Pannexin 1-Mediated ATP Signaling in the Trigeminal Spinal Subnucleus Caudalis Is Involved in Tongue Cancer Pain.

Pain is one of the most severe concerns in tongue cancer patients. However, the underlying mechanisms of tongue cancer pain are not fully understood. We investigated the molecular mechanisms of tongue cancer-induced mechanical allodynia in the tongue by squamous cell carcinoma (SCC) inoculation in rats. The head-withdrawal threshold of mechanical stimulation (MHWT) to the tongue was reduced following SCC inoculation, which was inhibited by intracisternal administration of 10Panx, an inhibitory peptide for pannexin 1 (PANX1) channels. Immunohistochemical analyses revealed that the expression of PANX1 was upregulated in the trigeminal spinal subnucleus caudalis (Vc) following SCC inoculation. The majority of PANX1 immunofluorescence was merged with ionized calcium-binding adapter molecule 1 (Iba1) fluorescence and a part of it was merged with glial fibrillary acidic protein (GFAP) fluorescence. Spike frequencies of Vc nociceptive neurons to noxious mechanical stimulation were significantly enhanced in SCC-inoculated rats, which was suppressed by intracisternal 10Panx administration. Phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) neurons increased significantly in the Vc after SCC inoculation, which was inhibited by intracisternal 10Panx administration. SCC inoculation-induced MHWT reduction and increased pERK-IR Vc neuron numbers were inhibited by P2X7 purinoceptor (P2X7R) antagonism. Conversely, these effects were observed in the presence of P2X7R agonist in SCC-inoculated rats with PANX1 inhibition. SCC inoculation-induced MHWT reduction was significantly recovered by intracisternal interleukin-1 receptor antagonist administration. These observations suggest that SCC inoculation causes PANX1 upregulation in Vc microglia and adenosine triphosphate released through PANX1 sensitizes nociceptive neurons in the Vc, resulting in tongue cancer pain.

Oxytocin-Dependent Regulation of TRPs Expression in Trigeminal Ganglion Neurons Attenuates Orofacial Neuropathic Pain Following Infraorbital Nerve Injury in Rats.

We evaluated the mechanisms underlying the oxytocin (OXT)-induced analgesic effect on orofacial neuropathic pain following infraorbital nerve injury (IONI). IONI was established through tight ligation of one-third of the infraorbital nerve thickness. Subsequently, the head withdrawal threshold for mechanical stimulation (MHWT) of the whisker pad skin was measured using a von Frey filament. Trigeminal ganglion (TG) neurons innervating the whisker pad skin were identified using a retrograde labeling technique. OXT receptor-immunoreactive (IR), transient receptor potential vanilloid 1 (TRPV1)-IR, and TRPV4-IR TG neurons innervating the whisker pad skin were examined on post-IONI day 5. The MHWT remarkably decreased from post-IONI day 1 onward. OXT application to the nerve-injured site attenuated the decrease in MHWT from day 5 onward. TRPV1 or TRPV4 antagonism significantly suppressed the decrement of MHWT following IONI. OXT receptors were expressed in the uninjured and Fluoro-Gold (FG)-labeled TG neurons. Furthermore, there was an increase in the number of FG-labeled TRPV1-IR and TRPV4-IR TG neurons, which was inhibited by administering OXT. This inhibition was suppressed by co-administration with an OXT receptor antagonist. These findings suggest that OXT application inhibits the increase in TRPV1-IR and TRPV4-IR TG neurons innervating the whisker pad skin, which attenuates post-IONI orofacial mechanical allodynia.

Fusobacterium nucleatum exacerbates chronic obstructive pulmonary disease in elastase-induced emphysematous mice.

Exacerbation of chronic obstructive pulmonary disease (COPD) is associated with disease progression and increased mortality. Periodontal disease is a risk factor for exacerbation of COPD, but little is known about the role of periodontopathic bacteria in this process. Here, we investigated the effects of intratracheal administration of Fusobacterium nucleatum, a periodontopathic bacteria species, on COPD exacerbation in elastase-induced emphysematous mice. The administration of F. nucleatum to elastase-treated mice enhanced inflammatory responses, production of alveolar wall destruction factors, progression of emphysema, and recruitment of mucin, all of which are symptoms observed in patients with COPD exacerbation. Hence, we propose that F. nucleatum may play a role in exacerbation of COPD.

