Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells

doi:10.21203/rs.3.rs-4205146/v1

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Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells

https://doi.org/10.21203/rs.3.rs-4205146/v1

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Journal Publication

published 07 Aug, 2024

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Background

The role of periodontal ligament stem cells (PDLSCs) in repairing periodontal destruction is crucial, but their functions can be impaired by excessive oxidative stress (OS). Nocardamine (NOCA), a cyclic siderophore, has been shown to possess anti-cancer and anti-bacterial properties. This study aimed to investigate the protective mechanisms of NOCA against OS-induced cellular dysfunction in PDLSCs.

Methods

The cytotoxicity of NOCA on PDLSCs was assessed using a CCK-8 assay. PDLSCs were then treated with hydrogen peroxide (H₂O₂) to induce OS. ROS levels, cell viability, and antioxidant factor expression were analyzed using relevant kits after treatment. Small molecule inhibitors U0126 and XAV-939 were employed to block ERK signaling and Wnt pathways respectively. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) activity staining and Alizarin Red S (ARS) staining of mineralized nodules. Expression levels of osteogenic gene markers and ERK pathway were determined via real-time quantitative polymerase chain reaction (RT-qPCR) or western blot (WB) analysis. β-catenin nuclear localization was examined by western blotting and confocal microscopy.

Results

NOCA exhibited no significant cytotoxicity at concentrations below 20 µM and effectively inhibited H₂O₂-induced OS in PDLSCs. NOCA also restored ALP activity, mineralized nodule formation, and the expression of osteogenic markers in H₂O₂-stimulated PDLSCs. Mechanistically, NOCA increased p-ERK level and promoted β-catenin translocation into the nucleus; however, blocking ERK pathway disrupted the osteogenic protection provided by NOCA and impaired its ability to induce β-catenin nuclear translocation under OS conditions in PDLSCs.

Conclusions

NOCA protected PDLSCs against H₂O₂-induced OS and effectively restored impaired osteogenic differentiation in PDLSCs by modulating the ERK/Wnt signaling pathway.

Periodontal destruction

Periodontal ligament stem cells

Oxidative stress

Nocardamine

Osteogenic differentiation

Periodontal tissue destruction, encompassing the periodontal ligament, alveolar bone, and gingiva, remains a significant health concern(1, 2). Excessive oxidative stress (OS) is a key driver of this destruction, implicated in major risk factors like chronic inflammation in periodontitis, hyperglycemia in diabetes, and mitochondrial dysfunction in senescence(3, 4). During OS progression, reactive oxygen species (ROS) skyrocket, disrupting the delicate balance within the endogenous antioxidant system(5). This OS onslaught impairs vital functions of periodontal ligament stem cells (PDLSCs), vital players in maintaining and repairing periodontal tissue(6, 7). Consequently, mitigating this excessive OS response emerges as a crucial strategy to restore PDLSC function and halt periodontal tissue destruction.

Marine-derived natural products offer a treasure trove of unexplored bioactivities with potential therapeutic applications(8, 9). Nocardamine (NOCA), also called desferrioxamine E, is a group of siderophores that act as ferric ion chelating agents excreted by microorganisms during iron deficiency conditions(10). NOCA is synthesized by various microorganisms and has been successfully reported with anti-cancer and anti-pathogenic microorganism properties(11, 12). However, its antioxidative capabilities and underlying mechanisms in PDLSCs remain largely uncharted.

In this study, we obtained NOCA from a Dysidea sp. Marine Sponge and demonstrated its ability to repair cellular dysfunction mediated by OS in PDLSCs. However, the detailed antioxidative effects and related molecular mechanisms of NOCA have not been studied yet. Therefore, our aim was to investigate the protective mechanism of NOCA on PDLSC function under OS conditions to provide an experimental basis for clinical treatment of periodontal destruction.

