ES apparatus and µsPES parameters
A pelvic floor rehabilitation instrument (PHENIX USB4, Electron-IC Concept Lignon Innovation Co., France) (Fig. 1A) was employed for research in vitro. This instrument with dozens of treatment schemes is a kind of TENS device using µsPES, which can treat PFDs (e.g., urinary retention and pelvic organ prolapse (POP)). Three schemes were chosen from this pelvic floor rehabilitation instrument. Scheme #1 with a frequency of 1/4/1 Hz, a pulse width of 270/230/270 µs, and 30 min duration could treat urinary retention by stimulating the pelvic floor muscles. Scheme #2 with a frequency of 30 Hz, a pulse width of 500µs, and 20 min duration could treat POP by stimulating type I muscle fibers. Scheme #3 with a frequency of 50 Hz, a pulse width of 250 µs, and 20 min duration could treat POP by stimulating type II muscle fibers. An oscilloscope (DSO7054A; Agilent Technologies Inc., Santa Clara, CA, USA) (Fig. 1C) was used to detect the waveform of the three schemes (Fig. 2).
Cell Lines and Cell Culture
Cervical squamous cell carcinoma SiHa cells and cervical adenocarcinoma HeLa cells were supplied by the Gynecology Laboratory of Peking University People’s Hospital (Beijing, China). Two groups of cells were both grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, UT, USA) and 1% antibiotics (100 U/ml penicillin/streptomycin; Gibco, New York, NY, USA). Cells were cultured at 37 °C with 5% CO2 and passaged after 3 days. SiHa cells and HeLa cells were harvested and resuspended in saline solution with a concentration of 106 cells/ml. Besides, 1000 µl cell suspension was placed in 2-mm gap cuvettes (Bio-Rad Laboratories Inc., Hercules, CA, USA) (Fig. 1B) and exposed to µsPES. Three µsPES-based schemes at current values of 20, 40, 60, 80, and 100 mA were applied to stimulate cervical squamous cell carcinoma SiHa cells and cervical adenocarcinoma HeLa cells. The temperature of the suspension was measured during the experiment.
Cell proliferation assay
A total of 2 × 103 cells/well were seeded into 96-well plates (Corning Inc., Corning, NY, USA) after treatment with three µsPES-based schemes at current values of 20, 40, 60, 80, and 100 mA each. 10 µl of the cell counting kit-8 (CCK-8) solution (Dojindo, Kumamoto, Japan) was added to each well and incubated at 37 °C for 4, 24, 48, and 72 h and then every 24 h if necessary. The absorbance was measured at 450 nm on a spectrophotometric plate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA) at each time-point. All experiments were performed in triplicate.
Western blot assay
SiHa cells and HeLa cells (treated and untreated cells) were harvested and lysed using cell lysis reagent containing NP40 and protease inhibitor cocktail for protein isolation after the treatment with µsPES scheme #2 at 100 mA current. Protein concentration was detected using the Bradford assay. Next, 30 ug of total protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA). After blocking with 5% non-fat dry milk, the membranes were incubated with the following primary antibodies: mouse anti-β-actin (Proteintech, Rosemont, IL, USA), rabbit anti-MAPK (Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-Phospho- MAPK (Cell Signaling Technology), rabbit anti-MEK1/2 (Cell Signaling Technology, Inc.), rabbit anti-Phospho-MEK1/2 (ImmunoWay Biotechnology Co., Plano, TX, USA), rabbit anti-Raf (Abcam, Cambridge, UK), Phospho-Raf (Cell Signaling Technology, Inc.), The blots were washed thrice, and antigen-antibody complexes were detected by a fluorescence method using fluorescent secondary anti-mouse (Cell Signaling Technology, Inc.) IgG and anti-rabbit IgG (Cell Signaling Technology, Inc.). Fluorescent signals were detected by a bicolor infrared laser imaging system (LI-COR Biosciences, Lincoln, NB, USA) and quantified using the ImageJ software.
Statistical analysis
All trials were repeated 3 times. Data were analyzed using a one-way analysis of variance (ANOVA) for making comparisons among multiple groups and a t-test for making comparisons between the two groups. Statistical analyses were performed using SPSS 26.0 software (IBM, Armonk, NY, USA). The results were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.