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Serotonin receptor subtype-2B signaling is associated with interleukin-18-induced cardiomyoblast hypertrophy in vitro

Chao-Yi Chen
Chao-Yi Chen
, 
Jyh-Gang Leu
Jyh-Gang Leu
, 
Kuan-Yu Lin
Kuan-Yu Lin
, 
Chin-Yu Shih
Chin-Yu Shih
 et 
Yao-Jen Liang
Yao-Jen Liang
   | 29 avr. 2022
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Article Category: Original Article
Publié en ligne: 29 avr. 2022
Pages: 79 - 87
DOI: https://doi.org/10.2478/abm-2022-0010
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© 2022 Chao-Yi Chen et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Pretreatment with 20 mM ECGC and 2 mM L-165,041 attenuated IL-18-induced inflammatory markers in H9c2 cardiomyoblasts. (A) NIK production significantly was inhibited by EGCG or L-165,041 (n = 4). (B) EGCG and L-165,041 significantly attenuated IL-18-induced nuNF-κB upregulation (n = 4). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. *P < 0.05 and **P < 0.01 when compared with control. #P < 0.05 when compared with IL-18 alone. ECGC (E), epigallocatechin gallate; IL-18, interleukin-18; L-165,041 (L), peroxisome proliferator-activated receptor delta (PPARd) agonist; NIK, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inducing kinase; nuNF-κB, nuclear NF-κB; SEM: standard error of mean.
Pretreatment with 20 mM ECGC and 2 mM L-165,041 attenuated IL-18-induced inflammatory markers in H9c2 cardiomyoblasts. (A) NIK production significantly was inhibited by EGCG or L-165,041 (n = 4). (B) EGCG and L-165,041 significantly attenuated IL-18-induced nuNF-κB upregulation (n = 4). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. *P < 0.05 and **P < 0.01 when compared with control. #P < 0.05 when compared with IL-18 alone. ECGC (E), epigallocatechin gallate; IL-18, interleukin-18; L-165,041 (L), peroxisome proliferator-activated receptor delta (PPARd) agonist; NIK, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inducing kinase; nuNF-κB, nuclear NF-κB; SEM: standard error of mean.

Figure 2

Effects of 20 mM ECGC and 2 mM L-165,041 treatment on IL-18-induced BNP mRNA and protein expressions in H9c2 cardiomyoblasts. Treatment with IL-18 significantly increased the BNP (n = 4) (A) mRNA and (B) protein expression (n = 5). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. **P < 0.01 when compared with control. #P < 0.05 and ##P < 0.01 when compared with IL-18. BNP, brain natriuretic peptide; ECGC (E), epigallocatechin gallate; IL-18, interleukin-18; L-165,041 (L), peroxisome proliferator-activated receptor delta (PPARd) agonist; SEM: standard error of mean.
Effects of 20 mM ECGC and 2 mM L-165,041 treatment on IL-18-induced BNP mRNA and protein expressions in H9c2 cardiomyoblasts. Treatment with IL-18 significantly increased the BNP (n = 4) (A) mRNA and (B) protein expression (n = 5). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. **P < 0.01 when compared with control. #P < 0.05 and ##P < 0.01 when compared with IL-18. BNP, brain natriuretic peptide; ECGC (E), epigallocatechin gallate; IL-18, interleukin-18; L-165,041 (L), peroxisome proliferator-activated receptor delta (PPARd) agonist; SEM: standard error of mean.

Figure 3

Pretreatment with 20 μM EGCG and 2 μM L-165,041 inhibited IL-18-induced HTR2B upregulation. (A) H9c2 cardiomyoblasts treated with IL-18 for up to 24 h, IL-18-induced HTR2B protein expression. Total protein was extracted and analyzed by western blotting for HTR2B protein expression (n = 4). (B) Serotonin (S) alone (10 μM) significantly increased the expression of HTR2B mRNA. H9c2 were treated IL-18 for 18 h. Pretreatment with EGCG and L-165,041 inhibited IL-18-induced HTR2B mRNA upregulation (n = 5) and (C) HTR2B protein expression (n = 5). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. *P < 0.05 and **P < 0.01 when compared with control. #P < 0.05 when compared to IL-18 treatment alone. HTR2B, 5-hydroxytryptamine receptor 2B; ECGC (E), epigallocatechin gallate; IL-18, Interleukin-18; L-165,041 (L), peroxisome proliferator-activated receptor delta (PPARδ) agonist; SB215505 (SB), HTR2B antagonist; serotonin, 5-hydroxytryptamine.
Pretreatment with 20 μM EGCG and 2 μM L-165,041 inhibited IL-18-induced HTR2B upregulation. (A) H9c2 cardiomyoblasts treated with IL-18 for up to 24 h, IL-18-induced HTR2B protein expression. Total protein was extracted and analyzed by western blotting for HTR2B protein expression (n = 4). (B) Serotonin (S) alone (10 μM) significantly increased the expression of HTR2B mRNA. H9c2 were treated IL-18 for 18 h. Pretreatment with EGCG and L-165,041 inhibited IL-18-induced HTR2B mRNA upregulation (n = 5) and (C) HTR2B protein expression (n = 5). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. *P < 0.05 and **P < 0.01 when compared with control. #P < 0.05 when compared to IL-18 treatment alone. HTR2B, 5-hydroxytryptamine receptor 2B; ECGC (E), epigallocatechin gallate; IL-18, Interleukin-18; L-165,041 (L), peroxisome proliferator-activated receptor delta (PPARδ) agonist; SB215505 (SB), HTR2B antagonist; serotonin, 5-hydroxytryptamine.