Reagents

NOCA is obtained from a Dysidea sp. Marine Sponge. Collagenase type I, dispase, MEM Alpha (aMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (Grand Island, NY, USA). β-Glycerophosphate, dexamethasone, L-ascorbic acid, indomethacin, 3-Isobutyl-1-methylxanthine, Alizarin Red S, Oil Red O, Alcian-blue staining solution and XAV-939 were purchased from Sigma-Aldrich (St Louis, MO). Cluster of differentiation 73 (CD73)/FITC, CD90/PerCP-Cy™5.5, and CD45/FITC were purchased from BD Pharmingen (San Diego, CA). Alkaline phosphatase color development kit, Dihydroethidium, ROS assay kit, Superoxide Dismutase (SOD) assay kit, Catalase (CAT) assay kit, Bovine Serum Albumin (BSA), β-catenin antibody, Lamin B1 antibody, DAPI staining solution, and U0126 were purchased from Beyotime (Shanghai, China). Primary antibody against SIRT1, COL1A1, phospho-Akt (p-Akt), Akt, p-ERK, ERK and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). RUNX2 antibody were purchased from Santa Cruz Biotechnology (USA). In addition, other reagents are explicitly stated.

Cell culture

PDLSCs were isolated from molar teeth obtained with informed consent from three healthy human donors (16–24 years old). Briefly, freshly harvested periodontal ligament tissue was cleaned, minced, and incubated in a solution containing 3 mg/mL collagenase type I and 4 mg/mL dispase at 37°C for 1 hour. The cell-containing solution was then cultured in 75 cm² cell culture flasks using standard medium consisting of aMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin under 5% CO₂ at 37°C. Upon reaching approximately 80% confluence, the cells were subcultured. For all experiments described below, PDLSCs from passages 3 to 5 were utilized.

Characterization of PDLSCs

Isolated PDLSCs were characterized for their mesenchymal stem cell (MSC) markers before experimentation. This study protocol was approved by the by the Ethics Committee of Longgang Otolaryngology Hospital, Institute of Otolaryngology, and Shenzhen Key Laboratory of Otolaryngology (2021 − 0159). Flow cytometry confirmed their expression of MSC markers CD73 and CD90 while lacking the hematopoietic marker CD45. To validate their multilineage differentiation potential, PDLSCs were cultured in osteogenic, adipogenic, and chondrogenic induction media. Osteogenic differentiation was induced in standard medium supplemented with 10 mM β-Glycerophosphate, 100 nM dexamethasone, and 50 µg/mL L-ascorbic acid. Adipogenic differentiation was induced in standard medium supplemented with 1 mM dexamethasone, 100 µM indomethacin, 500 µM 3-Isobutyl-1-methylxanthine, and 50 µg/mL L-ascorbic acid. Chondrogenic differentiation was induced using the Chondrogenic Differentiation Bullet Kit (Lonza, Walkersville, MD, USA, catalog number PT-3003 and PT-4124) according to the manufacturer's instructions. The medium was changed twice weekly. After three weeks of induction, the cells were fixed with 4% paraformaldehyde and stained with Alizarin Red S (ARS) for osteogenesis, Oil Red O (ORO) for adipogenesis, and Alcian blue (AB) for chondrogenesis.

Cell viability assay

PDLSCs were seeded at a density of 1 × 10⁴ cells in 100 µL of standard medium per well in 96-well plates and incubated for 24 hours. Subsequently, the cells were exposed to various concentrations of NOCA (0 µM, 1.25 µM, 2.5 µM, 5 µM, 10 µM, and 20 µM) in fresh complete α-MEM for an additional 24 hours. For analysis of oxidative stress protection, cells were pretreated for 4 hours with either 5 µM or 10 µM NOCA or 10 µM Que (Guangzhou Institute for Drug Control, Guangzhou, China) in standard medium before stimulation with 150 µM H₂O₂ (Sigma-Aldrich, St. Louis, MO) for 24 hours in standard medium. Cell viability was then assessed using the CKK-8 assay. Briefly, 10 µl of CKK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well and incubated for 2 hours. Finally, absorbance measurements were obtained at 450 nm using the SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).

Intracellular ROS and antioxidation assay

Intracellular ROS levels were assessed using both 5 µM Dihydroethidium (DHE) and 10 µM 2',7'-dichlorofluorescein diacetate (DCFH). Briefly, cells were incubated with the respective dye at 37°C for 20 minutes in the dark and then analyzed. DHE fluorescence was visualized using a fluorescence microscope (Leica DMI3000 B) with excitation/emission wavelengths of 503/561 nm. DCFH fluorescence was measured by flow cytometry (BD FACSAriaTM II). To explore potential mechanisms of ROS modulation, Superoxide Dismutase (SOD) activity and Catalase (CAT) activity were determined using corresponding assay kits (Beyotime) according to the manufacturer's instructions.

