Oxymatrine attenuates oxidized low‑density lipoprotein‑induced HUVEC injury by inhibiting NLRP3 inflammasome‑mediated pyroptosis via the activation of the SIRT1/Nrf2 signaling pathway

Abstract

Oxymatrine, a quinolizidine alkaloid isolated from the traditional Chinese herb Sophora flavescens Aiton, has been demonstrated to exert anti‑inflammatory and atherosclerotic effects, but the molecular mechanism has yet to be elucidated. Accumulating evidence indicates an important role of NLR family pyrin domain containing 3 (NLRP3) inflammasome‑mediated pyroptosis in the pathogenesis of atherosclerosis. The present study was undertaken to investigate whether oxymatrine attenuates oxidized low‑density lipoprotein (ox‑LDL)‑induced human umbilical vein endothelial cell (HUVEC) injury, an in vitro cell model of atherosclerosis, by inhibiting NLRP3 inflammasome‑mediated pyroptosis, and elucidate the role of the sirtuin (SIRT)1/nuclear factor‑erythroid 2‑related factor 2 (Nrf2) signaling pathway in this process. Cell viability and cytotoxicity were detected by CCK‑8 assay and a lactate dehydrogenase (LDH) assay kit. Cell apoptosis was detected by flow cytometry. Reactive oxygen species (ROS) generation was detected using a ROS assay kit. The malondialdehyde (MDA) content, mitochondrial membrane potential (MMP) level, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH‑Px) activities were determined using commercial kits. The inflammatory cytokines levels were measured by ELISA and protein expression was monitored by western blot analysis. The results revealed that oxymatrine alleviated ox‑LDL‑induced cytotoxicity and apoptosis. Concurrently, oxymatrine inhibited ox‑LDL‑induced NLRP3 inflammasome‑mediated pyroptosis in HUVECs, as evidenced by the significant decreases in the expression of NLRP3, apoptosis‑associated speck‑like protein containing a C‑terminal caspase recruitment domain (ASC), cleaved caspase‑1, interleukin (IL)‑1β and IL‑18 in HUVECs. In addition, NLRP3 siRNA transfection efficiently suppressed ox‑LDL‑induced pyroptosis and HUVEC injury. Furthermore, oxymatrine promoted SIRT1/Nrf2 signaling pathway activation in HUVECs subjected to ox‑LDL treatment, and SIRT1 deficiency induced by SIRT1 siRNA transfection abolished the protective effect of oxymatrine against ox‑LDL‑induced injury. SIRT1 siRNA also mitigated the oxymatrine‑induced decreases in ROS generation and MDA content, and the increases in MMP as well as the activities of SOD, CAT and GSH‑Px in HUVECs. Moreover, SIRT1 siRNA transfection blocked the inhibitory effect of oxymatrine on NLRP3 inflammasome‑mediated pyroptosis in ox‑LDL‑treated HUVECs. Collectively, these results indicated that oxymatrine may attenuate ox‑LDL‑induced HUVEC injury by inhibiting NLRP3 inflammasome‑mediated pyroptosis via activating the SIRT1/Nrf2 signaling pathway.

Keywords: NLRP3 inflammasome‑mediated pyroptosis; atherosclerosis; endothelial cell injury; oxymatrine; sirtuin 1/nuclear factor‑erythroid 2‑related factor 2 signaling pathway.

Figure 1

Effects of oxymatrine on cytotoxicity and apoptosis in ox-LDL-stimulated HUVECs. (A) HUVECs were pretreated with oxymatrine (2, 4 or 8 µM) for 2 h prior to treatment with ox-LDL (100 µg/ml) for 24 h, followed by the evaluation of cell viability. Data are expressed as the mean ± SD, n=3. ^**P<0.01 and ^NSP>0.05 vs. the control group; ^#P<0.05 and ^##P<0.01 vs. the ox-LDL group. Cells were pretreated with oxymatrine (4 µM) for 2 h prior to treatment with ox-LDL (100 µg/ml) for 24 h, followed by the determination of (B) LDH release, (C) apoptosis rate and (D) Bax and Bcl-2 expression. Quantitative analysis for (E) Bax/GAPDH and (F) Bcl-2/GAPDH. Data are expressed as the mean ± SD, n=3. ^**P<0.01 and ^NSP>0.05 vs. the control group; ^#P<0.05 and ^##P<0.01 vs. the ox-LDL group. OMT, oxymatrine; ox-LDL, oxidized low-density lipoprotein; NS, no statistical significance; HUVECs, human umbilical vein endothelial cells; LDH, lactate dehydrogenase.

