Senolytic effects of Moringa oleifera leaf extract in an in vitro model of Alzheimer’s disease

Senolytic effects of Moringa oleifera leaf extract in an in vitro model of Alzheimer’s disease | 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 Article Senolytic effects of Moringa oleifera leaf extract in an in vitro model of Alzheimer’s disease Nur Irlia Sofea Mohammad Zamani, Hamizah Shahirah Hamezah, Ahmed Mediani, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9082466/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Accumulation of senescent cells (SnCs) in the aging brain contributes to Alzheimer's disease (AD) progression through the secretion of senescence-associated secretory phenotype (SASP) that exacerbates neuroinflammation and neurodegeneration. Senolytic agents that selectively eliminate SnCs, have emerged as a potential therapeutic strategy, however safer natural alternatives remain underexplored. In this study, we aimed to investigate the senolytic potential of Moringa oleifera leaf (MOL) extract in an in vitro AD-senescence model using SH-SY5Y cells exposed to amyloid-β (Aβ 1−42 ) oligomers. SH-SY5Y cells exposed to Aβ oligomers exhibited senescent phenotype, characterised by increased senescence-associated β-galactosidase (SA-β-gal) positivity and upregulated nuclear expression of p21, p16, and γH2AX. Treatment with MOL extract significantly reduced the number of cells expressing senescence-associated molecular markers and induced apoptosis in senescent cells, while attenuating the secretion of pro-inflammatory SASP cytokines, including IL-8 and TNF-α. Overall findings suggest that MOL extract preferentially targets SnCs and mitigates SASP-associated inflammation. These results support the potential of MOL as a natural compound with senolytic activity and provide a foundation for further development into its therapeutic relevance in AD. Biological sciences/Biochemistry Biological sciences/Drug discovery Biological sciences/Neuroscience Senolytics Moringa oleifera Cellular senescence Alzheimer's disease Senescence-associated secretory phenotype Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The global population is rapidly aging, leading to increase in the prevalence of Alzheimer’s disease (AD). By 2050, more than 130 million people worldwide are expected to be affected [ 1 ], imposing substantial social, economic, and healthcare burdens. Current therapeutics approaches provide primarily symptomatic relief and modestly slow disease progression without addressing underlying pathogenic mechanisms [ 2 ], highlighting the need for mechanism-based strategies suitable for long-term use in aging-populations. Cellular senescence has emerged as a key biological link between aging and AD. Senescence is a stable state of cell cycle arrest induced by diverse stressors such as oxidative damage, mitochondrial dysfunction and amyloid-β (Aβ) toxicity. Senescent cells (SnCs) exhibit increased resistance to apoptosis through activation of senescent cell anti-apoptotic pathways (SCAPs) and secrete a range of pro-inflammatory factors collectively termed as senescence-associated secretory phenotypes (SASP). In the AD brain, senescent-associated phenotypes have been reported in astrocytes, microglia, and neurons, particularly near to Aβ plaques and neurofibrillary tangles (NFTs), where SASP-mediated chronic neuroinflammation disrupts synaptic function, accelerates neuronal loss, and exacerbates disease progression [ 3 – 5 ]. Despite increasing evidence linking senescence to AD pathology, the mechanisms by which Aβ induces senescence and its contribution to disease progression remain incompletely understood. Growing interest has focused on dietary bioactive compounds as multi-target interventions for age-related neurodegenerative diseases. Moringa oleifera is a nutrient-rich functional food known for its antioxidant, anti-inflammatory, and neuroprotective properties, largely attributed to its high flavonoid and phenolic content [ 6 , 7 ]. Phenolic compounds are widely recognised for their protective effects against chronic diseases through antioxidant and anti-inflammatory mechanisms [ 8 , 9 ]. In the context of AD, flavonoids, a major subclass of phenolic compounds are of particular interest due to their ability to cross the blood-brain barrier (BBB), improve cognitive function, modulate oxidative stress, inflammatory signalling and aging-associated molecular pathways [ 10 ]. However, the potential of Moringa oleifera to regulate cellular senescence in AD-relevant models remains largely unexplored. Therefore, this study aimed to investigate the senolytic potential of Moringa oleifera leaf (MOL) extract in an in vitro Aβ-induced senescence model, focusing on SnCs elimination and attenuation of SASP-associated inflammatory responses. Results Establishment of an in vitro AD-associated senescence model To elucidate the senolytic potential of MOL in AD, we established an in vitro AD-associated senescence model by exposing SH-SY5Y cells to 20 µM Aβ 1–42 oligomers (Fig. 1 a). Following Aβ exposure, we observed that SA-β-gal-positive cells were increased compared to untreated, non-senescent control cells (Fig. 1 b, c). Consistently, the expression of senescence markers, p16 (p < 0.001), p21 (p < 0.05) and γH2AX (p < 0.05) were significantly upregulated in Aβ-treated cells compared to non-senescent cells (Fig. 1 d, e), indicating activation of cell-cycle arrest pathways and DNA damage responses. Collectively, these findings confirm the successful induction of an Aβ-associated senescent phenotype in SH-SY5Y cells, validating this model for the evaluation of senolytic interventions. MOL extract induces apoptosis in Aβ-induced senescent SH-SY5Y cells To assess the senolytic potential of MOL, both young and senescent SH-SY5Y cells were treated with MOL and compared with the established senolytic cocktail dasatinib and quercetin (hereafter referred to as D + Q). MOL treatment significantly reduced the number of SnCs compared with young cells (p < 0.01) (Fig. 2 a, b). A similar reduction in the number of SnCs was observed following D + Q treatment (p < 0.05). Notably, the number of SnCs in both MOL and D + Q-treated group is significantly reduced compared to the number of SnCs in the untreated group (p < 0.01) (Fig. 2 a, b). This selective reduction indicates that MOL preferentially targets SnCs while preserving healthy, proliferative cells. No significant difference was observed between MOL and D + Q in their ability to reduce SnCs burden, suggesting that MOL exerts senolytic effects comparable to the reference senolytic treatment. To further investigate the mechanism underlying this selective elimination, caspase-3/7 activation was examined. SnCs treated with MOL showed pronounced morphological features of apoptosis, including cell rounding accompanied by positive caspase-3/7 activation signals (Fig. 2 c). Immunofluorescence analysis revealed a higher number of caspase-3/7–positive cells in MOL and D + Q treated groups compared with the untreated and DMSO vehicle-treated group (Fig. 2 d). Together, these findings demonstrate that MOL selectively induces apoptosis in senescent SH-SY5Y cells, consistent with senolytic activity. MOL extract reduced senescence-associated molecular markers To further evaluate the impact of MOL on senescence burden, the expression of senescence-associated molecular markers was examined following treatment. As expected, Aβ exposure increased SA-β-gal positivity (Fig. 3 a, b) and significantly upregulated p21, p16, and γH2AX compared with untreated controls, confirming the induction of cellular senescence (Fig. 4 a, b, c, d , e, f ). Treatment with MOL extract resulted in a non-significant reduction in SA-β-gal positivity (Fig. 3 a, b), suggesting partial attenuation of this hallmark of senescence. In contrast, the expression of p21, p16, and γH2AX were significantly downregulated following MOL treatment compared with untreated and DMSO vehicle-treated cells. Notably, these reductions were comparable to those observed with the senolytic positive control D + Q (Fig. 4 a - f). Collectively, these findings indicate that MOL effectively reduces senescence burden, with a pronounced impact on DNA damage signalling pathways, while exerting a more modest effect on SA-β-gal activity. MOL extract modulates