Fig 1: AntagomiR-135a-5p weakened the effect of OCA on mitigating vascular inflammation and calcification in CKD rats.To inhibit miR135a-5p, rats were injected with antagomiR135a-5p (80 µg/g body weight) through vena caudalis for three consecutive days before inducing chronic kidney disease (n = 8 per group). a The mRNA expression of TGFBR1 of aortas in indicated groups. b, c Calcified lesion areas of aortic (b) and aortic calcium content (c) in indicated groups. d Representative images of von Kossa staining of aortic ring in indicated groups (scale bar, 500 µm). e Representative images of micro-CT in indicated groups, arrows indicate calcified nodules. f Western blot analysis of NF-?B, TNF-a, TAK1, p-TAK1, TAB1, and p-I?Ba of aortas in indicated groups. g Representative immunofluorescence staining of p-I?Ba (pink), TAK1 (red) and TAB1 (green) of aortic rings in indicated groups (scale bar, 500µm). *P < 0.05, **P < 0.01, ns indicates not significant.
Fig 2: FXR activation inhibited osteogenic medium-induced TGFBR1/TAK1 pathway activation via increasing miR-135a-5p expression.a Potential sequences of the miR-135a-5p binding sites within the 3'-UTR of TGFBR1 was predicted by TargetScan Release 4.2 and RNAhybird. b The mRNA expression of TGFBR1 after overexpressing or inhibiting miR-135a-5p. c Western blot analysis of p-TAK1, TAB1, and p-I?Ba in TGF-ß1-cultured HASMCs in the absence or presence of miR-135a-5p mimic. d Representative immunofluorescence staining of TAKI (red) and p-I?Ba (green) in indicated groups (100×). e Western blot analysis of TAK1, p-TAK1, TAB1, and p-I?Ba in indicated groups. **P < 0.01, ns indicates not significant. Data were pooled as mean ± S.D. (error bars) from three or more independent experiments.
Fig 3: FXR activation inhibited TGFBR1/TAK1 pathways to attenuate osteogenic medium-induced HASMCs inflammation and calcification.HASMCs were cultured with osteogenic medium in the absence or presence of 3 µM OCA (FXR ligand) for 7 days. a Western blot results showed that FXR was activated by OCA. b Western blot analysis of the expression of TAK1, p-TAK1, TAB1, p-I?Ba, NF-?B, and TNF-a in indicated groups. c Representative immunofluorescence staining of NF-?B (green) and TNF-a (red) in indicated groups (100×). d Intercellular NF-?B content of HASMCs in indicated groups. e Representative images of alizarin red staining in indicated groups (100×). f Calcium content of HASMCs in indicated groups. g The mRNA expression of Runx2 and ALP in indicated groups. *P < 0.05, **P < 0.01. Data were pooled as mean ± S.D. (error bars) from three or more independent experiments.
Fig 4: TGFBR1/TAK1 pathway was activated by osteogenic medium, resulting in an increased pro-inflammatory cytokines expression and vascular calcification.HASMCs were cultured with osteogenic medium containing 2.0 mM Pi and 2.7 mM Ca. After 7 days, cells were stained with alizarin red (100×) a which identify calcium mineral as red. b Ca content assayed by calcium detection assay kit, which was normalized to protein concentration. c The mRNA expression of TGFBR1, TAK1, TAB1, and I?Ba in osteogenic medium-treated HASMCs. d Western blot analysis of the expression of TAK1, p-TAK1, TAB1, and p-I?Ba in osteogenic medium-treated HASMCs. e Western blot analysis of the expression of NF-?B, TNF-a, TAK1, p-TAK1, TAB1, and p-I?Ba in osteogenic medium-treated HASMCs in the absence or presence of TGFBR1 siRNA. f Representative immunofluorescence staining of NF-?B (green) and TNF-a (red) in indicated groups (100×). g Calcium content of HASMCs in indicated groups. h Representative images of alizarin red staining in indicated groups (100×). *P < 0.05, **P < 0.01. ns indicates not significant. Data were pooled as mean ± S.D. (error bars) from three or more independent experiments.
Fig 5: Schematic model for FXR activation attenuating TGFBR1/TAK1 pathway-mediated inflammation and calcification through increasing microRNA-135a-5p expression in osteogenic medium-cultured HASMCs.a TGFBR1 is activated in osteogenic medium-cultured HASMCs, and then activates TAK1, which binds with TAB1 and TAB2 to form a stable complex at the N-terminal kinase domain and the C-terminal region of TAK1. NF-?B pathway is the downstream of TAK1, which is reported to stimulate the expression of osteogenic transcription factor and osteogenic marker, resulting in calcification formation of HASMCs. MicroRNA-135a-5p is key regulator to inhibited TGFBR1, which is suppressed in osteogenic medium-cultured HASMCs. b FXR activation increases microRNA-135a-5p expression to inhibit TGFBR1/TAK1 pathway and further attenuates osteogenic medium-induced HASMCs inflammation and calcification.
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