Osteogenic differentiation assay

PDLSCs were cultured in osteogenic medium supplemented with 10 mM β-glycerophosphate, 100 nM dexamethasone, and 50 µg/mL L-ascorbic acid in standard medium. After 7 days and 14 days of culture, cells were fixed with 4% paraformaldehyde and assessed for osteogenic differentiation using Alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining, respectively. Images were acquired using a Sharp Corporation scanner and microscope. To further confirm osteogenic potential, the expression levels of Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), and Collagen type I alpha 1 chain (COL1A1) were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) using a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) and Western blot (WB) analysis using specific antibodies against RUNX2, ALP, and COL1A1.

RT-qPCR assay

Total RNA was extracted from PDLSC lysate using the Trizol reagent (Invitrogen, Carlsbad, CA). cDNA synthesis (1 µg) was then performed using the PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa, Tokyo, Japan) following the manufacturer's instructions. RT-qPCR was conducted using the SYBR® Premix Ex Taq TM II kit (TaKaRa) in a total reaction volume of 20 µL on a 7500 Real-Time PCR System. The specific primer sequences used for target gene amplification are listed in Table 1.

WB assay

Protein was extracted from cultured PDLSCs using either RIPA Lysis Buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) or the Nucleoprotein Extraction Kit (Sangon Biotech, Shanghai, China) and quantified by the BCA protein assay kit (Thermo Scientific, IL) to determine protein concentration. Subsequently, 40 µg of protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were then blocked with either 5% non-fat milk or 5% BSA for 1 hour at room temperature, followed by overnight incubation at 4°C with primary antibodies targeting specific proteins of interest. Afterward, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour, and finally visualized using PierceTM ECL Western Blotting Substrate (Thermo Scientific). The detection was performed using a Bio-Rad ChemiDocMP System, while quantification of the WB results was conducted utilizing ImageJ software.

Immunofluorescent staining

The cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 2% BSA for 1 hour. Afterward, they were incubated with β-catenin antibody at a dilution of 1:100 (or specify according to your experiment) overnight at 4°C. The next day, the cells were incubated with a fluorescently conjugated secondary antibody (Abcam, Cambridge, MA, USA) for 1 hour at room temperature. Nuclear staining was performed with DAPI staining solution for 10 minutes. Images were acquired using Laser Scanning Confocal Microscopy (Leica TCS SP5 II).

Statistical analysis

The statistical analysis results were reported as the mean ± standard error of the mean. Differences between each data set was assessed using the Student t test. Statistical significance was defined as P values < 0.05.

Characterization of PDLSCs

Flow cytometry analysis confirmed the isolated cells as mesenchymal stem cells, with 99.9% and 100% of cells positive for CD73 and CD90, respectively, and no detectable CD45 expression (Fig. 1A). Further characterization revealed their multipotent differentiation potential. When cultured in osteogenic medium, the cells formed mineralized nodules stained positively with ARS (Fig. 1B), displaying morphology consistent with bone matrix deposits. Adipogenic differentiation resulted in abundant lipid droplet formation, as evidenced by ORO staining (Fig. 1C). Chondrogenic induction led to the development of chondrocyte-like cells exhibiting blue-green staining with AB (Fig. 1D). These findings collectively provide strong evidence that the isolated cells possess multipotent differentiation potential, confirming their characteristics consistent with those of MSCs.

Effects of NOCA on cell viability

To assess the cytotoxicity of NOCA, PDLSCs were cultured with varying concentrations of NOCA for 24 hours. The results indicated that NOCA did not significantly impact cell viability at levels below 20 µM (Fig. 2). Therefore, subsequent experiments were conducted using concentrations lower than 20 µM.