SASP-associated cytokine secretion To determine whether MOL influences the inflammatory secretory profile of SnCs, the secretion of SASP-associated cytokines was measured following Aβ induction and subsequent treatments with DMSO vehicle, D + Q or MOL. After normalisation to total protein content, we observed MOL treatment significantly reduced IL-8 secretion compared with untreated and DMSO vehicle-treated cells (p < 0.001) (Fig. 5 a). TNF-α secretion was also markedly reduced following MOL treatment and falling below the assay’s limit of detection (4.69 pg/mL) (p < 0.01) (Fig. 5 b), suggesting strong suppression rather than assay insensitivity. In contrast, IL-1β levels showed an increase following MOL treatment (p < 0.05) (Fig. 5 c). These findings indicate that MOL does not uniformly suppress all SASP components but instead selectively modulates specific inflammatory mediators. Discussion Accumulating evidence from preclinical [ 11 – 13 ] and clinical studies [ 14 – 16 ] highlights cellular senescence as a key pathological process in AD, with SnCs enriched in Aβ–associated brain regions. Consistent with these observations, our findings demonstrate that MOL modulate several markers associated with senescence-related pathways in an AD cellular model. Although senolytic therapies have shown promise in improving cognitive function and reducing amyloid burden, their clinical translation remains limited due to potential adverse effects [ 17 – 19 ]. Here, our results provide experimental evidence that a naturally derived compound may exert senomodulatory effects. These findings support MOL as a potential safer alternative or complementary strategy to conventional senolytic approaches. In this study, we observed that Aβ exposure induces a senescent phenotype in SH-SY5Y cells, as indicated by the significant upregulation of p21, p16, and γH2AX, reflecting activation of cell-cycle arrest and DNA damage signalling pathways [ 18 , 20 ]. Previous studies have similarly reported elevated γH2AX expression in AD brains and neuronal models [ 21 , 22 ], which is associated with persistent DNA double-strand breaks and genomic instability during disease progression. Although γH2AX alone is not a definitive marker of cellular senescence, its concurrent induction with p21 and p16 suggests that Aβ-induced DNA damage activates senescence-associated cell-cycle arrest mechanisms. These molecular changes support the relevance of this model for recapitulating AD–associated cellular stress and provide a mechanistic basis for evaluating senolytic interventions. The observed marker profile is consistent with prior reports demonstrating that Aβ triggers neuronal senescence and genomic instability [ 3 , 23 , 24 ]. While SA-β-gal staining increased following Aβ exposure, the change did not reach statistical significance. This observation may reflect the relatively early stage of senescence induction in neuronal cells [ 25 ]. Evidence from other SH-SY5Y models indicate that SA-β-gal activity can be highly dependent on the type and duration of the senescence-inducing stimulus [ 26 – 28 ]. Furthermore, while SA-β-gal is widely used as a senescence marker, it has recognised limitations and may vary during early or partial senescence across different experimental contexts [ 29 ]. These findings suggest that early or mild senescence induction may not always produce statistically significant changes in SA-β-gal activity. MOL extract preferentially reduced the burden of SnCs, an effect typically observed with established senolytic drugs; which selectively eliminate SnCs while sparing young, non-senescent cells [ 30 ]. Consistent with this observation, MOL treatment activated caspase-3/7 and induced apoptotic morphological changes predominantly in SnCs, providing mechanistic evidence of senolytic activity. This mechanism aligns with those reported for established senolytic compounds such as ABT263 [ 31 ] and natural senolytic fisetin [ 32 ], which target the SCAPs and promote apoptotic cell death. The preservation of young cells following MOL treatment may also benefitted from its antioxidant and neuroprotective properties, which could mitigate oxidative and metabolic stress in non-SnCs [ 33 – 35 ]. D + Q is a well-established senolytic combination widely used in preclinical models and currently under clinical investigation [ 36 – 38 ]. The absence of a significant difference between MOL and D + Q in reducing the SnCs burden suggests that MOL exhibits senolytic efficacy comparable to that of this benchmark treatment in the present model. Although further validation in additional experimental systems is warranted, our findings provide the first evidence supporting MOL as a promising natural senolytic candidate. Beyond the elimination of SnCs, MOL robustly attenuated senescence-associated molecular pathways, consistent with previous studies reporting senotherapeutic effects of natural compounds [ 39 , 40 ]. MOL treatment reduced SA-β-gal positivity compared with the untreated, DMSO-vehicle treated, and D + Q-treated groups, which has been linked to improved cognitive function and reduced SnCs burden [ 41 , 42 ]. In parallel, MOL significantly decreased p16, p21, and γH2AX expression, indicating coordinated suppression of cell-cycle arrest and DNA damage signalling pathways. Notably, MOL most prominently reduced γH2AX expression, suggesting mitigation of Aβ-induced persistent DNA damage. Given that γH2AX reflects persistent DNA double-strand breaks, this observation raises the possibility that MOL may mitigate Aβ–induced genotoxic stress in this model. Although all markers were significantly reduced, differences in the magnitude of response suggest that MOL primarily exerts its effects through senolytic clearance, with secondary modulation of senescence-associated pathways. This pattern is consistent with a senolytic-dominant mechanism accompanied by secondary senomorphic activity. SnCs secrete a range of pro-inflammatory cytokines collectively known as the SASP, including IL-8, IL-1β, and TNF-α, which contribute to chronic inflammation and neurodegeneration [ 43 ]. The non-significant increase in TNF-α observed in DMSO vehicle-treated cells likely reflects vehicle-induced stress rather than a treatment-specific effect. In contrast, MOL treatment significantly reduced IL-8 and TNF-α secretions, consistent with the selective elimination or suppression of SnCs rather than global inhibition of inflammatory signalling. These observations support the anti-inflammatory and senescence-modulating effects of MOL and align with previous reports in other experimental models [ 44 , 45 ]. SASP components are regulated by inflammatory pathways, such as NF-κB and MAPK, which play central roles in AD-related neuroinflammation [ 46 , 47 ] and intersect with broader neurodegenerative signalling networks. As a key SASP mediator, IL-8 reinforces senescence and amplifies inflammatory signalling. Thus, its suppression strongly supports the senotherapeutic potential of MOL. In contrast, IL-1β increased following MOL treatment, which may reflect transient cytokine release associated with senolytic-induced cell death rather than enhanced inflammatory signalling [ 48 , 49 ]. In neuronal monocultures, the absence of professional phagocytes delayed the clearance of apoptotic SnCs, allowing IL-1β to persist in the extracellular environment. Although MOL has been reported to inhibit IL-1β production in other contexts [ 50 ], this effect may be masked by secondary cytokine release following SnCs elimination. Collectively, these findings suggest that MOL modulates SASP output in a manner consistent with senolytic activity, although the precise upstream molecular mechanisms remain to be defined. These findings highlight MOL as a promising natural senolytic candidate in the context of AD-associated cellular stress. However, several limitations should be acknowledged. First, this study employed a single neuroblastoma-derived cell line, which may not fully recapitulate the complexity of mature neurons or the brain microenvironment. Second, the use of a crude MOL extract precludes the identification of individual bioactive constituents and their specific molecular targets. Finally, the short-term in vitro nature of this study limits the assessment of its long-term efficacy and safety. Future studies should