Inhibiting microRNA-200a-3p attenuates pyroptosis via targeting the SIRT1/NF-κB/NLRP3 pathway in H₂O₂-induced HAEC

Oxidative stress induces HAEC injury

In this study, we first found that the expression of the pyroptosis-related protein was upregulated in aortia in HFD group (Figure 1A), and the miRNA-200a-3p level was significantly increased in HFD group (Figure 1B). In order to explore the mechanism, in vitro oxidative stress response was stimulated by cell treatment with H₂O_2. HAECs were treated with different concentrations of H₂O₂ (0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM) for 4 h, and cell viability was determined using the CCK-8 assay (Figure 1C). HAECs were treated with 0.4 mM H₂O₂ for different durations (0 h, 1 h, 2 h, 4 h, 8 h), and cell viability was detected using the CCK-8 assay (Figure 1D). LDH was released from HAECs treated with H₂O₂ (0.4 mM) at different times (Figure 1E). H₂O₂ stimulation also significantly increased intracellular ROS levels, contributing to the induction of cell damage (Figure 1F). Further, PI staining results revealed that the percentage of PI-positive cells increased in H₂O₂-treated HAECs in a time-dependent manner (Figure 1G). The changes of HAECs swelling and transparency of its cytoplasm can be seen with light microscope (Figure 1H).

Figure 1. Oxidative stress induces HAEC injury. (A) The pyroptosis-related proteins were assessed via western blot in aortia in apoE^-/- mice. (B) qRT-PCR results showing the miRNA-200a-3p expression increased in HFD group compared with that in NCD group. (C) HAECs were treated with different concentrations of H₂O₂ (0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM) for 4 h, and cell viability was detected using the CCK-8 assay. (D) HAECs were treated with 0.4 mM H₂O₂ for different durations (0 h, 1 h, 2 h, 4 h, 8 h), and cell viability was detected using the CCK-8 assay. (E) LDH released from HAECs treated with H₂O₂ (0.4 mM) at different times. (F) H₂O₂ stimulation significantly increased intracellular ROS, which contributed to the induction of cell damage. Plotting scale =100 μm. (G) PI staining results revealed increased percentage of PI positive cells in H₂O₂-treated HAECs in a time-dependent manner. Double staining of PI (red) and Hoechst 33342 (blue). Plotting scale = 200 μm. (H) Morphological changes of HAECs under light microscope. Plotting scale = 200 μm. (*p<0.05 vs control, **p<0.01 vs control, ***p<0.001 vs control). Data are presented as mean ± SD (n=3). HAECs, human aortic endothelial cells; CCK-8, cell-counting kit 8; LDH, lactate dehydrogenase; ROS, reactive oxygen species; PI, propidium iodide; SD, standard deviation.

Oxidative stress induces pyroptosis of HAECs

Pyroptosis is a novel form of cell death that is mediated by the inflammasome and caspase-1 activation. To determine whether H₂O₂ could induce the pyroptosis of HAECs, HAECs were treated with different concentrations of H₂O₂ for 4 h. Western blot analysis revealed that H₂O₂ upregulated the expression of the pyroptosis-related proteins in HAECs in a dose-dependent manner (Figure 2A). When HAECs were treated with H₂O₂ (0.4 mM) at different times, western blot analysis also revealed that H₂O₂ upregulated the expression of the pyroptosis-related proteins in HAECs (Figure 2B). To further determine whether H₂O₂-induced pyroptosis of HAECs is dependent on NLRP3 inflammation, a transfection experiment was conducted. siNLRP3 was found to decrease the expression levels of GSDMD-N, C-caspase-1, and IL-1β in HAECs treated with H₂O₂ (Figure 2C, 2D). Further, si-ASC decreased the expression levels of GSDMD-N and IL-1β in HAECs treated with H₂O₂ (Figure 2E, 2F). To investigate whether H₂O₂-induced pyroptosis of HAECs is caspase-1 dependent, a caspase-1 inhibitory experiment was conducted. The caspase-1 selective inhibitor, VX-765, decreased the level of activated caspase-1 and inhibited the maturation of IL-18 and IL-1β (Figure 2G). Thus, H₂O₂-induced pyroptosis of HAECs is dependent on NLRP3 inflammation.