NOCA attenuated H₂O₂-induced OS in PDLSCs

To investigate whether NOCA attenuated H₂O₂-induced oxidative stress in PDLSCs, we first evaluated ROS production using the DCFH and DHE assay. As expected, H₂O₂ treatment significantly increased ROS levels compared to control (p < 0.05). Pretreatment with either NOCA (5 µM and 10 µM) or Que (10 µM) reduced ROS levels, with NOCA at 10 µM decreasing ROS by 40% compared to H₂O₂ alone (p < 0.05) (Fig. 3A-D). We then assessed cell viability by CCK-8 assay and observed a significant decrease in viability after H₂O₂ incubation (p < 0.05). This reduction was dose-dependently reversed by NOCA pretreatment, with viability at 10 µM NOCA similar to control levels (p > 0.05) (Fig. 3E). Similarly, H₂O₂ significantly decreased SOD activity and CAT concentration by more than 30% compared to the control group (p < 0.05). NOCA treatment again provided dose-dependent protection, with 10 µM NOCA restoring SOD and CAT activity to near control levels (p > 0.05) (Fig. 3F, G). Finally, to explore a potential mechanism of NOCA's antioxidative effects, we analyzed the expression levels of Sirtuin 1 (SIRT1), a key transcription factor involved in antioxidant gene regulation(10). Notably, H₂O₂ treatment significantly suppressed both SIRT1 mRNA expression by 20% and SIRT1 protein levels by 50% compared to the control group (p < 0.05). However, pretreatment with increasing doses of NOCA dose-dependently restored SIRT1 levels to at least 80% of the control group (p < 0.05), as measured by RT-qPCR and WB (Fig. 3H-J). Collectively, these findings suggest that NOCA's ability to mitigate H₂O₂-induced OS in PDLSCs might be mediated, at least in part, through the upregulation of SIRT1 expression, leading to enhanced antioxidant activity and protection against cellular damage.

NOCA attenuated OS-impaired osteogenic differentiation in PDLSCs

To investigate whether NOCA could attenuate H₂O₂-induced impairment of osteogenic differentiation, PDLSCs were subjected to differentiation medium for 7 and 14 days following a 24-hour treatment with H₂O₂ and/or NOCA/Que. Osteogenic differentiation was assessed by ALP activity, calcium nodule formation, and the expression of key osteogenic markers. As expected, H₂O₂ treatment significantly reduced ALP activity, calcium nodule formation, and the expression of all three osteogenic markers (p < 0.05). Notably, both NOCA and Que improved ALP activity, with 10 µM NOCA to near control levels (p > 0.05). Similarly, NOCA/Que administration partially restored calcium nodule formation and increased the expression of osteogenic markers, suggesting their protective potential against H₂O₂-induced damage (Fig. 4). To elucidate the possible mechanisms underlying NOCA's protective effects, we further examined the activation of two well-known antioxidant pathways - Akt and ERK signaling pathways(13). Our findings revealed that after H₂O₂ treatment, p-Akt levels were increased by less than 20% compared to control (p < 0.05), while p-ERK levels were significantly decreased (p < 0.05). Notably, pretreatment with NOCA did not significantly alter p-Akt levels but partially restored p-ERK levels to near control (p < 0.05) during osteogenic differentiation in the presence of H₂O₂. These results suggest that NOCA's ability to mitigate H₂O₂-induced impairment of PDLSCs' osteogenic potential might be independently mediated through the ERK pathway, while bypassing the Akt signaling cascade (Fig. 5).

The ERK/Wnt pathway mediated the protective effects of NOCA on OS-impaired osteogenic differentiation in PDLSCs

To further elucidate the role of ERK signaling in NOCA's protective function, we pre-treated PDLSCs with the ERK inhibitor U0126 for 1 hour prior to subjecting them to NOCA and H₂O₂ treatment for 24 hours. This was followed by osteogenic differentiation induction for 7 and 14 days. Osteogenesis analysis revealed that 10 µM U0126 significantly decreased ALP activity and calcium nodule formation in NOCA-pretreated PDLSCs under H₂O₂-induced OS conditions (p < 0.05) (Fig. 6A, B). Additionally, RT-qPCR analysis showed downregulation of key osteogenic markers RUNX2, ALP, and COL1A1 upon U0126 treatment (Fig. 6C-H). As ERK is known to influence the Wnt signaling pathway, a downstream target of β-catenin in PDLSC osteogenesis(14), we investigated their potential crosstalk. Immunofluorescence analysis revealed that NOCA treatment significantly increased intranuclear β-catenin levels compared to H₂O₂ alone (p < 0.05). Notably, U0126 treatment significantly reduced nuclear β-catenin levels in both H₂O₂ and NOCA-treated PDLSCs (Fig. 7A-E). Furthermore, blocking the Wnt/β-catenin pathway using 5 µM XAV-939 significantly impaired osteogenesis potential in PDLSCs exposed to H₂O₂ and NOCA (Fig. 7F, G), thereby negating the protective effect exerted by NOCA on OS-induced impairment of osteogenic differentiation process. These findings collectively suggest that NOCA protects PDLSCs from OS-induced impairment of osteogenic differentiation through modulation of the ERK/Wnt signaling pathway, with β-catenin nuclear translocation playing a crucial role in this process.