employ primary neuronal cultures, co-culture systems, and well-characterised in vivo AD models to validate and extend these findings. Elucidating the molecular pathways modulated by MOL, including those involved in cellular senescence, oxidative stress, and inflammation, will provide deeper mechanistic insight, while identification of key bioactive compounds responsible for these effects will be essential to clarify their pharmacological potential. Collectively, these approaches will help establish the therapeutic and translational relevance of MOL as a senolytic strategy for targeting senescence-associated mechanism in AD. In conclusion, MOL extract exhibits senolytic activity in an in vitro AD-associated senescence model. MOL induces apoptosis in senescent neuronal cells, attenuates DNA damage-associated stress, and selectively modulates SASP-related inflammatory signalling. These findings position MOL as a promising natural senolytic and senescence-modulating agent in the context of AD-related neuroinflammation and cellular senescence. Methods Antibodies and materials Human neuroblastoma cell line SH-SY5Y was obtained from American Type Culture Collection (ATCC, CRL2266). The following primary antibodies were used in this study: p21 (Invitrogen, GT1032), p16 (Abcam, ab189034), and γH2AX (Cell Signaling Technology, 9718S). The Amyloid-β protein fragment (1–42) (Sigma Aldrich, A9810) was used for induction of AD-associated senescene model. Secondary antibodies included Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Thermo Fisher Scientific, A11008), and Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Thermo Fisher Scientific, A11001). Preparation and characterization of MOL extract MOL were provided by MR Moringa Sdn. Bhd (Terengganu, Malaysia) in November 2024. The plant material was authenticated by botanist Dr. Shamsul bin Khamis, and a voucher specimen (ID022/2022) was deposited at the Universiti Kebangsaan Malaysia Herbarium (UKMB). The preparation of MOL extract followed a previously described with minor modifications [ 51 ]. In brief, dried MOL were ground into a fine powder using a laboratory blender. A total of 1 L of 80% ethanol was added to 100 g of the powdered MOL. The mixture was sonicated at 90 W, 37 kHz for 1 h 30 minutes, followed by centrifugation at 10,000 rpm for 15 minutes. The supernatant was collected, and the solvent was evaporated at 40°C under partial vacuum, followed by freeze-drying to obtain the crude MOL extract. Phytochemical profiling of the MOL extract was performed using UHPLC-MS/MS, following a previously described method [ 51 ]. Detailed phytochemical composition and compound annotation have been reported previously. The analysis confirmed that the extract was rich in phenolic compounds (78%) [ 51 ]. Establishment of an in vitro AD-associated senescence model Amyloid-β (Aβ) 1–42 oligomers were prepared as previously described [ 52 ] to obtain aggregated Aβ species. SH-SY5Y cells were exposed to Aβ 1–42 to induce AD-related cellular senescence. Cell culture and treatment SH-SY5Y cells were cultured following a previous method with some modification [ 53 ]. Briefly, Dulbecco’s Modified Eagle Medium (DMEM, Nacalai Tesque) supplemented with 12% fetal bovine serum (FBS) and 1% penicillin-streptomycin were used to culture the cells at 37°C, in a humidified incubator with 5% CO 2 . Subsequent experiments were performed 24 h after seeding SH-SY5Y cells at a density of 2.5 x 10 4 cells/mL in 96-well plates and 7 x 10 5 cells/mL in 6-well plates. Cellular senescence was induced by exposure to 20 µM Aβ 1–42 oligomers for 48 h. Following senescence induction, cells were treated for 24 h with either vehicle control (0.008% DMSO), a senolytic positive control consisting cocktail of 3nM dasatinib and 3µM quercetin (D + Q), or 300µg/mL MOL extract. Apoptosis assay Apoptosis was assessed using the CellEvent™ Caspase-3/7 Detection Reagent (Invitrogen, C10423) according to the manufacturer’s instructions. Following treatment, cells were incubated with the detection reagent at 37°C in a CO 2 -free incubator for 30 minutes. Fluorescent images were captured using an inverted fluorescence microscope (Nikon, A1R). Positive and negative cells were defined based on fluorescence intensity. Signals from negative controls were set as a threshold, and cells with fluorescence intensity higher than the threshold were considered positive. Caspase 3/7-positive cells were quantified from five randomly selected fields at 40x magnification, and the values were averaged. Senescence-associated β-galactosidase staining SA-β-gal staining was performed using the cell senescence β-galactosidase staining kit (MedChemExpress, HY-K1089), following the manufacturer’s instructions. Stained cells were observed and imaged using an inverted light microscope (RaxVision). The number of SA-β-gal-positive cells was counted in five randomly selected fields, and the values were averaged. Immunofluorescence staining Cells were seeded onto sterile round glass cover slips. Following treatment, immunofluorescence staining was performed based as previously described method [ 54 ]. Cells were fixed with 4% paraformaldehyde, permeabilised with 0.1% Triton X-100, blocked with 2% FBS in PBS for 1 h, and then incubated with primary antibodies against p21 (1:500), p16 (1:200), and γH2AX (1:200) at 4 ° C overnight. After washing, cells were incubated with Alexa Fluor™ 488 secondary antibody at 1:1000 for 1 h at RT. Next, coverslips with cells were mounted using Prolong Gold Antifade Mountant with DAPI (Thermofisher Scientific, P36931). Images were visualized and captured using a fluorescence microscope (Nikon, A1R) under identical exposure settings for all experimental group. Nuclear positive cells were counted from five randomly selected fields at 40x magnification for each sample, and the values were averaged. Enzyme-linked immunosorbent assay (ELISA) Cell culture supernatant were centrifuged at 1000 x g for 20 min to remove cell debris. The concentrations of IL-8, TNF-α, and IL-1β secreted by SH-SY5Y cells were quantified using human ELISA kits for IL-8 (E-EL-H6008), TNF‐α (E-EL-H0109), and IL-1β (E-EL-H0149) purchased from Elabscience (Wuhan, CN) according to the manufacturer's protocol. Measurements below the limit of detection (LOD) were assigned a value of LOD/2, following standard practice for handling left-censored environment and analytical data [ 55 ]. Statistical analyses All data were obtained from three independent experiments and are presented as mean ± standard error mean (SEM). The data were analysed using an independent two-tailed t -test or one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test using the Statistical Package for the Social Sciences (SPSS) version 30. The p-values of *p < 0.05, **p < 0.01, ***p < 0.001 was considered statistically significant. Declarations Acknowledgements The authors would like to thank the staff of Clinical Research Laboratory at Manipal University College Malaysia (MUCM) for facilitating the research environment necessary to carry out this work. The authors gratefully acknowledge Dr. Hajah Maimunah Ropar and all staff at MR Moringa Sdn. Bhd. for providing the Moringa oleifera leaf samples for this study. Funding This work was supported by the Ministry of Higher Education Malaysia through the Fundamental Research Grant Scheme (FRGS/1/2024/SKK10/MUCM/02/1) and MUCM-MRB joint seed grant (MUCM-MRB/001/2024). Author contributions Conceptualization, N.I.S.M.Z and F.A.J.; data curation, N.I.S.M.Z, F.A.J. and H.S.H.; formal analysis, N.I.S.M.Z, M.Z.S and F.A.J.; funding acquisition, F.A.J; investigation, N.I.S.M.Z., H.S.H. and A.M; methodology, F.A.J, N.I.S.M.Z and M.G.; supervision, M.Z.S., H.S.H. and F.A.J; validation, F.A.J, H.S.H, and M.Z.S.; writing—original draft, N.I.S.M.Z.; writing—review and editing, F.A.J. and M.Z.S. All authors have read and agreed to the final version of this manuscript. Data availability All data generated or analyzed during this study are included in this published article. Conflict of interest The authors declare no competing interests. References Vos, T. et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1999-2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet. 396, 1204-1222 (2020). Yiannopoulou, K. G. & Papageorgiou, S. G. Current and Future Treatments in Alzheimer Disease: An Update. 