Figure 3
Effect of 1,25(OH) 2 D3 pretreatment on MRSA-induced cytokines expressions of hMSCs. (A) Human MSCs were preincubated with vitamin D (100 nM) for 24, 48 and 72 hours, followed by methicillin-resistant Staphylococcus aureus (MRSA)-stimulation for another 24 hours. Total RNA from hMSCs was isolated and processed for cDNA synthesis. Real-time quantitative PCR was performed with human specific primers of IL-8 (A i), TNFα (A ii), IL-6 (A iii), NR4A2 (A iv), Cathelicidin (A v), and VDR (A vi), using SYBR green assay. Endogenous control human GAPDH was used as a reference. The relative mRNA expression was determined by the delta-delta comparative threshold (ddCT) method. Data are from n = 3 independent experiments done in triplicate. †P < 0.05 compared to control. # P < 0.05 compared to MRSA. ††P < 0.05 compared to MRSA. ‡ P < 0.05 compared to control. ##P < 0.05 compared to control. Effect of 1,25(OH)2D3 preincubation on MRSA-stimulated IL-8, and TNFα secretion (B). Human MSCs were preincubated with 1,25(OH)2D3 (100 nM) for 48 hrs, followed by MRSA- stimulation for another 24 hrs. IL-8 (B i), and TNFα (B ii) protein secretion were determined by ELISA in supernatants. Data are from n = 3 independent experiments done in triplicate. * P < 0.0001 for MRSA vs. Cntl stimulation, ** P < 0.0003 for 1,25(OH)2D3 vs. MRSA stimulation.