Effects of Pulsed Radiofrequency Current and Thermal Condition on the Expression of β-Endorphin in Human Monocytic Cells

Abstract

Pulsed radiofrequency (PRF) current applied to peripheral nerves is a modality used in interventional pain medicine, but its underlying mechanisms remain unclear. This study aimed to investigate whether ex vivo exposure of human monocytic THP-1 cells to PRF current or to heat induces β-endorphin production.

Methods: THP-1 cells were exposed to PRF current for 15 min or incubated at elevated temperatures (42 °C to 50 °C) for 3 or 15 min. Flow cytometry was used to assess cell viability, and β-endorphin concentrations in culture supernatants were quantified by ELISA. In a separate experiment, cells were stimulated with lipopolysaccharide (LPS) to compare its effects on β-endorphin release.

Results: A 3 min exposure to temperatures ≥ 46 °C reduced THP-1 cell viability, whereas a 15 min exposure to PRF current or to heat at 42 °C did not impair viability. Both PRF current and mild heat significantly enhanced β-endorphin release. β-Endorphin levels in the supernatant of LPS-stimulated cells were comparable to those of cells exposed to PRF current.

Conclusions: Ex vivo application of PRF current or mild heat enhanced β-endorphin production from THP-1 cells without significant cytotoxicity. These preliminary findings warrant further investigation using primary human monocytes and in vivo models to assess therapeutic potential.

Keywords: THP-1 cells; apoptosis; beta-endorphin; monocytes; neuropathic pain; proopiomelanocortin (POMC); pulsed radiofrequency current; thermal effect.

Figure 1

Flow cytometry analysis of suspended THP-1 cells during subculture without centrifugation at 200× g. (a) Side scatter (SSC) plotted against forward light scatter (FSC) of the cell suspension. Normal THP-1 cells are gated within the circle and colored blue. (b) Fluorescence intensity of tetramethylrhodamine methyl ester (TMRM) plotted against FSC of the same cell suspension as shown in (a). CountBright counting beads are gated in a square and colored green. Particles with low TMRM fluorescence intensity are gated in a square and shown in purple. (c) FSC/SSC plot of the THP-1 cell suspension with a square gate for “total THP-1”. (d) Fluorescence intensity of TMRM plotted against fluorescence intensity of Annexin V-633 from the same cell suspension as shown in (c), where only particles gated in the “total THP-1” was indicated. Particles positively stained with Annexin V-633 are colored red. (a.u., arbitrary unit).

Figure 2

Suspension of THP-1 cells incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air for 24 h after application of 3 min heat or 15 min pulsed radiofrequency current was subjected to flow cytometry. (37 °C) Suspension of THP-1 cells was incubated at 37 °C for 3 min, and the culture continued at 37 °C for 24 h in a humidified atmosphere of 5% CO₂ in air. (42 °C, 44 °C, 46 °C, 48 °C, and 50 °C) Suspension of THP-1 cells was incubated for 3 min at the indicated temperatures, and the culture continued under the same conditions as in (37 °C). (PRF) The pulsed radiofrequency electric current (repeated 20 ms electric current at 480 kHz at 2 Hz) was applied to the sedimented cells for 15 min using a NeuroTherm NT500 RF generator (Abbott Laboratories, Chicago, IL, USA). After this application, the culture continued under the same conditions as in (37 °C). (Control) The electric probe for the PRF application was inserted into the cell suspension for 15 min without generating current. After this application, the culture continued under the same conditions as in (PRF). (a.u., arbitrary unit).

Figure 3

(PRF) Pulsed radiofrequency (PRF) current was applied for 15 min with a radiofrequency probe with a guiding needle, inserted into a 15 mL polypropylene conical tube containing sedimented THP-1 cells. During PRF application, the tube was placed in a dry thermal bath at 37 °C under ambient air conditions. (37 °C or 42 °C) Tubes were prepared in the same manner, with RF probe insertion, and incubated at either 37 °C or 42 °C without RF current application. Following treatment, cells were resuspended and incubated for 48 h at 37 °C. The concentration of β-endorphin in the supernatant of THP-1 cells was 7.1 ± 0.4 pg/mL in the 37 °C group, 10.1 ± 0.7 pg/mL in the 42 °C group, and 10.1 ± 1.1 pg/mL in the PRF group (mean ± SD, n = 6). Six individual data distribution and the mean of each group are presented in the graph. * Significantly different from the 37 °C group (p < 0.0001).

Figure 4

THP-1 cells were incubated in the absence or presence of 100 ng/mL lipopolysaccharide (LPS). Culture supernatants were collected on 1, 2, 4, and 6 days after incubation without LPS, and 48 h after medium replacement on day 4 for the LPS-stimulated cells. Note that the concentration of β-endorphin collected on day 1 was under the detection limit of the ELISA system. The β-endorphin concentration in the supernatant collected on day 2 was 4.7 ± 0.9 pg/mL, on day 4 was 9.1 ± 0.4 pg/mL, and on day 6 was 8.7 ± 1.1 pg/mL. The β-endorphin concentration in the supernatant collected at 48 h after the replacement of culture medium of THP-1 cells stimulated with LPS for 4 days was 14.2 ± 1.0 pg/mL (mean ± SD, n = 6). Six individual data distribution and the mean of each group are presented in the graph. * Significantly different from Day 2 (p < 0.0001). ^† Significantly different from LPS (p < 0.0001).