Figure 2. Oxidative stress induces HAEC pyroptosis. (A) HAECs were treated with 0, 0.2, 0.4, or 0.6 mM H₂O₂ for 4 h, and the pyroptosis-related proteins were assessed via western blot analysis. H₂O₂-induced HAEC pyroptosis in a dose-dependent manner. (B) HAECs were treated with H₂O₂ (0.4 mM) for different durations (0 h, 2 h, 4 h, 8 h) and the pyroptosis-related proteins were assessed via western blot. H₂O₂ induced HAEC pyroptosis in a time-dependent manner. (C) Western blot revealing the downregulation of the NLRP3 protein in HAECs treated with siNLRP3 via transient transfection. (D) Western blot demonstrating the decreased protein levels of GSDMD-N, C-caspase-1, and IL-1β in HAECs transduced with siNLRP3 compared with cells transduced with NC under H₂O₂ treatment. (E) Western blot demonstrating the downregulation of the ASC protein in HAECs treated with siASC via transient transfection. (F) Western blot demonstrating the decreased protein levels of GSDMD-N and IL-1β in HAECs transduced with si-ASC compared with cells transduced with NC under H₂O₂ treatment. (G) Western blot demonstrating the decreased protein levels of C-caspase-1, GSDMD-N, and IL-1β in HAECs treated with VX-765 compared with cells without VX-765 under H₂O₂ treatment. Data are presented as mean ± SD (n=3). (^*p<0.05 vs control, H₂O₂ or H₂O₂+siNC, ^**p<0.01 vs control, H₂O₂ or H₂O₂+siNC, ^***p<0.001 vs control, ns, no significance). HAECs, human aortic endothelial cells; NC, negative control.

Downregulation of p65 abolishes H₂O₂-induced pyroptosis of HAECs

p65 is known as an important transcription factor that binds to the promoter regions of target genes and mediates their transcription. Previous studies revealed that NF-κB activation is necessary for NLRP3 activation [23]. To explore whether p65 directly affects NLRP3 expression in H₂O₂-induced pyroptosis, the protein levels of p65 were first found to increase after H₂O₂ treatment in a time-dependent manner (Figure 3A). The effect of p65 deficiency on H₂O₂-induced pyroptosis was assessed. siRNA targeting p65 was used to reduce the protein expression of p65. Remarkably, si-p65 decreased H₂O₂-induced upregulation of LDH release from HAECs and the percentage of PI-positive cells (Figure 3D, 3E). To explore the role of p65 and NLRP3 in H₂O₂-induced pyroptosis, p65 was silenced with siRNA. The mRNA expression levels of NLRP3 and IL-1β decreased in HAECs transduced with si-p65 compared to those treated with H₂O₂ (Figure 3F). In addition, si-p65 decreased the H₂O₂-induced upregulation of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 protein in HAECs (Figure 3G). Nuclear plasma separation was performed to assess protein distribution in the cell nucleus and cytoplasm. Based on western blotting, H₂O₂ promoted the expression of p65 in the cytoplasm and nucleus (Figure 3H, 3I). In fact, the p65 protein levels in the nucleus were obviously increased.

