Fig. 1
Morphine induces the release of HSP70 from neurons by activating KATP channel a morphine induced the efflux of HSP70 into extracellular environment in a concentration-dependent manner in SH-SY5Y cells. Supernatants were collected 12 h after morphine (200 μM) and analyzed by western blot (n = 3). b morphine decreased the intracellular protein level of HSP70 in SH-SY5Y cells. Cell extracts were collected 12 h after morphine (200 μM) treatment and analyzed by immunoblot assay (n = 3). c Glibenclamide administration (200 μM, 15 min) prior to morphine (200 μΜ, 12 h) prevented the morphine-induced HSP70 release in SH-SY5Y cells. Supernatants were collected 12 h after morphine treatment and determined by western blot (n = 3). d Glibenclamide (200 μM) inhibited the decrease of intracellular HSP70 caused by morphine in SH-SY5Y cells. Cell extracts were collected 12 h after morphine treatment and analyzed by immunoblot assay (n = 3). e Consecutive administration of glibenclamide (0.4 and 2 μg/10 μL, i.t.) for 7 days inhibited the decrease of HSP70 induced by morphine (10 μg/10 μL, i.t.) in the spinal cord. The spinal samples were collected 1 h after the last morphine treated and determined by western blot (n = 4). f Consecutive administration of glibenclamide (2 μg/10 μL, i.t.) for 7 days inhibited the release of HSP70 induced by morphine (10 μg/10 μL, i.t.) in CSF. CSF was collected from rats 1 h after the last administration and determined by western blot (n = 4). Transferrin is used as a loading control. g, i Glibenclamide administration (200 μM, 15 min) prior to morphine (200 μΜ, 12 h) inhibited the release of HSP70 from SH-SY5Y cells induced by morphine. The green fluorescence indicated the intracellular level of HSP70 in SH-SY5Y cells (n = 4). Scale bar, 75 μm. h, j Kir6.2 siRNA downregulated the level of Kir6.2 on the cell membrane of SH-SY5Y cells. Kir6.2 siRNA or control siRNA was transfected to SH-SY5Y cells. Blue fluorescence labeled Kir6.2, red fluorescence labeled cell membrane, and green fluorescence indicated that FAM-labeled siRNA was successfully transfected into SH-SY5Y cells. The blue curves represent the line profile of Kir6.2, and the red curves represent the line profile of cell membrane. The fluorescence intensity of blue curve overlapped with red curve was utilized to represent the level of Kir 6.2 distributed on cell membrane. The data was obtained from four independent experiments (n = 4). Scale bar 10 μm. k Knockdown of Kir6.2 abolished morphine-induced release of HSP70. SH-SY5Y cells were transfected with Kir6.2 siRNA or control siRNA for 18 h, followed by 200 μM morphine treatment for 12 h. The efficiency of Kir6.2 knockdown was assessed by immunoblot assay (n = 3). a–k Data were analyzed by one-way ANOVA. h Data were analyzed by Student’s t test **P < 0.01, ***P < 0.001 vs. vehicle, ## P < 0.01, ### P < 0.001 vs. the morphine-treated group