Fig. 2

Extracellular HSP70 triggers inflammatory response dependent on TLR4 in microglia. a, b Recombinant mouse HSP70 (100 ng/mL, 12 h) upregulated the phosphorylation of p38 MAPK and NF-κB p65 in BV-2 cells. Cell extracts were collected and analyzed by western blot (n = 3). c, d The levels of Il1b and Tnfa mRNAs in response to HSP70 under treatment of TLR4 antagonist or p38 inhibitor were assessed in BV-2 cells. Cells were pretreated with TLR4 antagonist (TAK242, 10 μM) or p38 inhibitor (SB202190, 10 μM) for 15 min, followed by recombinant mouse HSP70 (100 ng/mL) treatment. Then, cell extracts were collected 12 h after HSP70 treatment and analyzed by qPCR (n = 3). e Recombinant mouse HSP70 (100 ng/mL, 12 h) increased the levels of pro-IL-1β and NLRP3 in BV-2 cells. Cell extracts were collected and analyzed by western blot (n = 3). f BV-2 cells were stimulated by recombinant mouse HSP70 (100 ng/mL) for 12 h, and then, the inflammasome was activated with 5 mM of ATP for 0.5 h, inducing the maturation of caspase-1 and IL-1β. Supernatants of BV-2 cells were collected and analyzed by western blot (n = 3). g, h Conditional medium collected from morphine-treated (200 μM, 12 h) SH-SY5Y cells incubated BV-2 cells for 12 h in presence of anti-HSP70 antibody (100 ng/mL) or normal IgM (100 ng/mL), and then, the cell extracts were collected and analyzed by qPCR (n = 3). a, b, e, and f Data were analyzed by Student’s t test. c, d, g, and h Data were analyzed by one-way ANOVA.*P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle, ^## P < 0.01, ^### P < 0.001 vs. the HSP70-treated group