Trans-cinnamaldehyde Attenuates H₂o₂-induced Cellular Senescence in Human Svgp12 Astroglial Cells

Trans-cinnamaldehyde Attenuates H₂o₂-induced Cellular Senescence in Human Svgp12 Astroglial 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 Trans-cinnamaldehyde Attenuates H₂o₂-induced Cellular Senescence in Human Svgp12 Astroglial Cells RANA NUR GURSU, Volkan YASAR, Hasan Emre BOLAT, Muslum Gok, Hulya ELBE This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8457575/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Aging is characterized by progressive functional decline driven by oxidative stress, inflammation, and cellular senescence. Astrocytes are particularly vulnerable to oxidative injury, making them a relevant cellular model for investigating aging-related neurodegenerative mechanisms. Trans-cinnamaldehyde (TCA), a major bioactive component of cinnamon, exhibits antioxidant and anti-inflammatory properties; however, its role in astroglial senescence remains poorly understood. Methods Oxidative stress-induced senescence was established in SVGp12 astroglial cells100 µM hydrogen peroxide (H2O2) was used to achieve this. Senescence was confirmed by morphological alterations, reduced cell viability (WST-8 assay), and senescence-associated β-galactosidase (SA-β-gal) staining. Following cytotoxicity screening, TCA was applied at a non-toxic concentration. HIF-1α expression was evaluated by immunocytochemistry (ICC) and semi-quantified using H-score analysis. Levels of KEAP1, TNF-α, IL-1β, IL-6 and NRF2 were measured by ELISA. Results H₂O₂ exposure significantly induced astroglial senescence, increased SA-β-gal positivity, elevated HIF-1α immunoreactivity, and upregulated proinflammatory cytokines and KEAP1/NRF2 signaling. TCA treatment made a big difference in cell viability, reduced the changes to cells that happen as they age, and decreased the expression of HIF-1α. Moreover, TCA markedlyattenuated the H₂O₂-induced increases in IL-6, KEAP1, TNF-α, NRF2 and IL-1β levels. Conclusion TCA effectively mitigates oxidative stress-induced senescence in astroglial cells by reducing HIF-1α expression, inflammatory cytokine production, and redox stress signaling. TCA may be a natural compound that can target astrocyte-associated ageing and neuroinflammation, according to these findings. Aging Cellular senescence Oxidative stress Astrocytes SVGp12 cells Trans-cinnamaldehyde Neuroinflammation Antioxidant activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Aging represents a progressive biological phenomenon characterized by long-term alterations in cellular performance and proliferative capacity, ultimately predisposing organisms to higher morbidity and mortality rates (Csekes & Račková, 2021 ). In most species, aging is accompanied by degenerative pathologies associated with the gradual loss of tissue and cellular integrity. At the cellular level, senescence represents a largely irreversible growth arrest induced by various stressors. The result of three things happening to senescent cells is a senescence-associated secretory phenotype (SASP): profound changes to their chromosomes, altered gene expression, and the release of pro-inflammatory cytokines and chemokines. (Campisi, 2013 ). Hayflick and Moorhead, who showed that normal human somatic cells can divide only a limited number of times in a lab setting (Hayflick & Moorhead, 1961 ), first introduced the idea of limited proliferative capacity. Helmut Sies introduced the term "oxidative stress" to describe an unequal balance between oxidant production and antioxidant defense mechanisms that results in biological damage. This unequal balance has been implicated in numerous pathological conditions, including cardiovascular disease, neurodegeneration, cancer, and aging-related disorders (Forman & Zhang, 2021 ). Supporting this concept, epidemiological studies have reported an inverse relationship between fruit and vegetable consumption and the incidence of inflammation, cancer, cardiovascular disease and aging-associated disorders, largely attributed to dietary antioxidants such as polyphenols, vitamins C and E, and carotenoids (Huang et al., 2005 ). Oxidative stress further modulates hypoxia-related and redox-dependent signaling cascades. Cellular adaptation to hypoxia is largely controlled by HIF-1, a heterodimeric transcription factor in which HIF-1α activity is selectively enhanced during hypoxic and oxidative stress through redox-dependent mechanisms involving mitochondrial reactive oxygen species (Semenza, 2001 ). Concurrently, nuclear factor erythroid 2-related factor 2 (NRF2), Kelch-like ECH-associated protein 1 (KEAP1) and the signaling pathway function as a regulator of cellular homeostasis of redox. In normal conditions, KEAP1 keeps NRF2 in the cytoplasm and causes it to be broken down by the cell's protein-breaking machine. However, oxidative stress promotes NRF2 release, nuclear translocation, and the induction of antioxidant and cytoprotective genes. Dysregulation of the NRF2- KEAP1 axis has been associated with aging, cellular senescence, and chronic inflammation, highlighting its importance in pathologies driven by oxidative stress (Baird & Yamamoto, 2020 ). An increase in β-galactosidase activity measurable at pH 6.0 has been established as a functional marker of the senescent cellular state and is referred to as senescence-associated β-galactosidase (SA-β-gal) (Lee et al., 2006 ). Additionally, key components of the SASP include inflammatory cytokines. Examples of these are interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). These are important mediators. They link oxidative stress, inflammation, and cellular senescence. Dysregulated production of these cytokines contributes to chronic inflammation and tissue dysfunction during aging (Lopez-Castejon & Brough, 2011 ; Kishimoto, 2006 ; Idriss & Naismith, 2000 ). In the central nervous system, astrocytes form a major glial population and are involved in a wide range of physiological processes, including regulation of neural homeostasis, support of neuronal viability, and coordination of local defense responses (Freeman, 2010 ). Their structural and functional plasticity enables them to regulate neural activity across molecular, cellular, and organ levels (Verkhratsky & Nedergaard, 2018 ). Increasing evidence indicates that astrocytes are particularly vulnerable to oxidative stress and contribute actively to neurodegenerative processes through their interactions with neurons and inflammatory signaling networks (Zhong et al., 2023 ). The SVGp12 cell line, derived from human fetal astroglial cells, has been widely employed as a neural model system in studies investigating astrocyte biology and stress responses (Henriksen et al., 2014 ). Due to its human astroglial origin, SVGp12 cells provide a valuable experimental platform for examining oxidative stress-induced cellular alterations relevant to neural aging. Neural aging is associated with reduced neuroplasticity, impaired neurogenesis, and increased susceptibility to neurodegeneration, reflecting both age-dependent and age-independent mechanisms (Koen & Rugg, 2019 ; Culig et al., 2022 ). These observations highlight the importance of cellular models that can recapitulate aging-related changes and facilitate the identification of potential protective interventions. In this context, naturally derived compounds with antioxidant and anti-inflammatory properties have attracted increasing