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Role of tumor necrosis factor-alpha in the central nervous system: a focus on autoimmune disorders. Frontiers in Immunology. Volume 14 - 2023, (2023). Zheng, Y. et al. Inflammatory signaling pathways in the treatment of Alzheimer's disease with inhibitors, natural products and metabolites (Review). Int J Mol Med. 52, (2023). Saliev, T. & Singh, P. B. Targeting Senescence: A Review of Senolytics and Senomorphics in Anti-Aging Interventions. Biomolecules. 15, (2025). Ma, W. W., Li, C. Q., Zhao, L., Wang, Y. S. & Xiao, R. NF-κB-mediated inflammatory damage is differentially affected in SH-SY5Y and C6 cells treated with 27-hydroxycholesterol. Food Sci Nutr. 7, 1685-1694 (2019). da Silva Parente, T. S. J., Sarandy, M. M., de Araújo, E. R. D., Gonçalves, R. V. & Zucolotto, S. M. Effect of Moringa oleifera on inflammatory diseases: an umbrella review of 26 systematic reviews. Frontiers in Pharmacology. 16, (2025). Azlan, U. K. et a l. Integrative metabolomics using untargeted UHPLC-MS/MS and chemometrics identifies optimal maturity stage of Moringa oleifera leaves from Kuala Terengganu, Malaysia. PeerJ 14, e20938 (2026). https://doi.org/10.7717/peerj.20938 Yeo, E. T. Y. et al. Piper sarmentosum Roxb. confers neuroprotection on beta-amyloid (Aβ)-induced microglia-mediated neuroinflammation and attenuates tau hyperphosphorylation in SH-SY5Y cells. Journal of Ethnopharmacology. 217, 187-194 (2018). Kaya, Z. B. et al. Optimizing SH-SY5Y cell culture: exploring the beneficial effects of an alternative media supplement on cell proliferation and viability. Scientific Reports. 14, 4775 (2024). Jam, F. A. et al. Neuroepithelial cell competition triggers loss of cellular juvenescence. Sci Rep. 10, 18044 (2020). Handelsman, D. J. & Ly, L. P. An Accurate Substitution Method To Minimize Left Censoring Bias in Serum Steroid Measurements. Endocrinology. 160, 2395-2400 (2019). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 23 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers agreed at journal 22 Mar, 2026 Reviewers agreed at journal 22 Mar, 2026 Reviewers agreed at journal 20 Mar, 2026 Reviewers invited by journal 20 Mar, 2026 Editor assigned by journal 20 Mar, 2026 Editor invited by journal 18 Mar, 2026 Submission checks completed at journal 15 Mar, 2026 First submitted to journal 15 Mar, 2026 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-9082466","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":610545097,"identity":"54013624-0e52-4d5f-a01b-e659e1c05ed5","order_by":0,"name":"Nur Irlia Sofea Mohammad Zamani","email":"","orcid":"","institution":"Manipal University College Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Nur","middleName":"Irlia Sofea Mohammad","lastName":"Zamani","suffix":""},{"id":610545098,"identity":"5f9cdf73-96b1-4d99-a6e7-4bf078f41040","order_by":1,"name":"Hamizah Shahirah Hamezah","email":"","orcid":"","institution":"Universiti Kebangsaan Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Hamizah","middleName":"Shahirah","lastName":"Hamezah","suffix":""},{"id":610545100,"identity":"0e4be35f-2640-49b7-a5bf-145fe2b54d25","order_by":2,"name":"Ahmed Mediani","email":"","orcid":"","institution":"Universiti Kebangsaan Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"","lastName":"Mediani","suffix":""},{"id":610545101,"identity":"fb28d477-39b1-473b-be0e-1958b22cea39","order_by":3,"name":"Mizanurfakhri Ghazali","email":"","orcid":"","institution":"Manipal University College Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Mizanurfakhri","middleName":"","lastName":"Ghazali","suffix":""},{"id":610545103,"identity":"508f952b-0238-411a-a20d-2f17db38ce97","order_by":4,"name":"Muhammad Zulfiqah Sadikan","email":"","orcid":"","institution":"Universiti Kuala Lumpur Royal College of Medicine Perak","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Zulfiqah","lastName":"Sadikan","suffix":""},{"id":610545104,"identity":"41e7f6d1-3af0-448d-95fd-f1ad7a461233","order_by":5,"name":"Faidruz Azura Jam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABIUlEQVRIie3RMUvDQBQH8HccJMtJHCNK/QRCuqQEh36VBsEuySQEFCmvFM4l2DVfQ4TMCQc6qHGN1CG3uBUEQVwUE411SdJV8P7L/Xnw4z04ABWVPxgdCZpVIQikKF8GQBFAaycs+SXU+iZkHQEw667VZR3RxXRxzCdAz24OT/LTx52BniI8BwL2olEzYe7MueMakNCPF97VE3NCF0mUCbDzZjIEl29NOSuvqYgmmJW4SDd4O2GGrIgJZL6Mj7yPktxLpO9dxPzaYgGJ/Jj6vCR5uYV0EjlzMBsxEi0vt/1zwZxIYhpmY2bfFi2HjeUDBpNef+5fvHivYjgwDtLiLdjv2dfNW1a0j3Wr/jOpJrDZTWD3p1irkZF0ExUVFZX/kk+evmOSpRTgQAAAAABJRU5ErkJggg==","orcid":"","institution":"Manipal University College Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Faidruz","middleName":"Azura","lastName":"Jam","suffix":""}],"badges":[],"createdAt":"2026-03-10 10:09:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9082466/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9082466/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105566141,"identity":"dd40bb31-ee0a-4dcd-b909-1b0aae990f85","added_by":"auto","created_at":"2026-03-27 12:55:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11651304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment of an \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e AD-associated senescence model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Experimental Scheme\u003c/p\u003e\n\u003cp\u003e(b) Representative image of\u003cstrong\u003e \u003c/strong\u003eSA-β-gal staining under 20x magnification.\u003c/p\u003e\n\u003cp\u003e(c) Quantification of SA-β-gal-positive cells.\u003c/p\u003e\n\u003cp\u003e(d) Immunofluorescent analysis of p21, p16, and γH2AX expression in young and senescent SH-SY5Y cells. Scale bars = 50 µm.\u003c/p\u003e\n\u003cp\u003e(e) Percentage of positive cells per field.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM from three independent experiments. Statistical analysis was performed using an independent two-tailed t-test for comparisons between two groups or one-way ANOVA followed by a Bonferroni’s post-hoc test for multiple comparisons (*p\u0026lt;0.05, ***p\u0026lt;0.001; ‘NS’ indicates not-significant (p\u0026gt;0.05)).\u003c/p\u003e","description":"","filename":"Figure1EstablishmentofinvitroADmodel.png","url":"https://assets-eu.researchsquare.com/files/rs-9082466/v1/264e9fa2d6d6943c0749045b.png"},{"id":105398201,"identity":"febef709-e24f-41ea-9f4d-638657c408b0","added_by":"auto","created_at":"2026-03-25 14:50:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10242753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMOL extract induces apoptosis in Aβ-induced senescent SH-SY5Y cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative images of young and senescence SH-SY5Y cells in untreated, D+Q and MOL-treated group under 10x magnification.\u003c/p\u003e\n\u003cp\u003e(b) Number of young and senescence SH-SY5Y cells.\u003c/p\u003e\n\u003cp\u003e(c) Representative images of caspase 3/7 expression in Aβ–induced senescent SH-SY5Y cells after 24 h MOL treatment. Scale bars = 50 µm.\u003c/p\u003e\n\u003cp\u003eData represented mean ± SEM from three independent experiments. Statistical analysis was performed using an independent two-tailed t-test for comparisons between two groups or one-way ANOVA followed by a Bonferroni’s post-hoc test for multiple comparisons (*p\u0026lt;0.05, **p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Figure2MoleliminatesSnCsthroughapoptosisinduction.png","url":"https://assets-eu.researchsquare.com/files/rs-9082466/v1/9791e5addee6a5f536391f6e.png"},{"id":105398203,"identity":"70f4e405-6efb-4a22-901c-17e8517b7b08","added_by":"auto","created_at":"2026-03-25 14:50:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21399198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of MOL treatment on SA-β-gal activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative images of SA-β-gal staining under 20x magnification.\u003c/p\u003e\n\u003cp\u003e(b) Quantification of SA-β-gal-positive cells.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM from three independent experiments. Statistical analysis was performed using an One-way ANOVA followed by a Bonferroni post-hoc test (“NS” indicates not significant (p\u0026gt;0.05)).