Figure 3. Effect of p65 on the H₂O₂-induced pyroptosis of HAECs. (A) Western blot demonstrating the increased protein levels of p65 upon H₂O₂ treatment in a time-dependent manner. (B) qRT-PCR results demonstrating the decreased mRNA expression of p65 in cells transduced with si-p65 compared with that in untransduced cells. (C) Western blot demonstrating the decreased protein expression of p65 in cells transduced with si-p65 compared with that in untransduced cells. (D) LDH release from HAECs treated with or without si-p65 following H₂O₂ treatment. (E) PI staining results of the increased percentage of PI positive cells in H₂O₂-treated HAECs. Double staining of PI (red) and Hoechst 33342 (blue). The percentage of PI positive cells in H₂O₂-treated HAECs decreased after transfection with si-p65. Plotting scale = 200 μm. (F) qRT-PCR results demonstrating the increased mRNA expression of NLRP3 and IL-1β in HAECs under H₂O₂ treatment, and decreased mRNA expression of NLRP3 and IL-1β in HAECs transduced with si-p65 compared with cells transduced with NC under H₂O₂ treatment. (G) Western blot results demonstrating the decreased protein expression of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 in HAECs transduced with si-p65 compared with cells transduced with NC under H₂O₂ treatment. Western blot demonstrating the increased protein expression of p65 in the cytoplasm (H) and nucleus (I) of H₂O₂-treated HAECs compared with NC-treated cells. The p65 protein levels in nucleus were obviously increased. (^*p<0.05 vs control or NC, ^**p<0.01 vs control or NC, ^***p<0.001 vs NC, ^#p<0.05 vs H₂O₂, ^##p<0.01 vs H₂O₂. ^###p<0.001 vs H₂O₂). Data are presented as mean ± SD (n=3). HAECs, human aortic endothelial cells; LDH, lactate dehydrogenase; qRT-PCR, quantitative real-time polymerase chain reaction; PI, propidium iodide; NC, negative control.

SIRT1 overexpression abolishes H₂O₂-induced pyroptosis of HAECs

Further exploration of the molecular mechanisms of p65-mediated pyroptosis revealed that acetylation of the p65 subunit plays an important role in the regulation of NF-κB transcriptional activity. SIRT1 inhibits the transcription of NF-κB by deacetylating the p65 protein at lysine 310; however, its role in HAEC pyroptosis has not been studied. The mRNA and protein levels of Sirt1 decreased after H₂O₂ treatment in a time-dependent manner (Figure 4A, 4B). The overexpression of SIRT1 in HAECs via transduction with the SIRT1 plasmid is shown in Figure 4C. SIRT1 overexpression decreased LDH release from HAECs treated with H₂O₂ and the percentage of PI-positive cells (Figure 4D, 4E). Moreover, SIRT1 overexpression decreased the protein expression of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 in HAECs treated with H₂O₂ (Figure 4F). Overall, SIRT1 protects HAECs from H₂O₂-induced injury.

Figure 4. Effect of SIRT1 on the H₂O₂-induced pyroptosis of HAECs. (A) qRT-PCR results demonstrating the decreased mRNA expression of Sirt1 upon H₂O₂ treatment in a time-dependent manner. (B) Western blot demonstrating the decreased protein levels of SIRT1 upon H₂O₂ treatment in a time-dependent manner. (C) Western blot demonstrating the overexpression of the SIRT1 protein in HAECs treated with ov-SIRT1 via transient transfection. (D) LDH release from HAECs treated with or without ov-SIRT1 following H₂O₂ treatment. (E) PI staining results demonstrating the increased percentage of PI positive cells in H₂O₂-treated HAECs. Double staining of PI (red) and Hoechst 33342 (blue). The percentage of PI positive cells in H₂O₂-treated HAECs decreased after transfection with ov-SIRT1. Plotting scale = 200 μm. (F) Western blot demonstrating the increased ratio of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, IL-18 in cells with H₂O₂ treatment compared with cells without H₂O₂ treatment. The ratio of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 decreased in cells transduced with ov-SIRT1 under H₂O₂ treatment compared with cells transduced with NC. (^*p<0.05 vs control or NC, ^**p<0.01 vs control or NC, ^***p<0.001 vs NC, ^#p<0.05 vs H₂O₂, ^##p<0.01 vs H₂O₂). Data are presented as mean ± SD (n=3). HAECs, human aortic endothelial cells; qRT-PCR, quantitative real-time polymerase chain reaction; LDH, lactate dehydrogenase; PI, propidium iodide; GSDMD, gasdermin-D; ASC, apoptosis-associated speck-like protein containing a CARD; NC, negative control.

