{"paper_id":"0719e92a-c5c8-4f37-a400-fb8f61816399","body_text":"Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells Hai-Peng HE, Mei-Zhen ZHAO, Wei-Hua JIAO, Zhi-Qiang LIU, Xian-Hai ZENG, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4205146/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Aug, 2024 Read the published version in Stem Cell Research & Therapy → Version 1 posted 4 You are reading this latest preprint version Abstract 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 (H 2 O 2 ) 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 H 2 O 2 -induced OS in PDLSCs. NOCA also restored ALP activity, mineralized nodule formation, and the expression of osteogenic markers in H 2 O 2 -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 H 2 O 2 -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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction 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. Materials and Methods 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 cm 2 cell culture flasks using standard medium consisting of aMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin under 5% CO 2 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 × 10 4 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 H 2 O 2 (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. Results 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. 1 A). Further characterization revealed their multipotent differentiation potential. When cultured in osteogenic medium, the cells formed mineralized nodules stained positively with ARS (Fig. 1 B), displaying morphology consistent with bone matrix deposits. Adipogenic differentiation resulted in abundant lipid droplet formation, as evidenced by ORO staining (Fig. 1 C). Chondrogenic induction led to the development of chondrocyte-like cells exhibiting blue-green staining with AB (Fig. 1 D). 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 H 2 O 2 -induced OS in PDLSCs To investigate whether NOCA attenuated H 2 O 2 -induced oxidative stress in PDLSCs, we first evaluated ROS production using the DCFH and DHE assay. As expected, H 2 O 2 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 H 2 O 2 alone (p < 0.05) (Fig. 3 A-D). We then assessed cell viability by CCK-8 assay and observed a significant decrease in viability after H 2 O 2 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. 3 E). Similarly, H 2 O 2 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. 3 F, 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, H 2 O 2 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. 3 H-J). Collectively, these findings suggest that NOCA's ability to mitigate H 2 O 2 -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 H 2 O 2 -induced impairment of osteogenic differentiation, PDLSCs were subjected to differentiation medium for 7 and 14 days following a 24-hour treatment with H 2 O 2 and/or NOCA/Que. Osteogenic differentiation was assessed by ALP activity, calcium nodule formation, and the expression of key osteogenic markers. As expected, H 2 O 2 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 H 2 O 2 -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 H 2 O 2 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 H 2 O 2 . These results suggest that NOCA's ability to mitigate H 2 O 2 -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 H 2 O 2 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 H 2 O 2 -induced OS conditions (p < 0.05) (Fig. 6 A, B). Additionally, RT-qPCR analysis showed downregulation of key osteogenic markers RUNX2, ALP, and COL1A1 upon U0126 treatment (Fig. 6 C-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 H 2 O 2 alone (p < 0.05). Notably, U0126 treatment significantly reduced nuclear β-catenin levels in both H 2 O 2 and NOCA-treated PDLSCs (Fig. 7 A-E). Furthermore, blocking the Wnt/β-catenin pathway using 5 µM XAV-939 significantly impaired osteogenesis potential in PDLSCs exposed to H 2 O 2 and NOCA (Fig. 7 F, 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. Discussion 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 H 2 O 2 -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 H 2 O 2 -induced OS. NOCA potently reduces ROS levels compared to H 2 O 2 -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 H 2 O 2 in PDLSCs. Therefore, we concluded that NOCA could reduce H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 -induced damage in PDLSCs( 23 , 24 ). Moreover, our study further corroborates this link by showing that H 2 O 2 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. H 2 O 2 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 H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 -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. Declarations 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). 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Supplementary Files 20240406Supplementarydata1Primersequences.docx 20240406Supplementarydata2westernblotuncropedgel.doc 20240406Supplementarydata3statisticalanalysis.docx AuthorChecklistFull.pdf Cite Share Download PDF Status: Published Journal Publication published 07 Aug, 2024 Read the published version in Stem Cell Research & Therapy → Version 1 posted Reviewers agreed at journal 11 Apr, 2024 Reviewers invited by journal 11 Apr, 2024 Editor assigned by journal 07 Apr, 2024 First submitted to journal 06 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4205146\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":290032184,\"identity\":\"5d6a1245-3921-4de7-b649-6307edc26057\",\"order_by\":0,\"name\":\"Hai-Peng HE\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Longgang Central Hospital of Shenzhen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hai-Peng\",\"middleName\":\"\",\"lastName\":\"HE\",\"suffix\":\"\"},{\"id\":290032185,\"identity\":\"afbd9c3a-d264-4dee-8758-2ab5ddf9cec8\",\"order_by\":1,\"name\":\"Mei-Zhen ZHAO\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Longgang Central Hospital of