Inhibition Of The Expression Of Nav1.8 By RNA Interference In Pain Study

Tetrodotoxin-resistant voltage-gate sodium channel a subunit—Nav1.8, expressed specifically in sensory neurons of dorsal root ganglion, underlies pain associated with tissue injury and inflammation. The present study intended to inhibit the expression of Navl.8 by application of a new biotechnic, RNA interference, and to discuss the potential of Navl.8 as a therapeutic target in treatment of pain.Part One Expression of tetrodotoxin-resistant sodium channel Nav1.8 in models of inflammatory and bone cancer pain in ratsObjective The aim of this investigation was to induce inflammatory and bone cancer pain in rats, and to compare the expression of tetrodotoxin-resistant sodium channels Navl.8 in inflammatory and cancer pain models. Method ①Inflammatory pain model was induced by intra-plantar injection of 4% carrageenan 150μl. Mechanical allodynia and thermal hyperalgesia were tested 3h, 6h, 12h, 24h, and 48h after inflammatory stimulation.② Female SD rats recieved intra-tibial injection of 5×10~3, 5×10~4 or 5×10~5 syngenetic Walker256 mammary gland carcinoma cells. Mechanical allodynia and thermal hyperalgesia were tested at 1d, 3ds, 5ds, 7ds, 10ds, and 14ds after cell injection. The development of the bone tumour and metastasis was monitored by histology. ③ The L5-6 dorsal root ganglion (DRG) were obtained from cancer and inflammatory pain rats. Expression of TTXr sodium channel Navl.8 mRNA was investigated by RT-PCR and quantitive-PCR. Result ①Intra-plantar injection of carrageenan rapidlyproduced edema, mechanical allodynia and thermal hyperalgesia within the injected hind paw. The above symptoms were not relieved at 48h post- injection. (2) Intra-tibial injections of walker256 cells produced a rapidly expanding tumor within the boundaries of the tibia, causing severe remodeling of the bone. No tumor was observed in the contralateral tibia. But rats receiving 5><105 walker256 cells showed significant body weight loss. One rat from this group developed lymph nodes and lung matastasis. Rats receiving intra-tibial injections of walker256 cells displayed gradual development of mechanical allodynia and thermal hyperalgesia, beginning from day 7-14 following injection of cells. These symptoms were not observed in rats receiving normal saline.(3)the mRNA expression of TTXr sodium channel Navl.8 in DRG were upregulated both in the Carrageenan and cancer-induced nocicetion. The increases were seen ipsilaterally. Conclusion The induction of bone cancer in rats by the syngeneic walker256 mammary tumour cell line provided a valid pre-clinical model for pain associated with bone metastases. Both the inflammatory and cancer pain resulted in an upregulation in the expression of the TTXr sodium channel Navl.8 in DRG, suggesting that Navl.8 may contribute to pain associated with imflammatory and cancer.Part Two Small interfering RNA inhibits tetrodotoxin-resistant sodium channel Navl.8 expression in cultured dorsal root ganglion neuronsObjective Duplexes of 21-nt RNAs, known as small interfering RNAs (SiRNAs), efficiently inhibit target gene expression by RNA interference(RNAi) when introduced into mammalin cells. The present study intended to develop a method to knock down the expression of tetrodotoxin -resistant sodium channel Navl.8 in cultured dorsal root ganglion neurons by using of in vitro transcripted siRNA. Method Based on the general roles of siRNA design, we synthesized 2pairs of siRNA targeting Navl.8 (named SiRNAa and SiRNAb) by in vitro transcription with T7 RNA polymerase. A mismatched duble-strand RNA was choosed as negative control RNA. SiRNA were transfected into primary cultured dorsal root ganglion neurons with cationic lipid based reagent Lipofectamine 2000. Expression of Navl.8 was determined with RT-PCR and Q-PCR 48h after the transfecion. The uptake of fluorocence-tagged negative control RNA was detected to evaluate the efficiency of the transfection. Result