Figure 6.

QHRD106 decreased microglial HMGB1 translocation and secretion by decreasing the binding between HMGB1 and HSP90AA1

(A–C) Primary microglial cells were treated with LPS for 4 h, the whole lysates were mixed with anti-HMGB1-coated Protein A/G PLUS-agarose beads, and the HMGB1-binding protein was subjected to SDS/PAGE. (A) The gel of the complex was stained by silver staining and analyzed by LC-MS/MS. (B) HSP90AA1 was identified in LPS-stimulated primary microglia. (C) The PPI network among the HMGB1-binding proteins in the LPS group was predicted with STRING tools.

(D) Primary microglial cells were treated with LPS for 4 h, and the interaction between HMGB1 and HSP90AA1 was observed using PLA.

(E and F) To observe the interaction between HMGB1 and HSP90AA1, whole-cell lysates in the control, saline, and QHRD106 groups were immunoprecipitated and immunoblotted.

(G–K) QHRD106 at 1 μM or GA at 0.2 μM was added.(G–I) The nucleocytoplasmic translocation of HMGB1 in primary microglia 4 h after LPS stimulation was evaluated by western blot analysis. GAPDH and Lamin B1 were used as cytoplasmic and nuclear loading controls, respectively. (J) Representative western blot images of medium HMGB1 and cell lysis GAPDH in primary microglia 24 h after LPS stimulation. (K) Quantification of medium HMGB1 expression.

All data are presented as the mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Tukey’s post hoc test. ∗p < 0.05, ∗∗p < 0.01 vs. the saline group; “ns” indicates no significance (p > 0.05).