Suppression of Gut Bacterial Translocation Ameliorates Vascular Calcification through Inhibiting Toll-Like Receptor 9-Mediated BMP-2 Expression

Figure 5

TLR9 activation promotes BMP-2 expression through activation of NF-κB signaling. (a) Real-time PCR analyzed the mRNA levels of BMP-2 in Ctrl, CRF, and CRF + Anti groups. Data are relative to β-actin level. ~12, vs. Ctrl. ^# vs. CRF. (b) Serum BMP-2 levels of Ctrl, CRF, and CRF + Anti groups were analyzed and quantified by ELISA; ~12, vs. Ctrl. ^# vs. CRF. HASMCs were treated with TLR9 ligand control (1 μg/mL) or ssDNA (10 μg/mL), with or without Pi for 7 days. (c) Real-time PCR analyzed the mRNA level of BMP-2. , vs. Ctrl. ^# vs. Pi. (d) Western blot analysis of BMP-2 protein level. GAPDH was a loading control. , vs. Ctrl. ^# vs. Pi. (e) HASMCs were treated with Pi or ssDNA for 24 h, and the supernatant BMP-2 was measured by ELISA; , vs. Ctrl. ^# vs. Pi. (f-h) HASMCs were treated with Pi, ssDNA, or PTN (PTN was preincubated for 30 minutes, 10 μmol/L) for (f) 30 minutes, and the IκBα protein level was measured by western blot. β-Actin was a loading control. (g) 24 h, and real-time PCR analyzed the mRNA levels of BMP-2. , vs. Ctrl, ^# vs. Pi. (h) 7 days, the calcium content was measured. , vs. Ctrl, ^# vs. Pi.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)