Medication-related osteonecrosis of the jaw after tooth extraction in patients receiving pharmaceutical treatment for osteoporosis: A retrospective cohort study.

Background/purpose: Bone resorption inhibitors, such as bisphosphonates (BPs) and anti-receptor activator of nuclear factor kappa B ligand antibodies (denosumab; Dmab), are used to treat osteoporosis and effectively reduce the risk of fracture. However, medication-related osteonecrosis of the jaw (MRONJ) has been reported as a rare adverse effect. Invasive tooth extraction procedures are reportedly a factor in the development of MRONJ. In this study, we aimed to retrospectively observe and clinically examine the effect of medication status on MRONJ development after tooth extraction in patients receiving drug treatment for osteoporosis. Materials and methods: This study was conducted among patients who visited our hospital between December 2015 and December 2021. We collected and analyzed the medical information of patients who underwent dental extractions while using osteoporosis medications, including oral and injectable BPs and Dmab. Results: Among antiresorptive medication users, 40 patients (70 teeth) underwent extraction. The mean duration of BP/Dmab use was 40.4 months, and the mean duration of drug holiday was 6.9 months. MRONJ after tooth extraction was not seen in BP users, but we observed two cases in Dmab users. A significant difference in MRONJ development was confirmed with the use of injectable compared with oral medication administration (odds ratio＝5.01). Conclusion: The use of injectable bone resorption inhibitors was associated with a higher risk of developing MRONJ. The route of administration, duration of medication, and withdrawal period should be carefully considered to prevent MRONJ after tooth extraction.

Nuclear localization of propiece IL-1α in HeLa cells.

PURPOSE: The study aimed to examine the nuclear localization of propiece interleukin (IL)-1α (ppIL-1α) and extracellular release rates of ppIL-1α, pIL-1α, and mIL-1α. METHODS: The subcellular localization of IL-1α molecules was observed in HeLa cells transfected with green fluorescent protein (GFP)-tagged IL-1α. Extracellular release efficiency was examined using N-terminal HiBiT-tagged IL-1α. The nuclear localization status of ppIL-1α was examined by incubating ppIL-1α transfectants with 0.1% Triton X-100 solution or with complete medium on ice. RESULTS: The results indicated the diffuse cytoplasmic and nuclear localization for m and p and ppIL-1, respectively. All IL-1α forms were released from the cells even in the steady state, and the release efficiency was 25%, 13%, and 8% for mIL-1α, pIL-1α, and ppIL-1α, respectively. Under oxidative stress condition, GFP-mIL-1α was totally diminished, but weak staining of GFP-pIL-1α and GFP-ppIL-1α was detected; nuclear localization of GFP-ppIL-1α was completely abolished by 0.1% Triton X-100 treatment, however, it remained in the nucleus after culture in complete medium on ice. CONCLUSION: The results of this study showed that ppIL-1α was localized in the nucleus and released extracellularly even in the steady state. Moreover, its cellular localization is not firm, and it is presumed to be floating in the nucleoplasm.

Periodontopathic Bacterium Fusobacterium nucleatum Affects Matrix Metalloproteinase-9 Expression in Human Alveolar Epithelial Cells and Mouse Lung.

BACKGROUND/AIM: Despite evidence of an association between pulmonary diseases and periodontopathic bacteria, the molecular mechanisms remain unknown. Matrix metalloproteinase-9 (MMP9) plays important roles in pneumonia, chronic obstructive pulmonary disease, and asthma; therefore, we assessed the effects of Fusobacterium nucleatum on MMP9 expression in mouse lung and A549 human alveolar epithelial cells. MATERIALS AND METHODS: Heat-killed F. nucleatum was administered to the trachea of mice or added to A549 cell cultures. MMP9 expression was determined using real-time PCR and western blotting. The involvement of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) in MMP9 expression was examined. RESULTS: F. nucleatum induced expression of MMP9 in mouse lung and bronchoalveolar lavage fluid. In A549 cells, F. nucleatum induced production of MMP9 protein and mRNA in a density-dependent manner; this was inhibited by inhibitors of extracellular-regulated kinase 1/2 and NF-κB, but not of p38 and Jun N-terminal protein kinase. CONCLUSION: F. nucleatum may contribute to the onset of pulmonary diseases via MMP9 expression through extracellular-regulated kinase 1/2 and NF-κB activation.

