Hypaphorine Attenuates Lipopolysaccharide-Induced Endothelial Inflammation via Regulation of TLR4 and PPAR-γ Dependent on PI3K/Akt/mTOR Signal Pathway

Abstract

Endothelial lesion response to injurious stimuli is a necessary step for initiating inflammatory cascades in blood vessels. Hypaphorine (Hy) from different marine sources is shown to exhibit anti-inflammatory properties. However, the potential roles and possible molecular mechanisms of Hy in endothelial inflammation have yet to be fully clarified. We showed that Hy significantly inhibited the positive effects of lipopolysaccharide (LPS) on pro-inflammatory cytokines expressions, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1) and vascular cellular adhesion molecule-1 (VCAM-1), as well as induction of the phosphorylation of Akt and mTOR in HMEC-1 cells. The downregulated peroxisome proliferator-activated receptor γ (PPAR-γ) and upregulated toll-like receptor 4 (TLR4) expressions in LPS-challenged endothelial cells were prevented by Hy. Inhibition of both PI3K and mTOR reversed LPS-stimulated increases in TLR4 expressions and decreases in PPAR-γ levels. Genetic silencing of TLR4 or PPAR-γ agonist pioglitazone obviously abrogated the levels of pro-inflammatory cytokines in LPS-treated HMEC-1 cells. These results suggest that Hy may exert anti-inflammatory actions through the regulation of TLR4 and PPAR-γ dependent on PI3K/Akt/mTOR signal pathways. Hy may be considered as a therapeutic agent that can potentially relieve or ameliorate endothelial inflammation-associated diseases.

Keywords: LPS; PPAR-γ; TLR4; endothelial cells; inflammation.

Figure 1

Effects of different doses of Hy on the mRNA expressions of TNF-α, IL-1β, VCAM-1 and MCP-1 response to LPS-treated HMEC-1 cells in vitro. (A) Effects of various concentrations of LPS (0, 100, 200 and 500 ng/mL for 48 h) on the TNF-α, IL-1β, VCAM-1 and MCP-1 levels in HMEC-1 cells; (B) Effects of different doses of Hy (0, 25, 50 and 100 μM for 48 h) on the mRNA expressions of TNF-α, IL-1β, VCAM-1 and MCP-1 HMEC-1 cells; (C) Effects of different doses of Hy (0, 25, 50 and 100 μM) on the TNF-α, IL-1β, VCAM-1 and MCP-1 expressions in LPS (500 ng/mL)-stimulated HMEC-1 cells. The HMEC-1 cells were pretreated with different doses of Hy for 6 h before LPS incubation for another 48 h. Values are mean ± S.D. * p < 0.05 vs. 0 ng/mL, 0 μM or control, † p < 0.05 vs. LPS. n = 4 for each group. LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; VCAM-1, vascular cellular adhesion molecule-1; MCP-1, monocyte chemoattractant protein 1.

Figure 2

Inhibition of TLR4 was involved in the anti-inflammatory action of Hy on HMEC-1 cells response to LPS. (A) Representative photos showing the effects of various concentrations of LPS (0, 100, 200 and 500 ng/mL for 48 h) on TLR4 protein levels in HMEC-1 cells; (B) Effects of various concentrations of LPS (0, 100, 200 and 500 ng/mL for 48 h) on TLR4 mRNA levels in HMEC-1 cells; (C) Effects of different doses of Hy (0, 25, 50 and 100 μM for 48 h) on TLR4 protein levels in HMEC-1 cells; (D) Effects of different doses of Hy (0, 25, 50 and 100 μM for 48 h) on TLR4 mRNA levels in HMEC-1 cells; (E) Representative photographs showing the effects of Hy (100 μM) on TLR4 protein expressions in LPS (500 ng/mL)-challenged HMEC-1 cells. The HMEC-1 cells were pretreated with Hy for 6 h before LPS incubation for another 48 h; (F) Effects of Hy (100 μM ) on TLR4 mRNA expressions in LPS (500 ng/mL)-challenged HMEC-1 cells; (G) Immunofluorescence staining showing the effects of Hy (100 μM ) on TLR4 protein expressions in HMEC-1 cells response to LPS (500 ng/mL), scale bar = 50 µm. Values are mean ± S.D. * p < 0.05 vs. 0 ng/mL or Veh, † p < 0.05 vs. Hy, ‡ p < 0.05 vs. Veh + LPS. n = 4 for each group. LPS, lipopolysaccharide; TLR4, toll-like receptor 4.

