PIEZO1 Ion Channels Mediate Mechanotransduction in Odontoblasts.

INTRODUCTION: Odontoblasts, terminally differentiated dentin-forming cells with their processes that penetrate into dentin, have been considered potential sensory cells. Current research suggests that odontoblasts sense external stimuli and transmit pain signals. PIEZO1, as a specific mechanically activated ion channel, may play an important role in mechanical transduction in odontoblasts. In this study, we devoted to investigating the functions and underlying molecular mechanisms of PIEZO1 ion channels in odontoblast mechanotransduction. METHODS: Human dental pulp stem cells were cultured in vitro and induced to differentiate into odontoblast-like cells (OLCs). The expression of PIEZO1 protein in pulp, dental pulp stem cells, and OLCs was detected by immunohistochemistry or immunofluorescence. The mechanical sensitivity of OLCs was detected by a constructed fluid shear stress model and examined by calcium fluorescence intensity. A single-cell mechanical stimulation model was used to detect the PIEZO1 electrophysiological properties of OLCs. Yoda1 (a PIEZO1-specific agonist), GsMTx4 (a PIEZO1 antagonist), and non-calcium ion extracellular solution were utilized to confirm PIEZO1 mechanotransduction in OLCs in both fluid shear stress and single-cell mechanical stimulation assays. The amount of ATP released by OLCs was measured under stimulation with Yoda1 and GsMTx4. Rat trigeminal ganglion neurons were cultured in vitro and detected by whole-cell patch-clamp recording under ATP stimulation. RESULTS: PIEZO1 ion channels were positively expressed in OLCs and odontoblastic bodies and processes but weakly expressed in dental pulp cells. After the treatment of OLCs with shearing stress or Yoda1, the fluorescence intensity of intracellular calcium ions increased rapidly but did not noticeably change after treatment with GsMTx4 or the non-calcium ion extracellular solution. When single-cell mechanical stimuli were applied to OLCs, the evoked inward currents were recorded by patch-clamp electrophysiology. The inward currents increased and current inactivation became slower after Yoda1 treatment, but these currents almost completely disappeared after the addition of GsMTx4. The amount of ATP released by OLCs increased significantly after Yoda1 stimulation, while GsMTx4 reversed the release of ATP. Whole-cell patch-clamp detection showed that ATP evoked slow inward currents and increased the frequency of action potentials of trigeminal ganglion neurons. CONCLUSIONS: Taken together, these findings indicated that odontoblasts evoked a fast inward current via PIEZO1 ion channels after the application of external mechanical stimuli and released ATP to transmit signals to adjacent cells. Thus, PIEZO1 ion channels in odontoblasts mediate mechanotransduction under various pathophysiological conditions in dentin.

RNA-seq analysis of palatal transcriptome changes in all-trans retinoic acid-induced cleft palate of mice.

Cleft palate is a common congenital maxillofacial malformation in newborns. All-trans retinoic acid (atRA) is an ideal exogenous stimulus to construct a mouse cleft palate model. However, the precise pathogenic mechanism remains to be elucidated. In our study, to explore the toxicity of atRA on palatal shelves during different stages of palate development, a total of 100 mg/kg atRA was administered to C57BL/6 mice at embryonic day 10.5 (E10.5). Mouse embryonic palatal shelves at E13.5, E14.5, E15.5, and E16.5 were collected for RNA extraction and histological treatment. Changes in gene expression were tested through RNA-seq. Selected differentially expressed genes (DEGs) related to metabolic pathways, such as Ptgds, Ttr, Cyp2g1, Ugt2a1 and Mgst3, were validated and analyzed by Quantitative real-time PCR (qRT-PCR). In addition, Gene Oncology analysis showed that transcriptional changes of genes from extracellular matrix (ECM) components, such as Spp1, and crystallin family might play important role in palatal shelves elevation (E13.5-E14.5). Therefore, the protein expression level of Ttr and Spp1 from E13.5 to E16.5 were tested by immunohistochemistry (IHC). Besides, the mRNA level of Spp1, were down-regulated at E16.5 and the protein were down-regulated at E15.5 and E16.5 in all-trans retinoic acid group, suggesting that atRA may involve in palatal bone formation by regulating Spp1. Overall, gene transcriptional profiles were obviously different at each time point of palate development. Thus, this study summarized some pathways and genes that may be related to palatogenesis and cleft palate through RNA-seq, to provide a direction for subsequent studies on the mechanism and targeted therapy of cleft palate.

[Effect of Yishenqinghuo compound on biological characteristics of rat bone marrow stromal cells].

OBJECTIVE: To evaluate the effect of Yishenqinghuo recipe on biological characteristic of rat bone marrow stromal cells (BMSCs) and search for the function and mechanism of this recipe in treatment of periodontitis. METHODS: 12- to 15-month-old SD rats were allocated into 5 groups. Group A: control group (with no periodontitis model, fed with the same dosage of saline as Group D); Group B: model group (with periodontitis model, fed with the same dosage of saline as Group D); Group C: high dosage group (with periodontitis model, fed with a high dosage of medicine); Group D: middle dosage group (with periodontitis model, fed with a middle dosage of medicine); and Group E: low dosage group (with periodontitis model, fed with a low dosage of medicine). Ten days later, serums were collected from all the five groups for in vitro cultivation of BMSCs. RESULTS: There was displayed a similarity in effect between the serum collected from Group C1 (serum collected 0.5 h after the last gavage of Group C) and that from Group A after the last gavage, the effect being the best in terms of proliferation of BMSCs. A comparison with the other groups had revealed a striking difference (P < 0.01), with Group B having turned out to be the worst. The serum from Group C2 (serum collected 1 h after the last gavage of Group C) after the last gavage could best enhance the generation and activity of ALP, having demonstrated a significant difference (P < 0.01) in comparison with the other groups. CONCLUSION: Yishenqinghuo recipe can improve the proliferation of BMSCs and facilitate the differentiation to osteoblast.

[Relation between the angiotensin II type 2 receptor polymorphism and essential hypertension in men].

OBJECTIVE: To identify single nucleotide polymorphisms (SNP) of the angiotensin II type 2 receptor (AGTR2) gene, and to determine whether the AGTR2 polymorphisms are associated with essential hypertension in a male Chinese population. METHODS: Direct DNA sequencing was performed in 20 subjects. 96 male hypertensive patients and 107 normal controls were included to assess the contribution of the SNP of AGTR2 gene to hypertension. RESULTS: Seven SNP of the AGTR2 gene were identified, of which 4 were reported for the first time. A case-control study including two polymorphisms (A1675G and T1334C) showed a significant increase in the A1675 allele frequency among male hypertensive subjects as compared with normotensive subjects (49.0% vs 34.6%, P < 0.05). CONCLUSION: The AGTR2 A1675G polymorphism might be involved in the development of essential hypertension in male Chinese.