Intramuscular electroporation with the pro-opiomelanocortin gene in rat adjuvant arthritis

Abstract

Endogenous opioid peptides have an essential role in the intrinsic modulation and control of inflammatory pain, which could be therapeutically useful. In this study, we established a muscular electroporation method for the gene transfer of pro-opiomelanocortin (POMC) in vivo and investigated its effect on inflammatory pain in a rat model of rheumatoid arthritis. The gene encoding human POMC was inserted into a modified pCMV plasmid, and 0-200 microg of the plasmid-POMC DNA construct was transferred into the tibialis anterior muscle of rats treated with complete Freund's adjuvant (CFA) with or without POMC gene transfer by the electroporation method. The safety and efficiency of the gene transfer was assessed with the following parameters: thermal hyperalgesia, serum adrenocorticotropic hormone (ACTH) and endorphin levels, paw swelling and muscle endorphin levels at 1, 2 and 3 weeks after electroporation. Serum ACTH and endorphin levels of the group into which the gene encoding POMC had been transferred were increased to about 13-14-fold those of the normal control. These levels peaked 1 week after electroporation and significantly decreased 2 weeks after electroporation. Rats that had received the gene encoding POMC had less thermal hypersensitivity and paw swelling than the non-gene-transferred group at days 3, 5 and 7 after injection with CFA. Our promising results showed that transfer of the gene encoding POMC by electroporation is a new and effective method for its expression in vivo, and the analgesic effects of POMC cDNA with electroporation in a rat model of rheumatoid arthritis are reversed by naloxone.

Figure 1

Time course of POMC gene injection and electroporation on the blood levels of ACTH. Results are means ± SEM. Statistical comparisons between groups were made by analysis of variance; individual comparisons were made with the post-hoc test. *P < 0.05.

Figure 2

Time course of POMC gene injection and electroporation on the blood levels of beta-endorphin. Results are means ± SEM. Statistical comparisons between groups were made by analysis of variance; individual comparisons were made with the post-hoc test. *P < 0.05.

Figure 3

Thermal hypersensitivity responses during CFA injection. The POMC and electroporation group (SG VI) suppressed the CFA-induced pain (*P < 0.05), showing a significant difference between rats receiving electroporation and CFA (SG VII), CFA only (SG VIII) and electroporation of pCMV vector alone (SG X), whereas naloxone reversed the analgesic effects of POMC (SG IX). Results are means ± SEM.

Figure 4

Paw swelling during CFA injection in control rats (SG VIII) and rats receiving POMC gene and electroporation (SG VI) and electroporation and paw CFA injection (SG VII). POMC gene injection and electroporation (SG VI) suppressed the swelling. Results are means ± SEM. Statistical comparisons between groups were made by analysis of variance; individual comparisons were made with the post-hoc test. *P < 0.05.

Figure 5

Effect of POMC injection on levels of endorphin in muscle at days 3, 5, 7 and 14 after injection with CFA. Results are means ± SEM. Statistical comparisons between groups were made by analysis of variance; individual comparisons were made with the post-hoc test. *P < 0.05, showing a significant difference between rats receiving electroporation (SG VII) and CFA only (SG VIII).

Figure 6

RNA levels of β-endorphin were measured at weeks 1, 2 and 3 after injection with PBS (SG III) or 200 μg of POMC cDNA (SG I) and electroporation. Results are means ± SEM. Statistical comparisons between groups were made by two-tailed unpaired t-test. *P < 0.05, showing a significant difference between the control groups and the POMC gene and electroporation group.

Figure 7

Confocal micrographs of POMC gene electroporation on endorphin expression in muscle. (A) Immunohistochemical detection of endorphin immunoreactivity in the negative control that omitted the primary antibody. A muscle nucleus is labelled by 4,6-diamidino-2-phenylindole (blue). (B) Overexpression of endorphin immunoreactivity (green) in the POMC-treated group. Scale bar, 40 μm. (C) High-magnification image (from (B)) of endorphin-positive puncta shows the detailed distribution of endorphin in a muscle cell. Scale bar, 20 μm. (D) Endorphin-positive puncta are absent from muscle electroporated with pCMV-Script vector alone. Scale bar, 20 μm. (E, F) No significant inflammatory cell infiltration or muscle damage on haematoxylin/eosin staining of POMC-electroporated muscle (E) and untreated muscle (F). Original magnification, ×200. Scale bar, 100 μm.