Long-term exposure of sucralose induces neuroinflammation and ferroptosis in human microglia cells via SIRT1/NLRP3/IL-1β/GPx4 signaling pathways

Abstract

Microglia serve as the primary defense mechanism in the brain. Artificial sweeteners are widely used as dietary supplements, though their long-term effects remain uncertain. In this study, we investigated the effects of sucralose on microglia during prolonged exposure via the neuroinflammatory and ferroptosis pathways. Initially, human microglial clone 3 (HMC3) cells were exposed to sucralose (0-50 mM) for 24, 48, and 72 h to investigate the short-term effects. Subsequently, HMC3 cells were treated with 1 mM sucralose for 7, 14, and 21 days to examine long-term effects. We measured levels of interleukin-1β (IL-1β), NOD-like receptor protein 3 (NLRP3), 8-hydroxydeoxyguanosine (8-OHdG), Sirtuin-1 (SIRT1), glutathione peroxidase-4 (GPx4), reduced glutathione (GSH), malondialdehyde (MDA), ferrous iron (Fe²⁺), and caspase 3/7. Additionally, we analyzed the impact of sucralose on cell morphology, migration, and expression levels of IL-1β, NLRP3, SIRT1, and GPx4. Sucralose inhibited cell viability and proliferation in HMC3 cells in a concentration- and time-dependent manner and induced membrane and nuclear abnormalities. Moreover, sucralose significantly reduced the cell migration rate. Long-term sucralose treatment decreased Fe²⁺, GPx4, GSH, and SIRT1 levels in HMC3 cells while increasing IL-1β, MDA, NLRP3, 8-OHdG, and caspase 3/7 activity. Sucralose treatment also enhanced microglial activation and neuroinflammation by upregulating IL-1β and NLRP3 and downregulating SIRT1 and GPx4, thereby inducing ferroptosis and suppressing cell viability. Consequently, high concentrations or long-term sucralose treatment may induce neuroinflammation and ferroptosis by targeting the SIRT1/NLRP3/IL-1β/GPx4 pathway in HMC3 cells.

Keywords: NLRP3; artificial sweeteners; ferroptosis; microglia; neuroinflammation; sirtuins; sucralose.

FIGURE 1

Sucralose treatment (in the range of 0–50 mM) affected cellular viability and proliferation in HMC3 cells up to 72 h. (a) MTT results in HMC3 cells; (b) BrdU incorporation in HMC3 cells. *p < .05, **p < .001, and ***p < .0001 vs. control group.

FIGURE 2

Long‐term sucralose treatment (1 mM) for 7, 14, and 21 days suppressed viability and proliferation in HMC3 cells. (a) MTT results in HMC3 cells; (b) BrdU incorporation in HMC3 cells. ***p < .0001 vs. control group.

FIGURE 3

Effects of sucralose treatment on cell morphology in HMC cells. (a1–c1) Untreated HMC3 cells; (a2) HMC3 cells treated with 1 mM sucralose for 7 days; (b2) HMC3 cells treated with 1 mM sucralose for 14 days; (c2) HMC3 cells treated with 1 mM sucralose for 21 days.

FIGURE 4

The impact of sucralose treatment on the cell migration rate in HMC3 cells. (a) Microscopic images of untreated cell migration in HMC3 cells. (b) Microscopic images of treated cell migration in HMC3 cells. (c) Cell migration rates in HMC3 cells. ***p < .0001 when compared to untreated/treated groups.

FIGURE 5

Long‐term sucralose treatment regulated IL‐1β, NLRP3, 8‐OHdG, SIRT1, MDA, GSH, GPx4 and Fe²⁺ levels in HMC3 cells. (a) IL‐1β levels in HMC3 cells; (b) NLRP3 levels in HMC3 cells; (c) 8‐OHdG levels in HMC3 cells; (d) MDA levels in HMC3 cells; (e) GPx4 levels in HMC3 cells; (f) GSH levels in HMC3 cells; (g) SIRT1 levels in HMC3 cells; (h) Fe²⁺ levels in HMC3. *p < .05, **p < .001, and ***p < .0001 vs. control group.

FIGURE 6

Effects of long‐term sucralose treatment on expression levels of IL‐1β, NLRP3, SIRT1, and GPx4 in HMC3 cells. (a) IL‐1β mRNA levels in HMC3 cells; (b) NLRP3 mRNA levels in HMC3 cells; (c) SIRT1 mRNA levels in HMC3 cells; (d) GPx4 mRNA levels in HMC3 cells. *p < .05, **p < .001, and ***p < .0001 vs. control group.

FIGURE 7

Long‐term sucralose treatment induced apoptosis in HMC3 cells. (a) Untreated HMC3 cells; (b) HMC3 cells treated with 1 mM sucralose for 7 days; (c) HMC3 cells treated with 1 mM sucralose for 14 days; (d) HMC3 cells treated with 1 mM sucralose for 21 days.