Combination of hyperlipidemia and 17ß-Estradiol induces TMJOA-like pathological changes in rats.

OBJECTIVE: We explored whether hyperlipidemia or combination of hyperlipidemia and E2 could induce TMJOA. MATERIALS AND METHODS: Four groups of female rats were treated with normal diet, normal diet with E2, high-fat diet, and high-fat diet with E2 (HFD/E2), respectively, to induce TMJOA till 8 weeks. Another three groups were then used for COX2 inhibitor celecoxib to block the induction of TMJOA. Primary condylar chondrocytes were treated with combination of E2, ox-LDL, and corresponding inhibitors for evaluating expressions of related molecules. RESULTS: Condylar cartilage proliferation with plenty of chondrocyte apoptosis and increased staining for LOX1, nuclear NF-κB, IL-1ß, and COX2 at 4 weeks and decreased condylar cartilage and increased subchondral bone density at 8 weeks were observed only in the HFD/E2 group. Celecoxib significantly alleviated the cartilage proliferation and apoptosis in the HFD/E2 group. Serum ox-LDL increased in both high-fat diet groups, while serum IL-1ß increased only in the HFD/E2 group. Combination of E2 and ox-LDL synergistically induced expressions of LOX1, phosphorylated NF-κB, IL-1ß, and COX2, while LOX1 inhibitor blocked the induction of phosphorylated NF-κB, and NF-κB inhibitor the induction of IL-1ß, and IL-1ß inhibitor the induction of COX2. CONCLUSION: Combination of hyperlipidemia and E2-induced TMJOA-like pathological changes through LOX1/NF-κB/IL-1ß/COX2-signaling pathway.

Prolonged PGE2 treatment increased TTX-sensitive but not TTX-resistant sodium current in trigeminal ganglionic neurons.

Prostaglandin E2 (PGE2) is an important inflammatory mediator for the initiation and maintenance of inflammatory and neuropathic pain. The acute effect of PGE2 on sodium currents has been widely characterized in sensory neurons; however, the prolonged effect of PGE2 remains to be determined. Here, we performed patch clamp recordings to evaluate the acute and prolonged effects of PGE2 on sodium currents in trigeminal ganglionic (TG) neurons from male Sprague-Dawley rats. We found that 24-h treatment with PGE2 (10 µM) increased the peak sodium current density by approximately 31% in a voltage-dependent manner and shifted the activation curve in a hyperpolarized direction but did not affect steady-state inactivation. Furthermore, treatment with PGE2 for 24 h increased the current density of tetrodotoxin-sensitive (TTX-S) but not TTX-resistant (TTX-R) channels significantly. Interestingly, TTX-S current was increased mostly in medium-sized, but not in small-sized, neurons after 24 h of treatment with PGE2. Moreover, the mRNA level of TTX-S Nav1.1 but not TTX-R Nav1.8 or Nav1.9 was significantly increased after 24 h of treatment with PGE2. In contrast, 5-min treatment with PGE2 (10 µM) increased the peak sodium current density by approximately 29% and increased TTX-R sodium currents, but not TTX-S currents, in both small- and medium-sized TG neurons. Our results presented a differential regulation of subtypes of sodium channels by acute and prolonged treatments of PGE2, which may help to better understand the mechanism of PGE2-mediated orofacial pain development.

Similarities and differences of estrogen in the regulation of temporomandibular joint osteoarthritis and knee osteoarthritis.

BACKGROUND: Temporomandibular joint osteoarthritis (TMJOA) and knee osteoarthritis (knee OA) are two kinds of common osteoarthritis (OA) that are characterized by chronic degeneration of soft and hard tissues around joints. Their gender and age differences suggest that there are similarities and differences between the pathogenic mechanisms of TMJOA and knee OA. OBJECTIVE: To review recent studies on the effect of estrogen on TMJOA and knee OA, and summarize their possible pathogenesis and molecular mechanisms. SOURCES: Articles up to present reporting the relationship of estrogen and TMJOA or knee OA are included. An extensive electronic search was conducted of databases including PubMed, Web of science core collection. CONCLUSION: According to epidemiological investigations, TMJOA primarily happens to females of puberty and childbearing age, while knee OA mainly affects postmenopausal women. Epidemiological investigation and experimental research suggest that estrogen may have a different effect on TMJ and on knee. Though estrogen regulates TMJOA and knee OA via estrogen-related receptors (ERR), their pathogenesis and pathway of estrogen regulation are different. To find out the accurate regulation of estrogen on TMJOA and knee OA, specific pathways and molecular mechanisms still need further exploration.

Labial gland-derived mesenchymal stem cells and their exosomes ameliorate murine Sjögren's syndrome by modulating the balance of Treg and Th17 cells.

BACKGROUND: Sjögren's syndrome (SS) is a chronic, systemic autoimmune disorder characterized by sicca syndrome and/or systemic manifestations. The disease severely affects the health and life of patients, and the treatment of SS has always been a clinical challenge and essentially palliative. Mesenchymal stem cells (MSCs) have been reported to exert immunomodulatory effects and as a potential novel therapeutic strategy for SS. Labial gland-derived MSCs (LGMSCs) are a population of resident stem cells in the labial gland, first isolated by our group. Exosomes released by MSCs contain a large variety of bioactive molecules and considered to function as an extension of MSCs. METHODS: LGMSCs were isolated from patients who were needed surgery to remove the lip mucocele and LGMSCs derived exosomes (LGMSC-Exos) were isolated by ultracentrifugation. The non-obese diabetic (NOD) mice were treated with LGMSCs or LGMSC-Exos by tail vein injection. The saliva flow rate of mice was determined and salivary glands were dissected and stained with hematoxylin and eosin. In vitro, peripheral blood mononuclear cells (PBMCs) from SS patients were cocultured with LGMSCs or LGMSC-Exos. Percentage of T helper 17 (Th17) cells and regulatory T (Treg) cells were determined by flow cytometry. The serum levels of cytokines in NOD mice and in the supernatant of the co-culture system by ELISA. RESULTS: Treatment with LGMSCs or LGMSC-Exos reduced inflammatory infiltration in the salivary glands, and restored salivary gland secretory function in NOD mice. Importantly, LGMSCs or LGMSC-Exos were demonstrated to inhibit the differentiation of Th17 cells but promote the induction of Treg cells in NOD mice and PBMCs from SS patients in vitro, accompanied by reduced interleukin 17 (IL-17), interferon gamma, and IL-6 levels and enhanced transforming growth factor beta and IL-10 secretion by T cells. CONCLUSIONS: LGMSCs are potential candidates for MSCs-based therapy and LGMSC-Exos might be utilized for establishing a new cell-free therapy against SS.

Effects of HDAC4 on IL-1ß-induced matrix metalloproteinase expression regulated partially through the WNT3A/ß-catenin pathway.

BACKGROUND: Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1ß)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/ß-catenin signaling pathway. METHODS: Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1ß and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1ß. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1ß, the translocation of ß-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/ß-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3ß) plasmids were assessed for their effects on HDAC4 levels using Western blotting. RESULTS: IL-1ß downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1ß-induced increases in MMP3 and MMP13. IL-1ß upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3ß rescued IL-1ß-induced downregulation of HDAC4 in SW1353 cells. CONCLUSION: HDAC4 exerted an inhibitory effect on IL-1ß-induced extracellular matrix degradation and was regulated partially by the WNT3A/ß-catenin signaling pathway.

Cancer-derived IgG involved in cisplatin resistance through PTP-BAS/Src/PDK1/AKT signaling pathway.

OBJECTIVES: This study aimed to explore whether knockdown of cancer-derived IgG (CIgG) could enhance cisplatin-induced anti-cancer effects. MATERIALS AND METHODS: Cancer-derived IgG was knocked down by siRNA or Tet-on shRNA in the absence or presence of cisplatin in WSU-HN6 or CAL27 cells. Cell proliferation, apoptosis, and mobility were evaluated using CCK-8, flow cytometry, and transwell assays, respectively. Molecular events were investigated using real-time PCR and Western blot assays. RESULTS: Knockdown of CIgG significantly promoted cisplatin-induced apoptosis and inhibition of cell proliferation, migration, and invasion. Cisplatin upregulated CIgG expression and phosphorylation of AKT and PDK1, while knockdown of CIgG downregulated phosphorylation of AKT and PDK1, and blocked cisplatin-induced upregulation of AKT and PDK1 phosphorylation. Moreover, knockdown of CIgG blocked cisplatin-induced upregulation of Src phosphorylation, and knockdown of Src blocked cisplatin-induced upregulation of AKT and PDK1 phosphorylation. Overexpression of Src upregulated AKT and PDK1 phosphorylation. Furthermore, knockdown of CIgG upregulated PTP-BAS mRNA and protein expression, whereas cisplatin downregulated PTP-BAS protein, but not mRNA expression; knockdown of PTP-BAS upregulated phosphorylation of Src, PDK1, AKT, and blocked CIgG knockdown-mediated enhancement of cisplatin-induced inhibition of cell proliferation. CONCLUSION: Knockdown of CIgG enhanced the anti-cancer effects of cisplatin through PTP-BAS/Src/PDK1/AKT signaling pathway in oral squamous cell carcinoma.

ΔNp63α promotes the expression and nuclear translocation of PTEN, leading to cisplatin resistance in oral cancer cells.

Pan-histone deacetylase (HDAC) inhibitors can induce the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein. However, the underlying mechanism by which this occurs remains unclear. In this study, we show that pan-HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, and sodium butyrate, were able to induce PTEN mRNA and protein expression via the acetylation of the transcription factor ΔNp63α by inhibiting HDAC1 and HDAC3. ΔNp63α enhanced PTEN promoter activity by binding two newly identified recognition sites on it. Unfortunately, the inhibition of HDAC1 or HDAC3 failed to activate PTEN, as knockdown of HDAC1 inhibited both membrane-bound and nuclear PTEN, and knockdown of HDAC3 only induced cytoplasmic PTEN. Furthermore, the overexpression of ΔNp63α downregulated membrane-bound PTEN but enhanced the nuclear translocation of PTEN, leading to the cisplatin resistance of oral cancer cells. PTEN accumulated in the nuclei of cancerous cells and normal cells when ΔNp63α was highly expressed in specimens from patients with squamous cell carcinoma of the tongue. However, inhibiting either HDAC1 or HDAC6 prevented the nuclear translocation of PTEN and attenuated cisplatin resistance. These results suggest that chemotherapeutic inhibitors of HDAC1 or HDAC6, together with cisplatin, might improve outcomes for patients with squamous cell carcinoma of the tongue.

Progesterone Attenuates Allodynia of Inflamed Temporomandibular Joint through Modulating Voltage-Gated Sodium Channel 1.7 in Trigeminal Ganglion.

Background: Women with temporomandibular disorders (TMDs) experience some amelioration of pain during pregnancy. Progesterone increases dramatically and steadily during pregnancy. Sodium channel 1.7 (Nav1.7) plays a prominent role in pain perceptions, as evidenced by deletion of Nav1.7 alone leading to a complete loss of pain. In a previous study, we showed that Nav1.7 in trigeminal ganglion (TG) is involved in allodynia of inflamed temporomandibular joint (TMJ). Whether progesterone modulates allodynia of inflamed TMJ through Nav1.7 in TG remains to be investigated. Methods: The effects of progesterone on sodium currents of freshly isolated TG neurons were examined using whole-cell recording. Female rats were ovariectomized and treated with increasing doses of progesterone for 10 days. Complete Freund's adjuvant was administered intra-articularly to induce TMJ inflammation. TMJ nociceptive responses were evaluated by head withdrawal thresholds. Real-time PCR and Western blotting were used to examine Nav1.7 mRNA and protein expression in TG. Immunohistofluorescence was used to examine the colocalization of progesterone receptors (PRα/ß) and Nav1.7 in TG. Results: Whole-cell recording showed that progesterone could attenuate sodium currents. Moreover, progesterone dose-dependently downregulated Nav1.7 mRNA expression and reduced the sensitivity of TMJ nociception in ovariectomized rats. Furthermore, treatment with progesterone attenuated allodynia of inflamed TMJ in a dose-dependent manner and repressed inflammation-induced Nav1.7 mRNA and protein expression in ovariectomized rats. The progesterone receptor antagonist, RU-486, partially reversed the effect of progesterone on allodynia of inflamed TMJ and TMJ inflammation-induced Nav1.7 mRNA and protein expression. Conclusion: Progesterone, by modulating trigeminal ganglionic Nav1.7, may represent a promising agent to prevent allodynia of inflamed TMJ.

Reducing macrophage numbers alleviates temporomandibular joint ankylosis.

Temporomandibular joint (TMJ) ankylosis is a severe joint disease mainly caused by trauma that leads to a series of oral and maxillofacial function disorders and psychological problems. Our series of studies indicate that TMJ ankylosis development is similar to fracture healing and that severe trauma results in bony ankylosis instead of fibrous ankylosis. Macrophages are early infiltrating inflammatory cells in fracture and play a critical role in initiating fracture repair. We hypothesize that the large numbers of macrophages in the early phase of TMJ ankylosis trigger ankylosed bone mass formation and that the depletion of these macrophages in the early phase could inhibit the development of TMJ ankylosis. By analysing human TMJ ankylosis specimens, we found large numbers of infiltrated macrophages in the less-than-1-year ankylosis samples. A rabbit model of TMJ bony ankylosis was established and large numbers of infiltrated macrophages were found at 4 days post-operation. Local clodronate liposome (CLD-lip) injection, which depleted macrophages, alleviated the severity of ankylosis compared with local phosphate-buffered saline (PBS)-loaded liposome (PBS-lip) injection (macrophage number, PBS-lips vs. CLD-lips: 626.03 ± 164.53 vs. 341.4 ± 108.88 n/mm2; ankylosis calcification score, PBS-lips vs. CLD-lips: 2.11 ± 0.78 vs. 0.78 ± 0.66). Histological results showed that the cartilage area was reduced in the CLD-lip-treated side (PBS-lips vs. CLD-lips: 6.82 ± 4.42% vs. 2.71 ± 2.78%) and that the Wnt signalling regulating cartilage formation was disrupted (Wnt5a expression decreased 60% and Wnt4 expression decreased 73%). Thus, our study showed that large numbers of macrophages infiltrated during the early phase of ankylosis and that reducing macrophage numbers alleviated ankylosis development by reducing cartilage formation.

RhoG/Rac1 signaling pathway involved in migration and invasion of salivary adenoid cystic carcinoma cells.

OBJECTIVES: This study aimed to explore whether RhoG/Rac1 was involved in migration and invasion of salivary adenoid cystic carcinoma (SACC). MATERIALS AND METHODS: RhoG and Rac1 were evaluated in two SACC cell lines, namely SACC-83 and SACC-LM, with low and high rates of lung metastasis, respectively. Functional changes were evaluated using cell proliferation, transwell, and wound-healing assays, and molecular events were investigated using real-time PCR and Western blot assays. RESULTS: RhoG and Rac1 were highly expressed and more activated in SACC-LM cells than in SACC-83 cells. RhoG overexpression promoted SACC-83 cell migration and invasion through activating Rac1. The knockdown of RhoG or Rac1 partially blocked epiregulin-induced migration and invasion in SACC-83 cells. Epiregulin-induced activation of RhoG/Rac1 in SACC-83 cells was blocked by a Src inhibitor, or an AKT inhibitor or AKT siRNA, or an ERK1/2 inhibitor. Moreover, the epiregulin-induced phosphorylation of AKT and ERK1/2 in SACC-83 cells was blocked by a Src inhibitor, and the epiregulin-induced phosphorylation of ERK1/2 was blocked by an AKT inhibitor or AKT siRNA. Overexpression of activated AKT induced activation of ERK1/2 and RhoG. CONCLUSIONS: RhoG/Rac1 signaling pathway was involved in SACC cell migration and invasion. RhoG/Rac1 at least partially mediated epiregulin/Src/AKT/ERK1/2 signaling to promote SACC cell migration and invasion.

Correction to: Reducing macrophage numbers alleviates temporomandibular joint ankylosis.

The article "Reducing macrophage numbers alleviates temporomandibular joint ankylosis", written by Lu Zhao, E Xiao, Linhai He, Denghui Duan, Yang He, Shuo Chen, Yi Zhang and Yehua Gan, was originally published electronically on the publisher's internet portal.

Connexin 43 contributes to temporomandibular joint inflammation induced-hypernociception via sodium channel 1.7 in trigeminal ganglion.

We previously demonstrated that sodium channel 1.7 (Nav1.7) in trigeminal ganglion (TG) was a critical factor in temporomandibular joint (TMJ) inflammation-induced hypernociception, but the mechanism underlying inflammation-induced upregulation of Nav1.7 remained unclear. Glial-neuron interaction plays a critical role in pain process and connexin 43 (Cx43), a gap junction protein expressed in satellite glial cells (SGCs) has been shown to play an important role in several pain models. In the present study, we investigate the role of Cx43 in TMJ inflammation-induced hypernociception and its possible impact on neuronal Nav1.7. We induced TMJ inflammation in rats by injecting complete Freund's adjuvant (CFA) into TMJ and observed a decrease in head withdraw threshold after 24 h. Electron microscopy showed morphological alterations of SGCs in TMJ-inflamed rats. The expression of Cx43, glial fibrillary acidic protein (GFAP), and Nav1.7 increased greatly compared with controls. In addition, pretreatment with Cx43 blockers in TMJ-inflamed rats could alleviate mechanical hypernociception, inhibit SGCs activation and IL-1ßrelease, and thus block the upregulation of Nav1.7. These findings indicate that the propagation of SGCs activation via Cx43 plays a critical role in Nav1.7-involved mechanical hypernociception induced by TMJ inflammation.

Chronic inflammation deteriorates structure and function of collagen fibril in rat temporomandibular joint disc.

Collagen is the building component of temporomandibular joint (TMJ) discs and is often affected by inflammation in temporomandibular disorders. The macromechanical properties of collagen are deteriorated by chronic inflammation. However, the mechanism by which inflammation influences disc function remains unknown. The relationship between the ultrastructure and nanomechanical properties of collagen in inflamed discs should be clarified. Seven-week-old female Sprague-Dawley rats were randomly divided into two groups. Chronic TMJ inflammation was induced by intra-articular injection of complete Freund's adjuvant, and samples were harvested after 5 weeks. Picrosirius staining revealed multiple colours under polarized light, which represented alternative collagen bundles in inflamed discs. Using atomic force microscopy scanning, the magnitude of Young's modulus was reduced significantly accompanied with disordered collagen fibril arrangement with porous architecture of inflamed discs. Transmission electron microscopy scanning revealed a non-uniform distribution of collagen fibres, and oversized collagen fibrils were observed in inflamed discs. Fourier transform infrared microspectroscopy revealed a decrease in 1 338 cm-1/amide II area ratio of collagen in different regions. The peak positions of amide I and amide II bands were altered in inflamed discs, indicating collagen unfolding. Our results suggest that sustained inflammation deteriorates collagen structures, resulting in the deterioration of the ultrastructure and nanomechanical properties of rat TMJ discs.

Carious status and supragingival plaque microbiota in hemodialysis patients.

OBJECTIVE: The aim of this study was to evaluate the carious status and the microbial profiles of supragingival plaque in patients with chronic kidney disease undergoing hemodialysis. METHODS: This study included 30 patients with chronic kidney disease undergoing hemodialysis as well as 30 control subjects. Dental examination was performed and the decayed-missing-filled-teeth was recorded. Supragingival plaque was taken and analyzed using 16S rRNA gene amplicon by Illumina MiSeq sequencing to detect microbial composition and community diversity and structure. RESULTS: The level of decayed-missing-filled-teeth was higher in the hemodialysis group than that in the control group. Microbial analysis showed a decrease in α diversity and a increase in relative abundance and prevalence of many acidogenic and aciduric caries related species in the supragingival plaque samples of the hemodialysis patients, including Streptococcus mutans, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus vaginalis, Scardovia wiggsiae F0424, and Actinomyces naeslundii. CONCLUSION: Our results suggested that the hemodialysis patients were more susceptible to caries. More attentions for caries prevention and treatment should be paid to improve their life quality, and even to reduce their cardiovascular events and survival.

Genistein Antagonizes 17ß-Estradiol Effects on Glutamate-Evoked Masseter Muscle Hypernociception in Rats.

Temporomandibular disorders (TMDs) predominantly affect women of reproductive ages, with pain as the main symptom. The aim of the present study was to examine the effects of 17ß-estradiol (E2) on glutamate-evoked hypernociception of masseter muscle and to examine whether genistein could antagonize the effects of E2 in female rats. Injection of glutamate into the masseter muscle dose-dependently decreased head withdrawal thresholds, a parameter for mechanical hypernociception. Head withdrawal thresholds in ovariectomized rats also decreased with increasing doses of E2 replacement, and were further aggravated by injection of glutamate (1M, 40µL) into the masseters. Genistein at doses of 7.5 and 15 mg/kg antagonized E2-induced hypernociception of masseter muscle, and at doses of 7.5, 15, and 30 mg/kg also antagonized E2 potentiation of glutamate-evoked hypernociception of masseter muscle. Genistein produced optimal antagonistic effects of E2 on nociception behavior at a dose of 15 mg/kg. On the molecular level, tyrosine phosphorylation of the NR2B subunit of the N-methyl-D-aspartate receptor (pNR2B) and phosphorylated mitogen-activated protein kinase (pERK1/2) were significantly upregulated in the hippocampus following glutamate injection and were further potentiated by E2 replacement. Genistein at dose of 15 mg/kg partially reversed E2-potentiated glutamate-evoked upregulation of pNR2B and pERK1/2 expression in the hippocampus. These results indicated that moderate doses of genistein could antagonize E2 enhanced glutamate-evoked hypernociception of masseter muscle possibly via N-methyl-D-aspartate receptor and ERK1/2 signaling pathways in the hippocampus.

Knockdown of Yin Yang 1 enhances anticancer effects of cisplatin through protein phosphatase 2A-mediated T308 dephosphorylation of AKT.

Cisplatin is still one of the first-line drugs for chemotherapy of head and neck squamous cell carcinoma (HNSCC) and shows a survival advantage for HNSCC. However, a substantial proportion of HNSCC eventually becomes resistance to cisplatin and the underlying mechanisms remain to be fully understood. Yin Yang 1 (YY1) is a multifunctional protein regulating both gene transcription and protein modifications and also plays a role in chemotherapy resistance. Here, we reported that knockdown of YY1 by lentivirus-mediated short hairpin RNA or tetracycline-inducible short hairpin RNA enhanced cisplatin-induced apoptosis and inhibition of cell proliferation, migration and invasion in the HNSCC cell lines, and inhibition of the xenograft tumor growth. The underlying mechanisms were revealed that knockdown of YY1 downregulated both S473 and T308 phosphorylation of AKT (protein kinase B), which was mainly responsible for cisplatin resistance, whereas overexpression of YY1 upregulated both S473 and T308 phosphorylation. Cisplatin upregulated YY1 mRNA and protein expression and both S473 and T308 phosphorylation of AKT. In the presence of cisplatin, knockdown of YY1 not only blocked cisplatin-induced increase in S473 and T308 phosphorylation of AKT, but still downregulated T308 phosphorylation. Moreover, protein phosphatase 2A (PP2A) antagonist, okadaic acid, upregulated T308, but not S473, phosphorylation, and simultaneously abolished YY1 knockdown-mediated enhancement of cisplatin-induced inhibition of cell proliferation. In addition, knockdown of YY1 promoted PP2A activity through upregulating mRNA and protein expressions of PP2A catalytic subunit alpha (PPP2CA) through the binding of YY1 in the promoter of PPP2CA. Conversely, activating PP2A by forskolin also promoted YY1 degradation and subsequently inhibited T308 phosphorylation. These results suggested that knockdown of YY1 enhanced anticancer effects of cisplatin through PP2A mediating T308 dephosphorylation of AKT, and that targeting YY1 or PP2A would enhance the efficiency of cisplatin chemotherapy in treatment of HNSCC.

