Eburicoic acid inhibits endothelial cell pyroptosis and retards the development of atherosclerosis through the Keap1/Nrf2/HO‑1/ROS pathway

Abstract

Atherosclerosis (AS)‑related coronary artery disease is the main cause of morbidity and mortality around the globe. Eburicoic acid, a triterpenoid compound from Antrodia camphorata, exerts anti‑inflammatory and anti‑hyperlipidemic effects, although its role in atherogenesis remains unknown. Endothelial cell pyroptosis‑caused chronic inflammatory response within vessel walls is a critical initial event in atherogenesis, making it a promising target to prevent AS. The present study was designed to investigate the effects of eburicoic acid on endothelial cell pyroptosis, AS progression and the underlying mechanisms. The results showed that with dose and time increased, treatment of human umbilical vascular endothelial cells (HUVECs) with eburicoic acid markedly decreased the expression of Kelch‑like ECH‑associated protein 1 (Keap1), NF‑E2‑related factor 2 (Nrf2), reactive oxygen species (ROS), NLR family pyrin domain‑containing protein 3 (NLRP3), cleaved caspase‑1, apoptosis‑associated speck‑like protein containing CARD (ASC), N‑terminal gasdermin‑D (GSDMD‑N), downregulated the secretion levels of pro‑inflammatory cytokines interleukin (IL) 1β, IL‑6 and IL‑18, inhibited caspase‑1 activity and lactate dehydrogenase release and improved plasma membrane integrity. By contrast, the expression of nuclear Nrf2, total Nrf2 and heme oxygenase‑1 (HO‑1) were increased by eburicoic acid treatment in HUVECs dose‑ and time‑dependently. Moreover, the inhibitory effects of eburicoic acid on HUVEC pyroptosis were mainly compromised by pre‑treatment with ROS agonist, HO‑1 small interfering (si)RNA, or Nrf2 siRNA. Finally, it was observed that administering high‑fat‑diet fed ApoE‑/‑ mice with eburicoic acid markedly increased Nrf2 and HO‑1 levels and reduced the expression of Keap1, NLRP3, cleaved caspase‑1, ASC and GSDMD‑N in aortas and ameliorated hyperlipidemia and inflammation in the serum, leading to smaller atherosclerotic plaques, less lipid accumulation and high content of collagen fiber within plaques. These findings identified eburicoic acid as a potent anti‑atherogenic natural product by suppressing endothelial cell pyroptosis via the Keap1/Nrf2/HO‑1/ROS pathway. Eburicoic acid may be considered an effective phytomedicine for treating AS.

Keywords: atherosclerosis; eburicoic acid; endothelial cell pyroptosis; heme oxygenase‑1; nuclear factor erythroid 2‑related factor 2.

Figure 1.

Eburicoic acid inhibits NLRP3 inflammasome activation and pyroptosis in ox-LDL-treated HUVECs in a dose-dependent manner. Cells were incubated with 100 µg/ml ox-LDL for 24 h, followed by treatment with 1, 5, 10 and 20 µg/ml eburicoic acid for 24 h. (A) Chemical structure of eburicoic acid; (B) RT-qPCR analyses of NLRP3, caspase-1, ASC and GSDMD mRNA levels. (C) western blotting analyses of NLRP3, pro-caspase-1, cleaved caspase-1, ASC and GSDMD-N protein levels; (D) ELISA Analyses of IL-1β, IL-6 and IL-18 secretion levels; (E) Determination of LDH release using an LDH release assay kit; (F) Spectrophotometry analysis of caspase-1 activity; (G) Representative images of Hoechst/PI staining. Data are represented as mean ± SD. ns, statistically insignificant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 20 µm. n=3. EA, eburicoic acid; NLRP3, NLR family pyrin domain-containing protein 3; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vascular endothelial cells; RT-qPCR, reverse transcription-quantitative PCR; ASC, apoptosis-associated speck-like protein containing CARD; GSDMD-N, N-terminal gasdermin-D; LDH, lactate dehydrogenase.

Figure 2.

