Fig. 4

The therapeutic effect of EX-Netrin1 was diminished by a PI3K inhibitor but reversed by a mTOR agonist. (A) ELISA was performed to detect the contents of TNF-α , IL-1β and IL-6 in the cell supernatant. (B-C) Fluorescence microscope was used to take representative images of neuronal cells and measure axonal length. Actin was stained red and nuclei were stained blue with DAPI. Scale bar: 20 μm. (D) Scanning electron microscope was used to observe pyroptotic morphology of the cells. Red arrows represent bubble like cell protrusions. Scale bar: 10 μm. (E) The levels of LDH released from cells were measured by ELISA. (F-G) The Unc5b/PI3K/Akt/mTOR pathway-related proteins were detected by western blot. (H) Confocal microscope was used to observe the binding of netrin-1 to Unc5b. Netrin-1 was stained red, Unc5b was stained green, and yellow fluorescence was observed when the two were combined. The picture above shows the control group. The picture below shows that cells are only treated by EX-Netrin1. Scale bar: 2 μm. (EX-MSCs group: LPS+EX-MSCs group; EX-NC group: LPS+EX-NC group; EX-Netrin1 group: LPS+EX-Netrin1 group; LY294002 group: LPS+EX-Netrin1+LY294002 group; MHY1485 group: LPS+EX-Netrin1+LY294002+MHY1485 group. Error bars showed means ± SD; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, vs. EX-MSCs group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, vs. EX-NC group; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, vs. EX-Netrin1 group; ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001, vs. LY294002 group)