The protective effects of propofol against CoCl₂-induced HT22 cell hypoxia injury via PP2A/CAMKIIα/nNOS pathway

HT22 cells were obtained from GuangZhou Jennio Bio- tech and maintained in DMEM (HyClone Laboratories, Logan, Utah, USA) with 5 mM glucose and 10% fetal bovine serum. Cells were incubated in a humidified atmosphere with 5% CO₂ at 37 °C and sub-cultured when reaching 90% confluence. The eighth passage was used in the present study.

Propofol (Sigma, St. Louis, MO, USA), PP2A inhibitor okadaic acid (Sigma, St. Louis, MO, USA), and PP2A activator FTY720 (Sigma, St. Louis, MO, USA) were dissolved in DMSO (Sigma, St. Louis, MO, USA). In order to avoid possible nonspecific effects, the final concentration of DMSO was adjusted to 0.01% for each solution. A 500 mM stock solution of CoCl₂ was prepared by dissolving CoCl₂ powder (Sigma-Aldrich, Dorset, UK) in serum-free DMEM.

HT22 cells were treated with CoCl₂ for 0, 1, 2, 6, 12 and 24 h respectively. By measuring cell viability, we determined the appropriate CoCl₂ treatment condition with significant effect on cell viability inhibition. During general anesthesia, the concentration of propofol in brain ranges from 4 to 20 μg/ml, which is about 20–100 μM [17]. Therefore, HT22 cells were pretreated with propofol for 2 h with different concentrations (5, 10, 25, 50 μM) to observe its protective effects, and the concentration of maximal protective effects was determined. In the following experiments, the optimal treatment time and concentration of CoCl₂ and propofol were used to investigate potential mechanisms.

Cell viability was maesured by cell counting kit-8 (CCK8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacture’s instruction. Briefly, 5 × 10³ cells per well were plated in 96-well plates and incubated in 37 °C. After designed treatments, 10 μl CCK-8 was added in each well and the 96-well plate was incubated in 37 °C for 2 h. Absorbance at a 450 nm wavelength of each well was determined by a microplate reader (Synergy H4, Bio-Tek). Accounting the mean value and standard deviation of optical density for every six wells was used to draw the cell viability curve.

After corresponding treatment, cells were harvested, washed with cold 1 × PBS, and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) for 30 min on ice, then centrifuged at 12,000 g for 15 min at 4 °C. The protein concentration was determined by BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amount (40 μg) of proteins obtained from different samples were separated by 8 or 10% SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The PVDF membranes were incubated with primary antibodies at 4 °C overnight after being blocked with 5% skim milk. The primary antibodies used were monoclonal antibody against β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PP2A (Cell Signaling Technology, Danvers, MA, USA), CAMKIIα (abcam, Cambridge, UK), pCAMKIIα (abcam, Cambridge, UK), nNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), pnNOS-Ser¹⁴¹² (abcam, Cambridge, UK), pnNOS-Ser⁸⁴⁷ (abcam, Cambridge, UK), BAX (Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (proteintech, Shanghai, China), caspase 3 (Cell Signaling Technology, Danvers, MA, USA). Thereafter, the PVDF membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). The protein bands were developed with the chemiluminescent reagents (Millipore, MA, USA). The software of image j was used to analyze the respective densities of the protein bands. In the present study, β-actin was used as loading control and the data were expressed as the ratio of specific protein expression to β-actin expression.