In the 2 pairs of siRNA targeting Navl.8, siRNAa introduced a significant decrease in Navl.8 expression (48.32±6.84%, compared with vehicle), while siRNAb produced a 23.32±3.19 percent decrease in Navl.8 expression compared with vehicle. Negative control RNA failed to cause any change in the expression of Navl.8. The efficiency of siRNA transfection into DRG neurons was about 30-50% based on the result from fluorosence-tagged siRNA. Conclusion SiRNA synthesized by in vitro transcription could inhibit endogenous Navl.8 gene expression in cultured primary DRG neurons, suggesting efficient RNAi activity in mammalin neurons. Inhibition of Navl.8 in specific pathological conditions (such as pain) by siRNAs may provide a potential therapeutic agent in treatment of diseases.Part Three Effect of intrathecal administration of small interfering RNA targeting Navl.8 in the model of inflammatory pain in ratsObjective To evaluate the in vivo gene silencing effect of TTXr sodium channel Navl.8 in DRG by intrathecal administration of chemical synthesized siRNA with 3' terminal methyl modification, and to investigate the antinociceptive effect of siRNA targeting Navl.8 in a rat model of inflammatory pain. Method ?Chemically synthesized siRNA targeting Navl.8 and negative control RNA, and methyl modified at 3' terminal. The sequence of the siRNA was testified in the previous study of our work. SiRNAs, negativecontrol RNA and normal saline were delivered as repeated daily bolus doses(25 jig to 100 ug, for 3 days) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats received intra plantar injection of 4% carrageenan 150ul to induce inflammatory pain. Mechanical allodynia and thermal hyperalgesia were tested 3h, 6h, 12h, 24h, and 48h after inflammatory stimulation. ?L5-6 DRG from rats pretreated with repeated i.th. administration of the siRNA was obtained twenty-four hours after the last siRNA injection. Navl.8 expression in the DRG was evaluated by using of RT-PCR and Q-PCR methods. Result and Conclusion Pretreatment with the siRNA to Navl.8, but not the negative control RNA or vehicle alone, attenuated carrageenan-induced inflammatory pain. Intrathecal administration of SiRNA was concomitant with a reduction in the Navl.8 transcripts in the lumbar DRG. Neither siRNA nor negative control RNA pretreatment altered expression of GAPDH in lumbar DRG, and had no effect on the baseline threshold of nociception. The antinociception effect of siRNA continued to 72h after the last RNA injection.Part Four Generation of Anti-rat Navl.8 Polyclonal Antibody by Genetic Immunization: A Preliminary StudyObjective TTXr sodium channel Navl.8 present in sensory neurons may be responsible for the excitability of nociceptors, and may underlie pain and tenderness associated with tissue injury and inflammation. Antibody is an important tool to study the role of Navl.8. This study aimed to produce mouse anti-Navl.8 polyclonal antibody with high throughput and high specificity by genetic immunization. Method: Intracellular high antigenicity fragment (S1837 - E1910) of Navl.8 was chosen by protein antigenicity prediction software (Accelrys) and its cDNA was amplified from rats DRG total RNA byRT - PCR , which was subsequently cloned into pBQAP - OVA/TT plasmid to construct recombinant plasmid pBQAP-OVA/TT-Navl.8 for genetic immunization. The mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi - GMCSF and pCMVi - F1T3L , which helped to enhance the antibody's generation. After 5 weeks, mice were sacrificed to obtain anti - Navl.8 antibody. The antibody was identified by Western blot analysis. Result The cDNA of Navl.8 intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP - OVA/TT - Navl.8 for genetic immunization was confirmed by restriction digestion and sequencing. The antibody could recognize a band of 250 kD Navl.8 from rat DRG protein in Western blot. Conclusion Genetic immunization can generate anti - Navl.8 polyclonal antibody with high throughput and specificity.