Eburicoic acid inhibits NLRP3 inflammasome activation and pyroptosis in ox-LDL-treated HUVECs in a time-dependent manner. Cells were incubated with 100 µg/ml ox-LDL for 24 h, followed by treatment with 10 µg/ml eburicoic acid. (A) RT-qPCR analyses of NLRP3, caspase-1, ASC and GSDMD mRNA levels; (B) western blotting analyses of NLRP3, pro-caspase-1, cleaved caspase-1, ASC and GSDMD-N protein levels; (C) ELISA Analyses of IL-1β, IL-6 and IL-18 secretion levels; (D) Determination of LDH release using an LDH release assay kit; (E) Spectrophotometry analysis of caspase-1 activity; (F) Representative images of Hoechst/PI staining. Data are represented as mean ± SD. ns, statistically insignificant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 20 µm. n=3. NLRP3, NLR family pyrin domain-containing protein 3; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vascular endothelial cells; RT-qPCR, reverse transcription-quantitative PCR; ASC, apoptosis-associated speck-like protein containing CARD; GSDMD-N, N-terminal gasdermin-D; IL, interleukin; LDH, lactate dehydrogenase EA, eburicoic acid.

Figure 3.

Eburicoic acid inhibits NLRP3 inflammasome activation and pyroptosis in ox-LDL-treated HUVECs by decreasing ROS production. (A) HUVECs were pre-treated with 100 µg/ml ox-LDL for 24 h and further incubated with 1, 5, 10 and 20 µg/ml eburicoic acid for 24 h or incubated with 10 µg/ml eburicoic acid for 6, 12, 24 and 48 h, respectively. Intracellular ROS production was detected using the peroxide-sensitive fluorescent probe DCFH-DA. (B-G) Cells were pre-treated with 100 µg/ml ox-LDL for 24 h. Then, cells were treated with 10 µg/ml eburicoic acid for 24 h, with or without co-incubation of 10 µM DMNQ (ROS agonist) for 6 h. (B) RT-qPCR analyses of NLRP3, caspase-1, ASC and GSDMD mRNA levels; (C) western blotting analyses of NLRP3, pro-caspase-1, cleaved caspase-1, ASC and GSDMD-N protein levels; (D) ELISA Analyses of IL-1β, IL-6 and IL-18 secretion levels; (E) Determination of LDH release using an LDH release assay kit; (F) Spectrophotometry analysis of caspase-1 activity; (G) Representative images of Hoechst/PI staining. Data are represented as mean ± SD. ns, statistically insignificant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 20 µm. n=3. NLRP3, NLR family pyrin domain-containing protein 3; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vascular endothelial cells; ROS, reactive oxygen species; RT-qPCR, reverse transcription-quantitative PCR; ASC, apoptosis-associated speck-like protein containing CARD; GSDMD-N, N-terminal gasdermin-D; IL, interleukin; LDH, lactate dehydrogenase EA, eburicoic acid.

Figure 4.

Eburicoic acid inhibits NLRP3 inflammasome activation and pyroptosis in ox-LDL-treated HUVECs via the HO-1/ROS pathway. HUVECs were pre-treated with 100 µg/ml ox-LDL for 24 h and further incubated with (A and B) 1, 5, 10 and 20 µg/ml eburicoic acid for 24 h or (C and D) incubated with 10 µg/ml eburicoic acid 6, 12, 24 and 48 h, respectively. The mRNA and protein levels of HO-1 were detected using RT-qPCR and western blotting, respectively. (E) Cells were transfected with HO-1 siRNA or scrambled control siRNA for 48 h. Western blotting was used to detect HO-1 protein level. Cells were pre-treated with 100 µg/ml ox-LDL for 24 h. Then, they were transfected with HO-1 siRNA for 48 h before treatment with 10 µg/ml eburicoic acid for 24 h. (F) RT-qPCR analyses of NLRP3, caspase-1, ASC and GSDMD mRNA levels. (G) Western blotting analyses of NLRP3, pro-caspase-1, cleaved caspase-1, ASC and GSDMD-N protein levels. (H) ELISA Analyses of IL-1β, IL-6 and IL-18 secretion levels. (I) Determination of LDH release using an LDH release assay kit. (J) Spectrophotometry analysis of caspase-1 activity. (K) Representative images of Hoechst/PI staining and ROS production. Data are represented as mean ± SD. ns, statistically insignificant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 20 µm. n=3. NLRP3, NLR family pyrin domain-containing protein 3; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vascular endothelial cells; HO-1, heme oxygenase-1; ROS, reactive oxygen species; si, small interfering; ASC, apoptosis-associated speck-like protein containing CARD; GSDMD-N, N-terminal gasdermin-D; IL, interleukin; LDH, lactate dehydrogenase; ROS, reactive oxygen species EA, eburicoic acid.