attention as potential modulators of aging-related cellular dysfunction. Cinnamon, obtained from the bark of the Cinnamomum genus, exhibits broad pharmacological activities, largely attributed to its active component cinnamaldehyde. Researchers have reported that trans-cinnamaldehyde (TCA) exerts antioxidant, anti-inflammatory, cardioprotective, anti-aging, and antitumor effects (Guo et al., 2024 ), highlighting its potential relevance in oxidative stress-associated conditions. Hydrogen peroxide (H₂O₂) is commonly used to model aging-related oxidative stress in vitro (Campisi, 2013 ; Shang et al., 2020 ). Expanding upon this methodology, the present study developed an H₂O₂-induced senescence model in SVGp12 astroglial cells to ascertain the protective effects of TCA. Based on these considerations, we hypothesized that TCA exerts senomorphic effects on astroglial cells by alleviating oxidative stress, modulating redox-sensitive signaling pathways, and attenuating senescence-associated inflammatory responses. The objective of this study was to assess the impact of TCA on oxidative stress-induced cellular senescence and its effect on HIF-1α, KEAP1/NRF2 signaling, and inflammatory cytokine expression (IL-1β, IL-6, and TNF-α) in SVGp12 cells. Researchers have studied TCA in many different experiments, and they have found that it has antioxidant and anti-inflammatory properties. However, scientists still don't know enough about how TCA might influence the process of astroglial senescence and the related pathways that respond to redox and stress. In particular, the integrated assessment of HIF-1α signaling, KEAP1/NRF2-mediated redox regulation, and senescence-associated inflammatory responses within an astroglial senescence model represents a distinctive aspect of the present study. By examining these pathways concurrently, this work contributes novel insight into the senomorphic potential of redox-modulating phytochemicals and their relevance to aging-related neuroinflammatory processes. Materials and Methods Cell Culture The human fetal astroglial cell line SVGp12 (ATCC® CRL-8621™, Manassas, VA, USA) was obtained from the American Type Culture Collection (ATCC). Cells at passages 2-4 were cultured in Dulbecco’s Modified Eagle (Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 11965-092) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen Life Technologies, Paisley, UK; Cat. No. 10270-106) and 1% penicillin/streptomycin (Multicell, Wisent Inc., St-Bruno, QC, Canada; Cat. No. 450-202-EL). Cells were maintained in a humidified incubator (MCO18AC-PE, Panasonic Healthcare Co., Ltd., Tokyo, Japan) at 37 °C with 5% CO₂ and were subcultured at approximately 80% confluence using 0.05% trypsin-EDTA (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. T3924). For cell viability assays, SVGp12 cells were seeded into 96-well plates (Thermo Scientific, Waltham, MA, USA) at a density of 5 × 10³ cells per well. For senescence-associated β-galactosidase (SA-β-gal) staining, cells were plated into 6-well plates (Thermo Scientific, Waltham, MA, USA) at a density of 1 × 10⁵ cells per well. Following senescence induction and determination of the appropriate trans-Cinnamaldehyde (TCA) (Aldrich, Sigma-Aldrich; Cat. No. C80687) concentration range, cells were seeded into 25 cm² culture flasks (Thermo Scientific, Waltham, MA, USA) at a density of 1.5 × 10⁶ cells per flask for subsequent ELISA analyses. For immunocytochemistry (ICC) experiments, cells were seeded onto sterile 13-mm glass coverslips (Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) placed in 24-well plates (NEST Biotechnology, Wuxi, China) at a density of 3 × 10⁴ cells per well. Viability Assessment of H₂O₂-Induced Senescence in SVGp12 Cells To generate the senescence model, human SVGp12 astroglial cells were plated into 96-well culture plates (Thermo Scientific, Waltham, MA, USA) at a volume of 100 µL per well, with four replicate wells allocated to each experimental group, and allowed to adhere overnight. The cells were subsequently exposed to hydrogen peroxide (H₂O₂) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. 216763) at final concentrations of 50, 100, and 200 µM for a duration of 2 h. After treatment, the H₂O₂-containing medium was aspirated and replaced with fresh complete growth medium, followed by an additional incubation period of 48 h. Cell viability was then evaluated using the Water-Soluble Tetrazolium Salt-8 (WST-8) assay. Briefly, 10 µL of CCK-8 reagent (Cell Counting Kit-8; Abbkine Scientific Co., Ltd., Wuhan, Hubei, China; Cat. No. KTA1020) was added to each well, and the plates were incubated for 2 h at 37 °C. Absorbance values were recorded at 450 nm using a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA). Cell viability was expressed as a percentage relative to untreated control cells. Senescence-Associated β-Galactosidase (SA-β-gal) Staining Senescence-associated β-galactosidase (SA-β-gal) activity was evaluated using a commercially available staining kit (Cell Senescence β-Galactosidase Staining Kit, MedChemExpress, Monmouth Junction, NJ, USA; Cat. No. HY-K1089-100T) in accordance with the supplier’s guidelines. Briefly, cells were gently washed twice with phosphate-buffered saline (PBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 10010-023) and subsequently fixed using the kit-provided fixative for 10–15 min at room temperature. Following fixation, the cells were rinsed again with PBS. A freshly prepared X-gal staining solution adjusted to pH 6.0 was then added to fully cover the cell monolayer, and the plates were incubated overnight at 37 °C in a dry, non-CO₂ incubator (Panasonic Healthcare Co., Ltd., Tokyo, Japan). After the incubation period, SA-β-gal-positive cells were identified by the presence of blue cytoplasmic staining and examined using an inverted light microscope (Primovert, Carl Zeiss Microscopy GmbH, Jena, Germany) fitted with a 20× Plan-Achromat objective. Representative images were acquired at 200× total magnification. For quantitative analysis, five randomly selected fields were analyzed per well, and a minimum of 500 cells per well were manually counted. In parallel, characteristic senescence-associated morphological changes, such as increased cell size and a flattened cellular appearance, were qualitatively documented. Immunocytochemistry and H-score analysis Immunocytochemical analysis was performed to evaluate the expression of HIF-1α in human SVGp12 cells across Negative Control, Control, Senescence, and Senescence + TCA groups. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. 