[Effects of lentivirus-mediated heat shock protein 70 gene on endoplasmic reticulum calcium homeostasis and calcium channels of PC12 cells induced by ischemia and hypoxia].

OBJECTIVE

To investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on calcium homeostasis and calcium channels of PC12 cells induced by ischemic and hypoxia and its mechanisms.

METHODS

PC12 cells at logarithmic phase were collected, and they were divided into recombined lentiviral infection group [infected by lentivirus containing HSP70 and green fluorescent protein (GFP) fluorescin gene], lentivirus control group (infected by lentivirus containing GFP without HSP70 gene) and non-infection group. PC12 cells were subjected ischemia/hypoxia for 4, 8, 12, 24 hours, and the cell activity was determined by methylthiazolyl tetrazolium (MTT) assay test in order to determine the best time for ischemia/hypoxia. The mRNA expressions of HSP70, muscle/endoplasmic reticulum Ca2+-ATP isoforms (SERCA2a, SERCA2b), ryanodine receptor 2 (RyR2), and inositol 1,4,5-triphosphate receptor 1 (IP3R1) were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the protein expressions of HSP70, SERCA, and IP3R were determined by Western Blot at 8 hours after ischemic/hypoxia. Flow cytometry was used to determine the levels of intracellular reactive oxygen (ROS) and intracellular Ca2+ ([Ca2+]i).

RESULTS

With the prolongation of time of ischemia/hypoxia, the cell viability in all groups showed an increase followed by a weakening, and peaked at 8 hours. The cell viability at 8 hours in lentiviral infection group was significantly higher than that of the non-infection group and lentivirus control group [A value (×10-2): 20.3±2.2 vs. 14.1±2.1, 15.0±1.6, both P < 0.01], the mRNA and protein expressions of HSP70 and SERCA in lentiviral infection group were significantly increased [HSP70 mRNA (2-ΔΔCt ): 0.785±0.018 vs. 0.428±0.019, 0.423±0.023; HSP70 protein (gray value): 2.72±0.20 vs. 1.56±0.36, 1.63±0.41; SERCA2a mRNA (2-ΔΔCt ): 0.971±0.037 vs. 0.367±0.014, 0.347±0.012; SERCA2b mRNA (2-ΔΔCt ): 8.869±0.162 vs. 3.015±0.091, 2.941±0.091; SERCA protein (gray value): 2.84±0.18 vs. 1.48±0.26, 1.52±0.29], and IP3R2 mRNA and protein expressions were significantly declined [IP3R2 mRNA (2-ΔΔCt ): 0.183±0.020 vs. 0.439±0.020, 0.433±0.040; IP3R2 protein (gray value): 1.15±0.12 vs. 1.91±0.20, 1.83±0.19], with statistically significant differences (all P < 0.01); no significant difference in RyR mRNA was found [2-ΔΔCt (×10-3): 1.97±0.63 vs. 2.02±0.22, 2.01±0.09, both P > 0.05]; the relative fluorescence intensity of ROS and [Ca2+]i in lentiviral infection group was significantly reduced (ROS: 30.54±1.23 vs. 58.03±1.97, 57.72±2.35; [Ca2+]i: 34.50±2.05 vs. 48.20±3.02, 46.80±2.75, all P < 0.01].

CONCLUSION

Exogenous HSP70 can maintain calcium homeostasis in the endoplasmic reticulum of PC12 cells, affect the Ca2+ channel protein regulated by calcium channel IP3R and calcium pump SERCA, which may cause hypoxia/ischemia intracellular injury.