Background: The purpose of the present study is to evaluate the mechanisms underlying tongue-referred pain associated with tooth pulp inflammation. Method: Using mechanical and temperature stimulation following dental surgery, we have demonstrated that dental inflammation and hyperalgesia correlates with increased immunohistochemical staining of neurons for TLR4 and HSP70. Results: Mechanical or heat hyperalgesia significantly enhanced in the ipsilateral tongue at 1 to 9 days after complete Freund’s adjuvant (CFA) application to the left lower molar tooth pulp compared with that of sham-treated or vehicle-applied rats. The number of fluorogold (FG)-labeled TLR4-immunoreactive (IR) cells was significantly larger in CFA-applied rats compared with sham-treated or vehicle-applied rats to the molar tooth. The number of heat shock protein (Hsp) 70-IR neurons in trigeminal ganglion (TG) was significantly increased on day 3 after CFA application compared with sham-treated or vehicle-applied rats to the molar tooth. About 9.2% of TG neurons were labeled with DiI applied to the molar tooth and FG injected into the tongue, and 15.4% of TG neurons were labeled with FG injected into the tongue and Alexa-labeled Hsp70-IR applied to the tooth. Three days after Hsp70 or lipopolysaccharide (LPS) application to the tooth in naive rats, mechanical or heat hyperalgesia was significantly enhanced compared with that of saline-applied rats. Following successive LPS-RS, an antagonist of TLR4, administration to the TG for 3 days, the enhanced mechanical or heat hyperalgesia was significantly reversed compared with that of saline-injected rats. Noxious mechanical responses of TG neurons innervating the tongue were significantly higher in CFA-applied rats compare with sham rats to the tooth. Hsp70 mRNA levels of the tooth pulp and TG were not different between CFA-applied rats and sham rats. Conclusions: The present findings indicate that Hsp70 transported from the tooth pulp to TG neurons or expressed in TG neurons is released from TG neurons innervating inflamed tooth pulp, and is taken by TG neurons innervating the tongue, suggesting that the Hsp70-TLR4 signaling in TG plays a pivotal role in tongue-referred pain associated with tooth pulp inflammation.

Nickel chloride administration prevents the growth of oral squamous cell carcinoma Nickel chloride administration prevents the growth of oral squamous cell carcinoma

The spontaneous IL-8 secretion observed in OSCC is partially dependent on the disregulated activity of transcription factor NF-κB. Nickel compounds are well established human carcinogens, however, little is known about the influence of nickel on the spontaneous secretion of IL-8 in oral squamous cell carcinoma (OSCC) cells. The aim of the present study was to investigate whether Ni²⁺ ions can influence on IL-8 secretion by OSCC. The IL-8 secretion was measured by ELISA. The expression of IL-8 mRNA was examined by real-time PCR. The NF-κB activity was measured by luciferase assay. The phosphorylation status and nuclear localization of NF-κB subunits were examined by Western blotting or Transfactor kit and immunofluorescence staining, respectively. The interaction of NF-κB p50 subunit and Ni²⁺ ions was examined by Ni²⁺-column pull down assay. The site-directed mutagenesis was used to generate a series of p50 mutants. Scratch motility assay was used to monitor the cell mobility. Our results demonstrated that, on the contrary to our expectations, Ni²⁺ ions inhibited the spontaneous secretion of IL-8. As IL-8 reduction was observed in a transcriptional level, we performed the luciferase assay and the data indicated that Ni²⁺ ions reduced the NF-κB activity. Measurement of p50 subunit in the nucleus and the immunofluorescence staining revealed that the inhibitory effect of Ni²⁺ ions was attributed to the prevention of p50 subunit accumulation to the nucleus. By Ni²⁺-column pull down assay, Ni²⁺ ions were shown to interact directly with His cluster in the N-terminus of p50 subunit. The inhibitory effect of Ni²⁺ ions was reverted in the transfectant expressing the His cluster-deleted p50 mutant. Moreover, Ni²⁺ ions inhibited the OSCC mobility in a dose dependent fashion. Taken together, inhibition of NF-κB activity by Ni²⁺ ion might be a novel therapeutic strategy for the treatment of oral cancer.