Radiation-induced malignant fibrous histiocytoma of the maxilla.

Malignant fibrous histiocytoma (MFH) originates from primitive mesenchymal cells and has the capacity for dual histiocytic and fibroblastic differentiation. We report on an MFH of the left maxilla that developed in a 79-year old woman 20 years after surgery and radiation for squamous cell carcinoma (SCC). Postoperative radiotherapy with 70 Gy was administered for a primary neoplasm of SCC of the left maxilla to a localized field through two lateral ports. This secondary neoplasm arose at the site of tumor resection (partial maxillectomy) within the irradiated field, and was resected. The development of sarcomas is a recognized complication of radiation therapy. The final diagnosis after the operation was MFH. The patient died of tumor recurrence at the skull base and within the cranium, 19 months after the operation. Radiation-induced sarcoma is well known, but radiation-induced MFH is relatively rare in the head and neck region. The details of this case are presented with a review of literature.

Porphyromonas gingivalis gingipains potentially affect MUC5AC gene expression and protein levels in respiratory epithelial cells.

Porphyromonas gingivalis (Pg) is a periodontopathic pathogen that may affect MUC5AC-related mucus hypersecretion along airway epithelial cells. Here, we attempted to establish whether Pg virulence factors (lipopolysaccharide, FimA fimbriae, gingipains) affect MUC5AC in immortalized and primary bronchial cells. We report that MUC5AC gene expression and protein levels are affected by Pg culture supernatant, but not by lipopolysaccharide or FimA fimbriae. Cells treated with either Pg single (Kgp or Rgp) or double (Kgp/Rgp) mutants had altered levels of MUC5AC gene expression and protein levels, and MUC5AC staining of double mutant-treated mouse lung cells showed that MUC5AC protein levels were unaffected. Taken together, we propose that Pg gingipains may be the primary virulence factor that influences both MUC5AC gene expression and protein levels.

Inhibition of prostaglandin E2 production by flavone and its related compounds.

We have previously reported that among 12 major ingredients of Sairei-to, Scutellariae radix inhibited prostaglandin E(2) (PGE(2)) production by lipopolysaccharide (LPS)-activated mouse macrophage-like RAW264.7 cells more efficiently than other ingredients, and wogonin, a major flavonoid from Scutellariae radix, showed greater inhibitory activity and membrane permeability than baicalein and baicalin. Here the effects of six other flavonoids, with similar structures, on membrane permeability and PGE(2) production were investigated. 7-Methoxyflavone inhibited the LPS-stimulated PGE(2) production to the greatest extent, followed by flavone>wogonin (5,7-dihydroxy-8-methoxyflavone)>> 7,8-dimethoxyflavone>chrysin (5,7-dihydroxyflavone)> baicalein (5,6,7-trihydroxyflavone)>>chromone. 7-Methoxyflavone also showed the highest membrane permeability, followed by flavone>chrysin>7,8-dimethoxy-flavone>wogonin>baicalein. When PGE(2) inhibitory activity was expressed per molecule incorporated into the cells, wogonin produced the greatest inhibition, further substantiating its anti-inflammatory potency.

Butyric Acid in Saliva of Chronic Periodontitis Patients Induces Transcription of the EBV Lytic Switch Activator BZLF1: A Pilot Study.

BACKGROUND/AIM: Epstein-Barr virus (EBV) associates with human chronic periodontitis (CP) progression. We previously demonstrated that butyric acid (BA), produced by periodontopathic bacteria, induced EBV lytic switch activator BZLF1 expression. We investigated whether short chain fatty acids (SCFAs) in CP patients' saliva enabled EBV reactivation. MATERIALS AND METHODS: Saliva was collected from seven CP patients and five periodontally healthy individuals. SCFAs were quantified using HPLC. BZLF1 mRNA and its pertinent protein ZEBRA were determined with Real-time PCR and western blotting. Histone H3 acetylation (AcH3) was further examined. RESULTS: BZLF1 mRNA expression and transcriptional activity in EBV-infected Daudi cells were induced only when treated with the CP saliva. Among SCFAs, BA alone correlated significantly with the BZLF1 transcription (r=0.88; p<0.02). As expected, CP patients' saliva induced AcH3. CONCLUSION: BA in saliva may play a role in EBV reactivation and hence contribute to EBV-related disease progression in CP patients.