Shenzhen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mei-Zhen\",\"middleName\":\"\",\"lastName\":\"ZHAO\",\"suffix\":\"\"},{\"id\":290032186,\"identity\":\"814702e2-39c8-4f73-9995-b8305f2b2506\",\"order_by\":2,\"name\":\"Wei-Hua JIAO\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shanghai Jiaotong University: Shanghai Jiao Tong University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wei-Hua\",\"middleName\":\"\",\"lastName\":\"JIAO\",\"suffix\":\"\"},{\"id\":290032187,\"identity\":\"22903931-5b77-448a-88e8-7aed3e167f90\",\"order_by\":3,\"name\":\"Zhi-Qiang LIU\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Longgang Central Hospital of Shenzhen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhi-Qiang\",\"middleName\":\"\",\"lastName\":\"LIU\",\"suffix\":\"\"},{\"id\":290032188,\"identity\":\"82d5b4a2-1195-4e01-8b70-210c18305e78\",\"order_by\":4,\"name\":\"Xian-Hai ZENG\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Longgang Central Hospital of Shenzhen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xian-Hai\",\"middleName\":\"\",\"lastName\":\"ZENG\",\"suffix\":\"\"},{\"id\":290032189,\"identity\":\"0891c5dc-0028-41d4-a188-b0b93de0ccd0\",\"order_by\":5,\"name\":\"Quan-Li LI\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Longgang Central Hospital of Shenzhen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Quan-Li\",\"middleName\":\"\",\"lastName\":\"LI\",\"suffix\":\"\"},{\"id\":290032190,\"identity\":\"a5b8913a-29d2-403d-a878-e4df8b40295a\",\"order_by\":6,\"name\":\"Tian-Yong HU\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Longgang Central Hospital of Shenzhen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tian-Yong\",\"middleName\":\"\",\"lastName\":\"HU\",\"suffix\":\"\"},{\"id\":290032191,\"identity\":\"7db7987d-c2c1-4d1c-95d3-2cf92f9eba79\",\"order_by\":7,\"name\":\"Bao-Hui CHENG\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACCSBmbGBgMGBgbHwAJEnT0mxAqhYGNgmi3MU/u/nYw6877OTMJZLbKn8U3JFnYD98dANeS+4cSzeWPZNsbDkjse02j8EzwwaetLQb+LQYSOSYSUu2MSduuAHUwmBwmLFBgseMgJb8b0At9WAthT8MDtsToSWHTfJj22GwFgYeg8OJBLVI3Egzk2Y8c9zY4MzDZmmgluQ2Qn7hn5H8TPLnjmo5g+PpDz/++HPYtp/98DG8WkCAmQeZx0ZIOQgw/iBG1SgYBaNgFIxcAAAcqUzwBpc8+QAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0000-0002-5065-9036\",\"institution\":\"Longgang Central Hospital of Shenzhen\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Bao-Hui\",\"middleName\":\"\",\"lastName\":\"CHENG\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-04-02 08:42:52\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4205146/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4205146/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1186/s13287-024-03812-2\",\"type\":\"published\",\"date\":\"2024-08-07T15:57:31+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":54853777,\"identity\":\"c07422d7-54bc-4cea-8d7e-54022b4117cb\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:29\",\"extension\":\"jpeg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":162450,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCharacterization of PDLSCs. (A) Flow cytometry analysis was performed to evaluate the expression of MSCs-related phenotypic markers, including CD73, CD90, and CD45. (B) Mineralized nodules were stained using ARS staining solution to assess osteogenic differentiation after 3 weeks of induction. (C) Lipid droplets were visualized by ORO staining to examine adipogenic differentiation after 3 weeks of induction. (D) Chondrocyte-like cells were identified through AB staining to investigate chondrogenic differentiation after 3 weeks of induction. (Scale bar = 100 µm).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/828521613c3014a93703b7d3.jpeg\"},{\"id\":54853770,\"identity\":\"a3d5b820-69ed-4f72-890e-3e1c0dba4e90\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:29\",\"extension\":\"jpeg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":36979,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFollowing treatment with various concentrations of NOCA ranging from 0 µM to 20 µM for a duration of 24 hours, cell viability was assessed using the CKK-8 assay. (A) Cytotoxic effects of NOCA on PDLSCs. (B) Chemical structure of NOCA. *P \\u0026lt; 0.05 vs NOCA 0 μM treated group, n = 3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/902941d2ce274d1081c2362b.jpeg\"},{\"id\":54853987,\"identity\":\"000c2e5d-dffd-468a-96a2-e68f29fc615a\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:29:29\",\"extension\":\"jpeg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":148274,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNOCA attenuated H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced OS in PDLSCs. PDLSCs were pretreated with NOCA or Que for 4 hours, followed by stimulation with 150 µM H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e for 24 hours. The cells were labeled with DHE or DCFH for 20 min. Standard medium served as a control group (CTL), and Que was chosen as a reference control. (A, C) ROS levels were visualized using fluorescence microscopy (Scale bar = 50 µm). (B, D) Flow cytometry was employed to detect ROS levels. (E) Cell viability assay, (F) SOD activity, and (G) CAT activity were measured using relevant kits. (H) SIRT1 mRNA expression was analyzed through RT-qPCR analysis. (I) SIRT1 protein expression evaluated via WB. (J) Relative band density of (I). *P \\u0026lt; 0.05, **P \\u0026lt; 0.01, ***P \\u0026lt; 0.001 vs NOCA 0 μM treated group, n = 3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/fa59db34b1fd4965a87838a2.jpeg\"},{\"id\":54853775,\"identity\":\"3fd7a766-c061-419e-b7b5-0398f0cd820f\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:29\",\"extension\":\"jpeg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":362955,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNOCA attenuated OS-impaired osteogenic differentiation in PDLSCs. After treatment with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and NOCA using a similar methodology as described above, the cells were subsequently induced in an osteogenic medium. Osteogenic media served as a control group (CTL), while Que was chosen as a reference control. (A) Images