SIRT1 overexpression abolishes pyroptosis by deacetylating p65 in H₂O₂-induced pyroptosis of HAECs

To further verify the relationship between SIRT1 and p65 in H₂O₂-induced HAEC pyroptosis, p65 acetylation levels in H₂O₂-induced HAECs were first determined. The protein ratio of Ac-p65 (Lys310)/p65 increased in H₂O₂-induced HAECs. Further, the overexpression of SIRT1 protein levels decreased the protein expression ratio of Ac-p65 (Lys310)/p65 in H₂O₂-induced HAECs (Figure 5A). To determine whether SIRT1 and p65 interact directly, endogenous IP western blotting was performed. Based on the result, the HAECs extracts were pulled down with the p65 antibody and the protein expression of SIRT1 was high (Figure 5B). To clarify whether SIRT1 affects the nuclear translocation of p65, western blotting was performed. SIRT1 overexpression increased p65 protein levels in the cytoplasm and decreased p65 protein levels in the nucleus (Figure 5C, 5D). The p65 staining results also showed that the level of p65 in the nucleus increased in H₂O₂-treated HAECs. However, ov-SIRT1 decreased the level of p65 in the nucleus in H₂O₂-treated HAECs (Figure 5E). Based on these results, SIRT1 overexpression abolished the inhibitory effect of H₂O₂ on HAEC pyroptosis by deacetylating p65 and inhibiting nuclear translocation.

Figure 5. Effect of SIRT1 deacetylation on p65 in H₂O₂-induced pyroptosis of HAECs. (A) Western blot demonstrating the increased protein expression ratio of Ac-p65 (Lys310)/p65 in the H₂O₂ group compared with the NC group. The overexpression of SIRT1 protein levels decreased the protein expression ratio of Ac-p65 (Lys310)/p65 compared with that in cells treated with H₂O₂. (B) Endogenous IP Western revealed the pulling down of the HAEC extracts with either a negative control (IgG) or RelA/p65 antibody, followed by SIRT1 or RelA/p65 immunoblot. (C) Western blot demonstrating the increased p65 protein levels in the cytoplasm of H₂O₂-treated HAECs compared with NC-treated cells. Overexpression of SIRT1 protein levels increased the p65 protein levels in the cytoplasm compared with that in cells treated with H₂O₂. (D) Western blot demonstrating increased p65 protein levels in the nucleus of H₂O₂-treated HAECs compared with NC-treated cells. Overexpression of SIRT1 protein levels decreased p65 protein levels in the nucleus compared with that in cells treated with H₂O₂. (E) Double staining of p65 (green) and DAPI (blue). DAPI staining results of the nucleus of HAECs. P65 staining results of increased p65 in the nucleus of H₂O₂-treated HAECs; the level decreased after transfection with ov-SIRT1 in H₂O₂-treated HAECs under a fluorescence microscope. Plotting scale =50 μm. (^*p<0.05 vs NC, ^**p<0.01 vs NC, ^#p<0.05 vs H₂O₂, ^##p<0.01 vs H₂O₂). Data are presented as mean ± SD (n=3). HAECs, human aortic endothelial cells; NC, negative control; IP, immunoprecipitation; DAPI, 4′,6-diamidino-2-phenylindole.

Downregulation of miRNA-200a-3p abolishes the effect of H₂O₂ on pyroptosis

To identify an effective target for anti-atherosclerosis therapy through SIRT1, we analyzed the possible direct targets of miR-200a-3p and SIRT1 using bioinformatics. First, the miRNA-200a-3p levels were found to be significantly increased in H₂O₂-induced HAECs in a time-dependent manner (Figure 3A). When the effect of miRNA-200a-3p on H₂O₂-induced pyroptosis was examined, the miR-200a-3p inhibitor was found to decrease the level of LDH release from HAECs treated with H₂O₂ and the percentage of PI-positive cells (Figure 6C, 6D). The miR-200a-3p inhibitor also decreased the protein expression of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 in H₂O₂-induced HAECs (Figure 6E)_. These results indicate that the miR-200a-3p inhibitor protected HAECs from H₂O₂-induced injury.