Effect of Scutellariae radix ingredients on prostaglandin E(2) production and COX-2 expression by LPS-activated macrophage.

We previously reported that Sairei-to concentration-dependently modified lipopolysaccharide (LPS)-stimulated prostaglandin E(2) (PGE(2)) production in mouse macrophage-like RAW264.7 cells. Among twelve major ingredients of Sairei-to, Scutellariae radix inhibited the LPS-stimulated PGE(2) production to the greatest extent, followed by Zingiberis rhizoma, Glycyrrhizae radix, Atractylodis lanceae rhizoma and Pinelliae tuber. Scutellariae radix contained several major flavonoids such as baicalin, baicalein and wogonin. We investigated the effect of these flavonoids on PGE(2) production and COX-2 expression by LPS-activated RAW264.7 cells. Wogonin inhibited PGE(2) production most efficiently, followed by baicalein and then baicalin, in the same order as their membrane permeability. It was unexpected that wogonin and all other compounds would fail to inhibit the expression of COX-2 at both protein and mRNA levels, suggesting the importance of re-evaluating the point of action of wogonin.

Search for Drugs Used in Hospitals to Treat Stomatitis.

Stomatitis is an inflammatory disease of the oral mucosa, often accompanied by pain. Usually it is represented by aphthous stomatitis, for which treatment steroid ointment is commonly used. However, in the cases of refractory or recurrent stomatitis, traditional herbal medicines have been used with favorable therapeutic effects. Chemotherapy, especially in the head and neck region, induces stomatitis at higher frequency, which directly affects the patient's quality of life and treatment schedule. However, effective treatment for stomatitis has yet to be established. This article presents the clinical report of Kampo medicines on the stomatitis patients in the Nihon university, and then reviews the literature of traditional medicines for the treatment of stomatitis. Among eighteen Kampo medicines, Hangeshashinto has been the most popular for the treatment of stomatitis, due to its prominent anti-inflammatory activity. It was unexpected that clinical data of Hangeshashinto on stomatitis from Chinese hospital are not available. Kampo medicines have been most exclusively administered to elder person, as compared to pediatric population. Supplementation of alkaline plant extracts rich in lignin-carbohydrate complex may further extend the applicability of Kampo medicines to viral diseases.

EBV LMP1 in Gingival Epithelium Potentially Contributes to Human Chronic Periodontitis via Inducible IL8 Production.

BACKGROUND/AIM: Human chronic periodontitis is a major health problem. Although some oral bacteria have been reported to be putative pathogens, Epstein-Barr virus (EBV) is reported to be associated with the progression of periodontitis. However, the role of EBV in the aetiology of periodontitis is unknown. Therefore, we investigated periodontal pathogenesis of EBV to confirm whether EBV-encoded latent membrane protein 1 (LMP1) induces Interleukin-8 (IL8) production in human gingival cells. MATERIALS AND METHODS: Real-time polymerase chain reaction, luciferase assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were performed for determining IL8 mRNA expression, nuclear factor kappa B (NF-ĸB) transcription, IL8 production, and the phosphorylation of NF-ĸB p65 and Inhibitor of kappa B alpha (IĸBα), respectively, in Ca9-22 human gingival epithelial cells. Two LMP1 mutants lacking C-terminal activating region (CATR) domains responsible for activating NF-ĸB were used. RESULTS: Extremely high IL8 production was induced by LMP1 in time- and dose-dependent manner, where simultaneous phosphorylation of NF-κB p65 and IĸBα and transcription of NF-ĸB were observed. On the contrary, IL8 production and NF-ĸB transcription were drastically inhibited by dominant negative mutant of IĸBα. Moreover, the LMP1 mutants failed to induce IL8 production. CONCLUSION: Our findings suggest that due to CATR domains, LMP1 contributes to the progression of periodontitis via IL8 production attributable to NF-ĸB activation.