158127) for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. T8787), and blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. A2153). Samples were incubated overnight at 4 °C with a primary antibody against HIF-1α (Santa Cruz Biotechnology, Dallas, TX, USA; sc-13515), followed by incubation with an HRP-conjugated secondary antibody (goat anti-mouse IgG-HRP; Santa Cruz Biotechnology, Dallas, TX, USA; Cat. No. sc-2005) for 1 hour at room temperature. Immunoreactivity was visualized using DAB chromogen (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. D5637), and nuclei were counterstained with hematoxylin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. H9627). For each experimental group, H-score values were determined by counting the number of cells exhibiting positive immunostaining in five randomly selected microscopic fields. Staining intensity was scored on a scale from 0 to 3, corresponding to no staining (0), weak (1), moderate (2), and strong staining (3), and the percentage of cells at each intensity level was estimated. The H-score was calculated by multiplying the percentage of positively stained cells by the corresponding intensity score and summing these values across all intensity categories, yielding a final score ranging from 0 to 300, with higher values indicating greater immunoreactivity, as previously described (Detre et al., 1995). The sections were examined using a light microscope (Eclipse 80i; Nikon Instruments Inc., Melville, NY, USA) equipped with a DS-Fi1 digital camera (5-megapixel CCD sensor, maximum resolution 2560 × 1920 pixels; Nikon Instruments Inc.) and a Digital Sight DS-L2 control unit (Embedded System Software version 450.1032.3220.100531). Images were captured and analyzed using the integrated Nikon image analysis system with a 12-bit depth. ELISA Analysis Cells were washed twice with phosphate-buffered saline (PBS; used for washing) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), collected, and homogenized in five volumes of ice-cold buffer consisting of 20 mM potassium phosphate (pH 7.3), 2 mM EDTA, 150 mM NaCl, and 0.2% Triton X-100. Homogenization was carried out on ice using a Pro-200 homogenizer (Pro Scientific, Oxford, CT, USA) at 25,000 rpm for two cycles of 20 s each. The lysates were centrifuged, and supernatants were aliquoted and stored at -80 °C for subsequent assays. Cell lysates were analyzed for IL-1β, IL-6, TNF-α, Keap1, and Nrf2 using human-specific ELISA kits (BT-Laboratory, Shanghai, China) in accordance with the manufacturer’s protocols. Briefly, standards and samples were loaded into 96-well plates, incubated with specific antibodies and streptavidin-HRP at 37 °C, and washed repeatedly. Following substrate reaction and termination, absorbance was measured at 450 nm using a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA). Concentrations were calculated from standard curves. Total protein content was determined by the bicinchoninic acid (BCA) method using a commercial assay kit (Pierce™ BCA Protein Assay Kit; Thermo Scientific, Waltham, MA, USA; Cat. No. 23225). Cell lysates and bovine serum albumin (BSA) standards were mixed with the prepared working reagent and incubated at 37 °C for 30 min. Following incubation, absorbance values were recorded at 562 nm using a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA). Protein concentrations were subsequently calculated based on the corresponding standard curve. Statistical Analysis All experiments were performed in three independent replicates. Results are expressed as mean values with corresponding standard deviations (SD). Data distribution was first examined for normality using the Shapiro–Wilk test, while variance homogeneity was evaluated with Levene’s test. Intergroup comparisons were conducted using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test when appropriate. Statistical analyses were carried out using SPSS software version 27 (IBM Corp., Armonk, NY, USA). In cases where the assumption of equal variances was not met, Welch’s ANOVA was applied, with subsequent post hoc evaluation using the Games–Howell test. For biochemical parameters, nonparametric comparisons were performed using the Kruskal–Wallis test, followed by Dunn’s correction for multiple comparisons. All statistical analyses were two-sided, and a p value of less than 0.05 was considered indicative of statistical significance. Results Immunocytochemical Evaluation of HIF-1α Expression Immunocytochemical analysis revealed low basal HIF-1α immunoreactivity in control human SVGp12 astroglial cells, whereas no specific staining was observed in the negative control group (Figure 2A,B). Exposure to H₂O₂ (100 µM) resulted in a marked increase in HIF-1α immunopositivity, accompanied by enlarged, senescence-associated cellular morphology (Figure 2C). In contrast, cells treated with trans-cinnamaldehyde (TCA) in the presence of H₂O₂ exhibited visibly reduced HIF-1α staining intensity compared with the senescence group (Figure 2D). Quantitative H-Score Analysis of HIF-1α Immunoreactivity H-score values obtained from the Control, Senescence, and TCA groups were first assessed for normality and variance homogeneity. The Shapiro-Wilk test indicated that the data were normally distributed (W = 0.978, p = 0.782), whereas Levene’s test revealed a violation of the homogeneity of variances assumption (F = 6.42, p = 0.005). Accordingly, Welch’s one-way ANOVA, which is robust to unequal variances, was employed for group comparisons. Welch’s ANOVA demonstrated a statistically significant difference in mean H-score values among the groups (F(2, 17.1) = 25.3, p < 0.001). Descriptive analysis showed that the Control group exhibited a mean H-score of 145 ± 4.82, the Senescence group 158 ± 4.97, and the TCA group 139 ± 9.29 (Table 1). Post hoc analysis using the Games–Howell test revealed that the Senescence group displayed significantly higher H-score values compared with the Control group (mean difference = +13.4, t = 6.12, p < 0.001). In contrast, the TCA-treated group exhibited significantly lower H-score values compared with the Senescence group (mean difference = −19.06, t = 5.72, p < 0.001). No statistically significant difference was observed between the Control and TCA groups (p = 0.234). Table 1: The mean levels of HIF-1α groups H score Control 145±4.82 Senescence 158±4.97 TCA 139±9.29 Data are presented as arithmetic mean ± standard deviation (SD). Induction of Cellular Senescence Assessed by SA-β-gal Staining The control human SVGp12 astroglial cells exhibited normal cell morphology and minimal SA-β-gal positivity, as revealed by senescence-associated β-galactosidase (SA-β-gal) analysis (Figure 3A). Treatment with 50 µM H₂O₂ did not result in a noticeable change in the proportion of SA-β-gal-positive cells or cellular morphology compared with the control group (Figure 3B). In contrast, exposure to 100 µM H₂O₂ led to a pronounced increase in SA-β-gal-positive cells, accompanied by characteristic senescence-associated morphological changes, including enlarged and flattened cell shapes (Figure 3C). These findings were consistently observed across multiple microscopic fields. Notably, treatment with 200 µM H₂O₂ resulted in a marked reduction in cell density, indicating reduced cell viability rather than a senescence-dominant phenotype (Figure 3D). Determination of Optimal H₂O₂ Concentration for Senescence Induction The combined WST-8 viability data (Figure 1) and SA-β-gal staining results (Figure 3) demonstrated that 100 µM H₂O₂ effectively induced a senescence phenotype in human SVGp12 astroglial cells without causing overt cytotoxicity. Therefore, this concentration was selected for subsequent experiments involving TCA administration under senescent conditions. Determination of the Therapeutic TCA Dose in Senescent Human SVGp12 Cells To determine the therapeutic dose range of TCA, human SVGp12 cells were treated with various concentrations of TCA (1 µM, 5 µM, 10 µM, 50 µM, and 100 µM). Cell viability was then assessed using the WST-8 assay (see Figure 4). The analysis revealed that 1 µM and 5 µM TCA were the only concentrations that did not produce a statistically significant decrease in cell viability compared with control cells (p > 0.05). Following senescence induction with 100 µM H₂O₂, the therapeutic window