of ALP activity staining, and (B) ARS staining were acquired using a scanner and microscope (Scale bar = 100 µm). (C-E) mRNA expression levels of RUNX2, ALP, and COL1A1 were analyzed by RT-qPCR. (F) The protein expression levels of COL1A1 and RUNX2 were analyzed by WB. (G, H) Relative band density of (F). *P \\u0026lt; 0.05, **P \\u0026lt; 0.01, ***P \\u0026lt; 0.001 vs NOCA 0 μM treated group, n = 3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/452a38799843a627b3e0bce9.jpeg\"},{\"id\":54853782,\"identity\":\"428f3ddd-4f3e-4e59-9be6-60377a62ec50\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:29\",\"extension\":\"jpeg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":90904,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffects of NOCA on the p-Akt and p-ERK in PDLSCs under OS. (A) The protein levels of p-Akt and Akt. (B) Relative band density of (A). (C) The protein levels of p-ERK and ERK. (D) Relative band density of (C). *P \\u0026lt; 0.05, **P \\u0026lt; 0.01 vs NOCA 0 μM treated group, n = 3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/6cf6bae21b84561c2b5a9aad.jpeg\"},{\"id\":54853988,\"identity\":\"a3fdc4ab-3532-4705-8337-86ca4393e227\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:29:30\",\"extension\":\"jpeg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":400890,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe impact of the ERK pathway on the protection of NOCA in OS-induced impairment of osteogenic differentiation in PDLSCs. Prior to treatment with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and NOCA for 24 h using a similar method as described above, PDLSCs was pre-treated with 10 μM U0126 for 1 h. Subsequently, the cells were induced in an osteogenic medium. Osteogenic media served as a control group (CTL). (A) ALP activity staining images, and (B) ARS staining images were acquired using a scanner and microscope (Scale bar = 100 µm). (C-E) The mRNA expression levels of RUNX2, ALP, and COL1A1 were analyzed by RT-qPCR. (F) The protein expression levels of COL1A1 and RUNX2 were examined by WB analysis. (G, H) Relative band density of (F). *P \\u0026lt; 0.05, **P \\u0026lt; 0.01, ***P \\u0026lt; 0.001 vs NOCA 0 μM treated group, n = 3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image6.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/8bc4eb3d14faa815c7a6f335.jpeg\"},{\"id\":54853783,\"identity\":\"7ba238b6-ab86-4adf-9318-b45deaed55e5\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:29\",\"extension\":\"jpeg\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":189463,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe Wnt/β-catenin pathway serves as the downstream target of ERK pathway. After treatment with U0126, H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and NOCA using a similar methodology as described above, the cells were cultured in osteogenic medium supplemented with or without 5 μM XAV-939. (A) The protein expression levels of p-ERK and ERK. (B) Relative band density of (A). (C) Confocal microscopy was employe to detect the results of immunofluorescence staining of β-catenin (Scale bar = 20 µm). (D) The protein levels of β-catenin. (E) Relative band density of (D). (F) ALP activity staining images, and (E) ARS staining images were acquired using a microscope (Scale bar = 100 µm). *P \\u0026lt; 0.05, **P \\u0026lt; 0.01 vs NOCA 0 μM treated group, n = 3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image7.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/dcb1851ef96f63f02788f093.jpeg\"},{\"id\":54853784,\"identity\":\"1a2eba30-5e4d-4cf7-97dc-dc1cd911248b\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:30\",\"extension\":\"jpeg\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":104523,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNOCA alleviates H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced oxidative stress and restores the damaged osteogenic potential of PDLSCs through preserved ERK/Wnt signaling pathway.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image8.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/7f931947cd0a4b5f3f5bb5b3.jpeg\"},{\"id\":62298814,\"identity\":\"af43269d-d438-4af8-b26d-c42d82360c6d\",\"added_by\":\"auto\",\"created_at\":\"2024-08-12 16:16:50\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2056839,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/ddd11b1d-b768-4441-846b-f81b7d866f8b.pdf\"},{\"id\":54853778,\"identity\":\"b849b4e0-a60d-441b-8d4b-504ddaba5857\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:29\",\"extension\":\"docx\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":12210,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"20240406Supplementarydata1Primersequences.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/8df148b35a61f06302e398e4.docx\"},{\"id\":54853786,\"identity\":\"124bd86f-eb48-4931-9a75-7c33a905922a\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:31\",\"extension\":\"doc\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":28209152,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"20240406Supplementarydata2westernblotuncropedgel.doc\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/170a27930f2d2fbddcdc2c2c.doc\"},{\"id\":54853781,\"identity\":\"7be2c270-a57f-4c7b-b758-5e22e284fcf8\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:29\",\"extension\":\"docx\",\"order_by\":7,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":21235,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"20240406Supplementarydata3statisticalanalysis.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/670078a43e3544cdc65eff6c.docx\"},{\"id\":54853780,\"identity\":\"7f62acb9-95e8-4e2c-90e4-aaf96a97aecf\",\"added_by\":\"auto\",\"created_at\":\"2024-04-17 17:21:29\",\"extension\":\"pdf\",\"order_by\":8,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":217666,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"AuthorChecklistFull.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4205146/v1/d9695d1e29dec713eaeafd7a.