HMGB1 Modulates TLR4, IL-10, and VEGF in JBMSCs

Human JBMSCs were seeded at a density of 1×10⁵ cells/cm² in 24-well plates and incubated in DMEM with 10% FBS. Three groups were included in this study. In the control group, the cells were cultured in conventional medium for 48 h. In the HMGB1 group, the cells were incubated in the medium containing HMGB1 (Biovision, CA, USA,) for 48 h.^{20 (link)} In the TAK-242 group, the cells were pretreated with 1 μM TAK-242 (Invitrogen, San Diego, CA, USA), a specific inhibitor of TLR4, for 2 h before HMGB1 (200 ng/mL) stimulation for 48 h. The mRNA of TLR4, IL-10, and VEGF were measured. Following manufacturer's protocol, total RNA was extracted from cells of each group using Trizol (Invitrogen, Carlsbad, CA, USA). Then, the first complementary DNA strand (cDNA) was synthesized by using SYBR(R) Prime Script™ RT reagent Kit (Takara, Osaka, Japan). Quantitative real-time PCR using SYBR Green I dye (Takara, Osaka, Japan) was used to analyze the levels of target mRNA of TLR4, IL-10, and VEGF. Figure 1 presents the primer pairs used in the experiment.