RESUMO
Cellular senescence severely limits the research and the application of dental pulp stem cells (DPSCs). A previous study conducted by our research group revealed a close implication of ROR2 in DPSC senescence, although the mechanism underlying the regulation of ROR2 in DPSCs remains poorly understood so far. In the present study, it was revealed that the expression of the ROR2-interacting transcription factor MSX2 was increased in aging DPSCs. It was demonstrated that the depletion of MSX2 inhibits the senescence of DPSCs and restores their self-renewal capacity, and the simultaneous overexpression of ROR2 enhanced this effect. Moreover, MSX2 knockdown suppressed the transcription of NOP2/Sun domain family member 2 (NSUN2), which regulates the expression of p21 by binding to and causing the 5-methylcytidine methylation of the 3'- untranslated region of p21 mRNA. Interestingly, ROR2 downregulation elevated the levels of MSX2 protein, and not the MSX2 mRNA expression, by reducing the phosphorylation level of MSX2 and inhibiting the RNF34-mediated MSX2 ubiquitination degradation. The results of the present study demonstrated the vital role of the ROR2/MSX2/NSUN2 axis in the regulation of DPSC senescence, thereby revealing a potential target for antagonizing DPSC aging.
Assuntos
Senescência Celular , Polpa Dentária , Senescência Celular/genética , Polpa Dentária/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Miller syndrome is a rare type of postaxial acrofacial dysostosis caused by biallelic mutations in the DHODH gene, which is characterized mainly by craniofacial malformations of micrognathia, orofacial clefts, cup-shaped ears, and malar hypoplasia, combined with postaxial limb deformities like the absence of fifth digits. METHODS: In this study, a prenatal case with multiple orofacial-limb abnormities was enrolled, and a thorough clinical and imaging examination was performed. Subsequently, genetic detection with karyotyping, chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) was carried out. In vitro splicing analysis was also conducted to clarify the impact of one novel variant. RESULTS: The affected fetus displayed typical manifestations of Miller syndrome, and WES identified a diagnostic compound heterozygous variation in DHODH, consisting of two variants: exon(1-3)del and c.819 + 5G > A. We conducted a further in vitro validation with minigene system, and the result indicated that the c.819 + 5G > A variant would lead to an exon skipping in mRNA splicing. CONCLUSIONS: These findings provided with the first exonic deletion and first splice site variant in DHODH, which expanded the mutation spectrum of Miller syndrome and offered reliable evidence for genetic counseling to the affected family.
Assuntos
Fenda Labial , Fissura Palatina , Di-Hidro-Orotato Desidrogenase , Micrognatismo , Feminino , Humanos , Gravidez , Di-Hidro-Orotato Desidrogenase/genética , População do Leste Asiático , Micrognatismo/genética , Diagnóstico Pré-NatalRESUMO
Background: Naringin is a naturally occurring flavanone that promotes osteogenesis. Owing to the high lipophilicity, poor in vivo bioavailability, and extensive metabolic alteration upon administration, the clinical efficacy of naringin is understudied. Additionally, information on the molecular mechanism by which it promotes osteogenesis is limited. Methods: In this study, we prepared TAT & RGD peptide-modified naringin-loaded nanoparticles (TAT-RGD-NAR-NPs), evaluated their potency on the osteogenic differentiation of human dental pulp stem cells (hDPSCs), and studied its mechanism of action through metabolomic analysis. Results: The particle size and zeta potential of TAT-RGD-NAR-NPs were 160.70±2.05 mm and -20.77±0.47mV, respectively. The result of cell uptake assay showed that TAT-RGD-NAR-NPs could effectively enter hDPSCs. TAT-RGD-NAR-NPs had a more significant effect on cell proliferation and osteogenic differentiation promotion. Furthermore, in metabolomic analysis, naringin particles showed a strong influence on the glycerophospholipid metabolism pathway of hDPSCs. Specifically, it upregulated the expression of PLA2G3 and PLA2G1B (two isozymes of phospholipase A2, PLA2), increased the biosynthesis of lysophosphatidic acid (LPA). Conclusion: These results suggested that TAT-RGD-NPs might be used for transporting naringin to hDPSCs for modulating stem cell osteogenic differentiation. The metabolomic analysis was used for the first time to elucidate the mechanism by which naringin promotes hDPSCs osteogenesis by upregulating PLA2G3 and PLA2G1B.