Figure 6. Effect of miRNA-200a-3p on H₂O₂-induced pyroptosis of HAECs. (A) HAECs were treated with H₂O₂ (0.4 mM) for 1 h, 2 h, 4 h, and 8 h; miRNA-200a-3p expression increased in all cases compared with that in control cells. (B) qRT-PCR results showing the decreased expression of miR-200a-3p in HAECs transduced with the miR-200a-3p inhibitor compared with negative control cells. (C) LDH release from HAECs treated with H₂O₂ and transduced with or without the miR-200a-3p inhibitor. (D) PI staining results showing the decreased percentage of PI positive cells in HAECs transduced with the miR-200a-3p inhibitor. (E) Western blot demonstrating increased ratio of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, IL-18 in cells treated with H₂O₂ compared with cells that were not treated with H₂O₂. The ratio of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, IL-18 decreased in cells transduced with the miR-200a-3p inhibitor under H₂O₂ treatment compared with cells transduced with the NC inhibitor. (^*p<0.05 vs control, ^**p<0.01 vs control or NC, ^***p<0.001 vs control or NC, ^#p<0.05 vs H₂O₂, ^##p<0.01 vs H₂O₂)_. Data are presented as mean ± SD (n=3). HAECs, human aortic endothelial cells; qRT-PCR, quantitative real-time polymerase chain reaction; LDH, lactate dehydrogenase; PI, propidium iodide; NC, negative control.

Downregulation of miRNA-200a-3p abolishes the effect of H₂O₂ on the pyroptosis of HAECs via target regulating SIRT1

To explore the relationship between miR-200a-3p and SIRT1 in H₂O₂-induced HAECs, miR-200a-3p was overexpressed in HAECs via transduction with miR-200a-3p mimics as depicted in Figure 7A. The miR-200a-3p mimics decreased the RNA and protein expression of SIRT1 in HAECs (Figure 7B, 7C), whereas the miR-200a-3p inhibitors increased the RNA and protein expression of SIRT1 in HAECs treated with H₂O₂ (Figure 7D, 7E). Based on bioinformatics and the existing literature, the relationship between miR-200a-3p and SIRT1 in H₂O₂-induced HAEC pyroptosis was assessed. A schematic representation of complementary miR-200a-3p and SIRT1-3'UTR sequences is provided in Figure 7F. The letters in red indicate the matched bases. By performing a luciferase reporter gene assay, we found that the miR-200a-3p mimic inhibited the expression of SIRT1. Finally, a rescue experiment was performed to verify the direct targeting of SIRT1 by miR-200a-3p. Western blot revealed the decreased expression of SIRT1 in HAECs (Figure 7G). Further, knockdown of miR-200a-3p was found to decrease the expression of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 in HAECs treated with H₂O₂. Knockdown of SIRT1 reversed the function of the miR-200a-3p inhibitors, which increased the expression of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 in HAECs treated with H₂O₂ (Figure 7H). Overall, miR-200a-3p can regulate SIRT1 expression at the post-transcriptional level by targeting the SIRT1 -3'UTR.

