Shenfu Injection protects PC12 cells from oxygen-glucose deprivation by the NOS/NO pathway

Abstract

Objective:To study whether the protective effect of Shenfu injection on PC12 cells damaged by Oxygen-glucose deprivation (OGD) involves NOS/NO pathway. Methods: PC12 cells co-incubated with a final concentration of 20.00mmol/L Na2S2O4 for 24 hours was used as an OGD induced damaged in vitro model of vascular dementia (VaD) . 50.00 μL/mL, 75.00 μL/mL, 100.00 μL/mL, 125.00 μL/mL, 150.00 μL/mL, 175.00μL/mL and 200.00μL/mL of SFI were co-incubated with 20.00 mmol•L-1 Na2S2O4-induced PC12 cells for 24 hour, 48 hour and 72 hour, respectively. Cell viability was determined by MTT assay to determine the appropriate concentration and timing of SFI. Griess method was used to detect NO content in the cell culture supernatant of the control group, OGD model group, 125.00 μL/mL, 150.00 μL/mL,and 175.00 μL/mL SFI group. QPCR was used to detect the expressions of iNOS and nNOS mRNA , and western blot was used to detect the protein expressions of iNOS and nNOS in these groups. Result: Compared with the OGD group, 50.00 μL/mL, 75.00 μL/mL, 100.00 μL/mL, 125.00 μL/mL, 150.00 μL/mL, 175.00 μL/mL and 200.00 μL/mL SFI can attenuate the inhibitory effect of Na2S2O4 on PC12 cells in a time and dose-dependent manner (P<0.05). Among them, the survival rate of PC12 cells in the 150.00μL/mL dose group was the highest, reaching 79.750 ±0.230. For the subsequent experiments, 125.00μL/mL, 150.00μL/mL, and 175μL/mL doses of SFI were elected to co-incubated with Na2S2O4-induced damaged PC12 cells for 24 hours. Compared with the control group, NO level in the supernatant of PC12 cells induced by Na2S2O4 were significantly increased. SFI decreased NO level in culture supernatant of PC12 cell induced by Na2S2O4 in a dose-dependent manner. Compared with the control group, the mRNA and protein expressions of iNOS and nNOS in PC12 cells was significantly increased after 24 hour induction by Na2S2O4 (P<0.05). Compared with the OGD group, the mRNA and protein expressions of iNOS and nNOS in the 125μL/mL, 150μL/mL and 175μL/mL SFI dose groups were significantly lower (P<0.05). Conclusion: These findings suggest that the protective effect of SFI on PC12 cells damaged by Na2S2O4 may be related to its down-regulation of iNOS and nNOS gene and protein expression, thereby reducing the excessive production of NO in PC12 cells.