mM) were measured. The values were normalized to the cell count. j–m Bar graphs quantifying basal respiration, maximal respiration, ATP production and spare respiration capacity; n = 5 mice per group. All the data represent biological replicates. The measured data are presented as the mean ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

EC membrane tension and transient receptor potential vanilloid 4 (TRPV4) mechanosensitive channels participate in Ox-LDL/HFD-induced endothelial damage. a Imaging of the aortic intima of the ND, HFD, and HFD+PEMFs groups in contact mode via atomic force microscopy (AFM). The ultrastructures of fresh intima tissue are depicted in each image. Changes in the surface roughness of vascular ECs in each group; n = 5 mice per group. b Young’s modulus values of vascular ECs in each group; n = 5 mice per group. c Topographical images of HUVECs taken with AFM in the following HUVEC groups: the NC group, the Ox-LDL group, and the Ox-LDL+PEMFs group. d Young’s modulus values of HUVECs in each group; n = 5 independent experiments per group. e A Flipper-TR kit was used to observe cell membrane tension; n = 20 cells per group. f Time course of HUVEC membrane currents measured at baseline and after the application of GSK1016790A (80 nM) and HC067047 (100 nM). The ramp pulses were applied every 10 s from −100 to +100 mV for 1 s from a holding potential of 0 mV. g Representative current–voltage curves taken at the three time points corresponding to the black, red, and blue dots in (a). h Peak current amplitudes measured at +100 mV under baseline conditions (control), after GSK1016790A application and after HC067047 application. Each dot represents the peak current amplitude measured from one HUVEC; n = 9 cells per group. All the data represent biological replicates. The measured data are presented as the mean ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 7

TRPV4 hyperactivation exacerbates cellular stiffness and mitochondrial dysfunction, whereas TRPV4 inhibition confers cytoprotection. a Topographical images of HUVECs from the following HUVEC groups: Ox-LDL group, Ox-LDL + HC067047 group, Ox-LDL+PEMFs group, Ox-LDL+PEMFs+HC067047 group, and Ox-LDL+PEMFs+GSK1016790A group. b, c Cell height and cell body area were calculated from topographical images of HUVECs; n = 5 independent experiments per group. d Young’s modulus values of HUVECs in each group; n = 5 independent experiments per group. e A Flipper-TR kit was used to observe cell membrane tension; n = 20 cells per group. f, g Mitochondrial membrane potential was detected by JC-1 staining; n = 5 independent experiments per group. Scale bar = 100 μm. h OCRs of HUVECs subjected to sequential injections of oligomycin (1 mM), FCCP (0.5 mM), and rotenone/antimycin A (0.5 mM) were measured. The values were normalized to the cell count. i–l Bar graphs quantifying basal respiration, maximal respiration, ATP production, and spare respiratory capacity; n = 5 independent experiments per group. m Western blot analysis of TRPV4, NLRP3, ASC, and p20 caspase-1 protein expression in HUEVCs; n = 5 independent experiments per group. All the data represent biological replicates. The measured data are presented as the mean ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 8

TRPV4 activation exacerbates atherogenesis, whereas TRPV4 inhibition has the same effect on PEMFs. a Oil red O staining was used to determine the lesion burden in the aortas of the ApoE-/- mice in the following groups: the HFD group, HFD + HC067047 group, HFD+PEMFs group, HFD+PEMFs+HC067047 group, and HFD+PEMFs+ GSK1016790A group; n = 5 mice per group. b Evans blue staining was used to assess aortic permeability in ApoE-/- mice; n = 5 mice per group. Scale bar = 100 μm. c HE staining was used to visualize the plaque area in the aortic arch. n = 5 mice per group. Scale bar = 100 μm. d–h Serum levels of IL-1β, IL-6, IL-18, MCP-1, and TNF-α were detected by ELISA; n = 5 mice per group. i Western blot analysis of TRPV4, NLRP3, ASC, and p20 caspase-1 in aortic tissues; n = 5 mice per group. All the data represent biological replicates. The measured data are presented as the mean ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001