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. 2020 Apr 14;12(7):6415-6435.
doi: 10.18632/aging.103038. Epub 2020 Apr 14.

Electrical stimulation inhibits Val-boroPro-induced pyroptosis in THP-1 macrophages via sirtuin3 activation to promote autophagy and inhibit ROS generation

Lin Cong  1 , Ziyu Gao  1 , Yinghong Zheng  1 , Ting Ye  1 , Zitong Wang  1 , Pengyu Wang  1 , Manman Li  1 , Bowen Dong  1 , Wei Yang  1 , Quanfeng Li  1 , Shupei Qiao  2 , Cao Wang  2 , Yijun Shen  2 , Hong Li  1 , Weiming Tian  2 , Liming Yang  1   3
Affiliations

Electrical stimulation inhibits Val-boroPro-induced pyroptosis in THP-1 macrophages via sirtuin3 activation to promote autophagy and inhibit ROS generation

Lin Cong et al. Aging (Albany NY). .

Abstract (V体育平台登录)

The incidence of atherosclerosis (AS), a major contributor to cardiovascular disease, is steadily rising along with an increasingly older population worldwide. Pyroptosis, a form of inflammatory programmed cell death, determines the release of pro-inflammatory mediators by endothelial cells, smooth muscle cells, and atheroma-associated macrophages and foam cells, thereby playing a critical role in AS progression. Canonical pyroptosis is mediated by inflammasome formation, activation of caspase-1, and maturation and release of proinflammatory cytokines. Electrical stimulation (ES) is a noninvasive, safe therapy that has been shown to alleviate symptoms in several health conditions VSports手机版. Here, we investigated the anti-inflammatory and anti-pyroptotic effects of ES in human THP-1 macrophages treated with the dipeptidyl peptidase inhibitor Val-boroPro (VbP). We found that ES downregulated NOD-like receptor family protein 3 (NLRP3) inflammasome, ASC, and caspase-1 expression and abrogated the release of Interleukin-1β (IL-1β) and Interleukin-18 (IL-18), indicating effective pyroptosis inhibition. These changes were paralleled by a reduction in reactive oxygen species (ROS) production, reversal of VbP-induced sirtuin3 (Sirt3) downregulation, deacetylation of ATG5, and induction of autophagy. These findings suggest that ES may be a viable strategy to counteract pyroptosis-mediated inflammation in AS by raising Sirt3 to promote autophagy and inhibit ROS generation. .

Keywords: ROS; electrical stimulation; macrophages; pyroptosis; sirtuin3 V体育安卓版. .