Figure 7. Effect of miRNA-200a-3p on H₂O₂-induced pyroptosis of HAECs via SIRT1. (A) qRT-PCR results showing the increased expression of miR-200a-3p in HAECs transduced with the miR-200a-3p mimics compared with negative control cells. (B, C) qRT-PCR and western blot results demonstrating the decreased SIRT1 RNA and protein expression in HAECs transduced with miR-200a-3p mimics. (D, E) qRT-PCR results showing the decreased SIRT1 RNA and protein expression in HAECs treated with H₂O₂ compared with cells that were not treated with H₂O₂. SIRT1 expression increased in HAECs transduced with the miR-200a-3p inhibitors compared with cells transduced with the NC inhibitor under H₂O₂ treatment. (F) Schematic of the complementary miR-200a-3p and SIRT1-3'UTR sequences. HEK-293T cells were co-transfected with wild-type or mutant SIRT1-3'UTR reporter constructs and miR-200a-3p mimics or corresponding negative controls (^**p<0.01 vs SIRT1-3'UTR-wt+NC mimics group, ns, not significant vs SIRT1-3'UTR-mut+NC mimics group)_. (G) Western blot demonstrating the decreased expression of SIRT1 in HAECs compared with that in negative control cells with or without H₂O₂ treatment. (H) Western blot demonstrating the decreased ratio of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 in cells transduced with miR-200a-3p inhibitor under H₂O₂ treatment compared with that in cells transduced with the NC inhibitor. The ratio of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 increased in cells transduced with the miR-200a-3p inhibitor and si-SIRT1 under H₂O₂ treatment compared with that in cells transduced with the miR-200a-3p inhibitor. (^*p<0.05 vs NC, ^**p<0.01 vs NC, ^***p<0.001 vs NC, ^#p<0.05 vs H₂O_2,^##p<0.01 vs H₂O₂, ^&p<0.05 vs H₂O₂+Anti-miR-200a-3p, ^&&p<0.01 vs H₂O₂+ Anti-miR-200a-3p.). Data are presented as mean ± SD (n=3). HAECs, human aortic endothelial cells; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control.

Mice

Eight-week-old male apoE^-/- mice (C57BL/6 background) were purchased from Changchun Yisi Company. The mice were randomly divided into two groups (n=6/group), the normal chow diet (NCD) group, and the high fat diet (HFD) group. The mice were fed a high fat diet (D12109C, Research Diet: 40 % fat, 1.25% cholesterol, and 0.5% cholic acid) for 12 weeks to establish an atherosclerosis model.

Cell culture and treatment

HAECs were obtained from Cell Research (Shanghai, China). The cells were grown in Endothelial Cell medium (ScienCell Research Laboratories, USA) supplemented with 5% fetal bovine serum, 1% (v/v) penicillin/streptomycin, and 1% endothelial cell growth factors at 37° C with 5% CO₂. The cells were digested and passaged with pancreatin (1:250, Solarbio, China). When the cells reached 80% confluence, they were treated with 0.4 mM H₂O₂ for 1 h, 2 h, 4 h, or 8 h. To inhibit caspase 1 activity, the cells were treated with the 30 μM of the caspase 1 inhibitor, VX-765 (Beyotime, China), for 1 h.

Cell viability assay

Cell viability was measured using the Cell Counting Kit-8 (TransGen Biotech, China), according to the manufacturer’s instructions. HAECs were seeded in 96-well plates at a density of 5,000 cells/well and exposed to various concentrations of the compounds for the indicated times. Thereafter, 10 μl of working reagent was added to each well and incubated for 2 h at 37° C. The absorbance was measured using a microplate reader (Synergy HT, Bio-Tek, USA) at a wavelength of 450 nm. Optical density is proportional to the number of living cells in the plate.

Lactate dehydrogenase (LDH) activity

The LDH activity was measured using an LDH assay kit. For every sample, the working buffer consisted of INX, enzyme solution, and lactic acid solution. A total of 120 μl cell culture supernatant and 60 μl of working buffer were mixed together and incubated at 37° C for 30 min. Finally, the absorbance was measured at 490 nm using a microplate reader.

Detection of ROS

An ROS assay kit (Beyotime) was used to detect the accumulation of ROS in HAECs, according to the manufacturer’s instructions. Briefly, HAECs were cultured on coverslips in 24-well plates at a density of 2 × 10⁴ cells/well. Cells were loaded with DCFH-DA (10 μM) in serum-free medium in the dark at 37° C for 20 min after pretreatment, and then washed three times with PBS. The fluorescence was examined using a confocal microscope (Olympus, Japan).