\u003c/p\u003e","description":"","filename":"Figure3EffectofMOLtreatmentonSAgalactivity.png","url":"https://assets-eu.researchsquare.com/files/rs-9082466/v1/b78ad7549aa56e9e45aaf330.png"},{"id":105398199,"identity":"93e748f5-3879-45a0-abaf-761dee9921a1","added_by":"auto","created_at":"2026-03-25 14:50:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10529827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMOL reduces senescence-associated markers expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Percentage of p21-positive cells per field.\u003c/p\u003e\n\u003cp\u003e(b) Percentage of p16-positive cells per field.\u003c/p\u003e\n\u003cp\u003e(c) Percentage of yH2AX positive cells per field.\u003c/p\u003e\n\u003cp\u003eRepresentative image of (d) p21, (e) p16, and (f) yH2AX across all treatment groups. Scale bars = 50 µm. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was performed using an One-way ANOVA followed by a Bonferroni post-hoc test (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure4MOLreducessenescenceassociatedmarkerexpression.png","url":"https://assets-eu.researchsquare.com/files/rs-9082466/v1/6010c3bd7c1b429299bd1810.png"},{"id":105398198,"identity":"cf7a1964-4d57-4cda-b02e-cda1df390b58","added_by":"auto","created_at":"2026-03-25 14:50:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1166497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMOL modulates SASP cytokine secretion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe normalised expression of (a) IL-8, (b) TNF-α, and (c) IL-1β in SH-SY5Y cells after 24 h treatment was determined using ELISA. Data represented mean ± SEM from three independent experiments (one-way ANOVA followed by Bonferroni’s post-hoc test *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure5MOLmodulatesSASPcytokinesecretion.png","url":"https://assets-eu.researchsquare.com/files/rs-9082466/v1/c61f5ccedc126400e7c6eceb.png"},{"id":105728145,"identity":"e4c72dc8-be7c-4c92-a909-fe3bac7ac742","added_by":"auto","created_at":"2026-03-30 11:10:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":47765309,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9082466/v1/8d4e3473-ed9d-4a1b-be14-666671c4f46f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Senolytic effects of Moringa oleifera leaf extract in an in vitro model of Alzheimer’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe global population is rapidly aging, leading to increase in the prevalence of Alzheimer\u0026rsquo;s disease (AD). By 2050, more than 130\u0026nbsp;million people worldwide are expected to be affected [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], imposing substantial social, economic, and healthcare burdens. Current therapeutics approaches provide primarily symptomatic relief and modestly slow disease progression without addressing underlying pathogenic mechanisms [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], highlighting the need for mechanism-based strategies suitable for long-term use in aging-populations.\u003c/p\u003e \u003cp\u003eCellular senescence has emerged as a key biological link between aging and AD. Senescence is a stable state of cell cycle arrest induced by diverse stressors such as oxidative damage, mitochondrial dysfunction and amyloid-β (Aβ) toxicity. Senescent cells (SnCs) exhibit increased resistance to apoptosis through activation of senescent cell anti-apoptotic pathways (SCAPs) and secrete a range of pro-inflammatory factors collectively termed as senescence-associated secretory phenotypes (SASP). In the AD brain, senescent-associated phenotypes have been reported in astrocytes, microglia, and neurons, particularly near to Aβ plaques and neurofibrillary tangles (NFTs), where SASP-mediated chronic neuroinflammation disrupts synaptic function, accelerates neuronal loss, and exacerbates disease progression [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite increasing evidence linking senescence to AD pathology, the mechanisms by which Aβ induces senescence and its contribution to disease progression remain incompletely understood.\u003c/p\u003e \u003cp\u003eGrowing interest has focused on dietary bioactive compounds as multi-target interventions for age-related neurodegenerative diseases. \u003cem\u003eMoringa oleifera\u003c/em\u003e is a nutrient-rich functional food known for its antioxidant, anti-inflammatory, and neuroprotective properties, largely attributed to its high flavonoid and phenolic content [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Phenolic compounds are widely recognised for their protective effects against chronic diseases through antioxidant and anti-inflammatory mechanisms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the context of AD, flavonoids, a major subclass of phenolic compounds are of particular interest due to their ability to cross the blood-brain barrier (BBB), improve cognitive function, modulate oxidative stress, inflammatory signalling and aging-associated molecular pathways [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the potential of \u003cem\u003eMoringa oleifera\u003c/em\u003e to regulate cellular senescence in AD-relevant models remains largely unexplored. Therefore, this study aimed to investigate the senolytic potential of \u003cem\u003eMoringa oleifera\u003c/em\u003e leaf (MOL) extract in an \u003cem\u003ein vitro\u003c/em\u003e Aβ-induced senescence model, focusing on SnCs elimination and attenuation of SASP-associated inflammatory responses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEstablishment of an\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eAD-associated senescence model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the senolytic potential of MOL in AD, we established an \u003cem\u003ein vitro\u003c/em\u003e AD-associated senescence model by exposing SH-SY5Y cells to 20 \u0026micro;M Aβ\u003csub\u003e1\u0026ndash;42\u003c/sub\u003e oligomers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Following Aβ exposure, we observed that SA-β-gal-positive cells were increased compared to untreated, non-senescent control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). Consistently, the expression of senescence markers, p16 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), p21 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and γH2AX (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were significantly upregulated in Aβ-treated cells compared to non-senescent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e), indicating activation of cell-cycle arrest pathways and DNA damage responses. Collectively, these findings confirm the successful induction of an Aβ-associated senescent phenotype in SH-SY5Y cells, validating this model for the evaluation of senolytic interventions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMOL extract induces apoptosis in Aβ-induced senescent SH-SY5Y cells\u003c/h2\u003e \u003cp\u003eTo assess the senolytic potential of MOL, both young and senescent SH-SY5Y cells were treated with MOL and compared with the established senolytic cocktail dasatinib and quercetin (hereafter referred to as D\u0026thinsp;+\u0026thinsp;Q). MOL treatment significantly reduced the number of SnCs compared with young cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). A similar reduction in the number of SnCs was observed following D\u0026thinsp;+\u0026thinsp;Q treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, the number of SnCs in both MOL and D\u0026thinsp;+\u0026thinsp;Q-treated group is significantly reduced compared to the number of SnCs in the untreated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). This selective reduction indicates that MOL preferentially targets SnCs while preserving healthy, proliferative cells. No significant difference was observed between MOL and D\u0026thinsp;+\u0026thinsp;Q in their ability to reduce SnCs burden, suggesting that MOL exerts senolytic effects comparable to the reference senolytic treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the mechanism underlying this selective elimination, caspase-3/7 activation was examined. SnCs treated with MOL showed pronounced morphological features of apoptosis, including cell rounding accompanied by positive caspase-3/7 activation signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Immunofluorescence analysis revealed a higher number of caspase-3/7\u0026ndash;positive cells in MOL and D\u0026thinsp;+\u0026thinsp;Q treated groups compared with the untreated and DMSO