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
VbP induces pyroptosis in THP-1 macrophages. (A) Detection of cell viability (CCK-8 assay) after (1) 72 h exposure to different concentrations of VbP, and (2) exposure to 15 μM VbP for various times. (B) ELISA results showing secretion of IL-18 (1) and IL-1β (2) by macrophages treated for 72 h with different concentrations of VbP. (C) Dose-dependent expression of pyroptosis-related proteins in untreated (control) and VbP-treated macrophages (72 h exposure). (D) Time-dependent expression of pyroptosis-related proteins in untreated (control) and VbP-treated (15 μM) macrophages. (E) Dose-response analysis of NLRP1 inflammasome expression in VbP-treated macrophages (72 h exposure). (F) Time-response analysis of NLRP1 expression in macrophages exposed to 15 μM VbP. n = 3; *P<0.05, **P<0.01, and ***P<0.001 vs. control cells.
Figure 2
Figure 2
ES inhibits VbP-induced pyroptosis in THP-1 macrophages. (A) Pattern diagram of the ES device. (B) Circuit diagram of the ES device. (C) Viability assay results in control THP-1 macrophages after ES at (1) different voltages (9 min exposure) and (2) 30 mv/cm for various exposure times. (D) Viability assay results in macrophages exposed to VbP (15 μM, 72 h) and subsequently treated with ES at (1) different voltages (9 min exposure) and (2) 20 mv/cm for different exposure times. (E) Viability measurements at different time points after ES (20 mv/cm for 9 min) in macrophages treated with VbP (15 μM, 72 h). (F) Western blot analysis of NLRP3 and caspase-1 protein expression at different time points after ES. (G) Dose-response analysis of the expression of pyroptosis-related proteins in macrophages treated with ES (9 min exposure) after VbP treatment (15 μM, 72 h). (H) Time-dependent analysis of the expression of pyroptosis-related proteins in macrophages treated with ES (20 mv/cm) after VbP exposure (15 μM, 72 h). n =3; *P<0.05, **P<0.01, and ***P<0.001 vs. control cells.
Figure 3
Figure 3
ES inhibits caspase-1-dependent pyroptosis of THP-1 macrophage induced by vbp. (A) Western blot analysis of the effect of caspase-1 inhibition on pyroptosis-related proteins. (B) RT-qPCR analysis of the effect of caspase-1 inhibition on pyroptosis-related mRNAs. (C) Representative western blot results showing downregulation of caspase-1 expression following siRNA-mediated caspase-1 knockdown. (D) Western blot analysis of the effect of caspase-1 knockdown on pyroptosis-related proteins. n = 3; *P<0.05, **P<0.01, and ***P<0.001 vs. control cells; #P<0.05, ##P<0.01, and ###P<0.001 vs. VbP-treated cells.
Figure 4
Figure 4
ES inhibits ROS production in VbP-treated THP-1 macrophages. (A) TEM analysis of ultrastructural alterations in THP-1 macrophages. Red arrows indicate cell swelling and nuclear pyknosis (scale bar: 10 μm). (B) DCFH-DA staining showing ROS generation in control macrophages following ES at different voltages (scale bar: 100 μm). (C) DCFH-DA staining showing ROS generation in VbP-treated macrophages following ES at various voltages (scale bar: 100 μm). (D) DCFH-DA staining showing ROS generation in VbP-treated macrophages exposed to NAC and ES (scale bar: 100 μm). (E) Effect of ES, alone or combined with NAC, on NLRP3 immunofluorescence in VbP-treated macrophages (scale bar: 20 μm). (F) ELISA analysis of the effects of NAC and ES on (1) IL-18 and (2) IL-1β secretion by THP-1 macrophages. (G) RT-qPCR analysis of the effects of NAC and ES on pyroptosis-related mRNAs. (H) Western blot analysis of the effects of NAC and ES on pyroptosis-related proteins. n =3; *P<0.05, **P<0.01, and ***P<0.001 vs. control cells; #P<0.05, ##P<0.01, and ###P<0.001 vs. VbP-treated cells.
Figure 5
Figure 5
ES rescues Sirt3 expression to induce autophagy in VbP-treated THP-1 macrophages. (A) Western blot analysis of Sirt3 expression in macrophages treated with VbP and ES. (B) Analysis of the effects of ES and the Sirt3 inhibitor 3-TYP on Sirt3 expression. (C) DCFH-DA staining showing the effect of 3-TYP on ROS generation by macrophages (scale bar: 100 μm). (D) TEM images showing autophagosome assembly (red arrows) in VbP-treated macrophages exposed to ES (scale bar: 10 μm). (E) Visualization of AVOs by AO staining. The autophagy inducer Rapa (1 μM) was used as positive control. (F) DMC staining showing induction of autophagic vacuoles by ES in VbP-treated macrophages. This effect was blocked by pre-treatment with the autophagy inhibitor 3-MA (10 mM) (scale bar: 50 μm). (G) Immunofluorescence analysis of LC3 and Lamp2 expression indicating induction of autolysosome formation by ES and inhibition of this process by 3-TYP in VbP-treated macrophages (scale bar: 20 μm). (H) Western blotting analysis of the effects of ES and 3-TYP on ATG5, Sirt3, and LC3 expression in VbP-treated macrophages. n = 3; *P<0.05, **P<0.01, and ***P<0.001 vs. control cells; #P<0.05, ##P<0.01, and ###P<0.001 vs. VbP-treated cells.
Figure 6
Figure 6
ES prevents VbP-induced oxidative stress in THP-1 macrophages. (A) Western blot analysis of the expression of 4-HNE adducts, catalase, and MnSOD. (B) ELISA determination of 4-HNE secretion. (C) Quantification of catalase activity (colorimetric detection). (D) Flow cytometry analysis of ROS generation in DCFH-DA labeled macrophages. n = 3; *P<0.05, **P<0.01, and ***P<0.001 vs. control cells; #P<0.05, ##P<0.01, and ###P<0.001 vs. VbP-treated cells.
Figure 7
Figure 7
ES induces Sirt3-mediated deacetylation of ATG5 in VbP-treated macrophages. (A) Representative western blots of ATG5 expression following immunoprecipitation with an anti-Sirt3 antibody (top), Sirt3 expression following immunoprecipitation with an anti-ATG5 antibody (middle) and ATG5 and Sirt3 expression in total cell lysates (bottom). (B) Representative western blot and quantitative analysis of acetylated-ATG5 in macrophages treated with VbP, 3-TYP, and ES. n =3; ***P<0.001 vs. control cells; #P<0.05 and ###P<0.001 vs. VbP-treated cells.
Figure 8
Figure 8
ES inhibits VbP-induced pyroptosis in foam cells. (A) Representative images of Oil Red O-stained THP-1 macrophages following incubation with ox-LDL for 6, 12, 24, and 48 h (scale bar: 50 μm). (B) Detection of foam cell viability by CCK8 assays: (1) Dose-response assay results (72 h incubation); (2) Time-response assay results (5 μM VbP). (C) Detection of NLRP1 and NLRP3 expression by western blotting in foam cells treated with different concentrations of VbP (72 h exposure). (D) Dose-response and (E) time-response RT-qPCR analysis of pyroptosis-related mRNAs in foam cells treated with different concentrations of VbP (72 h) or 10 μM VbP for up to 120 h. (F) Viability measurements in VbP-treated foam cells exposed to ES at (1) different voltages (9 min exposure) and (2) 20 mv/cm for various exposure times. (G) Viability measurements at different time points after ES (20 mv/cm for 9 min) in foam cells treated with VbP (10 μM for 72 h). (H) Analysis of NLRP3 and caspase-1 expression by western blotting at different time points after 9 min, 20 mv/cm ES. (I) Dose-response and (J) time-response RT-qPCR analysis of pyroptosis-related mRNAs in foam cells exposed to ES. (K) RT-qPCR analysis showing the effects of VbP, VX-765, and ES on caspase-1, IL-18, and IL-1β mRNA levels in foam cells. n = 3; *P<0.05, **P<0.01, and ***P<0.001 vs. control cells; ##P<0.01 and ###P<0.001 vs. VbP-treated cells.
Figure 9
Figure 9
Schematic representation of the effects of ES on VbP-treated THP-1 macrophages. VbP exposure induces ROS production, which promotes NLRP3 inflammasome formation leading to activation of caspase-1. Activated caspase-1 triggers pyroptosis through membrane pore formation, DNA fragmentation, and release of mature IL-1β and IL-18. The ensuing sterile inflammatory response contributes in turn to the progression of AS. Meanwhile, ES normalizes Sirt3 expression and promotes autophagy in VbP-treated macrophages. This reduces ROS production and prevents oxidative stress, which inhibits NLRP3 inflammasome formation and prevents pyroptosis.
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