Effects of 1,25-Dihydroxy vitamin D-3 on TNF-alpha induced inflammation in human chondrocytes and SW1353 cells: a possible role for toll-like receptors

The purpose of this study is to evaluate anti-inflammatory and chondro-protective effects of 1,25(OH)(2)D-3 in human chondrocytes and SW1353 cells via investigating expressions of MMPs, TIMPs, VDR, and intracellular signalling pathway mediators such as TLR-2 and -4. The HC and SW1353 cells were treated with 1,25(OH)(2)D-3 at 10, 100, and 1000 nM concentrations in the absence/presence of TNF-alpha (20 ng/mL) for 48 h. The mRNA expressions of MMP-1, -2, -3, -9, and -13, TIMP-1 and -2, VDR, TLR-2 and -4 in HC and SW1353 cells were detected by qPCR after treatments. The cytotoxicity and cell proliferation analyses were assessed by LDH and WST-1 assay, respectively. Protein levels of MMPs, TIMPs, and VDR were analysed by immunocytochemistry and ELISA methods. TNF-alpha markedly increased cytotoxicity for 24, 48, 72 h (p < 0.05) and vitamin D treatment was shown to diminish the cytotoxic effect of TNF-alpha. Cell proliferations increased by Vitamin D in a dose-dependent manner. mRNA expressions of MMP-1, -2, -3, -9, and -13, TLR-2 and -4 genes decreased with 1,25(OH)(2)D-3 treatment (p < 0.05). VDR, TIMP-1 and -2 levels elevated after TNF-alpha exposure compared with the control group in HC cells (p < 0.05). Protein expression levels were determined using Western blotting, ELISA and immunocytochemistry. 1,25(OH)(2)D-3 via binding to VDR, reversed the effects of TNF-alpha by inhibiting TLR-2 and 4. Decreased levels of VDR, TIMP-1 and -2 after TNF-alpha treatment were elevated by 1,25(OH)(2)D-3 proportional with increasing 1,25(OH)(2)D-3 doses. 1,25(OH)(2)D-3 and TNF-alpha co-treatment decreased MMP-1, -2, -3, -9, and -13 levels were after TNF-alpha exposure.