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L-cystathionine inhibits oxidized low density lipoprotein-induced THP-1-derived macrophage inflammatory cytokine monocyte chemoattractant protein-1 generation via the NF-κB pathway.

Zhu M, Du J, Liu AD, Holmberg L, Chen SY, Bu D, Tang C, Jin H - Sci Rep (2015)

Bottom Line: Compared with the ox-LDL group, 0.3 mmol/L and 1.0 mmol/L L-cystathionine significantly inhibited the expression of THP-1-derived macrophage MCP-1.Mechanistically, 0.3 mmol/L and 1.0 mmol/L L-cystathionine suppressed phosphorylation and nuclear translocation of the NF-κB p65 protein, as well as the DNA binding activity and DNA binding level of NF-κB with the MCP-1 promoter, which resulted in a reduced THP-1-derived macrophage MCP-1 generation.This study suggests that L-cystathionine could inhibit the expression of MCP-1 in THP-1-derived macrophages induced by ox-LDL via inhibition of NF-κB p65 phosphorylation, nuclear translocation, and binding of the MCP-1 promoter sequence after entry into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Peking University First Hospital, Beijing 100034, P. R. China.

ABSTRACT
This study aimed to explore whether and how L-cystathionine had any regulatory effect on the inflammatory response in THP-1-derived macrophages cultured in vitro under oxidized low-density lipoprotein (ox-LDL) stimulation. The human monocyte line THP-1 cell was cultured in vitro and differentiated into macrophages after 24 hours of PMA induction. Macrophages were pretreated with L-cystathionine and then treated with ox-LDL. The results showed that compared with the controls, ox-LDL stimulation significantly upregulated the expression of THP-1-derived macrophage MCP-1 by enhancing NF-κB p65 phosphorylation, nuclear translocation and DNA binding with the MCP-1 promoter. Compared with the ox-LDL group, 0.3 mmol/L and 1.0 mmol/L L-cystathionine significantly inhibited the expression of THP-1-derived macrophage MCP-1. Mechanistically, 0.3 mmol/L and 1.0 mmol/L L-cystathionine suppressed phosphorylation and nuclear translocation of the NF-κB p65 protein, as well as the DNA binding activity and DNA binding level of NF-κB with the MCP-1 promoter, which resulted in a reduced THP-1-derived macrophage MCP-1 generation. This study suggests that L-cystathionine could inhibit the expression of MCP-1 in THP-1-derived macrophages induced by ox-LDL via inhibition of NF-κB p65 phosphorylation, nuclear translocation, and binding of the MCP-1 promoter sequence after entry into the nucleus.

No MeSH data available.


Related in: MedlinePlus

L-cystathionine decreased DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage. The cells were pretreated with L-cystathionine (0.1 mmol/L, 0.3 mmol/L or 1.0 mmol/L, respectively) for 30 min, and were then stimulated with 50 mg/L ox-LDL for 30 min. (A) The effect of L-cystathionine on DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage detected by EMSA. (B) The effect of L-cystathionine on DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage detected by ELISA. (C) The effect of L-cystathionine on DNA binding level of NF-κB p65 with MCP-1 promoter induced by ox-LDL in the THP-1-derived macrophage measured by ChIP. (D, E, F) The basal DNA binding activity of NF-κB p65 in the THP-1-derived macrophage without ox-LDL treatment. L-Cth: L-cystathionine. **P < 0.01 compared with control group, ##p < 0.01 compared with ox-LDL group. Data are presented as mean ± SD (n = 3) of three independent experiments performed in triplicate.
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f4: L-cystathionine decreased DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage. The cells were pretreated with L-cystathionine (0.1 mmol/L, 0.3 mmol/L or 1.0 mmol/L, respectively) for 30 min, and were then stimulated with 50 mg/L ox-LDL for 30 min. (A) The effect of L-cystathionine on DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage detected by EMSA. (B) The effect of L-cystathionine on DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage detected by ELISA. (C) The effect of L-cystathionine on DNA binding level of NF-κB p65 with MCP-1 promoter induced by ox-LDL in the THP-1-derived macrophage measured by ChIP. (D, E, F) The basal DNA binding activity of NF-κB p65 in the THP-1-derived macrophage without ox-LDL treatment. L-Cth: L-cystathionine. **P < 0.01 compared with control group, ##p < 0.01 compared with ox-LDL group. Data are presented as mean ± SD (n = 3) of three independent experiments performed in triplicate.

Mentions: To study whether L-cystathionine has an inhibitory effect on the DNA binding activity of NF-κB, we used electrophoretic mobility shift assay (EMSA) to measure the effect of L-cystathionine on the DNA binding activity of p65 in the THP-1-derived macrophage. THP-1- derived macrophages were pretreated with L-cystathionine for 30 min, and then were stimulated with 50 mg/L ox-LDL for 30 min. The results indicated that ox-LDL significantly increased the DNA binding activity of p65 in the THP-1-derived macrophage. However, DNA binding activity of NF-κB p65 in the THP-1-derived macrophage was dose-dependently inhibited when the cells were pretreated with L-cystathionine (Fig. 4A). Moreover, we also used the ELISA method to measure the DNA binding activity of NF-κB p65. The results showed that ox-LDL markedly enhanced the DNA binding activity of p65 in the THP-1-derived macrophage. DNA binding activity of NF-κB p65 was dose-dependently inhibited in the cells pretreated with L-cystathionine (Fig. 4B). However, both EMSA and ELISA results showed that L-cystathionine had no effect on the DNA binding activity of p65 without ox-LDL stimulation (Fig. 4D, Fig. 4E).