vehicle-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Together, these findings demonstrate that MOL selectively induces apoptosis in senescent SH-SY5Y cells, consistent with senolytic activity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMOL extract reduced senescence-associated molecular markers\u003c/h3\u003e\n\u003cp\u003eTo further evaluate the impact of MOL on senescence burden, the expression of senescence-associated molecular markers was examined following treatment. As expected, Aβ exposure increased SA-β-gal positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b) and significantly upregulated p21, p16, and γH2AX compared with untreated controls, confirming the induction of cellular senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b, c, d ,\u003cb\u003ee, f\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTreatment with MOL extract resulted in a non-significant reduction in SA-β-gal positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b), suggesting partial attenuation of this hallmark of senescence. In contrast, the expression of p21, p16, and γH2AX were significantly downregulated following MOL treatment compared with untreated and DMSO vehicle-treated cells. Notably, these reductions were comparable to those observed with the senolytic positive control D\u0026thinsp;+\u0026thinsp;Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea - f). Collectively, these findings indicate that MOL effectively reduces senescence burden, with a pronounced impact on DNA damage signalling pathways, while exerting a more modest effect on SA-β-gal activity.\u003c/p\u003e\n\u003ch3\u003eMOL extract modulates SASP-associated cytokine secretion\u003c/h3\u003e\n\u003cp\u003eTo determine whether MOL influences the inflammatory secretory profile of SnCs, the secretion of SASP-associated cytokines was measured following Aβ induction and subsequent treatments with DMSO vehicle, D\u0026thinsp;+\u0026thinsp;Q or MOL. After normalisation to total protein content, we observed MOL treatment significantly reduced IL-8 secretion compared with untreated and DMSO vehicle-treated cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). TNF-α secretion was also markedly reduced following MOL treatment and falling below the assay\u0026rsquo;s limit of detection (4.69 pg/mL) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), suggesting strong suppression rather than assay insensitivity. In contrast, IL-1β levels showed an increase following MOL treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). These findings indicate that MOL does not uniformly suppress all SASP components but instead selectively modulates specific inflammatory mediators.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccumulating evidence from preclinical [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and clinical studies [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] highlights cellular senescence as a key pathological process in AD, with SnCs enriched in Aβ\u0026ndash;associated brain regions. Consistent with these observations, our findings demonstrate that MOL modulate several markers associated with senescence-related pathways in an AD cellular model. Although senolytic therapies have shown promise in improving cognitive function and reducing amyloid burden, their clinical translation remains limited due to potential adverse effects [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Here, our results provide experimental evidence that a naturally derived compound may exert senomodulatory effects. These findings support MOL as a potential safer alternative or complementary strategy to conventional senolytic approaches.\u003c/p\u003e \u003cp\u003eIn this study, we observed that Aβ exposure induces a senescent phenotype in SH-SY5Y cells, as indicated by the significant upregulation of p21, p16, and γH2AX, reflecting activation of cell-cycle arrest and DNA damage signalling pathways [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Previous studies have similarly reported elevated γH2AX expression in AD brains and neuronal models [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which is associated with persistent DNA double-strand breaks and genomic instability during disease progression. Although γH2AX alone is not a definitive marker of cellular senescence, its concurrent induction with p21 and p16 suggests that Aβ-induced DNA damage activates senescence-associated cell-cycle arrest mechanisms. These molecular changes support the relevance of this model for recapitulating AD\u0026ndash;associated cellular stress and provide a mechanistic basis for evaluating senolytic interventions. The observed marker profile is consistent with prior reports demonstrating that Aβ triggers neuronal senescence and genomic instability [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile SA-β-gal staining increased following Aβ exposure, the change did not reach statistical significance. This observation may reflect the relatively early stage of senescence induction in neuronal cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Evidence from other SH-SY5Y models indicate that SA-β-gal activity can be highly dependent on the type and duration of the senescence-inducing stimulus [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, while SA-β-gal is widely used as a senescence marker, it has recognised limitations and may vary during early or partial senescence across different experimental contexts [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These findings suggest that early or mild senescence induction may not always produce statistically significant changes in SA-β-gal activity.\u003c/p\u003e \u003cp\u003eMOL extract preferentially reduced the burden of SnCs, an effect typically observed with established senolytic drugs; which selectively eliminate SnCs while sparing young, non-senescent cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consistent with this observation, MOL treatment activated caspase-3/7 and induced apoptotic morphological changes predominantly in SnCs, providing mechanistic evidence of senolytic activity. This mechanism aligns with those reported for established senolytic compounds such as ABT263 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and natural senolytic fisetin [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], which target the SCAPs and promote apoptotic cell death. The preservation of young cells following MOL treatment may also benefitted from its antioxidant and neuroprotective properties, which could mitigate oxidative and metabolic stress in non-SnCs [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. D\u0026thinsp;+\u0026thinsp;Q is a well-established senolytic combination widely used in preclinical models and currently under clinical investigation [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The absence of a significant difference between MOL and D\u0026thinsp;+\u0026thinsp;Q in reducing the SnCs burden suggests that MOL exhibits senolytic efficacy comparable to that of this benchmark treatment in the present model. Although further validation in additional experimental systems is warranted, our findings provide the first evidence supporting MOL as a promising natural senolytic candidate.\u003c/p\u003e \u003cp\u003eBeyond the elimination of SnCs, MOL robustly attenuated senescence-associated molecular pathways, consistent with previous studies reporting senotherapeutic effects of natural compounds [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. MOL treatment reduced SA-β-gal positivity compared with the untreated, DMSO-vehicle treated, and D\u0026thinsp;+\u0026thinsp;Q-treated groups, which has been linked to improved cognitive function and reduced SnCs burden [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In parallel, MOL significantly decreased p16, p21, and γH2AX expression, indicating coordinated suppression of cell-cycle arrest and DNA damage signalling pathways. Notably, MOL most prominently reduced γH2AX expression, suggesting mitigation of Aβ-induced persistent DNA damage. Given that γH2AX reflects persistent DNA double-strand breaks, this observation raises the possibility that MOL may mitigate Aβ\u0026ndash;induced genotoxic stress in this model. Although all markers were significantly reduced, differences in the magnitude of response suggest that MOL primarily exerts its effects through senolytic clearance, with secondary modulation of senescence-associated pathways. This pattern is consistent with a senolytic-dominant mechanism accompanied by secondary senomorphic activity.