Nutr Res Pract.  2024 Oct;18(5):602-616. 10.4162/nrp.2024.18.5.602.

Protaetia brevitarsis larvae extract protects against lipopolysaccharidesinduced ferroptosis and inflammation by inhibiting acid sphingomyelinase

• Park WJ
• Oh E
• Kim Y

• KMID: 2560047
• DOI: http://doi.org/10.4162/nrp.2024.18.5.602

Abstract

BACKGROUND/OBJECTIVES
Inflammation and ferroptosis are implicated in various diseases and lipopolysaccharides (LPS) have been linked with these disorders. Recently, many edible insects, such as Gryllus bimaculatus, Protaetia brevitarsis larvae (PB) and Tenebrio molitor larvae, have been recommended as alternative foods because they contain lots of nutritional sources. In this study, we explored the potential of PB extract in preventing LPS-induced inflammation and ferroptosis in Hep3B cells.
MATERIALS/METHODS
PB powder was extracted using 70% ethanol and applied to Hep3B cells. Co-treatment with LPS was conducted to induce ferroptosis and inflammation. The antiinflammatory and anti-ferroptosis mechanisms of the PB extract were confirmed using Western blot, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction analysis.
RESULTS
PB extract effectively prevented LPS-induced cell death and restored LPS-induced inflammatory cytokine production, NF-κB signaling, endoplasmic reticulum (ER) stress and ferroptosis. Interestingly, PB extract reduced LPS-induced ceramide increase and acid sphingomyelinase (ASMase) expression. The use of the ASMase inhibitor, desipramine, also demonstrated a reduction in these pathways, highlighting the pivotal role of ASMase in inflammation and ferroptosis. Treatment with each inhibitor revealed that ferroptosis causes ER stress and that NF-κB and MAP kinase pathways are involved in inflammation.
CONCLUSION
PB emerges as a potential functional food with inhibitory effects on LPS-induced inflammation and ferroptosis, making it a promising candidate for nutritional interventions.

Keyword

Edible Insects; ceramide; sphingomyelin phosphodiesterase; ferroptosis; inflammation

Figure

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  Fig. 1 Effect of PB extract on Hep3B cell death. Hep3B cells were seeded at 3 × 104 cells/well in a 96-well plate and 4 × 105 cells/well in a 6-well plate. They were treated with various concentrations of PB extract for 48 h. (A) MTT cell viability assay and (B) representative western blots of c-caspase-3 were examined. Values are expressed as mean ± SEM compared to control group. All experiments were performed in triplicate.PB, Protaetia brevitarsis; c-caspase-3, cleaved caspase-3.**P < 0.01, ***P < 0.001.

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  Fig. 2 PB extract reduces LPS-induced inflammation, ferroptosis and cell death. Hep3B cells were seeded at 3 × 104 cells/well in a 96-well plate and 4 × 105 cells/well in a 6-well plate. They were treated with PB extract (100 µg/mL) for 24 h, followed by 20 µg/mL LPS for another 24 h. TNF-α (A), IL-1β (B), and IL-6 (C) in cell culture media were measured using commercial enzyme-linked immunosorbent assay kits (n = 4). (D) MTT cell viability assay, (E) GSH levels, and (F) MDA levels were examined (n = 4). (G) Representative western blots of indicated proteins. Values are expressed as mean ± SEM compared to control group. All experiments were performed in triplicate.TNF, tumor necrosis factor; Con, control (untreated); PB, Protaetia brevitarsis; LPS, lipopolysaccharides; IL, interleukin; GSH, glutathione; MDA, malondialdehyde; PERK, protein kinase R-like endoplasmic reticulum kinase; eIF2α, eukaryotic translation initiation factor 2A; Slc7a11, soluble carrier family 7 member 11; GPX4, glutathione peroxidase 4.*P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 3 PB extract reduces LPS-induced acid sphingomyelinase expression. Hep3B cells were seeded at 3 × 104 cells/well in a 96-well plate and 4 × 105 cells/well in a 6-well plate. They were treated with PB extract (100 µg/mL) for 24 h, followed by 20 µg/mL LPS for another 24 h. (A) mRNA levels of ASMase, NSMase1, and NSMase2 after 20 µg/mL LPS treatment for 24 h in Hep3B cells. mRNA levels of ASMase (B), and ceramide levels (C) after cotreatment LPS with PB extract, (n = 4). Values are expressed as mean ± SEM compared to control group. All experiments were performed in triplicate.Con, control (untreated); LPS, lipopolysaccharides; ASMase, acid sphingomyelinase; NSMase, neutral sphingomyelinase; PB, Protaetia brevitarsis.*P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 4 Desipramine reduces LPS-induced inflammation, ferroptosis and cell death. Hep3B cells were seeded at 3 × 104 cells/well in a 96-well plate and 4 × 105 cells/well in a 6-well plate. They were treated with desipramine (2 µM) for 24 h, followed by 20 µg/mL LPS for another 24 h. TNF-α (A), IL-1β (B), and IL-6 (C) in cell culture media were measured using commercial enzyme-linked immunosorbent assay kits (n = 4). (D) MTT cell viability assay, (E) GSH levels, and (F) MDA levels were examined (n = 4). (G) Representative western blots of indicated proteins. Values are expressed as mean ± SEM compared to control group. All experiments were performed in triplicate.TNF, tumor necrosis factor; Con, control (untreated); Desi, desipramine; LPS, lipopolysaccharides; IL, interleukin; GSH, glutathione; MDA, malondialdehyde; PERK, protein kinase R-like endoplasmic reticulum kinase; eIF2α, eukaryotic translation initiation factor 2A; Slc7a11, soluble carrier family 7 member 11; GPX4, glutathione peroxidase 4; ER, endoplasmic reticulum.*P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 5 PB extract and desipramine decrease erastin-induced ferroptosis and cell death. Hep3B cells were seeded at 3 × 104 cells/well in a 96-well plate and 4 × 105 cells/well in a 6-well plate. They were treated with PB extract (100 µg/mL) or desipramine (2 µM) for 24 h, followed by 20 µM erastin for another 24 h. (A) Representative western blots of indicated proteins after erastin treatment in PB extract- or desipramine-pretreated Hep3B cells. GSH levels (B), MDA levels (C), MTT cell viability assay (D), ceramide levels (E), and TNF-α (F) were measured after erastin treatment in PB extract- or desipramine-pretreated Hep3B cells (n = 4). Values are expressed as mean ± SEM compared to control group. All experiments were performed in triplicate.Con, control (untreated); PB, Protaetia brevitarsis; Desi, desipramine; Slc7a11, soluble carrier family 7 member 11; GPX4, glutathione peroxidase 4; GSH, glutathione; MDA, malondialdehyde; TNF, tumor necrosis factor.*P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 6 Ferroptosis/ER stress and NF-κB play an important role in LPS-induced cell death. Hep3B cells were seeded at 3 × 104 cells/well in a 96-well plate and 4 × 105 cells/well in a 6-well plate. They were treated with various inhibitors (4-PBA [ER stress inhibitor, 5 mM], SB203580 [p38 inhibitor, 10 µM], SP600125 [JNK inhibitor, 10 µM], PDTC [NF-κB inhibitor, 10 µM], and ferrostatin-1 [ferroptosis inhibitor, 1 µM]) for 24 h, followed by 20 µg/mL LPS for another 24 h. TNF-α (A), IL-1β (B), and IL-6 (C) in cell culture media were measured using commercial enzyme-linked immunosorbent assay kits (n = 4). MTT cell viability assay (D), GSH levels (E), and MDA levels (F) were examined (n = 4). (G) Representative western blots of indicated proteins. Values are expressed as mean ± SEM compared to control group. All experiments were performed in triplicate.TNF, tumor necrosis factor; Con, control (untreated); LPS, lipopolysaccharides; IL, interleukin; GSH, glutathione; MDA, malondialdehyde; ERK, protein kinase R-like endoplasmic reticulum kinase; eIF2α, eukaryotic translation initiation factor 2A; Slc7a11, soluble carrier family 7 member 11; GPX4, glutathione peroxidase 4; NF, nuclear factor.**P < 0.01, ***P < 0.001.