of TCA was further evaluated. Among the tested concentrations, only the Senescence and Senescence + 1 µM TCA groups exhibited no statistically significant difference in cell viability (p > 0.05). Accordingly, 1 µM TCA was selected as the optimal therapeutic concentration for subsequent experiments (Figure 5). Effects of TCA on Proinflammatory Cytokines and KEAP1-NRF2 Signaling Analysis of proinflammatory cytokines demonstrated that IL-1β, IL-6, and TNF-α levels were markedly elevated in the senescence group compared with control cells (Figure 6). TCA treatment mitigated the senescence-associated inflammatory response, decreasing the levels of all three cytokines relative to the senescence group, with a statistically significant reduction observed exclusively in TNF-α (p < 0.05). In addition, expression levels of the redox-regulatory proteins KEAP1 and NRF2 were increased under senescent conditions compared with controls. Notably, TCA administration resulted in a substantial reduction in the expression of both KEAP1 and NRF2 (Figure 7). Collectively, these findings indicate that oxidative stress-induced senescence in astroglial cells is associated with pronounced activation of inflammatory signaling and redox-responsive pathways, whereas TCA treatment partially mitigates these senescence-associated alterations. Discussion The establishment of an oxidative stress-induced senescence model in human SVGp12 astroglial cells using hydrogen peroxide was successful in the present study. Exposure to 100 µM H₂O₂ induced characteristic senescent morphological alterations and a marked increase in SA-β-gal positivity without a significant reduction in cell viability, indicating the induction of premature senescence rather than overt cytotoxicity. These findings align with those of other studies that have identified oxidative stress as a significant factor in the aging of neural and glial cells. In particular, Shang et al. (2020) demonstrated that H₂O₂-induced oxidative stress promotes astrocytic senescence, as evidenced by increased SA-β-gal activity, senescence-associated molecular markers, and characteristic morphological changes, closely resembling the phenotype observed in our model. Similarly, Gao et al. (2021) reported that H₂O₂-induced oxidative stress elicits senescence-like features in astrocytes, supporting the use of H₂O₂-based models for investigating astroglial senescence and aging-related mechanisms in vitro. At a broader level, these observations align with the concept that cellular senescence represents a stress-responsive program frequently driven by oxidative damage and contributing to cellular aging and age-associated pathological conditions (Campisi, 2013). These reports and our results show that human SVGp12 astroglial cells change a lot when they are exposed to 100 µM H2O2. This change is called "senescence," and it is shown by increased SA-β-gal positivity and changes in cell shape. The cells don't die a lot when this happens. Following the induction of cellular senescence, TCA treatment markedly improved cell viability and alleviated senescence-associated phenotypic alterations, indicating a cytoprotective effect against oxidative stress–mediated injury. These findings are consistent with accumulating evidence demonstrating that cinnamaldehyde possesses potent antioxidant and anti-inflammatory properties and effectively mitigates oxidative damage across a broad range of neural and non-neural cellular models (Karimirad et al., 2025). In line with this evidence, TCA has been shown to exert robust neuroprotective effects in multiple neural injury models. In PC12 cells, cinnamaldehyde attenuates oxidative and metabolic stress by reducing intracellular ROS levels, suppressing apoptotic signaling, and improving cellular viability under toxic conditions (Rashidi et al., 2021). Similarly, in models of dopaminergic degeneration, cinnamaldehyde mitigates oxidative stress-mediated neuronal damage and preserves neuronal integrity, as demonstrated in 6-hydroxydopamine–induced dopaminergic injury models (Pyo et al., 2013). Collectively, these studies highlight the broader neuroprotective potential of cinnamaldehyde against oxidative stress-driven cellular injury. Consistent with this framework, our findings demonstrate that TCA significantly improves cell viability in senescent human SVGp12 cells, supporting its protective role against oxidative stress–induced cellular damage within an astroglial senescence model. One of the key mechanistic insights emerging from this study is the suppression of HIF-1α expression following TCA treatment in senescent astroglial cells. HIF-1α functions as a critical regulator of cellular responses to hypoxic and oxidative stress conditions, and sustained activation of stress-adaptive signaling pathways has been closely associated with metabolic imbalance, mitochondrial perturbation, and stabilization of the senescent state. Within this framework, the reduction of HIF-1α observed in our model suggests that TCA may facilitate a partial reversal of stress-imposed transcriptional and metabolic constraints, thereby promoting the re-establishment of cellular homeostasis. This interpretation is supported by prior evidence indicating that chronic metabolic and mitochondrial stress signaling reinforces senescence-associated metabolic remodeling, whereas attenuation of these stress-responsive pathways mitigates oxidative stress-related cellular dysfunction (Wiley et al., 2016). Consistently, Bellot et al. (2009) demonstrated that prolonged activation of HIF-1α–dependent metabolic pathways under sustained stress conditions supports short-term cellular survival while imposing long-term constraints on cellular homeostasis. Senescence-associated inflammatory signaling is regulated through complex and partially independent pathways. Previous studies have demonstrated that SASP components are not uniformly regulated during senescence but are instead controlled at different levels through distinct regulatory networks (Coppé et al., 2010). In particular, IL-1 dependent signaling functions as a pivotal upstream regulator of the senescence-associated secretory phenotype, enabling sustained inflammatory signaling and stabilization of the senescent program, even in contexts where downstream cytokines such as IL-6 or TNF-α are partially modulated (Acosta et al., 2013). Accordingly, the relatively preserved IL-1β expression observed in our model may reflect its central regulatory role in maintaining senescence-associated inflammatory responses in aging-related neural environments. In line with this regulatory hierarchy, our experimental data indicate that TCA treatment significantly reduced TNF-α levels and partially attenuated IL-6 expression, whereas IL-1β levels remained comparatively preserved in senescent human SVGp12 cells. The KEAP1-NRF2 pathway operates as a tightly regulated redox-responsive system. Elevated KEAP1 and NRF2 levels in senescent cells may reflect prolonged oxidative stress driving continuous, yet insufficient, compensatory signaling. In contrast, the reduction of both proteins following TCA treatment likely indicates a decreased requirement for antioxidant activation due to improved redox balance, consistent with adaptive regulation of NRF2 signaling (Baird & Yamamoto, 2020). This coordinated reduction is more consistent with alleviation of oxidative stress rather than impairment of antioxidant defense mechanisms, as discussed in the