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( − )-Epigallocatechin Gallate Targets Notch to Attenuate the Inflammatory Response in the Immediate Early Stage in Human Macrophages

View Article: PubMed Central - PubMed

ABSTRACT

Inflammation plays important roles at different stages of diabetes mellitus, tumorigenesis, and cardiovascular diseases. (−)-Epigallocatechin gallate (EGCG) can attenuate inflammatory responses effectively. However, the immediate early mechanism of EGCG in inflammation remains unclear. Here, we showed that EGCG attenuated the inflammatory response in the immediate early stage of EGCG treatment by shutting off Notch signaling and that the effect did not involve the 67-kDa laminin receptor, the common receptor for EGCG. EGCG eliminated mature Notch from the cell membrane and the nuclear Notch intercellular domain, the active form of Notch, within 2 min by rapid degradation via the proteasome pathway. Transcription of the Notch target gene was downregulated simultaneously. Knockdown of Notch 1/2 expression by RNA interference impaired the downregulation of the inflammatory response elicited by EGCG. Further study showed that EGCG inhibited lipopolysaccharide-induced inflammation and turned off Notch signaling in human primary macrophages. Taken together, our results show that EGCG targets Notch to regulate the inflammatory response in the immediate early stage.

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Notch1/2 knockdown impaired the effects of (−)-epigallocatechin gallate (EGCG) on inflammation. After knockdown of Notch1 or Notch2 through infection by lentivirus particles, inflammation experiments were done the same way as for wild-type THP-1-derived macrophages. Knockdown of Notch1 or Notch2 was confirmed by western blotting (A). Notch1/2 knockdown macrophages were pretreated with EGCG (50 μg/mL) for 30 min before exposure to lipopolysaccharide (LPS) (200 EU/mL) for 3 h. Expression of the inflammatory cytokines in the culture medium was measured by a RayBio C-Series human inflammation antibody array: Notch1 knockdown (B) and Notch2 knockdown (D). Chips were scanned and analyzed: Notch1 knockdown (C) and Notch2 knockdown (E). Notch intercellular domain (NICD)-green fluorescent protein (GFP)-overexpressed macrophages were pretreated with EGCG (50 μg/mL) for 30 min and exposed to LPS (200 EU/mL) for 3 h. Levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the culture medium were measured by enzyme-linked immunosorbent assay (F), and cell lysates were analyzed by western blotting and a probe with antibody against Notch1 or GFP separately (G). Data are represented as mean ± SD (n = 3). Differences between the two groups were assessed by two-way ANOVA using GraphPad. Representatives of three independent experiments with similar results are shown. Results are represented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). n.s., not significant.
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Figure 5: Notch1/2 knockdown impaired the effects of (−)-epigallocatechin gallate (EGCG) on inflammation. After knockdown of Notch1 or Notch2 through infection by lentivirus particles, inflammation experiments were done the same way as for wild-type THP-1-derived macrophages. Knockdown of Notch1 or Notch2 was confirmed by western blotting (A). Notch1/2 knockdown macrophages were pretreated with EGCG (50 μg/mL) for 30 min before exposure to lipopolysaccharide (LPS) (200 EU/mL) for 3 h. Expression of the inflammatory cytokines in the culture medium was measured by a RayBio C-Series human inflammation antibody array: Notch1 knockdown (B) and Notch2 knockdown (D). Chips were scanned and analyzed: Notch1 knockdown (C) and Notch2 knockdown (E). Notch intercellular domain (NICD)-green fluorescent protein (GFP)-overexpressed macrophages were pretreated with EGCG (50 μg/mL) for 30 min and exposed to LPS (200 EU/mL) for 3 h. Levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the culture medium were measured by enzyme-linked immunosorbent assay (F), and cell lysates were analyzed by western blotting and a probe with antibody against Notch1 or GFP separately (G). Data are represented as mean ± SD (n = 3). Differences between the two groups were assessed by two-way ANOVA using GraphPad. Representatives of three independent experiments with similar results are shown. Results are represented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). n.s., not significant.

Mentions: To investigate whether the effects of EGCG on cellular responses to LPS stimulation are dependent on Notch, we undertook shRNA knockdown of Notch 1/2 in THP-1 cells. THP-1 cells were infected with Notch1/2 shRNA lentiviral particles (Santa Cruz Biotechnology) and screened with puromycin (5 μg/mL) to silence expression of Notch1 or Notch2. Notch 1/2 silencing was confirmed by western blotting (Figure 5A). Notch 1/2-silenced THP-1 cells were treated with PMA (10 ng/mL) for 48 h to obtain macrophages that were similar to wild-type macrophages. Notch 1/2-silenced macrophages were stimulated with LPS (200 EU/mL) and pretreated (or not) for 30 min with EGCG. Expression of the inflammatory cytokines in samples 3 h after LPS addition was measured using a human inflammation antibody array (RayBio). Results showed that Notch1/2 knockdown impaired the attenuated effects of EGCG on release of inflammatory cytokines (Figures 5B–E). Further analyses of the inflammation array results revealed that Notch1 knockdown blocked the attenuated effects of EGCG in 30 of 40 of the inflammatory cytokines and that knockdown of Notch2 blocked the attenuated effects of EGCG in 31 of the 40 inflammatory cytokines (Figures 5B–E; Tables S3 and S4 in Supplementary Material). These results demonstrated that Notch1/2 knockdown greatly impaired the inflammation-attenuated effects of EGCG and that Notch1/2 is a major target for EGCG for reduction of the inflammatory response in the early stage.