Hoechst 33342/propidium iodide (PI) fluorescent staining

Pyroptosis was assessed using Hoechst 33342/PI double fluorescent staining. HAECs were cultured in 6-well plates at a density of 3 × 10⁵ cells per well and transfected with various constructs or treated with H₂O₂. The cells were then stained with 20 μL Hoechst 33342 solution at 37° C in the dark for 10 min, followed by 5 μL PI at 25° C in the dark for 15 min. The stained cells were observed under a confocal laser-scanning microscope (Olympus).

Cell transfection

HAECs were seeded in six-well plates. When the cells reached 50% confluence, they were transfected with Sirt1 small interfering RNA (siRNA), p65 siRNA, NLRP3 siRNA, ASC siRNA, SIRT1-overexpressing pcDNA3.1-plasmid (100 nM), miRNA-200a-3p mimics, miRNA-200a-3p inhibitor, and negative control siRNA using Lipofectamine 3000 transfection reagent, according to the manufacturer’s instructions. At 48 h after transfection, the cells were treated with H₂O₂ for 4 h. Finally, protein expression was determined via western blot analysis, and the gene transfection efficiency was monitored using PCR or western blotting. The sequences of the NLRP3 siRNA used in our experiments are sense, 5′- GUGCAUUGAAGACAGGAAUTT-3′, and antisense, 5′-AUUCCUGUCUUCAA-UGCACTT-3′. The sequences of the ASC siRNA are sense, 5′-GGCAAUCCCACCA-AAUCAUTT-3′, and antisense, 5′-AUGAUUUGGUGG-GAUUGCCTT-3′. The sequences of the RelA/p65 siRNA are sense, 5′- GCACCAUCAACUAUGAUGATT-3′, and antisense, 5′-UCAUCAUAGUUGA-UGGUGCTT-3′. The sequences of the SIRT1 siRNA are sense, 5′-GCUGAUGAACC-GCUUGCUATT-3′, and antisense, 5′- UAGCAAGCGGUUCAUCAGCTT-3′. The sequences of the MiRNA-200a-3p mimics 5′-UAACACUGUCUGGUAACGAUGU AUCGUUACCAGACAGUGUUAUU-3′. The sequences of the miR-200a-3p inhibitor: 5′-ACAUCGUUACCAGAC-AGUGUUA-3′.

Quantitative real-time PCR (qRT-PCR)

TRIzol reagent (Ambion, USA) was used to extract total RNA from control, treated, and transfected cells. The concentration (ng/μL) of miRNA was quantified and evaluated for purity using a NanoDrop ultraviolet spectrophotometer (Thermo Fisher Scientific, USA). RNA was reverse-transcribed into cDNA using an mRNA reverse transcription kit and an miRNA reverse transcription kit (TransGen Biotech), according to the manufacturer’s instructions. SYBR Premix Ex Taq was used for DNA amplification and an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, USA) was used to quantify the relative RNA expression. After amplification, the threshold cycle (Ct) was determined and the relative mRNA or miRNA levels were calculated using the 2-ΔΔ Ct method. GAPDH was used as an internal control for data normalization. U6 was used to standardize miRNAs. qRT-PCR was performed using the primers listed as follows:

U6 (human) F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAA-TTTGCGT-3′; miR-200a-3p (human): 5′-TAACACTGTCTGGTAACGATGT-3′. SIRT1 (human) F:5′-TAGCCTTGTCAGATAAGGAAGGA -3’, R: 5’–ACAGCTT-CACAGTCAACTTTGT -3′; p65(human) F: 5′-ATGTGGAGATCATTGAGCAGC -3′, R: 5′-CCTGGTCCTGTGTAGCCATT -3′; NLRP3 (human) F: 5’ –GATCTTCGCTG-CGATCAACAG -3’, R: 5’ -CGTGCATTATCTGAACCCCAC -3’; IL-1β(human) F: 5’ -ATGATGGCTTATTACAGTGGCAA -3’, R: 5’ -GTCGGAGATTCGTAGCTGGA -3’; GAPDH (human) F: 5’ -GGAGCGAGATCCCTCCAAAAT -3’, R: 5’ –GGCTGTTG-TCATACTTCTCATGG -3’.