context of dynamic NRF2 regulation (Cuadrado et al., 2019). In our model, senescence induction was accompanied by elevated KEAP1 and NRF2 expression, whereas TCA treatment resulted in concurrent reductions in both proteins, supporting an overall attenuation of oxidative burden. Consistent with our in vitro observations, in vivo studies have identified senescent astrocytes as active contributors to neuroinflammatory environments in the aging brain. Clearance of these cells has been shown to mitigate neuroinflammation and functional decline, underscoring the pathological relevance of astroglial senescence beyond cell culture models (Bussian et al., 2018). Similarly, antioxidant-deficient Sod1⁻/⁻ mouse models demonstrate that persistent oxidative stress promotes cellular senescence and inflammatory activation, which can be attenuated by interventions that reduce oxidative burden (Zhang et al., 2017). These observations further support a central role for redox imbalance in senescence-associated tissue dysfunction. Taken together, our findings demonstrate that oxidative stress–induced senescence in astroglial cells is accompanied by coordinated alterations in inflammatory signaling, redox regulation, and stress-responsive transcriptional pathways. The ability of TCA to attenuate these senescence-associated changes supports its potential role as a senomorphic agent that restores cellular homeostasis by reducing oxidative and inflammatory burden rather than eliminating senescent cells. Accordingly, the present study provides experimental evidence that TCA exerts senomorphic effects in astroglial cells through modulation of oxidative stress, inflammatory signaling, and HIF-1α associated pathways, highlighting redox-modulating phytochemicals as promising candidates for therapeutic strategies targeting age-related neurodegenerative processes. Conclusion In conclusion, the present study demonstrates that trans-cinnamaldehyde (TCA) improves cell viability and attenuates oxidative stress-induced senescence in human SVGp12 astroglial cells. These effects are accompanied by a reduction in HIF-1α expression, partial suppression of proinflammatory cytokine production, and modulation of KEAP1-NRF2 redox signaling, indicating an overall restoration of cellular homeostasis under senescent conditions. Importantly, TCA appears to exert senomorphic rather than senolytic effects by alleviating oxidative and inflammatory burden without eliminating senescent cells. Collectively, these results underscore the promise of TCA as a redox-modifying phytochemical that may be significant for addressing astroglial senescence and age-related neuroinflammatory processes. To validate these observations and better define the therapeutic applicability of TCA in age-related neurodegenerative conditions, more studies using in vivo models and expanded molecular analyses are needed. Abbreviations ANOVA Analysis of Variance ATCC American Type Culture Collection BCA Bicinchoninic Acid BSA Bovine Serum Albumin CCK-8 Cell Counting Kit-8 DMEM Dulbecco’s Modified Eagle Medium FBS Fetal Bovine Serum HIF-1α Hypoxia-Inducible Factor 1 AlphaİC ICC Immunocytochemistry PBS Phospate Buffered Saline IL-1β Interleukin-1 Beta IL-6 Interleukin-6 KEAP1 Kelch-like ECH-associated protein 1 NRF2 Nuclear factor erythroid 2–related factor 2 PBS Phosphate-Buffered Saline ROS Reactive Oxygen Species SA-β-gal Senescence-Associated β-galactosidase SASP Senescence-Associated Secretory Phenotype SD Standard Deviation SOD1 Superoxide dismutase TCA Trans-Cinnamaldehyde TNF-α Tumor Necrosis Factor Alpha WST-8 Water-Soluble Tetrazolium Salt-8 Declarations Acknowledgements None. Author Contributions Study design: RNG, HEB, VY. Data collection and analysis: RNG, HEB, VY, MG. Writing: RNG, HEB, VY, MG. Supervision: HE. All authors (RNG, HEB, VY, MG, HE) revised the manuscript and approved the submitted version. Corresponding Author: Rana Nur Gursu Funding None. Conflict of interest The authors declare no competing interests. Ethical approvements As this study was conducted using a commercially available cell line, ethical approval was not necessary. Informed consent The manuscript has neither been previously published nor is under consideration by any other journal. The authors have all approved the content of the paper. References Acosta, J. C., Banito, A., Wuestefeld, T., Georgilis, A., Janich, P., Morton, J. P., Athineos, D., Kang, T. W., Lasitschka, F., Andrulis, M., Pascual, G., Morris, K. J., Khan, S., Jin, H., Dharmalingam, G., Snijders, A. P., Carroll, T., Capper, D., Pritchard, C., … Gil, J. (2013). A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nature Cell Biology, 15(8), 978–990. https://doi.org/10.1038/ncb2784 ATCC. (n.d.). SVGp12 (SV40 transformed human fetal glial cells). Retrieved September 12, 2025, from https://www.atcc.org/products/crl-8621 Baird, L., & Yamamoto, M. (2020). The molecular mechanisms regulating the KEAP1-NRF2 pathway. Molecular and Cellular Biology, 40(13), e00099-20. https://doi.org/10.1128/MCB.00099-20 Bellot, G., Garcia-Medina, R., Gounon, P., Chiche, J., Roux, D., Pouysségur, J., & Mazure, N. M. (2009). Hypoxia-induced autophagy is mediated through HIF-1 induction of BNIP3 and BNIP3L. Cell Metabolism, 9(5), 419–431. https://doi.org/10.1016/j.cmet.2009.03.009 Bussian, T. J., Aziz, A., Meyer, C. F., Swenson, B. L., van Deursen, J. M., & Baker, D. J. (2018). Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline. Nature, 562(7728), 578–582. https://doi.org/10.1038/s41586-018-0543-y Campisi, J. (2013). Aging, cellular senescence, and cancer. Annual Review of Physiology, 75, 685–705. https://doi.org/10.1146/annurev-physiol-030212-183653 Coppé, J. P., Desprez, P. Y., Krtolica, A., & Campisi, J. (2010). The senescence-associated secretory phenotype: The dark side of tumor suppression. Annual Review of Pathology, 5, 99–118. https://doi.org/10.1146/annurev-pathol-121808-102144 Csekes, E., & Račková, L. (2021). Skin aging, cellular senescence and natural polyphenols. International Journal of Molecular Sciences, 22(23), 12641. https://doi.org/10.3390/ijms222312641 Cuadrado, A., Rojo, A. I., Wells, G., Hayes, J. D., Cousin, S. P., Rumsey, W. L., Attucks, O. C., Franklin, S., Levonen, A. L., Kensler, T. W., & Dinkova-Kostova, A. T. (2019). Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases. Nature Reviews Drug Discovery, 18(4), 295–317. https://doi.org/10.1038/s41573-018-0008-x Culig, L., Chu, X., & Bohr, V. A. (2022). Neurogenesis in aging and age-related neurodegenerative diseases. Ageing Research Reviews, 79, 101636. https://doi.org/10.1016/j.arr.2022.101636 Forman, H. J., & Zhang, H. (2021). Targeting oxidative stress in disease: Promise and limitations of antioxidant therapy. Nature Reviews Drug Discovery, 20(9), 689–709. https://doi.org/10.1038/s41573-021-00233-1 Freeman, M. R. (2010). Specification and morphogenesis of astrocytes. Science, 330(6005), 774–778. https://doi.org/10.1126/science.1190928 Gao, L., Zhang, Y., Wang, L., Li, C., Yu, J., & Liu, Y. (2021). Baicalein delays H₂O₂-induced astrocytic senescence through inhibition of SASP and JAK2/STAT1/NF-κB signaling. ACS Chemical Neuroscience, 12(13), 2320–2335. https://doi.org/10.1021/acschemneuro.1c00024 Guo, J., Yan, S., Jiang, X., Su, Z., Zhang, F., Xie, J., Hao, E., & Yao, C. (2024). Advances in pharmacological effects and mechanism of action of cinnamaldehyde. Frontiers in Pharmacology, 15, 1365949. https://doi.org/10.3389/fphar.2024.1365949 Hayflick, L., & Moorhead, P. S. (1961). The serial cultivation of human diploid cell strains. Experimental Cell Research, 25, 585–621. Henriksen, S., Tylden, G. D., Dumoulin, A., Sharma, B. N., Hirsch, H. H., & Rinaldo, C. H. (2014). The human fetal glial cell line SVG p12 contains infectious BK polyomavirus. Journal of Virology, 88(9), 5355–5365. https://doi.org/10.1128/JVI.00696-14 Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53(6), 1841–1856. https://doi.org/10.1021/jf030723c Idriss, H. T., & Naismith, J. H. (2000). TNF alpha and the TNF receptor superfamily: Structure–function relationship(s). Microscopy Research and Technique, 50(3), 184–195. https://doi.org/10.1002/1097-0029(20000801)50:33.0.CO;2-H Karimirad, R., Inbaraj, B. S., & Chen, B.