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  Fig. 7 N-acetylcysteine only inhibits ferroptosis/ER stress, but not inflammatory cytokine production. Hep3B cells were seeded at 3 × 104 cells/well in a 96-well plate and 4 × 105 cells/well in a 6-well plate. They were treated with 10 mM NAC for 24 h, followed by 20 µg/mL LPS for another 24 h. TNF-α (A), IL-1β (B), and IL-6 (C) in cell culture media were measured using commercial enzyme-linked immunosorbent assay kits (n = 4). MTT cell viability assay (D), GSH levels (E), and MDA levels (F) were examined (n = 4). (G) Representative western blots of indicated proteins. Values are expressed as mean ± SEM compared to control group. All experiments were performed in triplicate.TNF, tumor necrosis factor; Con, control (untreated); NAC, N-acetylcysteine; LPS, lipopolysaccharides; IL, interleukin; GSH, glutathione; MDA, malondialdehyde; PERK, protein kinase R-like endoplasmic reticulum kinase; eIF2α, eukaryotic translation initiation factor 2A; GPX4, glutathione peroxidase 4; Slc7a11, soluble carrier family 7 member 11; ER, endoplasmic reticulum.**P < 0.01, ***P < 0.001.

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  Fig. 8 PB extract reduces LPS-induced inflammation, ferroptosis and cell death in THLE-2 cells. THLE-2 cells were seeded at 3 × 104 cells/well in a 96-well plate and 4 × 105 cells/well in a 6-well plate. They were treated with PB extract (100 µg/mL) for 24 h, followed by 20 µg/mL LPS for another 24 h. TNF-α (A), IL-1β (B), and IL-6 (C) in cell culture media were measured using commercial enzyme-linked immunosorbent assay kits (n = 4). (D) MTT cell viability assay, (E) GSH levels, (F) MDA levels, (G) ASMase expression, and (H) ceramide levels were examined (n = 4). (I) Representative western blots of indicated proteins. Values are expressed as mean ± SEM compared to control group. All experiments were performed in triplicate.TNF, tumor necrosis factor; Con, control (untreated); PB, Protaetia brevitarsis; LPS, lipopolysaccharides; IL, interleukin; GSH, glutathione; MDA, malondialdehyde; ASMase, acid sphingomyelinase; PERK, protein kinase R-like endoplasmic reticulum kinase; eIF2α, eukaryotic translation initiation factor 2A; Slc7a11, soluble carrier family 7 member 11; GPX4, glutathione peroxidase 4.*P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 9 Schematic showing the effects of PB extract in LPS-induced cell death. LPS induced ASMase activation, initiating a cascade that triggers ferroptosis, NF-κB, and MAP kinase pathways. Ferroptosis promotes ER stress, while NF-κB and MAP kinase pathways contribute to increased inflammation. Both pathways finally lead to cell death. PB extract inhibits ASMase activation, effectively inhibiting all these pathways and preventing cell death.LPS, lipopolysaccharides; PB, Protaetia brevitarsis; ASMase, acid sphingomyelinase; NF, nuclear factor; ER, endoplasmic reticulum.

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