Western blot analysis

Total protein was extracted from endothelial cells using procedures described elsewhere. High-efficiency RIPA protein lysate (TransGen Biotech) was used to lyse HAECs. The cell lysates were then separated on a 10% or 12% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore, USA). The protein concentrations were determined using the BCA Protein Assay kit (Thermo Fisher Scientific). Thereafter, the membrane was probed overnight at 4° C with primary antibodies against NLRP3 (Abcam), ASC (Abcam), Caspase 1 (Abcam), GSDMD-N (Abcam), NF-κB p65(CST), acetyl-NF-κB p65(Lys310) (CST), SIRT1 (CST), IL-18 (Abcam), and IL-1β (Abcam), which were all diluted at 1:1000 in phosphate buffered saline. Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000, Goat Anti-Mouse IgG HRP Affinity Purified PAb, USA; Goat Anti-Rabbit IgG HRP Affinity Purified PAb, USA), goat anti-mouse IgG HRP affinity purified systems, and goat anti-rabbit IgG HRP were applied to the membranes for 50 min at 25° C. Thereafter, the ECL fluorescent developer (Thermo Fisher Scientific) and LAS3000 Imager (Fuji Photo Film Co, Ltd.) were used to detect the protein bands. ImageJ software was used to calculate the intensity of the protein bands. The PVDF membranes were washed with membrane stripping buffer 1 (GenStar, China) for 30 min and then incubated with antibodies overnight. GAPDH (Bioss Technology, China) was used as the internal reference.

Nuclear and cytoplasmic protein extraction

Nuclear and cytoplasmic protein was extracted from endothelial cells using extraction kit (TransGen Biotech). The endothelial cells were collected and lysed with CPEB I and CPEB II, and supernatant was collected as cytoplasmic protein after centrifugation. CPEB I was added to the precipitate, and the supernatant was discarded after centrifugation. NPEB was added to the precipitate, and the supernatant was collected as nuclear protein after centrifugation. The samples were stored at -20° C for subsequent experiments.

Coimmunoprecipitation (CO-IP)

Cells were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitor complexes. Whole cell lysates (400 μl) were incubated overnight with an anti-p65 antibody (6 μl) and IgG (2 μl) at 4° C. Subsequently, 40 μl of protein A/G magnetic bead slurry was transferred to a 1.5-ml tube. The beads were washed three times with PBST, added to whole-cell lysates, and mixed for 2 h at room temperature to yield immune complexes. The bound proteins were eluted via boiling with loading buffer and evaluated via western blotting.

Immunofluorescence

Immunofluorescence staining was performed to detect the expression of p65 in endothelial cells. Briefly, the cells were fixed with 4% paraformaldehyde for 30 min, penetrated with 0.3% Triton X-100 for 1 h, and then blocked with goat serum for 1 h. Subsequently, the cells were incubated overnight with the anti-p65 antibody at 4° C, followed by Alexa Fluor-conjugated secondary antibody (Invitrogen, USA) in the dark for 40 min. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime) for 5 min. Finally, cells were imaged using a laser scanning confocal microscope (Olympus).

Luciferase reporter assays

Luciferase reporters containing wild-type or mutated SIRT1 plasmids were constructed using GP-miRGLO vectors (GenePharma, China). The luciferase vector (100 ng) containing the pcDNA3.1-SIRT1 plasmid was co-transfected with miR-200a-3p mimic and negative control (scramble sequence of miR-200a-3p) into human embryonic kidney 293T (HEK 293T) cells using Lipofectamine 3000 (Invitrogen), with 10 ng of Renilla luciferase reporter used as an internal control. After 48 h, the cells were collected and lysed. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System (TransGen Biotech), according to the manufacturer’s instructions.

Statistical analysis

All experiments were independently repeated at least three times, and the results are expressed as mean ± standard deviation (SD). Statistical analysis was carried out using GraphPad software (version 8.0). The average fluorescence intensity was analyzed using ImageJ. P-values were determined using analysis of variance. Statistical significance was set at P < 0.05.