Effect of Sairei-to and its ingredients on prostaglandin E2 production by mouse macrophage-like cells.

Sairei-to and its twelve ingredients were investigated for their activity to stimulate prostaglandin E2 (PGE2) production by unstimulated and lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW 264.7 cells. LPS significantly stimulated the production and extracellular secretion of PGE2 by RAW 264.7 cells. Sairei-to concentration-dependently modified the LPS-stimulated PGE2 production. Among Sairei-to ingredients, Scutellariae radix inhibited the LPS-stimulated PGE2 production to the greatest extent, followed by Zingiberis rhizoma, Glycyrrhizae radix, Atractylodis lanceae rhizoma and Pinelliae tuber. On the other hand, Bulpeuri radix, Alismatis rhizoma, Zizyphi fructus, Polyporus, Hoelen, ginseng radix and Cinnamomi cortex further enhanced the LPS-stimulated PGE2 production. Western blot analysis demonstrated that Sairei-to unexpectedly enhanced the expression of cyclooxygenase-2 (COX-2) protein level, but did not significantly affect phospholipase A2 protein level. The present study suggests that the modification of the enzyme activity of COX-2 may be involved in the concentration-dependent effect of Sairei-to on PGE2 production by macrophages.

Bimodal effect of glycyrrhizin on macrophage nitric oxide and prostaglandin E2 production.

Glycyrrhizin is a major constituent of Kanzo, a popular herbal medicine used in food and cosmetics. Glycyrrhizin alone did not stimulate nitric oxide (NO) or prostaglandin E2 (PGE2) production by RAW 264.7 cells, but modified lipopolysaccharide (LPS)-stimulated NO and PGE2 production in a bimodal fashion: it was stimulatory at lower concentrations, whereas it was inhibitory at higher concentrations. Electron-spin resonance spectroscopy showed that glycyrrhizin slightly scavenged the superoxide anion generated by hypoxanthine-xanthine oxidase reaction, but did not scavenge the DPPH and NO radicals. The present study demonstrates the concentration-dependent action of glycyrrhizin on both PGE2 and NO production by activated macrophages.

COX-2 as possible target for the inhibition of PGE2 production by Rikko-san in activated macrophage.

We have previously reported that Rikko-san (RKS) inhibited the lipopolysaccharide (LPS)-stimulated prostaglandin (PG) E2 in mouse macrophage-like RAW264.7 cells without affecting the expression of cyclooxygenase (COX)-2. Here RKS inhibition of the enzyme activity of both COX-1 and COX-2 proteins was investigated. Western blot analysis showed that RKS did not significantly change the S-nitrosylated COX-2 protein level. On the other hand, RKS inhibited the PG production catalyzed by purified COX-2, more effectively than that catalyzed by purified COX-1. These results suggest that RKS inhibits the PGE2 production by selectively inhibiting the COX-2 activity in activated macrophages.

[A pilot study of rebamipide-gargle for chemoradiotherapy-induced mucositis in oral cancer patients].

BACKGROUND: Mucositis induced by chemoradiotherapy is one of the serious side effects of cancer therapy for oral cancer. It is caused by toxic free radicals(activated oxygen)produced by these therapeutic modalities. Rebamipide is a novel anti-ulcer drug which possesses various cytoprotective activities such as free radical scavenging, induction of prostaglandin-E and acceleration of ulcer healing. We report the results of a pilot study on rebamipidegargle for inhibition of mucositis induced by chemo-radiotherapy. METHOD: The present study was conducted on 13 patients(7 men and 6 women; age range 53-88)with oral cancer. They received radiotherapy(30-60 Gy)for the oro-facial area and chemotherapy(docetaxel: 11 cases; UFT: 1 case; radiotherapy alone: 1 case)with simultaneous addition of 1% rebamipidegargle treatment(10-15 times/day)to prevent the onset of mucositis. Informed consent was obtained prior to entry. RESULTS: Nine cases had grade 1-2 according to the WHO criteria, and 4 patients were classified as grade 3-4. No adverse reactions that could be caused by the rebamipide gargle were observed. CONCLUSIONS: These results suggested that rebamipide gargle could inhibit the occurrence of stomatitis induced by chemoradiotherapy.

