Research Paper Volume 13, Issue 5 pp 6712—6723

CPI-1189 protects neuronal cells from oxygen glucose deprivation/re-oxygenation-induced oxidative injury and cell death

CPI-1189 inhibits OGDR-induced oxidative injury in neuronal cells. Stable SH-SY5Y cells with CRISPR/Cas9-p38α-KO-GFP (ko-p38α cells) were pretreated with or without CPI-1189 (100 nM, 1h pretreatment), control cells were transduced with the empty vector (“Cas9-C”), cells were subjected to OGDR procedure and cultured for applied time periods; Expression of listed proteins was shown (A); Cell viability and death were tested by CCK-8 (B) and Trypan blue staining (C) assays, respectively. SH-SY5Y cells (D–H) or primary murine cortical neurons (K–N) were pretreated for 1h with CPI-1189 (100 nM), followed by OGDR or hydrogen peroxide (H2O2, 300 μM) stimulation, cells were then cultured for applied time periods, cellular ROS contents (CellROX dye intensity, D, K), lipid peroxidation (by recording TBAR activity, E), and GSH/GSSG ratio (F–L) were tested. For cells with H2O2 stimulation, cell viability and death were tested by CCK-8 (G–M) and Trypan blue staining (H–N) assays, respectively. The primary murine cortical neurons were pretreated for 1h with SB203580 (5 μM) or plus CPI-1189 (100 nM), followed by OGDR stimulation and cells were then cultured for 48h; Cell viability and death were tested by CCK-8 (I) and Trypan blue staining (J) assays, respectively. * P vs. “Mock” cells. #P vs. cells with OGDR stimulation/H2O2 treatment but “DMSO (0.1%)” pretreatment. ** P B, C, I, J). Quantified values were mean ± standard deviation (SD, n=5). Experiments were repeated three times, with similar results obtained. Scale bar= 100 μm (D).

Figure 2. CPI-1189 inhibits OGDR-induced oxidative injury in neuronal cells. Stable SH-SY5Y cells with CRISPR/Cas9-p38α-KO-GFP (ko-p38α cells) were pretreated with or without CPI-1189 (100 nM, 1h pretreatment), control cells were transduced with the empty vector (“Cas9-C”), cells were subjected to OGDR procedure and cultured for applied time periods; Expression of listed proteins was shown (A); Cell viability and death were tested by CCK-8 (B) and Trypan blue staining (C) assays, respectively. SH-SY5Y cells (DH) or primary murine cortical neurons (KN) were pretreated for 1h with CPI-1189 (100 nM), followed by OGDR or hydrogen peroxide (H2O2, 300 μM) stimulation, cells were then cultured for applied time periods, cellular ROS contents (CellROX dye intensity, D, K), lipid peroxidation (by recording TBAR activity, E), and GSH/GSSG ratio (FL) were tested. For cells with H2O2 stimulation, cell viability and death were tested by CCK-8 (GM) and Trypan blue staining (HN) assays, respectively. The primary murine cortical neurons were pretreated for 1h with SB203580 (5 μM) or plus CPI-1189 (100 nM), followed by OGDR stimulation and cells were then cultured for 48h; Cell viability and death were tested by CCK-8 (I) and Trypan blue staining (J) assays, respectively. * P < 0.05 vs. “Mock” cells. #P < 0.05 vs. cells with OGDR stimulation/H2O2 treatment but “DMSO (0.1%)” pretreatment. ** P < 0.05 (B, C, I, J). Quantified values were mean ± standard deviation (SD, n=5). Experiments were repeated three times, with similar results obtained. Scale bar= 100 μm (D).