Assuntos
Flavanonas , Nanopartículas , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Polpa Dentária , Flavanonas/farmacologia , Produtos do Gene tat/genética , Fosfolipases A2 do Grupo IB/metabolismo , Fosfolipases A2 do Grupo III/metabolismo , Humanos , Lipossomos , Oligopeptídeos/metabolismo , Osteogênese , Células-TroncoRESUMO
Background: Bone tissue defect, one of the common orthopaedicdiseases, is traumatizing and affects patient's lifestyle. Although autologous and xenograft bone transplantations are performed in bone tissue engineering, clinical development of bone transplantation is limited because ofvarious factors, such as varying degrees of immune rejection, lack of bone sources, and secondary damage to bone harvesting. Methods: We synthesised a heparinised gelatine-hydroxyapatite-tricalcium phosphate (HG-HA-TCP) scaffold loaded with sustained-release vascular endothelial growth factor (VEGF) analysed their structure, mechanical properties, and biocompatibility. Additionally, the effects of HG-HA-TCP (VEGF) scaffolds on osteogenic differentiation and vascularisation of stem cells from human exfoliated deciduous teeth (SHED) in vitro and bone regeneration in vivo were investigated. Results: HG-HA-TCP scaffold possessed good pore structure, mechanical properties, and biocompatibility. HG-HA-TCP scaffold loaded with VEGF could effectively promote SHED proliferation, migration, and adhesion. Moreover, HG-HA-TCP (VEGF) scaffold increased the expression of osteogenesis- and angiogenesis-related genes and promoted osteogenic differentiation and vascularisation in cells. In vivo results demonstrated that VEGF-loaded HG-HA-TCP scaffold improved new bone regeneration and enhanced bone mineral density, revealed byhistological, micro-CT and histochemical straining analyses. Osteogenic and angiogenic abilities of the three biological scaffolds wereranked as follows: HG-HA-TCP (VEGF) > G-HA-TCP (VEGF) > G-HA-TCP. Conclusion: HG-HA-TCP (VEGF) scaffold with good biocompatibility could create an encouraging osteogenic microenvironment that could accelerate vessel formation and osteogenesis, providing an effective scaffold for bone tissue engineering and developing new clinical treatment strategies for bone tissue defects.
RESUMO
Dental pulp stem cells (DPSCs) play a vital role in tooth restoration, regeneration, and homeostasis. The link between DPSC senescence and tooth aging has been well-recognized. ROR2 plays an important role in aging-related gene expression. However, the expression and function of ROR2 in DPSC aging remain largely unknown. In this study, we found that ROR2 expression was significantly decreased in aged pulp tissues and DPSCs. The depletion of ROR2 in young DPSCs inhibits their self-renewal capacity, while its overexpression in aged DPSCs restores their self-renewal capacity. Interestingly, we found that sphingomyelin (SM) is involved in the senescence of DPSCs regulated by ROR2. Mechanistically, we confirmed that ROR2 inhibited the phosphorylation of STK4, which promoted the translocation of Forkhead Box O1 (FOXO1) to the nucleus. STK4 inhibition or knockdown of FOXO1 markedly increased the proliferation of DPSCs and upregulated the expression of SMS1, which catalyzed SM biogenesis. Moreover, FOXO1 directly bound to the SMS1 promoter, repressing its transcription. Our findings demonstrated the critical role of the ROR2/STK4-FOXO1/SMS1 axis in the regulation of SM biogenesis and DPSC senescence, providing a novel target for antagonizing tooth aging.