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003ePeriodontal tissue destruction, encompassing the periodontal ligament, alveolar bone, and gingiva, remains a significant health concern(\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). 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(\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e). During OS progression, reactive oxygen species (ROS) skyrocket, disrupting the delicate balance within the endogenous antioxidant system(\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e). This OS onslaught impairs vital functions of periodontal ligament stem cells (PDLSCs), vital players in maintaining and repairing periodontal tissue(\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e). Consequently, mitigating this excessive OS response emerges as a crucial strategy to restore PDLSC function and halt periodontal tissue destruction.\\u003c/p\\u003e \\u003cp\\u003eMarine-derived natural products offer a treasure trove of unexplored bioactivities with potential therapeutic applications(\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e). 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(\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). NOCA is synthesized by various microorganisms and has been successfully reported with anti-cancer and anti-pathogenic microorganism properties(\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e). However, its antioxidative capabilities and underlying mechanisms in PDLSCs remain largely uncharted.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we obtained NOCA from a \\u003cem\\u003eDysidea\\u003c/em\\u003e 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.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eReagents\\u003c/h2\\u003e \\u003cp\\u003eNOCA is obtained from a \\u003cem\\u003eDysidea\\u003c/em\\u003e 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\\u0026trade;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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell culture\\u003c/h2\\u003e \\u003cp\\u003ePDLSCs were isolated from molar teeth obtained with informed consent from three healthy human donors (16\\u0026ndash;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\\u0026deg;C for 1 hour. The cell-containing solution was then cultured in 75 cm\\u003csup\\u003e2\\u003c/sup\\u003e cell culture flasks using standard medium consisting of aMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin under 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e at 37\\u0026deg;C. Upon reaching approximately 80% confluence, the cells were subcultured. For all experiments described below, PDLSCs from passages 3 to 5 were utilized.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCharacterization of PDLSCs\\u003c/h2\\u003e \\u003cp\\u003eIsolated 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\\u0026thinsp;\\u0026minus;\\u0026thinsp;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 \\u0026micro;g/mL L-ascorbic acid. Adipogenic differentiation was induced in standard medium supplemented with 1 mM dexamethasone, 100 \\u0026micro;M indomethacin, 500 \\u0026micro;M 3-Isobutyl-1-methylxanthine, and 50 \\u0026micro;g/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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell viability assay\\u003c/h2\\u003e \\u003cp\\u003ePDLSCs were seeded at a density of 1 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e cells in 100 \\u0026micro;L 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 \\u0026micro;M, 1.25 \\u0026micro;M, 2.5 \\u0026micro;M, 5 \\u0026micro;M, 10 \\u0026micro;M, and 20 \\u0026micro;M) 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 \\u0026micro;M or 10 \\u0026micro;M NOCA or 10 \\u0026micro;M Que (Guangzhou Institute for Drug Control, Guangzhou, China) in standard medium before stimulation with 150 \\u0026micro;M H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e (Sigma-Aldrich, St. Louis, MO) for 24 hours in standard medium. Cell viability was then assessed using the CKK-8 assay. Briefly, 10 \\u0026micro;l 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).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIntracellular ROS and antioxidation assay\\u003c/h2\\u003e \\u003cp\\u003eIntracellular ROS levels were assessed using both 5 \\u0026micro;M Dihydroethidium (DHE) and 10 \\u0026micro;M 2',7'-dichlorofluorescein diacetate (DCFH). Briefly, cells were incubated with the respective dye at 37\\u0026deg;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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eOsteogenic differentiation assay\\u003c/h2\\u003e \\u003cp\\u003ePDLSCs were cultured in osteogenic medium supplemented with 10 mM β-glycerophosphate, 100 nM dexamethasone, and 50 \\u0026micro;g/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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eRT-qPCR assay\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA was extracted from PDLSC lysate using the Trizol reagent (Invitrogen, Carlsbad, CA). cDNA synthesis (1 \\u0026micro;g) 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\\u0026reg; Premix Ex Taq TM II kit (TaKaRa) in a total reaction volume of 20 \\u0026micro;L on a 7500 Real-Time PCR System. The specific primer sequences used for target gene amplification are listed in Table\\u0026nbsp;1.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWB assay\\u003c/h2\\u003e \\u003cp\\u003eProtein 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 \\u0026micro;g 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\\u0026deg;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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunofluorescent staining\\u003c/h2\\u003e \\u003cp\\u003eThe 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\\u0026deg;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).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eThe statistical analysis results were reported as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard error of the mean. Differences between each data set was assessed using the Student t test. Statistical significance was defined as P values\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003eCharacterization of PDLSCs\\u003c/h2\\u003e\\n\\u003cp\\u003eFlow 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.