Figure 2

Effects of oxymatrine on NLRP3 inflammasome-mediated pyroptosis in ox-LDL-stimulated HUVECs. HUVECs were pretreated with oxymatrine (4 µM) for 2 h prior to treatment with ox-LDL (100 µg/ml) for 24 h. (A) Western blot analysis for NLRP3 and ASC protein expression. Quantitative analysis for (B) NLRP3/GAPDH and (C) ASC/GAPDH. (D) Western blot analysis for cleaved caspase-1, caspase-1, IL-1β and IL-18 protein expression levels. Quantitative analysis for (E) cleaved caspase-1/caspase-1, (F) IL-1β/GAPDH and (G) IL-18/GAPDH. Data are expressed as the mean ± SD, n=3. ^*P<0.05, ^**P<0.01 and ^NSP>0.05 vs. the control group; ^#P<0.05 and ^##P<0.01 vs. the ox-LDL group. HUVECs were transfected with siNLRP3 or siNC for 24 h, followed by measurement of (H) NLRP3, ASC, cleaved caspase-1, caspase-1, IL-1β and IL-18 protein expression. Data are expressed as the mean ± SD, n=3. ^*P<0.05, ^**P<0.01, ***P<0.001 vs. siNC group. Cells were transfected with siNLRP3 or siNC before treatment with oxymatrine (4 µM) for 2 h prior to treatment with ox-LDL (100 µg/ml) for 24 h. Determination of (I) cell viability, (J) LDH release and (K) apoptosis rate. Data are expressed as the mean ± SD, n=3. ^**P<0.01 vs. the control group; ^#P<0.05 and ^##P<0.01 vs. the ox-LDL + siNC group. OMT, oxymatrine; ox-LDL, oxidized low-density lipoprotein; NS, no statistical significance; siNLRP3, small interfering ribonucleic acid (siRNA) for NLRP3; siNC, siRNA for negative control; HUVECs, human umbilical vein endothelial cells; LDH, lactate dehydrogenase; NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain; Cleaved cas-1, Cleaved caspase-1.

Figure 3

Effects of oxymatrine on the SIRT1/Nrf2 signaling pathway in ox-LDL-treated HUVECs. HUVECs were pretreated with oxymatrine (4 µM) for 2 h prior to treatment with ox-LDL (100 µg/ml) for 24 h. (A) Western blot analysis for SIRT1, C-Nrf2 and HO-1 protein expression. Quantitative analysis for (B) SIRT1, (C) C-Nrf2 and (D) HO-1 relative to GAPDH. (E) Western blot analysis for N-Nrf2 protein expression. (F) Quantitative analysis for Nrf2 relative to histone H3. Data are expressed as the mean ± SD, n=3. ^**P<0.01 and ^NSP>0.05 vs. the control group; ^#P<0.05 and ^##P<0.01 vs. the ox-LDL group. OMT, oxymatrine; ox-LDL, oxidized low-density lipoprotein; NS, no statistical significance; HUVECs, human umbilical vein endothelial cells; SIRT, sirtuin; Nrf2, nuclear factor-erythroid 2-related factor 2; C-Nrf2, cytoplasmic Nrf2; N-Nrf2, nuclear Nrf2; HO-1, heme oxygenase 1.

