OBJECTIVE: To investigate the changes in the expression of voltage-gated potassium channel subunit KCNA2 in the dorsal root ganglion (DRG) neurons of rats with osteoarthritis (OA) pain induced by sodium monoiodoacetate and explore the mechanism. METHODS: A total of 156 adult male Sprague-Dawley rats were randomly divided into blank control group, saline group and intra-articular monoiodoacetate injection-induced OA group. The paw withdrawal mechanical threshold (PWMT) was measured before and at 1, 2, 4, and 6 weeks after monoiodoacetate injection. At 4 weeks after the injection, the pathological changes in the knee joints were analyzed using HE staining and Safranin O-Fast Green staining, and the expression of activating transcription factor 3 (ATF-3) and inducible nitric oxide synthase (iNOS) in the DRG neurons were detected by immunofluorescence staining. The expression of mRNA in the DRG neurons was detected by RT-qPCR at 1, 2, 4 and 6 weeks after the injection. The expression of KCNA2 in the DRG was measured by Western blotting, and the methylation level of promoter region was measured by MSPCR at 4 weeks after the injection. RESULTS: The PWMT of the rats in OA group was significantly decreased at 2, 4, and 6 weeks after the injection as compared with the baseline ( < 0.05 or < 0.001) as well as the control group ( < 0.05 or < 0.001). Four weeks after the intra-articular injection, fractures and defects on the surface of the articular cartilage, bone hyperplasia, and blurred tidal line were observed in the rats in OA group, but no obvious pathological changes were detected in the control or saline groups. Compared with those in the control group, the expressions of ATF-3 and iNOS were significantly increased ( < 0.01) at 4 weeks after injection; the expression of mRNA at 2, 4 and 6 weeks and the expression of KCNA2 protein at 4 weeks were all significantly decreased ( < 0.05 or < 0.01), and the methylation level of gene was significantly increased at 4 weeks after the injection in OA group ( < 0.01). CONCLUSIONS The expression of KCNA2 is decreased in the DRG neurons of rats with OA pain likely as a result of enhanced methylation of promoter region.
Animals ; Disease Models, Animal ; Ganglia, Spinal ; Knee Joint ; Kv1.2 Potassium Channel ; metabolism ; Male ; Osteoarthritis ; complications ; metabolism ; Pain ; etiology ; metabolism ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley

To detect the phosphorylation of potassium channel protein (Kv1.2 and Kv1.5) in rat cardiac muscle cells accurately, we applied the combined method of immunoprecipitation and Western blot in this study. Compared with using Western blot alone, the combination of immunoprecipitation and Western blot displayed high sensitivity to detect the activation of potassium channel proteins. Because of its simplicity, quickness and reproducibility, we find that this method was promising for detecting the phosphorylation of Kv1.2 and Kv1.5 proteins or other potassium channel proteins in rat cardiac muscle cells.
Animals ; Blotting, Western ; methods ; Cell Membrane ; metabolism ; Female ; Immunoprecipitation ; methods ; Kv1.2 Potassium Channel ; analysis ; genetics ; metabolism ; Kv1.5 Potassium Channel ; analysis ; genetics ; metabolism ; Male ; Myocytes, Cardiac ; cytology ; metabolism ; Phosphorylation ; Rats ; Rats, Sprague-Dawley