\u003c/p\u003e \u003cp\u003eSnCs secrete a range of pro-inflammatory cytokines collectively known as the SASP, including IL-8, IL-1β, and TNF-α, which contribute to chronic inflammation and neurodegeneration [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The non-significant increase in TNF-α observed in DMSO vehicle-treated cells likely reflects vehicle-induced stress rather than a treatment-specific effect. In contrast, MOL treatment significantly reduced IL-8 and TNF-α secretions, consistent with the selective elimination or suppression of SnCs rather than global inhibition of inflammatory signalling. These observations support the anti-inflammatory and senescence-modulating effects of MOL and align with previous reports in other experimental models [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. SASP components are regulated by inflammatory pathways, such as NF-κB and MAPK, which play central roles in AD-related neuroinflammation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and intersect with broader neurodegenerative signalling networks. As a key SASP mediator, IL-8 reinforces senescence and amplifies inflammatory signalling. Thus, its suppression strongly supports the senotherapeutic potential of MOL. In contrast, IL-1β increased following MOL treatment, which may reflect transient cytokine release associated with senolytic-induced cell death rather than enhanced inflammatory signalling [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In neuronal monocultures, the absence of professional phagocytes delayed the clearance of apoptotic SnCs, allowing IL-1β to persist in the extracellular environment. Although MOL has been reported to inhibit IL-1β production in other contexts [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], this effect may be masked by secondary cytokine release following SnCs elimination. Collectively, these findings suggest that MOL modulates SASP output in a manner consistent with senolytic activity, although the precise upstream molecular mechanisms remain to be defined.\u003c/p\u003e \u003cp\u003eThese findings highlight MOL as a promising natural senolytic candidate in the context of AD-associated cellular stress. However, several limitations should be acknowledged. First, this study employed a single neuroblastoma-derived cell line, which may not fully recapitulate the complexity of mature neurons or the brain microenvironment. Second, the use of a crude MOL extract precludes the identification of individual bioactive constituents and their specific molecular targets. Finally, the short-term \u003cem\u003ein vitro\u003c/em\u003e nature of this study limits the assessment of its long-term efficacy and safety. Future studies should employ primary neuronal cultures, co-culture systems, and well-characterised \u003cem\u003ein vivo\u003c/em\u003e AD models to validate and extend these findings. Elucidating the molecular pathways modulated by MOL, including those involved in cellular senescence, oxidative stress, and inflammation, will provide deeper mechanistic insight, while identification of key bioactive compounds responsible for these effects will be essential to clarify their pharmacological potential. Collectively, these approaches will help establish the therapeutic and translational relevance of MOL as a senolytic strategy for targeting senescence-associated mechanism in AD.\u003c/p\u003e \u003cp\u003eIn conclusion, MOL extract exhibits senolytic activity in an \u003cem\u003ein vitro\u003c/em\u003e AD-associated senescence model. MOL induces apoptosis in senescent neuronal cells, attenuates DNA damage-associated stress, and selectively modulates SASP-related inflammatory signalling. These findings position MOL as a promising natural senolytic and senescence-modulating agent in the context of AD-related neuroinflammation and cellular senescence.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies and materials\u003c/h2\u003e \u003cp\u003eHuman neuroblastoma cell line SH-SY5Y was obtained from American Type Culture Collection (ATCC, CRL2266). The following primary antibodies were used in this study: p21 (Invitrogen, GT1032), p16 (Abcam, ab189034), and γH2AX (Cell Signaling Technology, 9718S). The Amyloid-β protein fragment (1\u0026ndash;42) (Sigma Aldrich, A9810) was used for induction of AD-associated senescene model. Secondary antibodies included Goat anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Cross-Adsorbed Secondary Antibody, Alexa Fluor\u0026trade; 488 (Thermo Fisher Scientific, A11008), and Goat anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Cross-Adsorbed Secondary Antibody, Alexa Fluor\u0026trade; 488 (Thermo Fisher Scientific, A11001).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation and characterization of MOL extract\u003c/h3\u003e\n\u003cp\u003eMOL were provided by MR Moringa Sdn. Bhd (Terengganu, Malaysia) in November 2024. The plant material was authenticated by botanist Dr. Shamsul bin Khamis, and a voucher specimen (ID022/2022) was deposited at the Universiti Kebangsaan Malaysia Herbarium (UKMB). The preparation of MOL extract followed a previously described with minor modifications [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In brief, dried MOL were ground into a fine powder using a laboratory blender. A total of 1 L of 80% ethanol was added to 100 g of the powdered MOL. The mixture was sonicated at 90 W, 37 kHz for 1 h 30 minutes, followed by centrifugation at 10,000 rpm for 15 minutes. The supernatant was collected, and the solvent was evaporated at 40\u0026deg;C under partial vacuum, followed by freeze-drying to obtain the crude MOL extract. Phytochemical profiling of the MOL extract was performed using UHPLC-MS/MS, following a previously described method [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Detailed phytochemical composition and compound annotation have been reported previously. The analysis confirmed that the extract was rich in phenolic compounds (78%) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eEstablishment of an\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eAD-associated senescence model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmyloid-β (Aβ)\u003csub\u003e1\u0026ndash;42\u003c/sub\u003e oligomers were prepared as previously described [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] to obtain aggregated Aβ species. SH-SY5Y cells were exposed to Aβ\u003csub\u003e1\u0026ndash;42\u003c/sub\u003e to induce AD-related cellular senescence.\u003c/p\u003e\n\u003ch3\u003eCell culture and treatment\u003c/h3\u003e\n\u003cp\u003eSH-SY5Y cells were cultured following a previous method with some modification [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Briefly, Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Nacalai Tesque) supplemented with 12% fetal bovine serum (FBS) and 1% penicillin-streptomycin were used to culture the cells at 37\u0026deg;C, in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequent experiments were performed 24 h after seeding SH-SY5Y cells at a density of 2.5 x 10\u003csup\u003e4\u003c/sup\u003e cells/mL in 96-well plates and 7 x 10\u003csup\u003e5\u003c/sup\u003e cells/mL in 6-well plates. Cellular senescence was induced by exposure to 20 \u0026micro;M Aβ\u003csub\u003e1\u0026ndash;42\u003c/sub\u003e oligomers for 48 h. Following senescence induction, cells were treated for 24 h with either vehicle control (0.008% DMSO), a senolytic positive control consisting cocktail of 3nM dasatinib and 3\u0026micro;M quercetin (D\u0026thinsp;+\u0026thinsp;Q), or 300\u0026micro;g/mL MOL extract.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assay\u003c/h2\u003e \u003cp\u003eApoptosis was assessed using the CellEvent\u0026trade; Caspase-3/7 Detection Reagent (Invitrogen, C10423) according to the manufacturer\u0026rsquo;s instructions. Following treatment, cells were incubated with the detection reagent at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e-free incubator for 30 minutes. Fluorescent images were captured using an inverted fluorescence microscope (Nikon, A1R). Positive and negative cells were defined based on fluorescence intensity. Signals from negative controls were set as a threshold, and cells with fluorescence intensity higher than the threshold were considered positive. Caspase 3/7-positive cells were quantified from five randomly selected fields at 40x magnification, and the values were averaged.