The association between oxidative stress (OS) and periodontal tissue destruction has been reported(15). Dysregulation of the cellular redox balance, characterized by an imbalance between ROS production and antioxidant defenses, leads to cellular damage and dysfunction. This underscores the therapeutic potential of natural antioxidants, particularly those derived from marine organisms, in combating OS-related periodontal disease(16, 17). Here, we investigated the antioxidant effect and underlying mechanism of NOCA, a compound isolated from the marine sponge Dysidea sp., on H₂O₂-induced OS in human periodontal ligament stem cells (PDLSCs). Que, a known antioxidant drug, served as a reference control(18).

OS, characterized by an imbalance between ROS production and antioxidant defenses, significantly contributes to periodontal tissue destruction(19). We investigated the antioxidant effects of NOCA, a marine sponge-derived compound, in PDLSCs exposed to H₂O₂-induced OS. NOCA potently reduces ROS levels compared to H₂O₂-treated controls. This ROS scavenging ability is further bolstered by upregulation of key antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). NOCA treatment increases SOD activity and CAT activity, effectively neutralizing superoxide anions and hydrogen peroxide, respectively. SIRT1, a nicotinamide adenine dinucleotide-dependent deacetylase, is involved in OS response by reducing ROS levels and regulating several antioxidant genes, such as SOD and CAT(20, 21). Previous studies reported that SIRT1 activation for repairing OS-induced cellular dysfunction involves multiple defense mechanisms(22). Our studies demonstrated that NOCA reversed the impaired expression of SIRT1 in PDLSCs caused by H₂O₂ in PDLSCs. Therefore, we concluded that NOCA could reduce H₂O₂-induced OS by enhanced the expression of antioxidant factors.

While most studies exploring OS damage to PDLSCs focus on cell viability and osteogenic potential, both vital factors in periodontal tissue regeneration(3), the underlying mechanisms remain incompletely understood. Counteracting excessive OS is crucial for restoring H₂O₂-induced damage to cell viability and osteogenic differentiation in PDLSCs(3). Prior studies have demonstrated that manipulating key regulators like Tripartite Motif 16 or recombinant Klotho protein can alleviate OS, thereby rescuing PDLSC function(21, 22). Our study builds upon these findings by revealing that NOCA, a marine sponge-derived compound, not only enhances cell viability but also promotes osteogenic differentiation of PDLSCs specifically under H₂O₂-induced OS. Notably, NOCA exhibits no effect on osteogenic potential under normal conditions (data not shown), suggesting its targeted action against OS-mediated dysfunction.

While the Akt signaling pathway plays a crucial role in antioxidant defense by stimulating the Nrf2 pathway(21), our data suggest NOCA's protective effect in PDLSCs is mediated primarily through the ERK signaling cascade. Pretreatment with NOCA showed no significant impact on Akt signaling, but interestingly, it significantly upregulated p-ERK, the active form of the ERK pathway. This finding aligns with previous reports demonstrating that activation of p-ERK alleviates H₂O₂-induced damage in PDLSCs(23, 24). Moreover, our study further corroborates this link by showing that H₂O₂ suppressed p-ERK levels, while NOCA pretreatment reversed this suppression, restoring p-ERK activity. Notably, blocking the ERK pathway using U0126 abolished NOCA's protective effect on osteogenic differentiation, underscoring the critical role of ERK signaling in NOCA's mechanism of action. Therefore, our findings suggest that NOCA primarily modulates the ERK signaling pathway, rather than Akt, to counteract OS-induced impairment of osteogenic differentiation in PDLSCs. This specific targeting of the ERK pathway highlights a potentially unique mechanism of action for NOCA, warranting further investigation of its downstream targets and potential therapeutic applications.