L-cystathionine inhibits oxidized low density lipoprotein-induced THP-1-derived macrophage inflammatory cytokine monocyte chemoattractant protein-1 generation via the NF-κB pathway.

Zhu M, Du J, Liu AD, Holmberg L, Chen SY, Bu D, Tang C, Jin H - Sci Rep (2015)

L-cystathionine decreased DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage. The cells were pretreated with L-cystathionine (0.1 mmol/L, 0.3 mmol/L or 1.0 mmol/L, respectively) for 30 min, and were then stimulated with 50 mg/L ox-LDL for 30 min. (A) The effect of L-cystathionine on DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage detected by EMSA. (B) The effect of L-cystathionine on DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage detected by ELISA. (C) The effect of L-cystathionine on DNA binding level of NF-κB p65 with MCP-1 promoter induced by ox-LDL in the THP-1-derived macrophage measured by ChIP. (D, E, F) The basal DNA binding activity of NF-κB p65 in the THP-1-derived macrophage without ox-LDL treatment. L-Cth: L-cystathionine. **P < 0.01 compared with control group, ##p < 0.01 compared with ox-LDL group. Data are presented as mean ± SD (n = 3) of three independent experiments performed in triplicate.
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Related In: Results  -  Collection

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f4: L-cystathionine decreased DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage. The cells were pretreated with L-cystathionine (0.1 mmol/L, 0.3 mmol/L or 1.0 mmol/L, respectively) for 30 min, and were then stimulated with 50 mg/L ox-LDL for 30 min. (A) The effect of L-cystathionine on DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage detected by EMSA. (B) The effect of L-cystathionine on DNA binding activity of NF-κB p65 induced by ox-LDL in the THP-1-derived macrophage detected by ELISA. (C) The effect of L-cystathionine on DNA binding level of NF-κB p65 with MCP-1 promoter induced by ox-LDL in the THP-1-derived macrophage measured by ChIP. (D, E, F) The basal DNA binding activity of NF-κB p65 in the THP-1-derived macrophage without ox-LDL treatment. L-Cth: L-cystathionine. **P < 0.01 compared with control group, ##p < 0.01 compared with ox-LDL group. Data are presented as mean ± SD (n = 3) of three independent experiments performed in triplicate.
Mentions: To study whether L-cystathionine has an inhibitory effect on the DNA binding activity of NF-κB, we used electrophoretic mobility shift assay (EMSA) to measure the effect of L-cystathionine on the DNA binding activity of p65 in the THP-1-derived macrophage. THP-1- derived macrophages were pretreated with L-cystathionine for 30 min, and then were stimulated with 50 mg/L ox-LDL for 30 min. The results indicated that ox-LDL significantly increased the DNA binding activity of p65 in the THP-1-derived macrophage. However, DNA binding activity of NF-κB p65 in the THP-1-derived macrophage was dose-dependently inhibited when the cells were pretreated with L-cystathionine (Fig. 4A). Moreover, we also used the ELISA method to measure the DNA binding activity of NF-κB p65. The results showed that ox-LDL markedly enhanced the DNA binding activity of p65 in the THP-1-derived macrophage. DNA binding activity of NF-κB p65 was dose-dependently inhibited in the cells pretreated with L-cystathionine (Fig. 4B). However, both EMSA and ELISA results showed that L-cystathionine had no effect on the DNA binding activity of p65 without ox-LDL stimulation (Fig. 4D, Fig. 4E).

Bottom Line: Compared with the ox-LDL group, 0.3 mmol/L and 1.0 mmol/L L-cystathionine significantly inhibited the expression of THP-1-derived macrophage MCP-1.Mechanistically, 0.3 mmol/L and 1.0 mmol/L L-cystathionine suppressed phosphorylation and nuclear translocation of the NF-κB p65 protein, as well as the DNA binding activity and DNA binding level of NF-κB with the MCP-1 promoter, which resulted in a reduced THP-1-derived macrophage MCP-1 generation.This study suggests that L-cystathionine could inhibit the expression of MCP-1 in THP-1-derived macrophages induced by ox-LDL via inhibition of NF-κB p65 phosphorylation, nuclear translocation, and binding of the MCP-1 promoter sequence after entry into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Peking University First Hospital, Beijing 100034, P. R. China.

ABSTRACT
This study aimed to explore whether and how L-cystathionine had any regulatory effect on the inflammatory response in THP-1-derived macrophages cultured in vitro under oxidized low-density lipoprotein (ox-LDL) stimulation. The human monocyte line THP-1 cell was cultured in vitro and differentiated into macrophages after 24 hours of PMA induction. Macrophages were pretreated with L-cystathionine and then treated with ox-LDL. The results showed that compared with the controls, ox-LDL stimulation significantly upregulated the expression of THP-1-derived macrophage MCP-1 by enhancing NF-κB p65 phosphorylation, nuclear translocation and DNA binding with the MCP-1 promoter. Compared with the ox-LDL group, 0.3 mmol/L and 1.0 mmol/L L-cystathionine significantly inhibited the expression of THP-1-derived macrophage MCP-1. Mechanistically, 0.3 mmol/L and 1.0 mmol/L L-cystathionine suppressed phosphorylation and nuclear translocation of the NF-κB p65 protein, as well as the DNA binding activity and DNA binding level of NF-κB with the MCP-1 promoter, which resulted in a reduced THP-1-derived macrophage MCP-1 generation. This study suggests that L-cystathionine could inhibit the expression of MCP-1 in THP-1-derived macrophages induced by ox-LDL via inhibition of NF-κB p65 phosphorylation, nuclear translocation, and binding of the MCP-1 promoter sequence after entry into the nucleus.

No MeSH data available.


Related in: MedlinePlus