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). Further characterization revealed their multipotent differentiation potential. When cultured in osteogenic medium, the cells formed mineralized nodules stained positively with ARS (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB), displaying morphology consistent with bone matrix deposits. Adipogenic differentiation resulted in abundant lipid droplet formation, as evidenced by ORO staining (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). Chondrogenic induction led to the development of chondrocyte-like cells exhibiting blue-green staining with AB (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). These findings collectively provide strong evidence that the isolated cells possess multipotent differentiation potential, confirming their characteristics consistent with those of MSCs.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003eEffects of NOCA on cell viability\\u003c/h2\\u003e\\n\\u003cp\\u003eTo 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 \\u0026micro;M (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Therefore, subsequent experiments were conducted using concentrations lower than 20 \\u0026micro;M.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003eNOCA attenuated H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced OS in PDLSCs\\u003c/h2\\u003e\\n\\u003cp\\u003eTo investigate whether NOCA attenuated H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced oxidative stress in PDLSCs, we first evaluated ROS production using the DCFH and DHE assay. As expected, H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e treatment significantly increased ROS levels compared to control (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Pretreatment with either NOCA (5 \\u0026micro;M and 10 \\u0026micro;M) or Que (10 \\u0026micro;M) reduced ROS levels, with NOCA at 10 \\u0026micro;M decreasing ROS by 40% compared to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e alone (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA-D). We then assessed cell viability by CCK-8 assay and observed a significant decrease in viability after H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e incubation (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). This reduction was dose-dependently reversed by NOCA pretreatment, with viability at 10 \\u0026micro;M NOCA similar to control levels (p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05) (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE). Similarly, H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e significantly decreased SOD activity and CAT concentration by more than 30% compared to the control group (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). NOCA treatment again provided dose-dependent protection, with 10 \\u0026micro;M NOCA restoring SOD and CAT activity to near control levels (p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05) (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF, 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(\\u003cspan class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). Notably, H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e treatment significantly suppressed both SIRT1 mRNA expression by 20% and SIRT1 protein levels by 50% compared to the control group (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). However, pretreatment with increasing doses of NOCA dose-dependently restored SIRT1 levels to at least 80% of the control group (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), as measured by RT-qPCR and WB (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eH-J). Collectively, these findings suggest that NOCA's ability to mitigate H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-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.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003eNOCA attenuated OS-impaired osteogenic differentiation in PDLSCs\\u003c/h2\\u003e\\n\\u003cp\\u003eTo investigate whether NOCA could attenuate H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced impairment of osteogenic differentiation, PDLSCs were subjected to differentiation medium for 7 and 14 days following a 24-hour treatment with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and/or NOCA/Que. Osteogenic differentiation was assessed by ALP activity, calcium nodule formation, and the expression of key osteogenic markers. As expected, H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e treatment significantly reduced ALP activity, calcium nodule formation, and the expression of all three osteogenic markers (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Notably, both NOCA and Que improved ALP activity, with 10 \\u0026micro;M NOCA to near control levels (p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05). Similarly, NOCA/Que administration partially restored calcium nodule formation and increased the expression of osteogenic markers, suggesting their protective potential against H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced damage (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). 