-H. (2025). Recent Advances on the Analysis and Biological Functions of Cinnamaldehyde and Its Derivatives. Antioxidants, 14(7), 765. https://doi.org/10.3390/antiox14070765 Kishimoto, T. (2006). Interleukin-6: Discovery of a pleiotropic cytokine. Arthritis Research & Therapy, 8(Suppl 2), S2. https://doi.org/10.1186/ar1916 Koen, J. D., & Rugg, M. D. (2019). Neural dedifferentiation in the aging brain. Trends in Cognitive Sciences, 23(7), 547–559. https://doi.org/10.1016/j.tics.2019.04.012 Lee, B. Y., Han, J. A., Im, J. S., Morrone, A., Johung, K., Goodwin, E. C., Kleijer, W. J., DiMaio, D., & Hwang, E. S. (2006). Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell, 5(2), 187–195. https://doi.org/10.1111/j.1474-9726.2006.00199.x Lopez-Castejon, G., & Brough, D. (2011). Understanding the mechanism of IL-1β secretion. Cytokine & Growth Factor Reviews, 22(4), 189–195. https://doi.org/10.1016/j.cytogfr.2011.10.001 Nowakowski, P., Markiewicz-Żukowska, R., Gromkowska-Kępka, K., Naliwajko, S. K., Moskwa, J., Bielecka, J., Grabia, M., Borawska, M., & Socha, K. (2021). Mushrooms as potential therapeutic agents in the treatment of cancer: Evaluation of anti-glioma effects of Coprinus comatus, Cantharellus cibarius, Lycoperdon perlatum and Lactarius deliciosus extracts. Biomedicine & Pharmacotherapy, 133, 111090. https://doi.org/10.1016/j.biopha.2020.111090 Osemwegie, O., Butler, L., Subbiah, S., & Smith, E. (2021). Effects of in vitro exposure of perfluorooctanoic acid and monocrotophos on astroglia SVG p12 cells. Journal of Applied Toxicology, 41(6), 1010–1021. https://doi.org/10.1002/jat.4129 Pyo JH, Jeong YK, Yeo S, Lee JH, Jeong MY, Kim SH, Choi YG, Lim S. Neuroprotective effect of trans-cinnamaldehyde on the 6-hydroxydopamine-induced dopaminergic injury. Biol Pharm Bull. 2013;36(12):1928-35. doi: 10.1248/bpb.b13-00537. PMID: 24292051. Rashidi, R., Moallem, S. A., Moshiri, M., Hadizadeh, F., & Etemad, L. (2021). Protective Effect of Cinnamaldehyde on METH-induced Neurotoxicity in PC12 Cells via Inhibition of Apoptotic Response and Oxidative Stress. Iranian journal of pharmaceutical research : IJPR, 20(2), 135-143. https://doi.org/10.22037/ijpr.2020.111891.13411. Ruan, Y., Luo, H., Tang, J., Ji, M., Yu, D., Yu, Q., Cao, Z., Mai, Y., Zhang, B., Chen, Y., Liu, J., & Liao, W. (2024). Curcumin inhibits oxidative stress and autophagy in C17.2 neural stem cells through ERK1/2 signaling pathways. Aging Medicine, 7(5), 559–570. https://doi.org/10.1002/agm2.12361 Semenza, G. L. (2001). HIF-1 and mechanisms of hypoxia sensing. Current Opinion in Cell Biology, 13(2), 167–171. https://doi.org/10.1016/S0955-0674(00)00194-0 Shang, D., Hong, Y., Xie, W., Tu, Z., & Xu, J. (2020). Interleukin-1β drives cellular senescence of rat astrocytes induced by oligomerized amyloid β peptide and oxidative stress. Frontiers in Neurology, 11 , Article 929. https://doi.org/10.3389/fneur.2020.00929 Tsikas, D., & Theodoridis, G. (2016). Analytical tools and protocols in oxidative stress. Journal of Chromatography B, 1019, 1–3. https://doi.org/10.1016/j.jchromb.2016.03.015 Verkhratsky, A., & Nedergaard, M. (2018). Physiology of astroglia. Physiological Reviews, 98(1), 239–389. https://doi.org/10.1152/physrev.00042.2016 Wiley, C. D., Velarde, M. C., Lecot, P., Liu, S., Sarnoski, E. A., Freund, A., Shirakawa, K., Lim, H. W., Davis, S. S., Ramanathan, A., Gerencser, A. A., Verdin, E., & Campisi, J. (2016). Mitochondrial dysfunction induces senescence with a distinct secretory phenotype. Cell Metabolism, 23(2), 303–314. https://doi.org/10.1016/j.cmet.2015.11.011 Zhang Y, Unnikrishnan A, Deepa SS, Liu Y, Li Y, Ikeno Y, Sosnowska D, Van Remmen H, Richardson A. A new role for oxidative stress in aging: The accelerated aging phenotype in Sod1 -/- mice is correlated to increased cellular senescence. Redox Biol. 2017 Apr;11:30-37. doi: 10.1016/j.redox.2016.10.014. Epub 2016 Nov 2. PMID: 27846439; PMCID: PMC5109248. Zhong, Y., Zhang, B., Huang, Y., Du, J., Liang, B., Li, Z., Ye, R., Wang, B., Xian, H., Yang, X., Rong, W., Guo, X., Yang, X., & Huang, Z. (2023). CircBCL11B acts as a ceRNA to facilitate 1,2-dichloroethane-induced astrocyte swelling via miR-29b-3p/AQP4 axis in SVG p12 cells. Toxicology Letters, 373, 88–97. https://doi.org/10.1016/j.toxlet.2023.04.003 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8457575","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":571047324,"identity":"43694664-b37c-4d35-aba8-ef3701fa56f8","order_by":0,"name":"RANA NUR GURSU","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYJACxgYgIcHA3HDwQwWQxczcQKwWxsbDEmdAWhiJ19J8gLcNzscNzNvPHvw4o+awvGR7Y8MByXm10fztQC0/Krbh1CJzJi9ZcsOxw4azeQ42HCjcdjx3xmHGBsaeM7dxapFgyDGQfMB2mHGeRCLQlm3HchuAWpgZ2/Bo4X9j/PPBv8P2YC28c47lzieoRSLHTHJj2+HE2WAtDTW5GwhreWNmObMvPXlmz8GGwxLHDuRuBGo5iNcv/DnGN3u+WdvOON58+OOHmrrceecPH3zwowK3FihohjEOg8kDhNQDQR0GYxSMglEwCkYBHAAAy4tk673oIDwAAAAASUVORK5CYII=","orcid":"","institution":"Muğla University","correspondingAuthor":true,"prefix":"","firstName":"RANA","middleName":"NUR","lastName":"GURSU","suffix":""},{"id":571047327,"identity":"8a5e0d73-611d-4887-899c-9ff69fcaa156","order_by":1,"name":"Volkan YASAR","email":"","orcid":"","institution":"Muğla University","correspondingAuthor":false,"prefix":"","firstName":"Volkan","middleName":"","lastName":"YASAR","suffix":""},{"id":571047328,"identity":"ac884078-fb4f-450e-bcbf-1b4b853bda2b","order_by":2,"name":"Hasan Emre BOLAT","email":"","orcid":"","institution":"Muğla University","correspondingAuthor":false,"prefix":"","firstName":"Hasan","middleName":"Emre","lastName":"BOLAT","suffix":""},{"id":571047329,"identity":"b3de2aae-9f21-4b8e-86d5-034e42f57239","order_by":3,"name":"Muslum Gok","email":"","orcid":"","institution":"Muğla University","correspondingAuthor":false,"prefix":"","firstName":"Muslum","middleName":"","lastName":"Gok","suffix":""},{"id":571047330,"identity":"c49411de-7607-406f-a54d-aa9f3450a1ed","order_by":4,"name":"Hulya ELBE","email":"","orcid":"","institution":"Muğla University","correspondingAuthor":false,"prefix":"","firstName":"Hulya","middleName":"","lastName":"ELBE","suffix":""}],"badges":[],"createdAt":"2025-12-26 20:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8457575/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8457575/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99798239,"identity":"773e610c-ef47-4361-8c45-c994615fcf86","added_by":"auto","created_at":"2026-01-08 