Assuntos
Polpa Dentária/metabolismo , Proteína Forkhead Box O1/antagonistas & inibidores , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Esfingomielinas/biossíntese , Células-Tronco/metabolismo , Regulação para Baixo , HumanosRESUMO
BACKGROUND: Antimicrobial peptides (AMP), as a small molecular polypeptide with a broad antibacterial spectrum and high efficiency, have attracted more and more attention. Few pieces of research on the effect of the antimicrobial peptide on osteoblast under inflammatory conditions have so far been reported. The main aim of this work was to investigate the antiapoptosis effect of the antimicrobial peptide on MC3T3-E1 cells induced by TNF-α and its related mechanism. METHODS: Rat MC3T3-E1 cells were co-cultured with different concentrations of antibacterial peptide DP7 and TNF-α.MTS assay, cell scratch test, alkaline phosphatase activity, and alizarin red staining assay were used to determine osteoblast viability in this experiment. Annexin V-FITC/PI double staining cells and flow cytometry were used to analyze apoptosis and Western blot assay detection to show mitogen-activated protein kinase (MAPK) protein expression in rat MC3T3-E1 cells. Then, Realtime polymerase chain reaction (PCR) was used to examine the caspase-3 gene expression. Also, ELISA detection was used to clarify the anti-apoptotic effect of the p38 MAPK inhibitor, SB203580, on cells' apoptosis. RESULTS: Antimicrobial peptide could promote the proliferation, migration, and osteogenic ability of MC3T3-E1 cells induced by TNF-α, but inhibit cell apoptosis rate (P<0.05), and the effect was concentration-dependent. Western blot results showed after TNF-αtreatment, the expression of p-p38 MAPK in the MC3T3-E1 cells increased after TNF-α and antimicrobial peptide cotreatment, TNF-α induced p-p38 MAPK phosphorylation was inhibited, and the difference was statistically significant (P<0.05). Realtime PCR results showed that the gene expression of caspase-3 mRNA was up-regulated after TNF-α treatment, while their expression was down-regulated after cultured with TNF-α and antimicrobial peptide. Elisa's analysis showed that cell apoptosis increased after TNF-α treatment alone, and cell apoptosis was reduced to the normal levels when combined with antimicrobial peptide, and cell apoptosis induced by TNF-α was partially abolished when combined with SB203580. CONCLUSIONS: Antimicrobial peptide DP7 could inhibit MC3T3-E1 cells apoptosis induced by TNF-α, and the effect was concentration-dependent. The antiapoptosis activation of the antimicrobial peptide on MC3TE-E1 cells may be related to the inhibition of the p38 MAPK pathway.
RESUMO
BACKGROUND: Intratumoral injection is a palliative treatment that aims at further improvement in the survival and quality of life of patients with advanced or recurrent carcinomas, or cancer patients with severe comorbidities or those with a poor performance status. METHODS: In this study, a solvent-injection method was used to prepare paclitaxel-cholesterol complex-loaded lecithin-chitosan nanoparticles (PTX-CH-loaded LCS_NPs) for intratumoral injection therapy, and the physicochemical properties of NPs were well characterized. RESULTS: The particle size and zeta potential of PTX-CH-loaded LCS_NPs were 142.83±0.25 nm and 13.50±0.20 mV, respectively. Release behavior of PTX from PTX-CH-loaded LCS_NPs showed a pH-sensitive pattern. The result of cell uptake assay showed that PTX-CH-loaded LCS_NPs could effectively enter cells via the energy-dependent caveolae-mediated endocytosis and macropinocytosis in company with the Golgi apparatus. Meanwhile, PTX-CH-loaded LCS_NPs had a better ability to induce cell apoptosis than PTX solution. The in vivo antitumor results suggested that PTX-CH-loaded LCS_NPs effectively inhibited mouse mammary cancer growth and metastasis to distant organs and significantly improved the survival rate of tumor-bearing mice by intratumoral administration. CONCLUSION: In general, our study demonstrated that PTX-CH-loaded LCS_NPs used for palliative treatment by intratumoral injection showed improved safety and antitumor efficacy, which provided an alternative approach in the field of palliative chemotherapy.