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(\\u003cspan class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e). Our findings revealed that after H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e treatment, p-Akt levels were increased by less than 20% compared to control (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), while p-ERK levels were significantly decreased (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Notably, pretreatment with NOCA did not significantly alter p-Akt levels but partially restored p-ERK levels to near control (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) during osteogenic differentiation in the presence of H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e. These results suggest that NOCA's ability to mitigate H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced impairment of PDLSCs' osteogenic potential might be independently mediated through the ERK pathway, while bypassing the Akt signaling cascade (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003eThe ERK/Wnt pathway mediated the protective effects of NOCA on OS-impaired osteogenic differentiation in PDLSCs\\u003c/h2\\u003e\\n\\u003cp\\u003eTo 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 H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e treatment for 24 hours. This was followed by osteogenic differentiation induction for 7 and 14 days. Osteogenesis analysis revealed that 10 \\u0026micro;M U0126 significantly decreased ALP activity and calcium nodule formation in NOCA-pretreated PDLSCs under H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced OS conditions (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA, B). Additionally, RT-qPCR analysis showed downregulation of key osteogenic markers RUNX2, ALP, and COL1A1 upon U0126 treatment (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC-H). As ERK is known to influence the Wnt signaling pathway, a downstream target of \\u0026beta;-catenin in PDLSC osteogenesis(\\u003cspan class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e), we investigated their potential crosstalk. Immunofluorescence analysis revealed that NOCA treatment significantly increased intranuclear \\u0026beta;-catenin levels compared to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e alone (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Notably, U0126 treatment significantly reduced nuclear \\u0026beta;-catenin levels in both H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and NOCA-treated PDLSCs (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA-E). Furthermore, blocking the Wnt/\\u0026beta;-catenin pathway using 5 \\u0026micro;M XAV-939 significantly impaired osteogenesis potential in PDLSCs exposed to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and NOCA (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eF, 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 \\u0026beta;-catenin nuclear translocation playing a crucial role in this process.\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe association between oxidative stress (OS) and periodontal tissue destruction has been reported(\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e). 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(\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). Here, we investigated the antioxidant effect and underlying mechanism of NOCA, a compound isolated from the marine sponge \\u003cem\\u003eDysidea\\u003c/em\\u003e sp., on H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced OS in human periodontal ligament stem cells (PDLSCs). Que, a known antioxidant drug, served as a reference control(\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eOS, characterized by an imbalance between ROS production and antioxidant defenses, significantly contributes to periodontal tissue destruction(\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e). We investigated the antioxidant effects of NOCA, a marine sponge-derived compound, in PDLSCs exposed to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced OS. NOCA potently reduces ROS levels compared to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-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(\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). Previous studies reported that SIRT1 activation for repairing OS-induced cellular dysfunction involves multiple defense mechanisms(\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). Our studies demonstrated that NOCA reversed the impaired expression of SIRT1 in PDLSCs caused by H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e in PDLSCs. Therefore, we concluded that NOCA could reduce H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced OS by enhanced the expression of antioxidant factors.\\u003c/p\\u003e \\u003cp\\u003eWhile most studies exploring OS damage to PDLSCs focus on cell viability and osteogenic potential, both vital factors in periodontal tissue regeneration(\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e), the underlying mechanisms remain incompletely understood. Counteracting excessive OS is crucial for restoring H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced damage to cell viability and osteogenic differentiation in PDLSCs(\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e). Prior studies have demonstrated that manipulating key regulators like Tripartite Motif 16 or recombinant Klotho protein can alleviate OS, thereby rescuing PDLSC function(\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). 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 H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced OS. Notably, NOCA exhibits no effect on osteogenic potential under normal conditions (data not shown), suggesting its targeted action against OS-mediated dysfunction.\\u003c/p\\u003e \\u003cp\\u003eWhile the Akt signaling pathway plays a crucial role in antioxidant defense by stimulating the Nrf2 pathway(\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e), 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 H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced damage in PDLSCs(\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e). Moreover, our study further corroborates this link by showing that H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e 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.