The Anti-Inflammatory Activity of HMGB1 A Box Is Enhanced When Fused with C-Terminal Acidic Tail

Figure 3

The C tail-fused A box could suppress TNF-𝛼 and IL-6 release in vitro and in vivo more efficiently than A box alone. (a) and (b) The C tail-fused A box could inhibit HMGB1-induced TNF-𝛼 and IL-6 release in vitro more efficiently than A box alone. THP-1 cells in 96-well plates were treated with rHMGB1 (0.05 μmol/L) and various concentrations of A box, A box+C tail, A box+(Gly₄Ser)₃+C tail, or DHFR control proteins (as indicated) for 6 hours in the presence of polymyxin B (6 units per pg of LPS). The levels of TNF-𝛼 and IL-6 in the cultured supernatants were measured by ELISA. Each experiment was performed in triplicate and repeated three times. (c) and (d) The C tail-fused A box could reduce serum TNF-𝛼 and IL-6 levels in the mice subjected to endotoxemia more efficiently than A box alone. BALB/c mice (5 mice/group), subjected to LD₇₅ doses of LPS (15 mg/kg), were treated at 0 and 12 hours after LPS injection with A box, A box+C tail, A box+(Gly₄Ser)₃+C tail or DHFR control proteins (40 nmol per mouse). The sera of the mice were obtained from orbital blood 6, 12, and 18 hours after LPS injection respectively. The serum levels of TNF-𝛼 and IL-6 were determined by ELISA. Each experiment was repeated three times. The results showed that the A box alone and the two C tail-fused A box proteins could significantly inhibit TNF-𝛼 and IL-6 release in vitro and in vivo. The A box+(Gly₄Ser)₃+C tail fusion protein had the most efficient inhibitory activity, followed by the A box+C tail fusion protein and the A box. Data shown are the means ± standard deviation and were analyzed with one-way ANOVA. a: 𝑃<.05 versus DHFR; b: 𝑃<.05 versus A box; c: 𝑃<.05 versus A box+C tail.

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