13:47:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15479259,"visible":true,"origin":"","legend":"","description":"","filename":"maindocumentRANANURG.26.12.docx","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/cb4a9b66baafc7ef46c852f2.docx"},{"id":99774123,"identity":"c3d805e7-f01c-493a-abd9-a4a36cf3c0b5","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6846,"visible":true,"origin":"","legend":"","description":"","filename":"67757525373b4015802ca326b1977d78.json","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/0fe4c8e210a962c3750457c2.json"},{"id":99798836,"identity":"1e966173-0202-4b29-9fb5-be3348145e23","added_by":"auto","created_at":"2026-01-08 13:48:57","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113551,"visible":true,"origin":"","legend":"","description":"","filename":"67757525373b4015802ca326b1977d781enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/7cb54bec162f1bb1f8c146c4.xml"},{"id":99799303,"identity":"99e96141-464c-4ed7-9c50-ad32a18258a9","added_by":"auto","created_at":"2026-01-08 13:49:27","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12736,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/b35bdf74e5e047466abeef39.png"},{"id":99774135,"identity":"1d04434b-8436-4451-9761-4e2bedf3384f","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6682060,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/963a02732606bbc7dedd7bef.png"},{"id":99798541,"identity":"de60163e-4056-408d-b80a-83315f11a28c","added_by":"auto","created_at":"2026-01-08 13:48:34","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8566498,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/a50d053e97e862717091b9aa.png"},{"id":99774131,"identity":"e36c600a-8273-40b4-8b9b-05039a355400","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20327,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/9ed249b58c4d83e1652ac709.png"},{"id":99797629,"identity":"42f10a9f-fa2d-4337-9c9f-6524310f6a0b","added_by":"auto","created_at":"2026-01-08 13:46:11","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12944,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/fa42279c96d63e0614e0f193.png"},{"id":99798626,"identity":"7412e263-8749-41f5-8b3d-23ed9a2b3cb9","added_by":"auto","created_at":"2026-01-08 13:48:40","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":145292,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/ba220d4822ef4049f1eea5a9.jpeg"},{"id":99799233,"identity":"08664c53-1db2-4907-b40a-80dc1ee10302","added_by":"auto","created_at":"2026-01-08 13:49:22","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":77438,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/559744bbae0b0c9e170b7ace.jpeg"},{"id":99798152,"identity":"d4aaf2b8-bd9c-4d6c-8525-1951bffaa532","added_by":"auto","created_at":"2026-01-08 13:47:25","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4097,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/631803d9c88a40f5baf47a0f.png"},{"id":99798896,"identity":"e3f003f8-74da-4f6e-a976-40c576045c30","added_by":"auto","created_at":"2026-01-08 13:49:00","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":849233,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/0b06f1f7bdbc75df84620566.png"},{"id":99774145,"identity":"bed9ec8f-58e4-47fd-a20a-5e209bca7a55","added_by":"auto","created_at":"2026-01-08 09:34:21","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":784324,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/8568db338e852d4f5fdc531f.png"},{"id":99798507,"identity":"13eb42a5-c654-4ae1-bf15-534092c044df","added_by":"auto","created_at":"2026-01-08 13:48:27","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3574,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/9035b2d2f6985382b660bbf5.png"},{"id":99774139,"identity":"7500d678-37c6-4544-bed7-7fde5b927787","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2712,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/0868948dd67a56cccfeaabd0.png"},{"id":99774146,"identity":"ddf144ce-2832-4931-ba73-222f5e1c1380","added_by":"auto","created_at":"2026-01-08 09:34:21","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15413,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/d716c8732a391e621fbd9adc.png"},{"id":99774133,"identity":"341944f6-0693-4e29-8718-a46f32a30ba0","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14103,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/2d582d5fb6e063e2496c679b.png"},{"id":99774143,"identity":"5a1956e9-9647-4f76-b57e-84fd6f4b0ac2","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111588,"visible":true,"origin":"","legend":"","description":"","filename":"67757525373b4015802ca326b1977d781structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/fdb79638938643853bea66ad.xml"},{"id":99774140,"identity":"0b2cedf8-1f51-494b-b8cc-43dcc12ef23a","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121289,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/7f975bfc50a91a1635690073.html"},{"id":99774121,"identity":"77edf20d-1686-467a-b3cc-09fded6e8bea","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15138,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of human SVGp12 astroglial cells following H₂O₂ exposure. Cells were treated with increasing concentrations of H₂O₂ (50, 100, and 200 µM) for 2 h, and viability was assessed using the WST-8 assay. Exposure to 100 µM H₂O₂ did not result in a statistically significant reduction in cell viability compared with control cells (p \u0026gt; 0.05), supporting its selection for senescence induction without overt cytotoxicity.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/31b75bacb587d3e2d593f9ec.png"},{"id":99774125,"identity":"efb020e9-bbf8-42ce-b250-2f867819d22a","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4454751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunocytochemistry (ICC) staining of HIF-1α in human SVGp12 astroglial cells. \u003c/strong\u003e(A) Control group showing baseline HIF-1α immunoreactivity with normal cell morphology;\u003cbr\u003e\n(B) Negative control (without primary antibody) showing no specific ICC staining;\u003cbr\u003e\n(C) Senescence group (H₂O₂, 100 µM) exhibiting increased HIF-1α immunopositivity accompanied by enlarged, senescence-associated cellular morphology;\u003cbr\u003e\n(D) TCA + H₂O₂ group showing reduced HIF-1α staining intensity compared with the senescence group, consistent with attenuation of oxidative stress-induced senescence.\u003cbr\u003e\nScale bar: 100 µm (20×).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/74b4e84b48ac605a64b56b07.png"},{"id":99774128,"identity":"d8cba0f3-94bc-45db-b39c-2aaf99c88b1d","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5574947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSA-β-gal staining of human SVGp12 astroglial cells.\u003cbr\u003e\n \u003c/strong\u003e(A) Control group showing normal morphology with minimal SA-β-gal positivity;\u003cbr\u003e\n(B) 50 µM H₂O₂ group exhibiting morphology comparable to control cells with no apparent increase in SA-β-gal-positive cells;\u003cbr\u003e\n(C) 100 µM H₂O₂ group showing enlarged, flattened senescent cells with marked SA-β-gal positivity;\u003cbr\u003e\n(D) 200 µM H₂O₂ group exhibiting reduced cell density, indicating predominant cytotoxic effects rather than a senescence-dominant phenotype.\u003cbr\u003e\nScale bar: 100 µm (20×).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/6b1cd71a5cc72a74b8a94642.png"},{"id":99799520,"identity":"f5d958c8-9464-4ecb-a7c7-b957f16f6067","added_by":"auto","created_at":"2026-01-08 13:49:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24598,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of human SVGp12 astroglial cells following trans-cinnamaldehyde (TCA) exposure. Cells were treated with increasing concentrations of TCA (1, 5, 10, 50, and 100 µM) for 24 h, and the viability of the control (C) and vehicle control (D; DMSO) groups was measured using the WST-8 assay. Both control groups showed comparable viability. Data are presented as mean ± 95% confidence interval (CI).