Figure 5.

Eburicoic acid promotes Nrf2 nuclear translocation in ox-LDL-treated HUVECs. HUVECs were pre-treated with 100 µg/ml ox-LDL for 24 h. (A and B) Cells were incubated with 1, 5, 10 and 20 µg/ml eburicoic acid for 24 h. The protein levels of cytoplasmic Keap1 and Nrf2 and nuclear Nrf2 were detected using western blotting. (C and D) Cells were incubated with 10 µg/ml eburicoic acid for 6, 12, 24 and 48 h, respectively. The protein levels of cytoplasmic Keap1 and Nrf2 and nuclear Nrf2 were detected using western blotting. (E-H) Cells were incubated with various doses of eburicoic acid for 24 h or 10 µg/ml eburicoic acid for various durations. The mRNA and protein levels of total Nrf2 were detected using RT-qPCR and western blotting, respectively. Data are represented as mean ± SD. ns, statistically insignificant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 20 µm. n=3. Nrf2, NF-E2-related factor 2; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vascular endothelial cells; Keap1, Kelch-like ECH-associated protein 1; RT-qPCR, reverse transcription-quantitative PCR EA, eburicoic acid.

Figure 6.

Eburicoic acid inhibits NLRP3 inflammasome activation and pyroptosis in ox-LDL-treated HUVECs via the Nrf2/HO-1/ROS pathway. (A) Cells were transfected with Nrf2 siRNA or scrambled control siRNA for 48 h. western blotting was used to detect the Nrf2 protein level. (B-G) Cells were pre-treated with 100 µg/ml ox-LDL for 24 h. Then, they were transfected with Nrf2 siRNA for 48 h before treatment with 10 µg/ml eburicoic acid for 24 h. (B) RT-qPCR analyses of HO-1, NLRP3, caspase-1, ASC and GSDMD mRNA levels; (C) western blotting analyses of NLRP3, pro-caspase-1, cleaved caspase-1, ASC and GSDMD-N protein levels; (D) ELISA Analyses of IL-1β, IL-6 and IL-18 secretion levels; (E) Determination of LDH release using an LDH release assay kit; (F) Spectrophotometry analysis of caspase-1 activity; (G) Representative images of Hoechst/PI staining and ROS production. Data are represented as mean ± SD. ns, statistically insignificant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 20 µm. n=3. NLRP3, NLR family pyrin domain-containing protein 3; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vascular endothelial cells; Nrf2, NF-E2-related factor 2; HO-1, heme oxygenase-1; ROS, reactive oxygen species; si, small interfering; ASC, apoptosis-associated speck-like protein containing CARD; GSDMD-N, N-terminal gasdermin-D; LDH, lactate dehydrogenase; ROS, reactive oxygen species; RT-qPCR, reverse transcription-quantitative PCR EA, eburicoic acid.

Figure 7.

Eburicoic acid mitigates the progression of atherosclerotic plaques in HFD-fed apoE^−/− mice. apoE^−/− mice (n=15/each group) fed on an HFD were administered with 10 mg/kg eburicoic acid (dissolved in 0.5% CMV) or vehicle only by oral gavage once daily. (A-C) The AST, ALT, BUN and Scr levels were detected using the respective commercial kits; (D) western blotting analyses of Keap1, Nrf2, HO-1, NLRP3, cleaved caspase-1, ASC and GSDMD-N protein levels in the aorta; n=3. (E) Assessment of plasma TC, TG, HDL-C and LDL-C levels; n=3. (F) Determination of serum IL-1β, IL-6, TNF-α and IL-18 levels using ELISA assay; n=3. (G) Oil Red O, HE and Masson staining of cross-sections of the aortic root. Image Pro software analyzed the lipid accumulation, lesion area and collagen content within atherosclerotic plaques. n=15. Data are represented as mean ± SD. ns, statistically insignificant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 100 µm. HFD, high-fat-diet; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; Scr, serum creatinine; Keap1, Kelch-like ECH-associated protein 1; Nrf2, NF-E2-related factor 2; HO-1, heme oxygenase-1; NLRP3, NLR family pyrin domain-containing protein 3; TC, total cholesterol; TG, triglycerides; HDL-C high-density lipoprotein cholesterol LDL-C, low-density lipoprotein cholesterol IL, interleukin; EA, eburicoic acid.