Figure 4

Effects of siSIRT1 transfection on the protective effects of oxymatrine against ox-LDL-induced injury in HUVECs. HUVECs transfected with siSIRT1 or siCon were treated with oxymatrine (4 µM) for 2 h prior to treatment with ox-LDL (100 µg/ml) for 24 h. (A) Western blot analysis for protein expression. Quantitative analysis for (B) SIRT1 and (C) HO-1 relative to GAPDH. (D) Quantitative analysis for N-Nrf2 relative to histone H3. Determination of (E) cell viability, (F) LDH release and (G) apoptosis rate. (H) Western blot analysis for cleaved caspase-3 and caspase-3 protein expression. (I) Quantitative analysis for cleaved caspase-3 relative to caspase-3. Data are expressed as the mean ± SD, n=3. ^**P<0.01 vs. the ox-LDL group; ^NSP>0.05 vs. the OMT + ox-LDL group; ^#P<0.05 and ^##P<0.01 vs. the OMT + ox-LDL + siCon group. OMT, oxymatrine; ox-LDL, oxidized low-density lipoprotein; NS, no statistical significance; siSIRT1, siRNAs for SIRT1; siCon, siRNAs for negative control; HUVECs, human umbilical vein endothelial cells; LDH, lactate dehydrogenase; SIRT, sirtuin; Nrf2, nuclear factor-erythroid 2-related factor 2; C-Nrf2, cytoplasmic Nrf2; N-Nrf2, nuclear Nrf2; HO-1, heme oxygenase 1.

Figure 5

Effects of siSIRT transfection on the inhibitory effect of oxymatrine on oxidative stress in ox-LDL-treated HUVECs. HUVECs transfected with siSIRT1 or siCon were treated with oxymatrine (4 µM) for 2 h prior to treatment with ox-LDL (100 µg/ml) for 24 h. (A) DCFH-DA staining for ROS. Scale bar, 200 µm. (B) Quantitative analysis for ROS generation by flow cytometry. (C) Cell MDA assay kit for measuring MDA content. (D) JC-1 kit for measuring mitochondrial membrane potential. (E) SOD assay kit for measuring SOD activity. (F) CAT assay kit for measuring CAT activity. (G) GSH-Px assay kit for measuring GSH-Px activity. Data are expressed as the mean ± SD, n=3. ^*P<0.05 and ^**P<0.01 vs. the control group; ^#P<0.05 and ^##P<0.01 vs. the ox-LDL group; ^NSP>0.05 vs. the OMT + ox-LDL group; ^$P<0.05 and ^$$P<0.01 vs. the OMT + ox-LDL + siCon group. OMT, oxymatrine; ox-LDL, oxidized low-density lipoprotein; NS, no statistical significance; siSIRT1, siRNAs for SIRT1; siCon, siRNAs for negative control; HUVECs, human umbilical vein endothelial cells; SIRT, sirtuin; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; ROS, reactive oxygen species; DCFH-DA, 2′,7′-dichlorofluorescein diacetate.

Figure 6

Effects of siSIRT transfection on the inhibitory effect of oxymatrine against NLRP3 inflammasome-mediated pyroptosis in ox-LDL-treated HUVECs. HUVECs transfected with siSIRT1 or siCon were treated with oxymatrine (4 µM) for 2 h prior to treatment with ox-LDL (100 µg/ml) for 24 h. (A) Western blot analysis for protein expression. Quantitative analysis for (B) NLRP3 and (C) ASC relative to GAPDH. (D) Western blot analysis for protein expression. Quantitative analysis for (E) cleaved caspase-1/caspase-1, (F) IL-1β and (G) IL-18 relative to GAPDH. Data are expressed as the mean ± SD, n=3. ^**P<0.01 vs. the ox-LDL group; ^NSP>0.05 vs. the OMT + ox-LDL group; ^#P<0.05 and ^##P<0.01 vs. the OMT + ox-LDL + siCon group. OMT, oxymatrine; ox-LDL, oxidized low-density lipoprotein; NS, no statistical significance; siSIRT1, siRNAs for SIRT1; siCon, siRNAs for negative control; HUVECs, human umbilical vein endothelial cells; NLRP3, NLR family pyrin domain containing 3; SIRT, sirtuin; ASC, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain.