LS8 cell apoptosis induced by NaF through p-ERK and p-JNK - a mechanism study of dental fluorosis.

OBJECTIVE: To investigate the possible biological mechanism of dental fluorosis at a molecular level. MATERIAL AND METHODS: Cultured LS8 were incubated with serum-free medium containing selected concentrations of NaF (0 ∼ 2 mM) for either 24 or 48 h. Subcellular microanatomy was characterized using TEM; meanwhile, selected biomolecules were analysed using various biochemistry techniques. Transient transfection was used to modulate a molecular pathway for apoptosis. RESULTS: Apoptosis of LS8 was induced by NaF treatment that showed both time and concentration dependency. The activity of caspase-3, -8, -9 was found to be increased with NaF in a dose-dependent manner. Western blot revealed that the protein expression of p-ERK and p-JNK were decreased, while the expression of p-P38 was increased. Inhibition of the p-ERK and p-JNK pathways resulted in a similar decrease for caspase-3. CONCLUSION: During NaF-induced apoptosis of LS8, p-ERK and p-JNK were closely associated with induction of apoptosis, which might be a mechanism of dental fluorosis.

Costunolide enhances cisplatin-induced cytotoxicity in hypopharyngeal SCC FaDu cells by increasing the production of reactive oxygen species.

OBJECTIVE: The sesquiterpene lactone costunolide (CTL) has attracted much attention due to its antitumor effect on a variety of malignant tumors. However, the effect of CTL on hypopharyngeal squamous cell carcinoma (HSCC) remains unclear. This study aimed to examine the effects of this sesquiterpene lactone on HSCC FaDu cells. METHODS: The FaDu cell line was treated with CTL and/or cisplatin. CCK-8 was used to examine cell viability. Annexin-FITC/PI and Hoechst 33258 staining were used to measure apoptosis. Reactive oxygen species (ROS) were analysed by MitoSOX Red staining. Protein expression was examined by Western blotting. RESULTS: CTL (0, 2, 4, 6, 8, 10, 20, 30, and 40 µM) dose-dependently induced cytotoxicity in FaDu cells. CTL increased ROS production and induced apoptosis in FaDu cells. Moreover, CTL synergized with cisplatin to induce apoptosis in FaDu cells. In addition, the caspase inhibitor Z-DEVD-FMK attenuated CTL-induced apoptosis. Western blot analysis showed that CTL increased the expression levels of cleaved caspase-3 and Bax and decreased the expression levels of Bcl-2, phospho-AKT and phospho-NF-κB. CONCLUSION: In conclusion, these data demonstrate that CTL induced apoptosis and enhanced cisplatin-induced cytotoxicity in HSCC FaDu cells.

Puerarin attenuates cisplatin-induced apoptosis of hair cells through the mitochondrial apoptotic pathway.

Puerarin, one of the main components of Pueraria lobata, has been reported to possess a wide range of pharmacological activities, including anti-inflammatory, antioxidative and anti-apoptotic effects. However, the role of puerarin in ototoxic drug-induced hair cell injury has not been well characterized. This study explored whether puerarin protects against cisplatin-induced hair cell damage and its potential mechanisms. The viability of puerarin-treated HEI-OC1 cells was assessed by CCK8 assay. Reactive oxygen species (ROS) was estimated with flow cytometric analysis using Cellrox Green fluorescent probe. Apoptosis-related protein levels were detected by western blot analysis. Immunostaining of the organ of Corti was performed to determine mice cochlear hair cell survival. Our results showed that puerarin improved cell viability and suppressed apoptosis in the cisplatin-damaged HEI-OC1 cells and cochlear hair cells. Mechanistic studies revealed that puerarin attenuated mitochondrial apoptosis pathway by regulating apoptotic related proteins, such as Bax and cleaved caspase-3, and attenuated ROS accumulation after cisplatin damage. Moreover, puerarin was involved in regulating the Akt pathway in HEI-OC1 cells in response to cisplatin. Our results demonstrated that puerarin administration decreased the sensitivity to apoptosis dependent on the mitochondrial apoptotic pathway by reducing ROS generation, which could be used as a new protective agent against cisplatin-induced ototoxicity.

NaF reduces KLK4 expression by decreasing Foxo1/Runx2 expression in LS8 cells.

OBJECTIVE: This study aimed to investigate the effect of high fluoride on runt-related transcription factor 2 (Runx2) expression and to explore the possible relationship among Runx2, forkhead box o1 (Foxo1) and kallikrein 4 (KLK4) in high fluoride-treated ameloblasts. DESIGN: Ameloblast-like cells (LS8 cells) were exposed to various concentrations of sodium fluoride (NaF) for up to 48 h. Runx2 expression was downregulated by gene silencing, and Foxo1 expression was up- and downregulated by gene overexpression and silencing, respectively. The mRNA and protein levels of Runx2, Foxo1, KLK4 and matrix metalloproteinase 20 (MMP20) were detected by qRT-PCR and western blotting. RESULTS: Runx2 expression was decreased in a dose- and time-dependent manner in NaF-treated LS8 cells. The knockdown of Runx2 markedly decreased KLK4 expression in LS8 cells under NaF conditions. However, the variation trend of MMP20 was unclear. In addition, forced Foxo1 expression led to significant upregulation of Runx2 in LS8 cells under NaF conditions. In contrast, the knockdown of Foxo1 markedly decreased the Runx2 protein levels under NaF conditions. Moreover, Foxo1 downregulation markedly decreased runx2 mRNA levels, and this inhibition in LS8 cells was intensified when combined with NaF treatment. CONCLUSION: The results indicated that NaF reduces Runx2 expression in LS8 cells and that decreased Foxo1/Runx2 expression induced by high fluoride is a cause of low KLK4 expression.

NaF Reduces KLK4 Gene Expression by Decreasing Foxo1 in LS8 Cells.

Decreased expression and increased phosphorylation of Forkhead box o1 (Foxo1) in ameloblasts were observed both in vivo and in vitro when treated by fluoride. The present study aims to investigate the possible relationship between Foxo1 and enamel matrix proteinases, matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), in NaF-treated ameloblasts. Ameloblast-like cells (LS8 cells) were exposed to NaF at selected concentration (0/2 mM) for 24 h. Gene overexpression and silencing experiments were used to up- and down-regulate Foxo1 expression. The expression levels of Foxo1, MMP20, and KLK4 were detected by quantitative real-time PCR and western blot. Dual luciferase reporter assay was performed to evaluate the regulation of Foxo1 on the transcriptional activity of KLK4 promoter. The results showed that KLK4 expression was decreased in LS8 cells treated by NaF, while MMP20 expression was not changed. Foxo1 activation led to significantly up-regulation of KLK4 in LS8 cells under NaF condition. Knockout of Foxo1 markedly decreased klk4 expression in mRNA level, and intensified inhibition occurred in LS8 cells when combined with NaF treatment. However, the variation trend of MMP20 was not clear. Dual luciferase reporter assay showed that Foxo1 activation enhanced the transcriptional activity of KLK4 promoter. These findings suggest that the decrease of Foxo1 expression induced by high fluoride was a cause for low KLK4 expression.

Foxo1 Attenuates NaF-Induced Apoptosis of LS8 Cells through the JNK and Mitochondrial Pathways.

Fluoride-induced ameloblast apoptosis is a key event in dental fluorosis development. Forkhead box o1 (Foxo1) is a transcription factor involved in cell apoptosis. The present study aims to investigate the effect of Foxo1 on ameloblast apoptosis induced by fluoride in vitro and to explore its possible mechanism. Ameloblast-like cells (LS8 cells) were exposed to various concentrations of NaF for up to 48 h. Foxo1 activation was modulated using lentiviral vectors, and cell apoptosis was measured by flow cytometry. The expression levels of Foxo1, c-Jun N-terminal kinase (JNK), and some well-known regulators of the mitochondrial pathway of apoptosis (cytoplasmic cytochrome c, cleaved caspase-9, cleaved caspase-3, Bcl-2, and Bax) were detected by quantitative real-time PCR, western blot, and immunofluorescence assay. The results showed significantly decreased expression and increased phosphorylation of Foxo1 in NaF-treated LS8 cells. Further investigation revealed that forced Foxo1 activation with lentiviral vectors attenuated NaF-induced apoptosis of LS8 cells, markedly decreasing protein levels of cytoplasmic cytochrome c, cleaved caspase-9, and cleaved caspase-3 while increasing the Bcl-2/Bax ratio and JNK expression level. These findings suggest that Foxo1 attenuated NaF-induced apoptosis of LS8 cells via inhibiting the mitochondrial pathway and activating JNK.

RNA-binding protein Lin28 is associated with injured dentin-dental pulp complex in Sprague-Dawley rats.

Reactivation of Lin28 accelerates hair, cartilage, bone and mesenchyme regrowth after ear and digit injuries. However, the relationship of Lin28 to reparative dentin has been under investigation. The aim of the present study was to examine whether Lin28 participates in the reparative dentin process and lipopolysaccharide (LPS)-stimulated human dental pulp cells (HDPCs) and to identify the underlying signaling pathway mechanisms. The study established a wound-healing model of the dentin-dental pulp complex in vivo and LPS-induced dental pulp cell inflammation in vitro. In vivo, the results of hematoxylin and eosin staining demonstrated the obvious appearance of reparative dentin and odontoblast-like cells were arranged along the reparative dentin. Immunohistochemical examination demonstrated that Lin28 expression was increased by 72 h after cavity preparation but was decreased by 21 d after cavity preparation. In vitro, HDPCs were exposed to 100 ng/ml LPS for 24 h, and the expression of Lin28 was increased. Overexpression of Lin28 was associated with the downregulated expression of let-7b, let-7g and miR-98. These findings suggest that the wound-healing model was successfully established. Lin28 was involved in the reparative process of the dentin-dental pulp complex and HDPCs exposed to LPS, and Lin28/let-7 may be the underlying mechanism.

Optimizing concentration of titanium tetrafluoride solution for human dentine remineralization.

OBJECTIVE: The aim of the present study was to select the optimal concentration of TiF4 solution to facilitate the remineralization of early dentine caries lesions. DESIGN: Sixty human dentine specimens were cut and randomly divided into 6 groups (1%, 2%, 3%, 4% TiF4 groups, 2.712% NaF group and distilled deionized water (DDW) control group). Artificial dentine caries-like lesions were created. After being subjected to fluoride treatment and immersed in remineralizing solution for 2weeks, the specimens were observed by microCT, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Data were analysed using linear regression analysis (P<0.05). RESULTS: The lesion depths of the specimens treated by 2% TiF4 solution were statistically less than those of the other groups. Further, the greyscale values of these lesion areas were greater. The 3% and 4% TiF4 solutions caused further lesion demineralization. The 2.712% NaF solution seemed to be detrimental to remineralization during the experimental time, as the subsurface area remained hypomineralized with a thick precipitation layer on the surface. CONCLUSIONS: The 2% TiF4 solution demonstrated better remineralizing potency than did the other treatments.

Baicalin inhibits toll-like receptor 2/4 expression and downstream signaling in rat experimental periodontitis.

Periodontitis is a severe inflammatory response, leading to characteristic periodontal soft tissue destruction and alveolar bone resorption. Baicalin possesses potent anti-inflammatory activity; however, it is still unclear whether baicalin regulates toll-like receptor (TLR) 2/4 expression and downstream signaling during the process of periodontitis. In this study, the cervical area of the maxillary second molars of rats was ligated and inoculated with Porphyromonas gingivalis (P. gingivalis) for 4weeks to induce periodontitis. Some rats with periodontitis were treated intragastrically with baicalin (50, 100 or 200mg/kg/day) or vehicle for 4weeks. Compared with the sham group, the levels of TLR2, TLR4 and MyD88 expression and the p38 MAPK and NF-κB activation were up-regulated in the experimental periodontitis group (EPG), accompanied by marked alveolar bone loss and severe inflammation. Treatment with 100 or 200mg/kg/day baicalin dramatically reduced the alveolar bone loss, the levels of HMGB1, TNF-α, IL-1ß, and MPO expression, and the numbers of inflammatory infiltrates in the gingival tissues. Importantly, treatment with 100 or 200mg/kg/day baicalin mitigated the periodontitis-up-regulated TLR2, TLR4 and MyD88 expression, and the p38 MAPK and NF-κB activation. Hence, the blockage of the TLR2 and TLR4/MyD88/p38 MAPK/NF-κB signaling by baicalin may contribute to its anti-inflammatory effects in rat model of periodontitis. In conclusion, these novel findings indicate that baicalin inhibits the TLR2 and TLR4 expression and the downstream signaling and mitigates inflammatory responses and the alveolar bone loss in rat experimental periodontitis. Therefore, baicalin may be a potential therapeutic agent for treatment of periodontitis.