Veratridine modifies the gating of human voltage-gated sodium channel Nav1.7.

Veratridine is a lipid-soluble neurotoxin derived from plants in the family Liliaceae. It has been broadly investigated for its action as a sodium channel agonist. However, the effects of veratridine on subtypes of sodium channels, especially Nav1.7, remain to be studied. Here, we investigated the effects of veratridine on human Nav1.7 ectopically expressed in HEK293A cells and recorded Nav1.7 currents from the cells using whole-cell patch clamp technique. We found that veratridine exerted a dose-dependent inhibitory effect on the peak current of Nav1.7, with the half-maximal inhibition concentration (IC50) of 18.39 µM. Meanwhile, veratridine also elicited tail current (linearly) and sustained current [half-maximal concentration (EC50): 9.53 µM], also in a dose-dependent manner. Veratridine (75 µM) shifted the half-maximal activation voltage of the Nav1.7 activation curve in the hyperpolarized direction, from -21.64 ± 0.75 mV to -28.14 ± 0.66 mV, and shifted the half-inactivation voltage of the steady-state inactivation curve from -59.39 ± 0.39 mV to -73.78 ± 0.5 mV. An increased frequency of stimulation decreased the peak and tail currents of Nav1.7 for each pulse along with pulse number, and increased the accumulated tail current at the end of train stimulation. These findings reveal the different modulatory effects of veratridine on the Nav1.7 peak current and tail current.

Sexual dimorphism of estrogen-sensitized synoviocytes contributes to gender difference in temporomandibular joint osteoarthritis.

OBJECTIVES: Temporomandibular joint osteoarthritis (TMJOA) is approximately twice as prevalent in women than in men. Synoviocytes are believed to play a critical role in joint inflammation. However, it is unknown whether synoviocytes from different genders possess sexual dimorphisms that contribute to female-predominant TMJOA. MATERIALS AND METHODS: Freund's complete adjuvant combined with monosodium iodoacetate was used to induce TMJOA in female and male rats. Histologic and radiographic features were used to evaluate TMJOA. The expression of CD68, MCP-1, iNOS, and IL-1ß was detected by immunohistochemistry and real-time PCR. Primary fibroblast-like synoviocytes (FLSs) isolated from the synovial membrane of female and male rats were used for in vitro experiments. RESULTS: Female rats showed aggravated TMJOA features as compared to male rats. Increased expression of iNOS and IL-1ß was detected in synovial membrane from female TMJOA rats as compared to male rats. Furthermore, greater amounts of CD68-positive macrophage infiltration and increased MCP-1 expression around the synovial membrane were detected in female TMJOA rats compared to males. Primary cultured FLSs from female rats showed higher sensitivity to TNF-α treatment and recruited increased macrophage migration than male FLSs. More important, ovariectomy (OVX) by ablation in female rats repressed the sensitivity of female FLSs to TNF-α treatment due to the loss of estrogen production. Blockage of the estrogen receptor repressed estrogen-potentiated TNF-α-induced pro-inflammatory cytokine expression in OVX-FLSs. Moreover, the injection of estrogen receptor antagonists relieved the cartilage destruction and bone deterioration of TMJOA in female rats. CONCLUSION: Estrogen-sensitized synoviocytes in female rats may contribute to gender differences in the incidence and progression of TMJOA.

Glial interleukin-1ß upregulates neuronal sodium channel 1.7 in trigeminal ganglion contributing to temporomandibular joint inflammatory hypernociception in rats.