The Wnt/β-catenin signaling pathway plays a vital role in protecting against oxidative damage, particularly in PDLSCs. H₂O₂ disrupts this pathway by suppressing β-catenin, leading to impaired survival and bone formation capabilities(25). Conversely, activating the Wnt/β-catenin pathway safeguards PDLSCs from these detrimental effects(26). Notably, this pathway is downstream of ERK signaling and crucial for MSC proliferation, differentiation, and bone tissue homeostasis(13). Our study reveals NOCA's potent protective effect against H₂O₂-induced damage in PDLSCs through its modulation of the ERK/Wnt axis: (a) NOCA promotes its nuclear translocation under oxidative stress. (b) Blocking the Wnt pathway significantly diminishes NOCA's protective effect on PDLSC osteogenic differentiation under H₂O₂-induced OS. (c) ERK pathway inhibition also prevents NOCA from increasing nuclear β-catenin levels. These findings demonstrate that NOCA's ability to improve osteogenic potential in PDLSCs under OS hinges primarily on its preservation of the ERK/Wnt signaling pathway. This targeted action presents a unique mechanism of action and highlights NOCA's potential as a promising therapeutic candidate for alleviating oxidative stress-related periodontal diseases.

Despite these significant in vitro findings, the translational potential of NOCA as a therapeutic agent for oxidative stress-related periodontal diseases cannot be fully ascertained without in vivo studies. In animal models, factors such as bioavailability, pharmacokinetics, and the interaction of NOCA with the host's immune system and other physiological processes can be assessed. These are critical considerations for the development of NOCA as a clinical therapeutic.

In conclusion, our study demonstrates that NOCA, a marine sponge-derived compound, effectively counteracts H₂O₂-induced oxidative stress in PDLSCs, significantly restoring their impaired osteogenic potential. This protective effect appears to be mediated, at least in part, by NOCA's ability to preserve the ERK/Wnt signaling pathway, a key regulator of cell survival and differentiation. These findings suggest NOCA's potential as a promising natural therapeutic agent for alleviating periodontal tissue destruction associated with OS. However, further in vivo studies are necessary to validate NOCA's efficacy and safety in a physiological context and fully elucidate its therapeutic potential for clinical applications. Our future research will focus on these critical next steps.

Ethics approval and consent to participate

The experimental protocol of the study titled with “Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells” was approved by the Ethics Committee of Longgang Otolaryngology Hospital, Institute of Otolaryngology, and Shenzhen Key Laboratory of Otolaryngology (2021-0159) on Sep. 27, 2027.

Consent for publication

Consent to participate/consent for publication is not applicable to this study.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This work was supported by grants Natural Science Foundation of China (No. 81973915, 82004046 and 82204743), Guangdong Basic and Applied Basic Research Foundation (2023A1515012207 and 2022A1515111103), Shenzhen Innovation of Science and Technology Commission (No. JCYJ20200109144625016, JCYJ20210324142207019 and JCYJ20220531091602005), Longgang District Science and Technology Plan (No. LGKCYLWS2022010, LGKCYLWS2022003 and LWGJ2022-110), and Shenzhen Key Medical Discipline Construction Fund (No. SZXK039).

Acknowledgements

We acknowledge that the cell-based research of this study was performed at Shenzhen Key Laboratory of Otolaryngology and Shenzhen Institute of Otolaryngology.

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Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells

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Journal Publication

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Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and Methods

Reagents

Cell culture

Characterization of PDLSCs

Cell viability assay

Intracellular ROS and antioxidation assay

Osteogenic differentiation assay

RT-qPCR assay

WB assay

Immunofluorescent staining

Statistical analysis

Results

Characterization of PDLSCs

Effects of NOCA on cell viability

NOCA attenuated H₂O₂-induced OS in PDLSCs

NOCA attenuated OS-impaired osteogenic differentiation in PDLSCs

The ERK/Wnt pathway mediated the protective effects of NOCA on OS-impaired osteogenic differentiation in PDLSCs

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and Methods

Reagents

Cell culture

Characterization of PDLSCs

Cell viability assay

Intracellular ROS and antioxidation assay

Osteogenic differentiation assay

RT-qPCR assay

WB assay

Immunofluorescent staining

Statistical analysis

Results

Characterization of PDLSCs

Effects of NOCA on cell viability

NOCA attenuated H2O2-induced OS in PDLSCs

NOCA attenuated OS-impaired osteogenic differentiation in PDLSCs

The ERK/Wnt pathway mediated the protective effects of NOCA on OS-impaired osteogenic differentiation in PDLSCs

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

NOCA attenuated H₂O₂-induced OS in PDLSCs