Avenanthramide C Suppresses Matrix Metalloproteinase-9 Expression and Migration Through the MAPK/NF- κB Signaling Pathway in TNF-α-Activated HASMC Cells

FIGURE 1. Chemical structures of Avenanthramides and effects on cell viability in HASMC cells.(A) Avenanthramides: AvnA, AvnB and AvnC (Chemical structures) (B) Cell viability measured by MTT. 5 × 103 cell/well cells were treated with Avn C (0, 50, or 100 µM) and TNF-α (100 ng/ml) for 24 h.

FIGURE 2. Avn C inhibits TNF-α -stimulated migration and wound healing of HASMC cells (A) HASMC cells were scratched with a yellow pipette tip. The cells were treated with 100 ng/ml TNF-α with or without 100 μM Avn C for 24 h. Cells migrated into the wound area (B) The migration ability of HASMC cells was detected by a transwell assay. The cells were incubated with 100 ng/ml TNF-α with or without 100 μM Avn C for 24 h. Results shown are representative of three independent experiments. They are presented as mean ± SEM. *p < 0.05 and **p < 0.01, significant differences from TNF-α treated cells. NT, no treatment.

FIGURE 3. Effects of Avn C on TNF-α-induced MMP-9 and MMP-2 expression levels in HASMC cells.HASMC cells were co-treated with 0–100 μM Avn C and 100 ng/ml TNF-α for 24 h. Protein (A) and mRNA (B) levels respectively were examined by Western blotting and RT-PCR (C) Gelatin zymography results of MMP-9 enzyme activity levels. Results shown are representative of three independent experiments. They are presented as mean ± SEM. *p < 0.05 and **p < 0.01, significant differences from the TNF-α treated cells. NT, no treatment.

FIGURE 4. Effects of Avn C on TNF-α-induced cytokines secretion levels of IL-1β, IL-6 and TNF-α in HASMC cells (A) and (B) were not different in the secretion of IL-1 β and TNF- α cytokines with treatment by AvnC according to ELISA, but (C) Avn C attenuated TNF-α-induced IL-6 inflammatory cytokines. Results shown are representative of three independent experiments. Also, Avn C showed no effect on the total expression level of IL-1β and TNF-α, but it specifically decreased the expression level of IL-6 in TNF-α -stimulated HASMC cells by RT-PCR analysis (D). Results are presented was mean ± SEM. *p < 0.05 indicates a significant difference from TNF-α-treated cells. NT, no treatment; T, TNF-α; T + AvnC, TNF-α + Avn C.

FIGURE 5. Effects of Avn C on TNF-α-induced nuclear translocation of NF-kB in HASMC cells (A) Transfer of NF-κB to the nucleus was detected by immunofluorescence assay. HASMC cells were immune-stained by FITC and Hoechst. Scale bars, 5 μm. NE, Nuclear extracts; CE, Cytosolic extract; NT, no treatment (B) Nuclear extracts were subjected to translocation to the nuclear region of the NF-κB subunit by Western blot. They are presented as mean ± SEM. *p < 0.05 and **p < 0.01, significant differences from the TNF-α treated cells. NT, no treatment.

FIGURE 6. Avn C inhibits MAPK phosphorylation in TNF-α-activated HASMC cells.Levels of phosphorylated ERK, p38, and JNK were assessed by Western blotting. Levels of ERK, JNK, and p38 were indicated by loaded protein as individual control. Results shown are representative of three independent experiments. Results are presented as mean ± SEM. *p < 0.05 and **p < 0.01 indicate significant differences from TNF-α treated cells. NT, no treatment.

FIGURE 7. A schematic illustration of anti-atherosclerosis action of Avn C.