( − )-Epigallocatechin Gallate Targets Notch to Attenuate the Inflammatory Response in the Immediate Early Stage in Human Macrophages
Notch1/2 knockdown impaired the effects of (−)-epigallocatechin gallate (EGCG) on inflammation. After knockdown of Notch1 or Notch2 through infection by lentivirus particles, inflammation experiments were done the same way as for wild-type THP-1-derived macrophages. Knockdown of Notch1 or Notch2 was confirmed by western blotting (A). Notch1/2 knockdown macrophages were pretreated with EGCG (50 μg/mL) for 30 min before exposure to lipopolysaccharide (LPS) (200 EU/mL) for 3 h. Expression of the inflammatory cytokines in the culture medium was measured by a RayBio C-Series human inflammation antibody array: Notch1 knockdown (B) and Notch2 knockdown (D). Chips were scanned and analyzed: Notch1 knockdown (C) and Notch2 knockdown (E). Notch intercellular domain (NICD)-green fluorescent protein (GFP)-overexpressed macrophages were pretreated with EGCG (50 μg/mL) for 30 min and exposed to LPS (200 EU/mL) for 3 h. Levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the culture medium were measured by enzyme-linked immunosorbent assay (F), and cell lysates were analyzed by western blotting and a probe with antibody against Notch1 or GFP separately (G). Data are represented as mean ± SD (n = 3). Differences between the two groups were assessed by two-way ANOVA using GraphPad. Representatives of three independent experiments with similar results are shown. Results are represented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). n.s., not significant.
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Figure 5: Notch1/2 knockdown impaired the effects of (−)-epigallocatechin gallate (EGCG) on inflammation. After knockdown of Notch1 or Notch2 through infection by lentivirus particles, inflammation experiments were done the same way as for wild-type THP-1-derived macrophages. Knockdown of Notch1 or Notch2 was confirmed by western blotting (A). Notch1/2 knockdown macrophages were pretreated with EGCG (50 μg/mL) for 30 min before exposure to lipopolysaccharide (LPS) (200 EU/mL) for 3 h. Expression of the inflammatory cytokines in the culture medium was measured by a RayBio C-Series human inflammation antibody array: Notch1 knockdown (B) and Notch2 knockdown (D). Chips were scanned and analyzed: Notch1 knockdown (C) and Notch2 knockdown (E). Notch intercellular domain (NICD)-green fluorescent protein (GFP)-overexpressed macrophages were pretreated with EGCG (50 μg/mL) for 30 min and exposed to LPS (200 EU/mL) for 3 h. Levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the culture medium were measured by enzyme-linked immunosorbent assay (F), and cell lysates were analyzed by western blotting and a probe with antibody against Notch1 or GFP separately (G). Data are represented as mean ± SD (n = 3). Differences between the two groups were assessed by two-way ANOVA using GraphPad. Representatives of three independent experiments with similar results are shown. Results are represented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). n.s., not significant.
Mentions: To investigate whether the effects of EGCG on cellular responses to LPS stimulation are dependent on Notch, we undertook shRNA knockdown of Notch 1/2 in THP-1 cells. THP-1 cells were infected with Notch1/2 shRNA lentiviral particles (Santa Cruz Biotechnology) and screened with puromycin (5 μg/mL) to silence expression of Notch1 or Notch2. Notch 1/2 silencing was confirmed by western blotting (Figure 5A). Notch 1/2-silenced THP-1 cells were treated with PMA (10 ng/mL) for 48 h to obtain macrophages that were similar to wild-type macrophages. Notch 1/2-silenced macrophages were stimulated with LPS (200 EU/mL) and pretreated (or not) for 30 min with EGCG. Expression of the inflammatory cytokines in samples 3 h after LPS addition was measured using a human inflammation antibody array (RayBio). Results showed that Notch1/2 knockdown impaired the attenuated effects of EGCG on release of inflammatory cytokines (Figures 5B–E). Further analyses of the inflammation array results revealed that Notch1 knockdown blocked the attenuated effects of EGCG in 30 of 40 of the inflammatory cytokines and that knockdown of Notch2 blocked the attenuated effects of EGCG in 31 of the 40 inflammatory cytokines (Figures 5B–E; Tables S3 and S4 in Supplementary Material). These results demonstrated that Notch1/2 knockdown greatly impaired the inflammation-attenuated effects of EGCG and that Notch1/2 is a major target for EGCG for reduction of the inflammatory response in the early stage.

View Article: PubMed Central - PubMed

ABSTRACT

Inflammation plays important roles at different stages of diabetes mellitus, tumorigenesis, and cardiovascular diseases. (&minus;)-Epigallocatechin gallate (EGCG) can attenuate inflammatory responses effectively. However, the immediate early mechanism of EGCG in inflammation remains unclear. Here, we showed that EGCG attenuated the inflammatory response in the immediate early stage of EGCG treatment by shutting off Notch signaling and that the effect did not involve the 67-kDa laminin receptor, the common receptor for EGCG. EGCG eliminated mature Notch from the cell membrane and the nuclear Notch intercellular domain, the active form of Notch, within 2&thinsp;min by rapid degradation via the proteasome pathway. Transcription of the Notch target gene was downregulated simultaneously. Knockdown of Notch 1/2 expression by RNA interference impaired the downregulation of the inflammatory response elicited by EGCG. Further study showed that EGCG inhibited lipopolysaccharide-induced inflammation and turned off Notch signaling in human primary macrophages. Taken together, our results show that EGCG targets Notch to regulate the inflammatory response in the immediate early stage.

No MeSH data available.


Related in: MedlinePlus