Figure 4

Pretreatment with 0.1 mM SB215505, 20 mM EGCG, and 2 mM L-165,041 inhibited IL-18-induced H9c2 hypertrophy. H9c2 hypertrophy was inhibited by SB215505, EGCG, and L-165,041, as analyzed by flow cytometry. FSC reflects the relative size of cells. The relative average size for control was 282; IL-18 was 354; IL-18 + SB was 304; IL-18 + SB + EGCG was 272; and IL-18 + SB + L-165,041 was 273 (n = 4). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. *P < 0.05 when compared with control. #P < 0.05 and ##P < 0.01 when compared with IL-18. ECGC (E), epigallocatechin gallate; FSC, forward scatter; IL-18, interleukin-18; L-165,041 (L), peroxisome proliferator-activated receptor delta (PPARd) agonist; SB215505 (SB), HTR2B antagonist; SEM, standard error of mean; SSC, side scatter. HTR2B, 5-hydroxytryptamine receptor 2B.
Pretreatment with 0.1 mM SB215505, 20 mM EGCG, and 2 mM L-165,041 inhibited IL-18-induced H9c2 hypertrophy. H9c2 hypertrophy was inhibited by SB215505, EGCG, and L-165,041, as analyzed by flow cytometry. FSC reflects the relative size of cells. The relative average size for control was 282; IL-18 was 354; IL-18 + SB was 304; IL-18 + SB + EGCG was 272; and IL-18 + SB + L-165,041 was 273 (n = 4). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. *P < 0.05 when compared with control. #P < 0.05 and ##P < 0.01 when compared with IL-18. ECGC (E), epigallocatechin gallate; FSC, forward scatter; IL-18, interleukin-18; L-165,041 (L), peroxisome proliferator-activated receptor delta (PPARd) agonist; SB215505 (SB), HTR2B antagonist; SEM, standard error of mean; SSC, side scatter. HTR2B, 5-hydroxytryptamine receptor 2B.

Figure 5

siHTR2B significantly attenuated IL-18-induced HTR2B expression. The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). (A) Pretreatment with siHTR2B significantly decreased HTR2B mRNA expression (n = 5). Differences from control were assessed using a Mann–Whitney U test. (B) Total protein was extracted and analyzed by western blotting for HTR2B protein expression (n = 5). Pretreatment with siHTR2B significantly inhibited IL-18-induced HTR2B protein expression. Differences between groups were assessed using a Kruskal–Wallis test. **P < 0.01 when compared with control. #P < 0.05 when compared with IL-18. HTR2B, 5-hydroxytryptamine receptor 2B; IL-18, interleukin-18; SEM, standard error of mean.
siHTR2B significantly attenuated IL-18-induced HTR2B expression. The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines) ± SEM (error bars). (A) Pretreatment with siHTR2B significantly decreased HTR2B mRNA expression (n = 5). Differences from control were assessed using a Mann–Whitney U test. (B) Total protein was extracted and analyzed by western blotting for HTR2B protein expression (n = 5). Pretreatment with siHTR2B significantly inhibited IL-18-induced HTR2B protein expression. Differences between groups were assessed using a Kruskal–Wallis test. **P < 0.01 when compared with control. #P < 0.05 when compared with IL-18. HTR2B, 5-hydroxytryptamine receptor 2B; IL-18, interleukin-18; SEM, standard error of mean.

Figure 6

Pretreatment with siHTR2B significantly attenuated IL-18-induced MMP-3 and MMP-9 expression. Total protein was extracted and analyzed by western blotting for MMP-3 and MMP-9 expression (n = 5). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines; dashed, MMP-3; solid, MMP-9) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. *P < 0.05 and **P < 0.01 when compared with control. #P < 0.05 and ##P < 0.01 when compared with IL-18 alone. HTR2B, 5-hydroxytryptamine receptor 2B; IL-18, interleukin-18; MMP-3, matrix metalloproteinase-3; MMP-9, matrix metalloproteinase-9; SEM, standard error of mean.
Pretreatment with siHTR2B significantly attenuated IL-18-induced MMP-3 and MMP-9 expression. Total protein was extracted and analyzed by western blotting for MMP-3 and MMP-9 expression (n = 5). The data are normalized and shown as individual data (solid circles) and as mean (horizontal lines; dashed, MMP-3; solid, MMP-9) ± SEM (error bars). Differences between groups were assessed using a Kruskal–Wallis test. *P < 0.05 and **P < 0.01 when compared with control. #P < 0.05 and ##P < 0.01 when compared with IL-18 alone. HTR2B, 5-hydroxytryptamine receptor 2B; IL-18, interleukin-18; MMP-3, matrix metalloproteinase-3; MMP-9, matrix metalloproteinase-9; SEM, standard error of mean.
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