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSenescence-associated β-galactosidase staining\u003c/h2\u003e \u003cp\u003eSA-β-gal staining was performed using the cell senescence β-galactosidase staining kit (MedChemExpress, HY-K1089), following the manufacturer\u0026rsquo;s instructions. Stained cells were observed and imaged using an inverted light microscope (RaxVision). The number of SA-β-gal-positive cells was counted in five randomly selected fields, and the values were averaged.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eCells were seeded onto sterile round glass cover slips. Following treatment, immunofluorescence staining was performed based as previously described method [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Cells were fixed with 4% paraformaldehyde, permeabilised with 0.1% Triton X-100, blocked with 2% FBS in PBS for 1 h, and then incubated with primary antibodies against p21 (1:500), p16 (1:200), and γH2AX (1:200) at 4\u003csup\u003e\u0026deg;\u003c/sup\u003eC overnight. After washing, cells were incubated with Alexa Fluor\u0026trade; 488 secondary antibody at 1:1000 for 1 h at RT. Next, coverslips with cells were mounted using Prolong Gold Antifade Mountant with DAPI (Thermofisher Scientific, P36931). Images were visualized and captured using a fluorescence microscope (Nikon, A1R) under identical exposure settings for all experimental group. Nuclear positive cells were counted from five randomly selected fields at 40x magnification for each sample, and the values were averaged.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eCell culture supernatant were centrifuged at 1000 x g for 20 min to remove cell debris. The concentrations of IL-8, TNF-α, and IL-1β secreted by SH-SY5Y cells were quantified using human ELISA kits for IL-8 (E-EL-H6008), TNF‐α (E-EL-H0109), and IL-1β (E-EL-H0149) purchased from Elabscience (Wuhan, CN) according to the manufacturer's protocol. Measurements below the limit of detection (LOD) were assigned a value of LOD/2, following standard practice for handling left-censored environment and analytical data [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll data were obtained from three independent experiments and are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error mean (SEM). The data were analysed using an independent two-tailed \u003cem\u003et\u003c/em\u003e-test or one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test using the Statistical Package for the Social Sciences (SPSS) version 30. The p-values of *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the staff of Clinical Research Laboratory at Manipal University College Malaysia (MUCM) for facilitating the research environment necessary to carry out this work. The authors gratefully acknowledge Dr. Hajah Maimunah Ropar and all staff at MR Moringa Sdn. Bhd. for providing the \u003cem\u003eMoringa oleifera\u003c/em\u003e leaf samples for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ministry of Higher Education Malaysia through the Fundamental Research Grant Scheme (FRGS/1/2024/SKK10/MUCM/02/1) and MUCM-MRB joint seed grant (MUCM-MRB/001/2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization,\u0026nbsp;N.I.S.M.Z\u0026nbsp;and\u0026nbsp;F.A.J.; data curation,\u0026nbsp;N.I.S.M.Z, F.A.J. and H.S.H.; formal analysis,\u0026nbsp;N.I.S.M.Z, M.Z.S\u0026nbsp;and\u0026nbsp;F.A.J.; funding acquisition,\u0026nbsp; F.A.J; investigation, N.I.S.M.Z., H.S.H. and A.M; methodology, F.A.J, N.I.S.M.Z and M.G.; supervision, M.Z.S., H.S.H. and F.A.J; validation, F.A.J, H.S.H, and M.Z.S.; writing—original draft, N.I.S.M.Z.; writing—review and editing, F.A.J. and \u0026nbsp;M.Z.S. All authors have read and agreed to the final version of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\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\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eVos, T.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Global burden of 369 diseases and injuries in 204 countries and territories, 1999-2019: a systematic analysis for the Global Burden of Disease Study 2019. \u003cem\u003eThe Lancet.\u003c/em\u003e \u003cstrong\u003e396,\u003c/strong\u003e 1204-1222 (2020).\u003c/li\u003e\n \u003cli\u003eYiannopoulou, K. G. \u0026amp; Papageorgiou, S. G. 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An Accurate Substitution Method To Minimize Left Censoring Bias in Serum Steroid Measurements. \u003cem\u003eEndocrinology.\u003c/em\u003e \u003cstrong\u003e160,\u003c/strong\u003e 2395-2400 (2019).\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Senolytics, Moringa oleifera, Cellular senescence, Alzheimer's disease, Senescence-associated secretory phenotype","lastPublishedDoi":"10.21203/rs.3.rs-9082466/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9082466/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAccumulation of senescent cells (SnCs) in the aging brain contributes to Alzheimer's disease (AD) progression through the secretion of senescence-associated secretory phenotype (SASP) that exacerbates neuroinflammation and neurodegeneration. Senolytic agents that selectively eliminate SnCs, have emerged as a potential therapeutic strategy, however safer natural alternatives remain underexplored. In this study, we aimed to investigate the senolytic potential of \u003cem\u003eMoringa oleifera\u003c/em\u003e leaf (MOL) extract in an \u003cem\u003ein vitro\u003c/em\u003e AD-senescence model using SH-SY5Y cells exposed to amyloid-β (Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e) oligomers. SH-SY5Y cells exposed to Aβ oligomers exhibited senescent phenotype, characterised by increased senescence-associated β-galactosidase (SA-β-gal) positivity and upregulated nuclear expression of p21, p16, and γH2AX. Treatment with MOL extract significantly reduced the number of cells expressing senescence-associated molecular markers and induced apoptosis in senescent cells, while attenuating the secretion of pro-inflammatory SASP cytokines, including IL-8 and TNF-α. Overall findings suggest that MOL extract preferentially targets SnCs and mitigates SASP-associated inflammation. These results support the potential of MOL as a natural compound with senolytic activity and provide a foundation for further development into its therapeutic relevance in AD.\u003c/p\u003e","manuscriptTitle":"Senolytic effects of Moringa oleifera leaf extract in an in vitro model of Alzheimer’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 14:50:20","doi":"10.21203/rs.3.rs-9082466/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-23T09:18:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129278240309453215673242802149586837317","date":"2026-03-23T04:48:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73404104395612203255003512833327583966","date":"2026-03-23T01:17:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326298317419269564675970130514777736585","date":"2026-03-22T16:15:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148870221450977604559143763408821558271","date":"2026-03-20T13:22:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-20T10:51:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-20T10:49:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-18T08:59:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-15T06:38:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-15T06:34:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad7cf36b-05f9-47fc-8de6-42a5d02b75eb","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64956287,"name":"Biological sciences/Biochemistry"},{"id":64956288,"name":"Biological sciences/Drug discovery"},{"id":64956289,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-05-11T18:10:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 14:50:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9082466","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9082466","identity":"rs-9082466","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}