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. 2023 Mar 20:14:1139532.
doi: 10.3389/fphar.2023.1139532. eCollection 2023.

Effects of frog skin peptide temporin-1CEa and its analogs on ox-LDL induced macrophage-derived foam cells

Xue-Feng Yang  1   2 , Xin Liu  1 , Xiao-Yi Yan  1 , De-Jing Shang  1
Affiliations

Effects of frog skin peptide temporin-1CEa and its analogs on ox-LDL induced macrophage-derived foam cells

Xue-Feng Yang et al. Front Pharmacol. .

Abstract

Purpose: Atherosclerosis is one of the most important pathological foundations of cardiovascular and cerebrovascular diseases with high morbidity and mortality. Studies have shown that macrophages play important roles in lipid accumulation in the vascular wall and thrombosis formation in atherosclerotic plaques. This study aimed to explore the effect of frog skin antimicrobial peptides (AMPs) temporin-1CEa and its analogs on ox-LDL induced macrophage-derived foam cells. Methods: CCK-8, ORO staining, and intracellular cholesterol measurements were used to study cellular activity, lipid droplet formation and cholesterol levels, respectively. ELISA, real-time quantitative PCR, Western blotting and flow cytometry analysis were used to study the expression of inflammatory factors, mRNA and proteins associated with ox-LDL uptake and cholesterol efflux in macrophage-derived foam cells, respectively. Furthermore, the effects of AMPs on inflammation signaling pathways were studied. Results: Frog skin AMPs could significantly increase the cell viability of the ox-LDL-induced foaming macrophages and decrease the formation of intracellular lipid droplets and the levels of total cholesterol and cholesterol ester (CE). Frog skin AMPs inhibited foaming formation by reducing the protein expression of CD36, which regulates ox-LDL uptake but had no effect on the expression of efflux proteins ATP binding cassette subfamily A/G member 1 (ABCA1/ABCG1). Then, decreased mRNA expression of NF-κB and protein expression of p-NF-κB p65, p-IκB, p-JNK, p-ERK, p-p38 and the release of TNF-α and IL-6 occurred after exposure to the three frog skin AMPs. Conclusion: Frog skin peptide temporin-1CEa and its analogs can improve the ox-LDL induced formation of macrophage-derived foam cells, in addition, inhibit inflammatory cytokine release through inhibiting the NF-κB and MAPK signaling pathways, thereby inhibiting inflammatory responses in atherosclerosis.

Keywords: atherosclerosis; foam cells; frog skin peptide; inflammation; lipid metabolism.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Ox-LDL induced the foaming cell forming of THP-1 and RAW264.7 macrophages. Effects of ox-LDL on the cell viability of THP-1 and RAW264.7 (A); ox-LDL induced cell morphological changes and lipid droplet formation analyzed by ORO staining (B, 400X); Levels of total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) in ox-LDL induced foaming cells (C,D). *p < 0.05 vs. the control (ctrl) group.
FIGURE 2
FIGURE 2
Effects of frog skin AMPs on ox-LDL induced foam cells. Effects of frog skin AMPs on the cell viability of THP-1 cells (A), RAW264.7 cells (B) and ox-LDL induced foaming cells (C,D); Cell morphological changes and lipid droplet formation analyzed by ORO staining (E, 400X); Levels of total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) in the frog skin AMPs treated foam cells (F,G). *p < 0.05 vs. the ctrl group. # p < 0.05 vs. the ox-LDL group.
FIGURE 3
FIGURE 3
Effects of frog skin AMPs on lipid metabolism in ox-LDL induced foam cells. Gene expression of membrane proteins involved in ox-LDL uptake and cholesterol efflux by real-time quantitative PCR in foaming THP-1 cells (A,B) and GAPDH was used as an internal reference; Expression of proteins involved in ox-LDL uptake and cholesterol efflux by Western blot in foaming RAW264.7 cells (C–E) and ATP1A1 was used as an internal reference; Flow cytometry analysis of CD36 expression in foaming THP-1 cells (F,G). *p < 0.05 vs. the ctrl group. # p < 0.05 vs. the ox-LDL group.
FIGURE 4
FIGURE 4
Effects of frog skin AMPs on the inflammatory response in ox-LDL induced foam cells. Levels of TNF-α and IL-6 in foaming THP-1 cells (A,B); Levels of TNF-α and IL-6 in foaming RAW264.7 cells (C,D). *p < 0.05 vs. the ctrl group. # p < 0.05 vs. the ox-LDL group.
FIGURE 5
FIGURE 5
Effects of frog skin AMPs on NF-κB signaling pathway in ox-LDL induced foam cells. mRNA expression of NF-κB in the foaming THP-1 and RAW264.7 cells by real-time quantitative PCR (A,B); Expression of p-NF-κB p65, NF-κB p65, p-IκB and IκB protein in the foaming THP-1 cells treated with temporin-1CEa and its analogs by Western blot (C,D); Expression of p-NF-κB, NF-κB, p-IκB and IκB protein in the foaming RAW264.7 cells treated with LK2(6)A(L) by Western blot (E,F) and GAPDH was used as an internal reference. *p < 0.05 vs. the ctrl group. # p < 0.05 vs. the ox-LDL group.
FIGURE 6
FIGURE 6
Effects of frog skin AMPs on the protein expression of MAPK signaling pathway in ox-LDL induced foam cells. Expression of p-JNK, JNK, p-ERK, ERK, p-p38 and p38 protein in the foaming THP-1 cells treated with temporin-1CEa and its analogs by Western blot (A,B); Expression of p-JNK, JNK, p-ERK, ERK, p-p38 and p38 protein in the foaming RAW264.7 cells treated with LK2(6)A(L) by Western blot (C,D) and GAPDH was used as an internal reference. *p < 0.05 vs. the ctrl group. # p < 0.05 vs. the ox-LDL group.
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