Figure 3

Activation of PPAR-γ mediated the protective role of Hy against LPS-evoked inflammation in HMEC-1 cells. (A) Representative photos showing the effects of various concentrations of LPS (0, 100, 200 and 500 ng/mL for 48 h) on PPAR-γ protein levels in HMEC-1 cells; (B) Effects of various concentrations of LPS (0, 100, 200 and 500 ng/mL for 48 h) on PPAR-γ mRNA levels in HMEC-1 cells; (C) Effects of different doses of Hy (0, 25, 50 and 100 μM for 48 h) on PPAR-γ protein levels in HMEC-1 cells; (D) Effects of different doses of Hy (0, 25, 50 and 100 μM for 48 h) on PPAR-γ mRNA levels in HMEC-1 cells; (E) Representative photographs showing the effects of Hy (100 μM) on PPAR-γ protein expressions in LPS (500 ng/mL)-challenged HMEC-1 cells. The HMEC-1 cells were pretreated with Hy for 6 h before LPS incubation for another 48 h; (F) Effects of Hy (100 μM) on PPAR-γ mRNA expressions in LPS (500 ng/mL)-challenged HMEC-1 cells; (G) Immunofluorescence staining showing the effects of Hy (100 μM) on PPAR-γ expressions in HMEC-1 cells response to LPS (500 ng/mL), scale bar = 50 μm. Values are mean ± S.D. * p < 0.05 vs. 0 ng/mL or Veh, † p < 0.05 vs. Hy, ‡ p < 0.05 vs. Veh + LPS. n = 4 for each group. LPS, lipopolysaccharide; PPAR-γ, peroxisome proliferator-activated receptor γ.

Figure 4

Interaction of PPAR-γ with TLR4 in HMEC-1 cells in response to LPS. (A) Western blotting of TLR4 in HMEC-1 cells. The HMEC-1 cells were pre-incubated with pioglitazone (20 μM) for 6 h followed by LPS (500 ng/mL) stimulation for 48 h; (B) Relative density of TLR4 protein bands determined by densitometry of the blots, (C) TLR4 mRNA levels; (D) Western blotting of PPAR-γ in HMEC-1 cells. The HMEC-1 cells were transfected with 100 nM control siRNA or TLR4 siRNA for 24 h followed by LPS (500 ng/mL) stimulation for 48 h; (E) Relative density of PPAR-γ protein bands determined by densitometry of the blots; (F) PPAR-γ mRNA levels. Values are mean ± S.D. * p < 0.05 vs. control or Veh, † p < 0.05 vs. Con(control) siRNA, ‡ p < 0.05 vs. control + LPS or Veh + LPS. n = 4 for each group. LPS, lipopolysaccharide; TLR4, toll-like receptor 4, PPAR-γ, peroxisome proliferator-activated receptor γ.

Figure 5

PI3K/Akt/mTOR signaling pathway is involved in the inhibition effect of Hy on LPS-induced inflammation response in HMEC-1 cells. (A) Effects of LPS on the protein expressions of total Akt (T-Akt), phosphorylated-Akt (p-Akt), mTOR, and phosphorylated-mTOR (p-mTOR) at indicated doses (0, 100, 200 and 500 ng/mL) in HMEC-1 cells. The confluent HMEC-1 cells were stimulated with different doses of LPS (0, 100, 200 and 500 ng/mL) for 15 min; (B) Quantitative analysis of phosphorylated Akt and mTOR; (C) Effects of Hy on the expressions of Akt, p-Akt, mTOR, and p-mTOR at indicated doses (0, 25, 50 and 100 μM) in HMEC-1 cells. The HMEC-1 cells were starved for 12 h and then stimulated with different doses (0, 25, 50 and 100 μM) of Hy for 15 min; (D) Quantitative analysis of phosphorylated Akt and mTOR; (E) Effects of Hy on phosphorylated Akt and mTOR in HMEC-1 cells response to LPS. The HMEC-1 cells were pretreated with Hy for 6 h before LPS incubation for 15 min; (F) Quantitative analysis of phosphorylated Akt and mTOR protein bands determined by densitometry of the blots. Values are mean ± S.D. * p < 0.05 vs. 0 ng/mL or Veh, † p < 0.05 vs. Hy, ‡ p < 0.05 vs. Veh + LPS. n = 4 for each group. LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; VCAM-1, vascular cellular adhesion molecule-1; MCP-1, monocyte chemoattractant protein 1.

Figure 6

Negative correlation of PPAR-γ with TLR4 in LPS-stimulated HMEC-1 cells was dependent on PI3K/Akt/mTOR signaling pathway. The HMEC-1 cells were pretreated with LY294002 (10 μM), or mTOR inhibitor rapamycin (200 nM) for 6 h before LPS incubation for 48 h. The protein expressions of TLR4 (A) and PPAR-γ (C) were detected by Western blotting analysis. The mRNA expressions of TLR4 (B) and PPAR-γ (D) were detected by real time quantitative PCR. Values are mean ± S.D. * p < 0.05 vs. Veh, † p < 0.05 vs. Veh + LPS. n = 4 for each group. LPS, lipopolysaccharide; TLR4, toll-like receptor 4, PPAR-γ, peroxisome proliferator-activated receptor γ.