\\u003c/p\\u003e \\u003cp\\u003eThe Wnt/β-catenin signaling pathway plays a vital role in protecting against oxidative damage, particularly in PDLSCs. H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e disrupts this pathway by suppressing β-catenin, leading to impaired survival and bone formation capabilities(\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e). Conversely, activating the Wnt/β-catenin pathway safeguards PDLSCs from these detrimental effects(\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e). Notably, this pathway is downstream of ERK signaling and crucial for MSC proliferation, differentiation, and bone tissue homeostasis(\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e). Our study reveals NOCA's potent protective effect against H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-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 H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-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.\\u003c/p\\u003e \\u003cp\\u003eDespite 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.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, our study demonstrates that NOCA, a marine sponge-derived compound, effectively counteracts H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-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 \\u003cem\\u003ein vivo\\u003c/em\\u003e 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.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe experimental protocol of the study titled with \\u0026ldquo;Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells\\u0026rdquo; 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.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConsent to participate/consent for publication is not applicable to this study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll data generated or analyzed during this study are included in this published article.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe 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.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis 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).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe acknowledge that the cell-based research of this study was performed at Shenzhen Key Laboratory of Otolaryngology and Shenzhen Institute of Otolaryngology.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eBhattarai G, So H-S, Kieu TTT, Kook S-H, Lee J-C, Jeon Y-M. Astaxanthin Inhibits Diabetes-Triggered Periodontal Destruction, Ameliorates Oxidative Complications in STZ-Injected Mice, and Recovers Nrf2-Dependent Antioxidant System. Nutrients. 2021;13(10):3575.\\u003c/li\\u003e\\n\\u003cli\\u003eAquino-Martinez R, Khosla S, Farr JN, Monroe DG. Periodontal Disease and Senescent Cells: New Players for an Old Oral Health Problem? International Journal of Molecular Sciences. 2020;21(20):7441.\\u003c/li\\u003e\\n\\u003cli\\u003eJia L, Xiong Y, Zhang W, Ma X, Xu X. Metformin promotes osteogenic differentiation and protects against oxidative stress-induced damage in periodontal ligament stem cells via activation of the Akt/Nrf2 signaling pathway. Experimental Cell Research. 2020;386(2):111717.\\u003c/li\\u003e\\n\\u003cli\\u003eD\\u0026rsquo;Aiuto F, Nibali L, Parkar M, Patel K, Suvan J, Donos N. Oxidative Stress, Systemic Inflammation, and Severe Periodontitis. Journal of Dental Research. 2010;89(11):1241-6.\\u003c/li\\u003e\\n\\u003cli\\u003eMateos R, P\\u0026eacute;rez-Correa JR, Dom\\u0026iacute;nguez H. Bioactive Properties of Marine Phenolics. Marine Drugs. 2020;18(10):501.\\u003c/li\\u003e\\n\\u003cli\\u003eMaglioni S, Arsalan N, Hamacher A, Afshar S, Schiavi A, Beller M, et al. High-Content C. elegans Screen Identifies Natural Compounds Impacting Mitochondria-Lipid Homeostasis and Promoting Healthspan. Cells. 2022;11(1):100.\\u003c/li\\u003e\\n\\u003cli\\u003eNormant V, Josts I, Kuhn L, Perraud Q, Fritsch S, Hammann P, et al. Nocardamine-Dependent Iron Uptake in Pseudomonas aeruginosa: Exclusive Involvement of the FoxA Outer Membrane Transporter. ACS Chemical Biology. 2020;15(10):2741-51.\\u003c/li\\u003e\\n\\u003cli\\u003eConti R, Chagas FO, Caraballo-Rodriguez AM, Melo WGdP, do Nascimento AM, Cavalcanti BC, et al. Endophytic Actinobacteria from the Brazilian Medicinal Plant Lychnophora ericoides Mart. and the Biological Potential of Their Secondary Metabolites. Chemistry \\u0026amp; Biodiversity. 2016;13(6):727-36.\\u003c/li\\u003e\\n\\u003cli\\u003eKalinovskaya NI, Romanenko LA, Irisawa T, Ermakova SP, Kalinovsky AI. Marine isolate Citricoccus sp. KMM 3890 as a source of a cyclic siderophore nocardamine with antitumor activity. Microbiological Research. 2011;166(8):654-61.\\u003c/li\\u003e\\n\\u003cli\\u003eQin Z, Song J, Huang J, Jiang S, Zhang G, Huang M, et al. Mitigation of triptolide-induced testicular Sertoli cell damage by melatonin via regulating the crosstalk between SIRT1 and NRF2. Phytomedicine. 2023;118:154945.\\u003c/li\\u003e\\n\\u003cli\\u003eHuang M, Yan Y, Deng Z, Zhou L, She M, Yang Y, et al. Saikosaponin A and D attenuate skeletal muscle atrophy in chronic kidney disease by reducing oxidative stress through activation of PI3K/AKT/Nrf2 pathway. Phytomedicine. 2023;114:154766.\\u003c/li\\u003e\\n\\u003cli\\u003eLi W, Liu Y, Wang B, Luo Y, Hu N, Chen D, et al. Protective effect of berberine against oxidative stress-induced apoptosis in rat bone marrow-derived mesenchymal stem cells. Exp Ther Med. 2016;12(6):4041-8.\\u003c/li\\u003e\\n\\u003cli\\u003eLi W, Huang X, Yu W, Xu Y, Huang R, Park J, et al. Activation of Functional Somatic Stem Cells Promotes Endogenous Tissue Regeneration. Journal of dental research. 2022;101(7):802-11.\\u003c/li\\u003e\\n\\u003cli\\u003eLetsiou S, Bakea A, Le Goff G, Lopes P, Gardikis \\u0026Kappa;, Alonso C, et al. In vitro protective effects of marine-derived Aspergillus puulaauensis TM124-S4 extract on H2O2-stressed primary human fibroblasts. Toxicology in Vitro. 2020;66:104869.\\u003c/li\\u003e\\n\\u003cli\\u003eLi Y, Li J, Lin S-J, Yang Z-S, Jin H-X. Preparation of Antioxidant Peptide by Microwave- Assisted Hydrolysis of Collagen and Its Protective Effect Against H2O2-Induced Damage of RAW264.7 Cells. Marine Drugs. 2019;17(11):642.\\u003c/li\\u003e\\n\\u003cli\\u003eWei Y, Fu J, Wu W, Ma P, Ren L, Yi Z, et al. Quercetin Prevents Oxidative Stress-Induced Injury of Periodontal Ligament Cells and Alveolar Bone Loss in Periodontitis. Drug Des Devel Ther. 2021;15:3509-22.