BACKGROUND: The proinflammatory cytokine interleukin-1ß (IL-1ß) drives pain by inducing the expression of inflammatory mediators; however, its ability to regulate sodium channel 1.7 (Nav1.7), a key driver of temporomandibular joint (TMJ) hypernociception, remains unknown. IL-1ß induces cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). We previously showed that PGE2 upregulated trigeminal ganglionic Nav1.7 expression. Satellite glial cells (SGCs) involve in inflammatory pain through glial cytokines. Therefore, we explored here in the trigeminal ganglion (TG) whether IL-1ß upregulated Nav1.7 expression and whether the IL-1ß located in the SGCs upregulated Nav1.7 expression in the neurons contributing to TMJ inflammatory hypernociception. METHODS: We treated rat TG explants with IL-1ß with or without inhibitors, including NS398 for COX-2, PF-04418948 for EP2, and H89 and PKI-(6-22)-amide for protein kinase A (PKA), or with adenylate cyclase agonist forskolin, and used real-time PCR, Western blot, and immunohistofluorescence to determine the expressions or locations of Nav1.7, COX-2, cAMP response element-binding protein (CREB) phosphorylation, and IL-1ß. We used chromatin immunoprecipitation to examine CREB binding to the Nav1.7 promoter. Finally, we microinjected IL-1ß into the TGs or injected complete Freund's adjuvant into TMJs with or without previous microinjection of fluorocitrate, an inhibitor of SGCs activation, into the TGs, and evaluated nociception and gene expressions. Differences between groups were examined by one-way analysis of variance (ANOVA) or independent samples t test. RESULTS: IL-1ß upregulated Nav1.7 mRNA and protein expressions in the TG explants, whereas NS398, PF-04418948, H89, or PKI-(6-22)-amide could all block this upregulation, and forskolin could also upregulate Nav1.7 mRNA and protein expressions. IL-1ß enhanced CREB binding to the Nav1.7 promoter. Microinjection of IL-1ß into the TGs or TMJ inflammation both induced hypernociception of TMJ region and correspondingly upregulated COX-2, phospho-CREB, and Nav1.7 expressions in the TGs. Moreover, microinjection of fluorocitrate into the TGs completely blocked TMJ inflammation-induced activation of SGCs and the upregulation of IL-1ß and COX-2 in the SGCs, and phospho-CREB and Nav1.7 in the neurons and alleviated inflammation-induced TMJ hypernociception. CONCLUSIONS: Glial IL-1ß upregulated neuronal Nav1.7 expression via the crosstalk between signaling pathways of the glial IL-1ß/COX-2/PGE2 and the neuronal EP2/PKA/CREB/Nav1.7 in TG contributing to TMJ inflammatory hypernociception.

Progesterone attenuates temporomandibular joint inflammation through inhibition of NF-κB pathway in ovariectomized rats.

Sex hormones may contribute to the symptomatology of female-predominant temporomandibular disorders (TMDs) inflammatory pain. Pregnant women show less symptoms of TMDs than that of non-pregnant women. Whether progesterone (P4), one of the dominant sex hormones that regulates multiple biological functions, is involved in symptoms of TMDs remains to be explored. Freund's complete adjuvant were used to induce joint inflammation. We evaluated the behavior-related and histologic effects of P4 and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in the synovial membrane. Primary TMJ synoviocytes were treated with TNF-α or IL-1ß with the combination of P4. Progesterone receptor antagonist RU-486 were further applied. We found that P4 replacement attenuated TMJ inflammation and the nociceptive responses in a dose-dependent manner in the ovariectomized rats. Correspondingly, P4 diminished the DNA-binding activity of NF-κB and the transcription of its target genes in a dose-dependent manner in the synovial membrane of TMJ. Furthermore, P4 treatment showed decreased mRNA expression of proinflammatory cytokines, and partially reversed TNF-α and IL-1ß induced transcription of proinflammatory cytokines in the primary synoviocytes. Moreover, progesterone receptor antagonist RU-486 partially reversed the effects of P4 on NF-κB pathway. In conclusion, progesterone ameliorated TMJ inflammation through inhibition of NF-κB pathway.