Melatonin attenuates smoking-induced atherosclerosis by activating the Nrf2 pathway via NLRP3 inflammasomes in endothelial cells

Abstract

Substantial evidence suggests that the effects of smoking in atherosclerosis are associated with inflammation mediated by endothelial cells. However, the mechanisms and potential drug therapies for smoking-induced atherosclerosis remain to be clarified. Considering that melatonin exerts beneficial effects in cardiovascular diseases, we examined its effects on cigarette smoke-induced vascular injury. We found that cigarette smoke extract (CSE) treatment induced NLRP3-related pyroptosis in human aortic endothelial cells (HAECs). CSE also induced ROS generation and upregulated the Nrf2 pathway in HAECs. Furthermore, pretreatment of HAECs with Nrf2-specific siRNA and an Nrf2 activator revealed that Nrf2 can inhibit CSE-induced ROS/NLRP3 activation. Nrf2 also improved cell viability and the expression of VEGF and eNOS in CSE-treated HAECs. In balloon-induced carotid artery injury model rats exposed to cigarette smoke, melatonin treatment reduced intimal hyperplasia in the carotid artery. Mechanistic studies revealed that compared with the control group, Nrf2 activation was increased in the melatonin group, whereas ROS levels and the NLRP3 inflammasome pathway were inhibited. These results reveal that melatonin might effectively protect against smoking-induced vascular injury and atherosclerosis through the Nrf2/ROS/NLRP3 signaling pathway. Overall, these observations provide compelling evidence for the clinical use of melatonin to reduce smoking-related inflammatory vascular injury and atherosclerosis.

Keywords: Nrf2 pathway; atherosclerosis; cigarette smoking; melatonin; pyroptosis.

Figure 1

CSE treatment induced pyroptosis in HAECs. (A) Morphology of HAECs treated with and without CSE imaged using electron microscopy. Autophagosomes, cytoplasmic outflow and cell membrane break indicated by red arrows. (B, C) The protein levels of GSDMD and GSDMD-N were upregulated in HAECs after treatment with CSE, as indicated by western blot results. β-Actin was used as an internal control. **p < 0.01. The data are represented as mean ± SD (n = 3).

Figure 2

NLRP3 inflammasome pathway is involved in CSE-induced pyroptosis in HAECs. (A–H) The protein levels of ASC, Pro-Caspase-1, Caspase-1, Pro-IL-1β, IL-1β, Pro-IL-18 and IL-18 were upregulated in HAECs after treatment with CSE, as indicated by western blot results. (I, J) The protein levels of NLRP3 was increased in HAECs after treatment with CSE, as indicated by western blot results. β-Actin was used as an internal control. (K–M) The mRNA level of NLRP3, IL-1β, and IL-18 was increased in HAECs after treatment with CSE. *p < 0.05, **p < 0.01. The data are represented as mean ± SD (n = 3).

Figure 3

CSE induced ROS generation and upregulated NRF2 pathway in HAECs. (A, B) Cellular ROS level was measured with flow cytometry, and ROS level was promoted after CSE treatment. (C–G) Western blotting on protein level of n-Nrf2 and c-Nrf2 was performed to analyze Nrf2 nuclear translocation, suggesting that CSE increased Nrf2 nuclear translocation. The protein levels of HO-1 and NQO1 were upregulated in HAECs after treatment with CSE, as indicated by western blot results. β-Actin was used as an internal control. (H–J) The mRNA levels of Nrf2, HO-1 and NQO1 were upregulated in HAECs after treatment with CSE, as indicated by RT-PCR results. *p < 0.05, **p < 0.01, ns, not significant. The data are represented as mean ± SD (n = 3).

Figure 4

Nrf2 negatively regulated CSE-induced ROS level. (A–F) RT-PCR on mRNA level of Nrf2, HO-1 and NQO1 and western blotting on protein level of HO-1 and NQO1 was performed. (G, H) Cellular ROS level was measured with flow cytometry, and CSE-induced ROS level was negatively regulated by Nrf2 pathway. *p < 0.05, **p < 0.01, ns, not significant, compared with CSE group. The data are represented as mean ± SD (n = 3).

Figure 5

Nrf2 negatively regulated CSE-induced NLRP3 inflammasome activation. (A–C) The mRNA level of NLRP3, IL-1β and IL-18 was detected by RT-PCR. (D–K) NLRP3, Pro-Caspase-1, Caspase-1, Pro-IL-1β, IL-1β, Pro-IL-18 and IL-18 were detected by Western blot. *p < 0.05, **p < 0.01, ns, not significant, compared with CSE group. The data are represented as mean ± SD (n = 3).

Figure 6

Nrf2 upregulation improves endothelial cell viability and the expression of VEGF and eNOS. (A) The expression of VEGF proteins was measured by ELISA. (B) Effect of Nrf2 on cell growth of HAECs treated by CSE was analyzed by measuring cell viability. (C–E) The mRNA and protein level of eNOS were detected by RT-PCR and Western blot. *p < 0.05, **p < 0.01, ns, not significant, compared with CSE group. The data are represented as mean ± SD (n = 3).

Figure 7

Melatonin reduced intimal hyperplasia of rat carotid artery after balloon injury with cigarette smoking. (A, B) H&E and VVG stained sections of carotid arteries of each group. Intima and media were labeled with "I" and "M" respectively. (C) Quantitative graphs of Intima to Media (I/M) ratio and intimal area. **p < 0.01. The data are represented as mean ± SD (n = 3).

Figure 8

Melatonin promoted expression of Nrf2/HO-1 pathway and decreased ROS level in vivo. (A–C) The mRNA level of Nrf2, HO-1 and NQO1 was detected by RT-PCR. (D–G) The protein level of Nrf2, HO-1 and NQO1 were detected by Western blot. (H, I) Nrf-2 expression were detected by immunohistochemical staining (magnification ×400). Intima and media were labeled with "I" and "M" respectively. Nrf-2 mean density was analyzed in each group (a, control group; b, smoking group; c, melatonin group). (J, K) Representative images of carotid arteries stained with DHE (red), DAPI (blue) and merge images in (a–c) control, (d–f) smoking, and (g–i) melatonin groups. The fluorescent intensities of ROS were quantified. *p < 0.05, **p < 0.01. The data are represented as mean ± SD (n = 3).

Figure 9

Melatonin inhibited expression of NLRP3 inflammasome in vivo. (A–C) The mRNA level of NLRP3, IL-1β and IL-18 was detected by RT-PCR. (D–K) NLRP3, Pro-Caspase-1, Caspase-1, Pro-IL-1β, IL-1β, Pro-IL-18 and IL-18 were detected by Western blot. *p < 0.05, **p < 0.01. The data are represented as mean ± SD (n = 3).

Figure 10

Proposed model illustrating how melatonin protects smoking-related vascular inflammatory injury through Nrf2/ROS/NLRP3 axis. ROS induced by smoking increased NLRP3 transcription and subsequent inflammasome activation, which promoted secretion of mature IL-1β and IL-18. ROS also stimulated the nuclear translocation of Nrf2 as a negative feedback. Melatonin induces the nuclear translocation of Nrf2, downstream expression, and elimination of intracellular ROS, resulting in a decrease in the transcription level of NLRP3 and downstream inflammasome activation.