\\u003c/li\\u003e\\n\\u003cli\\u003eWarinhomhoun S, Muangnoi C, Buranasudja V, Mekboonsonglarp W, Rojsitthisak P, Likhitwitayawuid K, et al. Antioxidant Activities and Protective Effects of Dendropachol, a New Bisbibenzyl Compound from Dendrobium pachyglossum, on Hydrogen Peroxide-Induced Oxidative Stress in HaCaT Keratinocytes. Antioxidants. 2021;10(2):252.\\u003c/li\\u003e\\n\\u003cli\\u003eBhatti FUR, Kim SJ, Yi A-K, Hasty KA, Cho H. Cytoprotective role of vitamin E in porcine adipose-tissue-derived mesenchymal stem cells against hydrogen-peroxide-induced oxidative stress. Cell and Tissue Research. 2018;374(1):111-20.\\u003c/li\\u003e\\n\\u003cli\\u003eLi M, Yan J, Chen X, Tam W, Zhou L, Liu T, et al. Spontaneous up-regulation of SIRT1 during osteogenesis contributes to stem cells\\u0026rsquo; resistance to oxidative stress. Journal of Cellular Biochemistry. 2018;119(6):4928-44.\\u003c/li\\u003e\\n\\u003cli\\u003eLi J, Li M, Wang C, Zhang S, Gao Q, Wang L, et al. NaSH increases SIRT1 activity and autophagy flux through sulfhydration to protect SH-SY5Y cells induced by MPP~+. Cell Cycle. 2020;19(17):2216-25.\\u003c/li\\u003e\\n\\u003cli\\u003eZhao Y, Liu H, Xi X, Chen S, Liu D. TRIM16 protects human periodontal ligament stem cells from oxidative stress-induced damage via activation of PICOT. Experimental Cell Research. 2020;397(1):112336.\\u003c/li\\u003e\\n\\u003cli\\u003eChen H, Huang X, Fu C, Wu X, Peng Y, Lin X, et al. Recombinant Klotho Protects Human Periodontal Ligament Stem Cells by Regulating Mitochondrial Function and the Antioxidant System during H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e Induced Oxidative Stress. Oxidative Medicine and Cellular Longevity. 2019;2019:9261565.\\u003c/li\\u003e\\n\\u003cli\\u003eFeng Y, Fu X, Lou X, Fu B. Stromal cell-derived factor 1 protects human periodontal ligament stem cells against hydrogen peroxide-induced apoptosis. Mol Med Rep. 2017;16(4):5001-6.\\u003c/li\\u003e\\n\\u003cli\\u003eHao J, Yang H, Cao Y, Zhang C, Fan Z. IGFBP5 enhances the dentinogenesis potential of dental pulp stem cells via JNK and ErK signalling pathways. Journal of Oral Rehabilitation. 2020;47(12):1557-65.\\u003c/li\\u003e\\n\\u003cli\\u003eShin SY, Kim CG, Jho E-H, Rho M-S, Kim YS, Kim Y-H, et al. Hydrogen peroxide negatively modulates Wnt signaling through downregulation of \\u0026beta;-catenin. Cancer Letters. 2004;212(2):225-31.\\u003c/li\\u003e\\n\\u003cli\\u003eKook S-H, Lee D, Cho E-S, Heo JS, Poudel SB, Ahn Y-H, et al. Activation of canonical Wnt/\\u0026beta;-catenin signaling inhibits H2O2-induced decreases in proliferation and differentiation of human periodontal ligament fibroblasts. Molecular and Cellular Biochemistry. 2016;411(1):83-94.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"stem-cell-research-and-therapy\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scrt\",\"sideBox\":\"Learn more about [Stem Cell Research \\u0026 Therapy](http://stemcellres.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/scrt/default.aspx\",\"title\":\"Stem Cell Research \\u0026 Therapy\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Periodontal destruction, Periodontal ligament stem cells, Oxidative stress, Nocardamine, Osteogenic differentiation\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4205146/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4205146/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eThe 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.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e \\u003cp\\u003eThe cytotoxicity of NOCA on PDLSCs was assessed using a CCK-8 assay. PDLSCs were then treated with hydrogen peroxide (H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e) 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.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eNOCA exhibited no significant cytotoxicity at concentrations below 20 \\u0026micro;M and effectively inhibited H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced OS in PDLSCs. NOCA also restored ALP activity, mineralized nodule formation, and the expression of osteogenic markers in H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-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.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e \\u003cp\\u003eNOCA protected PDLSCs against H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e-induced OS and effectively restored impaired osteogenic differentiation in PDLSCs by modulating the ERK/Wnt signaling pathway.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-04-17 17:21:24\",\"doi\":\"10.21203/rs.3.rs-4205146/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2024-04-11T10:12:32+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-04-11T08:59:58+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-04-08T00:13:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Stem Cell Research \\u0026 Therapy\",\"date\":\"2024-04-06T06:37:52+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"stem-cell-research-and-therapy\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scrt\",\"sideBox\":\"Learn more about [Stem Cell Research \\u0026 Therapy](http://stemcellres.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/scrt/default.aspx\",\"title\":\"Stem Cell Research \\u0026 Therapy\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"8847b2e5-4a28-4721-b7e5-c6382ca706ae\",\"owner\":[],\"postedDate\":\"April 17th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-08-12T16:08:53+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4205146\",\"link\":\"https://doi.org/10.1186/s13287-024-03812-2\",\"journal\":{\"identity\":\"stem-cell-research-and-therapy\",\"isVorOnly\":false,\"title\":\"Stem Cell Research \\u0026 Therapy\"},\"publishedOn\":\"2024-08-07 15:57:31\",\"publishedOnDateReadable\":\"August 7th, 2024\"},\"versionCreatedAt\":\"2024-04-17 17:21:24\",\"video\":\"\",\"vorDoi\":\"10.1186/s13287-024-03812-2\",\"vorDoiUrl\":\"https://doi.org/10.1186/s13287-024-03812-2\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4205146\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4205146\",\"identity\":\"rs-4205146\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}