Activation of endogenous opioid gene expression in human keratinocytes and fibroblasts by pulsed radiofrequency energy fields

Figures & data

Table 1 Primer Sequences used for qRT-PCR.

Oligo Name	Primer sequence (5′ to 3′)
GADPH	GAGTCAACGGTTTGGTCGT TTGATTTTGGAGGATCTCG
PENK	AGCTGTCCCAACCCAGAGCTT CCTCCTCTGCATCCTTCTTC
POMC	CCCCTACAGGATGGAGCACT CGTTCTTGATGATGGCGTTT
PDYN	AAGCTCTCTGGTCATTCCT TCCCTCATCAGCTCAGACTC
ET-1	TCCCGTTCAGAAGACAGCTT CAGAGGCAAAGACAAGGAC
ETA RECEPTOR	TCGAGAAAGTGGCAAAAACAG TGAATTCATGGTTGCCAAGT
ETB RECEPTOR	TCCCGTTCAGAAGACAGCTT CAGAGGCAAAGACAAGGAC
COX-1	TTCGGTGTCCAGTTCCAATA ACCCCATAGTCCACCAACAT
COX-2	TCTGAAACCCACTCCAAACA AAGGCTTCCCAGCTTTTGTA
PTGES	GTGGCTATACCTGGACTT CTTGAAATGGTTCCCATCAG
PTGDS	GGCAGGTATGTCACCGTTCT TTCATCCTGGCCTTTTCATC
IL-10	AAGCCTGACCACGCTTTCTA ATGAAGTGGTTGGAATGA
IL-1β	TTCGACACATGGATAACGA TCTTTCAACACGCAGGACAG
IL-6	ATGCAATAACCACCCCTGAC GAGGTGCCCATGCTACATTT
NGF	ATACAGGCGCGGAACCACACTC TGCTCCTGTGAGTCCTGTTG
TNF-α	TCCTTCAGACCACCCTCAACC AGGCCCCAGTTTGAATTCTT

Figure 1 Effect of PRFE treatment on COX mRNA and prostaglandin D and E synthase expression in HEK and HDF in culture. (A and B) We evaluated the effect of PRFE treatment on levels of mRNA expression after 30 minutes of treatment with PRFE using standard conditions. Cells were harvested 4 hours following initiation of PRFE treatment and the RT-PCR products were analyzed by electrophoresis through 2% agarose gel. (C) Quantitative RT-PCR was used to determine the effect of PRFE on message levels for the various genes involved in prostaglandin synthesis (**P < 0.05 for control versus PRFE treatment) in HDF.

Abbreviations: COX, cyclooxygenase; GADPH, glyceraldehyde 3-phosphate dehydrogenase; HDF, human dermal fibroblasts; HEK, human epidermal keratinocytes; RT-PCR, reverse transcription polymerase chain reaction; PRFE, pulsed radiofrequency energy; PTGDS, prostaglandin D synthase; PTGES, prostaglandin E synthase.

Figure 2 Effect of PRFE fields on COX enzyme activity in HEK and HDF. Total COX enzyme activity was determined after PRFE field treatment. (A) COX was evaluated after PRFE treatment at the indicated times. Data shown are from at least two independent experiments performed in triplicate. (B) Effect at 4 hours following PRFE treatment (n = 6, ***P < 0.01 for HDF).

Note: No statistically significant differences in COX enzyme activity were found between HEK and untreated controls.
Abbreviations: CTL, control; COX, cyclooxygenase; HDF, human dermal fibroblasts; HEK, human epidermal keratinocytes; PRFE, pulsed radiofrequency energy.

Figure 3 PRFE increases endogenous opioid expression in HEK and HDF at the mRNA and peptide levels. (A) Endogenous opioid expression was determined in HEK and HDF. Cells were treated with PRFE and total RNA was isolated after 2 hours (n = 12, P < 0.01 for all opioids, control versus PRFE-treated). (B and C) Determination of PDYN and PENK opioid levels at 2 hours following PRFE treatment using enzyme-linked immunosorbent assay (error bars show standard error of the mean, two separate experiments in triplicate, P < 0.05).

Abbreviations: CTL, control; HDF, human dermal fibroblasts; HEK, human epidermal keratinocytes; PRFE, pulsed radiofrequency energy; POMC, proopiomelanocortin; PENK, proenkephalin; PDYN, prodynorphin.

Figure 4 Regulation of cytokines involved in the peripheral pain response.

Notes: Cytokine expression was determined using quantitative RT-PCR. HEK or HDF were treated with PRFE using standard conditions. Total RNA was isolated 2 hours following treatment. Levels are expressed as a fold increase or a fold decrease (n = 6, error bars show standard error of the mean, differences for control versus PRFE-treated are P < 0.05).
Abbreviations: HDF, human dermal fibroblasts; HEK, human epidermal keratinocytes; IL, interleukin; NGF-α, nerve growth factor alpha; TNF-α, tumor necrosis factor-alpha; RT-PCR, reverse transcription polymerase chain reaction.

Figure 5 Increased expression of endogenous opioids is regulated by an ET-1/ET_B receptor pathway after treatment with PRFE. (A) mRNA expression levels after treatment with PRFE using standard conditions. Cells were harvested after PRFE treatment at the indicated times and RT-PCR products were analyzed by electrophoresis through 2% agarose gel. (B) Quantitative RT-PCR was used to determine the effect of PRFE on message levels of the endogenous opioids, ET-1 and ET_B.

Notes: Differences between controls and PRFE-treated cells were significant (P < 0.05) for all opioid mRNAs in both HEK and HDF. Increases in ET_B mRNA were significant (P < 0.05) in HEK only.
Abbreviations: ET, endothelin; GADPH, glyceraldehyde 3-phosphate dehydrogenase; HDF, human dermal fibroblasts; HEK, human epidermal keratinocytes; PRFE, pulsed radiofrequency energy; POMC, pro-opiomelanocortin; PENK, proenkephalin; PDYN, prodynorphin; RT-PCR, reverse transcription polymerase chain reaction.

Figure 6 Antagonists of ET_A and ET_B receptors inhibit opioid expression after PRFE treatment of HEK and HDF.

Notes: HEK and HDF were pretreated with ET_A and ET_B receptor antagonists (BQ788 and BQ123, respectively) for 1 hour at 100 nM prior to PRFE treatment. Quantitative RT-PCR was performed for the endogenous opioids on RNA isolated 2 hours after PRFE treatment. Data are expressed as percent of untreated antagonist control (n = 6, P < 0.01).
Abbreviations: ET, endothelin; HDF, human dermal fibroblasts; HEK, human epidermal keratinocytes; PRFE, pulsed radiofrequency energy; POMC, proopiomelanocortin; PENK, proenkephalin; PDYN, prodynorphin; RT-PCR, reverse transcription polymerase chain reaction.