TGF-β1 suppression of miRNA-519b enhances HO-1 production.

<p>(A) OASFs were incubated with TGF-β1 for 0, 1, 3, 10, and 30 ng/ml. miRNA-519b expression levels were examined by qPCR assay (n = 4 per group). (B) OASFs were transfected with miR-519b mimic and stimulated with TGF-β1 for 24 hours. HO-1 mRNA expression was examined by qPCR assay (n = 4 per group). (C) OASFs were transfected with miRNA-519b mimic then stimulated with TGF-β1 for 24 hours. HO-1 expression levels were examined by Western blotting (n = 3 per group). (D) OASFs were co-transfected with miR-519b mimic and the HO-1-luciferase expression vector, then pretreated with Gö6976 and GF109203x before incubation with TGF-β1 for 24 hours. Luciferase activity was assayed (n = 4 per group). *: p<0.05 as compared with baseline. #: p<0.05 as compared with the TGF-β1-treated group.</p

PKCα is involved in TGF-β1-induced HO-1 production.

<p>(A&B) OASFs were pretreated with a PKC inhibitor (GF109203x; 10 μM) and a specific PKCα/β inhibitor (Gö6976; 10 μM), or transfected with PKCα siRNA for 24 hours, then incubated with TGF-β1 (10 ng/ml) for 24 hours. HO-1 mRNA levels were examined by qPCR (n = 4 per group). (C) OASFs were transfected with the HO-1-luciferase expression vector then pretreated with Gö6976 and GF109203x before incubation with TGF-β1 for 24 hours. Luciferase activity was assayed (n = 4 per group). (D) OASFs were incubated with TGF-β1 for indicated time intervals, and the extent of PKCα phosphorylation was examined by Western blotting (n = 3 per group). *: p<0.05 as compared with baseline. #: p<0.05 as compared with the TGF-β1-treated group.</p

Baseline characteristics of patients undergoing conventional or computer-assisted navigation TKA.

<p>Baseline characteristics of patients undergoing conventional or computer-assisted navigation TKA.</p

The binding of miRNA-519b to HO-1 3’ UTR mitigates TGF-β1-induced increases in HO-1 expression.

<p>(A) Diagram of the miRNA-519b binding site in the wild-type and mutant HO-1 3′UTRs. In order to evaluate miRNA-519b binding, the 3′UTR mutant was used. The “full mutant” lost all apparent binding of miRNA-519b. Cells were co-transfected with miRNA-519b and the wt-HO-1-3′UTR or mt-HO-1-3′UTR plasmids for 24 hours, and the relative luciferase activity was measured. (B) OASFs were transfected with the wt-HO-1-3′UTR plasmid with or without miRNA-519b mimic, then stimulated with TGF-β1. HO-1 promoter activity was expressed as the relative luciferase activity (n = 3 per group). (C) OASFs were transfected with the mt-HO-1-3′UTR plasmid with or without miRNA-519b mimic, then stimulated with TGF-β1. The relative luciferase activity reflected HO-1 promoter activity (n = 3 per group). (D) OASFs were pretreated with U73122, Gö6976 and GF109203x for 30 min or transfected with PLCγ and PKCα siRNA for 24 hours, then incubated with TGF-β1 (10 ng/ml) for 24 hours. MiR-519b expression was examined by qPCR (n = 4 per group). *: p<0.05 as compared with baseline. #: p<0.05 as compared with the TGF-β1-treated group.</p

TGF-β1 stimulates HO-1 expression in OASFs in a concentration-dependent manner.

<p>(A) Human OASFs were incubated with 0, 1, 3, 10, and 30 ng/ml of TGF-β1 for 24 hours, and HO-1 mRNA expression levels were examined by qPCR (n = 4 per group). (B) OASFs were incubated under various concentrations of TGF-β1 for 24 hours, and HO-1 expression levels were examined by Western blotting (n = 3 per group). (C) OASFs were transfected with the HO-1-luciferase expression vector and incubated with various concentrations of TGF-β1 for 24 hours. The Luciferase intensity reflects HO-1 promoter activity (n = 4 per group). *: p < 0.05 as compared with the control group.</p

Serum IL-10 concentrations (pg/mL) prior to TKA,then at 24 and 72 hours after TKA.

<p>Serum IL-10 concentrations (pg/mL) prior to TKA,then at 24 and 72 hours after TKA.</p

Concentrations of inflammatory markers in Hemovac drainage fluid fromeach TKA cohort.

<p>Concentrations of inflammatory markers in Hemovac drainage fluid fromeach TKA cohort.</p

Serum IL-6 concentrations (pg/mL) prior to TKA, then at 24 and 72 hours after TKA.

<p>Serum IL-6 concentrations (pg/mL) prior to TKA, then at 24 and 72 hours after TKA.</p

Serum TGF-β1 concentrations (pg/mL) prior to TKA,then at 24 and 72 hours after TKA.

<p>Serum TGF-β1 concentrations (pg/mL) prior to TKA,then at 24 and 72 hours after TKA.</p

Serum TNF-α concentrations (pg/mL) prior to TKA,then at 24 and 72 hours after TKA.

<p>Serum TNF-α concentrations (pg/mL) prior to TKA,then at 24 and 72 hours after TKA.</p