Somatotopic Organization and Temporal Characteristics of Cerebrocortical Excitation in Response to Nasal Mucosa Stimulation With and Without an Odor in the Rat: An Optical Imaging Study.

Nasal mucosa has roles in warming and humidifying inspired air and is highly sensitive to mechanical stimuli. Moreover, the upper part of the nasal mucosa expresses olfactory receptors processing olfactory information. Although the somatosensory map of the face in the primary (S1) and secondary (S2) somatosensory cortices is clearly documented, the map of the nasal mucosa and the effect of odors on their activities are largely unknown. This study aimed to identify the cortical regions in S1 and their temporal features in response to somatosensory stimulation of the nasal mucosa using an optical imaging technique in urethane-anesthetized rats. An air puff application response to nasal mucosa first occurred in a part of contralateral S1 and subsequently, spread toward the rostrally and ventrally adjacent sites. Upper pharynx stimulation initially activated this rostrally expanded site and the excitatory propagation from the initially activated region toward ventral region likely represented S2. Signal intensity and activated area increased dependent on air pressure. Nasal tip stimulation initially excited S1 region caudally adjacent to that of nasal mucosa. Moreover, the amplitude of S1 excitation was similar between air puff stimulation with and without an odor, amyl acetate. In contrast to contralateral S1, air puff stimulation with the odor showed a faint optical signal increase in the ipsilateral piriform cortex. These results suggest that somatosensory information from the nasal mucosa and skin, and upper pharynx are processed in spatially continuous regions of S1, and interaction between somatosensory and olfactory systems is relatively small in contralateral S1.

A large-scale study of treatment methods for foreign bodies in the maxillary sinus.

Recently, reports regarding a foreign body in the maxillary sinus have considerably increased, with the majority being iatrogenic cases resulting from dental treatment. This study involves an extensive review of the Japanese literature, including 112 papers from 1978 to 2017. These papers documented total 407 cases of a foreign body in the maxillary sinus. Among the 392 cases for which treatment details were available, the Caldwell-Luc approach was used for 216, the alveolar approach for 116, extraction using nasal endoscopy for 15, and extraction using oral endoscopy for eight. Spontaneous passage occurred in 19 cases, follow-up with medication was used in 17, and "other" was noted in one. This study determined that surgical removal remains the most common method for treating both tooth roots and other foreign bodies and that the Caldwell-Luc approach is used in majority of the surgeries. No marked differences were noted among the removal methods used in relation to the foreign body type.

Partial Protection of Paclitaxel-induced Neurotoxicity by Antioxidants.

BACKGROUND/AIM: In order to search for substances that reduce the neurotoxicity of paclitaxel, the sensitivity of differentiated rat neuronal PC12 cells to paclitaxel was compared to that of malignant and non-malignant cells, and the extent to which four antioxidants can alleviate paclitaxel-induced neurotoxicity was investigated. MATERIALS AND METHODS: Viability of cells was determined by the MTT method. Cytotoxicity was evaluated as the concentration that reduced cell viability by 50% (CC50). Tumor specificity of paclitaxel was determined as the ratio of CC50 against non-malignant cells to that against malignant cells. RESULTS: Paclitaxel was three-fold more cytotoxic towards human oral squamous cell carcinoma cell lines (Ca9-22, HSC-2, HSC-3. HSC-4) than human normal epithelial and mesenchymal (human gingival fibroblast, human periodontal ligament fibroblast, human pulp cell) normal cells, confirming its antitumor potential. However, paclitaxel at as low a concentration as 5 ng/ml significantly reduced neurite formation in nerve growth factor-induced differentiated PC12 cells, although complete killing of cells was not achieved even at 2,000-fold higher concentration (10 µM). Paclitaxel-induced neurotoxicity was enhanced with the prolongation of incubation time and reduction of inoculation cell density. Four antioxidants, namely docosahexaenoic acid, acetyl-L-carnitine hydrochloride, N-acetyl-L-cysteine and sodium ascorbate, only partially protected PC12 cells from paclitaxel-induced toxicity. CONCLUSION: The present study suggests the involvement of both oxidative and other mechanisms in paclitaxel-induced neurotoxicity.