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/364f1ab84856357c8e99b5e7.png"},{"id":99774127,"identity":"273bea4e-08b0-4fe3-a84f-7089de2f0aaf","added_by":"auto","created_at":"2026-01-08 09:34:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16895,"visible":true,"origin":"","legend":"\u003cp\u003eThe experimental groups consisted of Control, Senescence, Senescence + 1 µM TCA, and Senescence + 5 µM TCA.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/08f3484f05f64cfc45a019f7.png"},{"id":99799013,"identity":"4b01febc-539d-4daa-a642-63bfe14c4b65","added_by":"auto","created_at":"2026-01-08 13:49:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53840,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations of IL-1β, IL-6, and TNF-α in control, senescence, and TCA-treated groups (Data are presented as mean ± SD and p \u0026lt; 0.05 was considered statistically significant).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/37ce3432c70c7f5034fbc4ec.png"},{"id":99798517,"identity":"7b2597b7-3054-4336-bc33-581c65823fe3","added_by":"auto","created_at":"2026-01-08 13:48:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":32489,"visible":true,"origin":"","legend":"\u003cp\u003eKEAP1 and NRF2 expression in control, senescence, and TCA-treated groups (Data are presented as mean ± SD and p \u0026lt; 0.05 was considered statistically significant).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/f50be8c8588832672c7d6f9e.png"},{"id":100369180,"identity":"d4f4fe8a-880a-4698-ba7d-f2ba62daf444","added_by":"auto","created_at":"2026-01-16 07:58:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14891818,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8457575/v1/04c42257-f6e9-4b74-9928-ddc48076c2d7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTrans-cinnamaldehyde Attenuates H₂o₂-induced Cellular Senescence in Human Svgp12 Astroglial Cells\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAging represents a progressive biological phenomenon characterized by long-term alterations in cellular performance and proliferative capacity, ultimately predisposing organisms to higher morbidity and mortality rates (Csekes \u0026amp; Račkov\u0026aacute;, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In most species, aging is accompanied by degenerative pathologies associated with the gradual loss of tissue and cellular integrity. At the cellular level, senescence represents a largely irreversible growth arrest induced by various stressors. The result of three things happening to senescent cells is a senescence-associated secretory phenotype (SASP): profound changes to their chromosomes, altered gene expression, and the release of pro-inflammatory cytokines and chemokines. (Campisi, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Hayflick and Moorhead, who showed that normal human somatic cells can divide only a limited number of times in a lab setting (Hayflick \u0026amp; Moorhead, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1961\u003c/span\u003e), first introduced the idea of limited proliferative capacity.\u003c/p\u003e \u003cp\u003eHelmut Sies introduced the term \"oxidative stress\" to describe an unequal balance between oxidant production and antioxidant defense mechanisms that results in biological damage. This unequal balance has been implicated in numerous pathological conditions, including cardiovascular disease, neurodegeneration, cancer, and aging-related disorders (Forman \u0026amp; Zhang, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Supporting this concept, epidemiological studies have reported an inverse relationship between fruit and vegetable consumption and the incidence of inflammation, cancer, cardiovascular disease and aging-associated disorders, largely attributed to dietary antioxidants such as polyphenols, vitamins C and E, and carotenoids (Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOxidative stress further modulates hypoxia-related and redox-dependent signaling cascades. Cellular adaptation to hypoxia is largely controlled by HIF-1, a heterodimeric transcription factor in which HIF-1α activity is selectively enhanced during hypoxic and oxidative stress through redox-dependent mechanisms involving mitochondrial reactive oxygen species (Semenza, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Concurrently, nuclear factor erythroid 2-related factor 2 (NRF2), Kelch-like ECH-associated protein 1 (KEAP1) and the signaling pathway function as a regulator of cellular homeostasis of redox. In normal conditions, KEAP1 keeps NRF2 in the cytoplasm and causes it to be broken down by the cell's protein-breaking machine. However, oxidative stress promotes NRF2 release, nuclear translocation, and the induction of antioxidant and cytoprotective genes. Dysregulation of the NRF2- KEAP1 axis has been associated with aging, cellular senescence, and chronic inflammation, highlighting its importance in pathologies driven by oxidative stress (Baird \u0026amp; Yamamoto, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn increase in β-galactosidase activity measurable at pH 6.0 has been established as a functional marker of the senescent cellular state and is referred to as senescence-associated β-galactosidase (SA-β-gal) (Lee et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Additionally, key components of the SASP include inflammatory cytokines. Examples of these are interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). These are important mediators. They link oxidative stress, inflammation, and cellular senescence. Dysregulated production of these cytokines contributes to chronic inflammation and tissue dysfunction during aging (Lopez-Castejon \u0026amp; Brough, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kishimoto, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Idriss \u0026amp; Naismith, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the central nervous system, astrocytes form a major glial population and are involved in a wide range of physiological processes, including regulation of neural homeostasis, support of neuronal viability, and coordination of local defense responses (Freeman, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Their structural and functional plasticity enables them to regulate neural activity across molecular, cellular, and organ levels (Verkhratsky \u0026amp; Nedergaard, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Increasing evidence indicates that astrocytes are particularly vulnerable to oxidative stress and contribute actively to neurodegenerative processes through their interactions with neurons and inflammatory signaling networks (Zhong et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe SVGp12 cell line, derived from human fetal astroglial cells, has been widely employed as a neural model system in studies investigating astrocyte biology and stress responses (Henriksen et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Due to its human astroglial origin, SVGp12 cells provide a valuable experimental platform for examining oxidative stress-induced cellular alterations relevant to neural aging.\u003c/p\u003e \u003cp\u003eNeural aging is associated with reduced neuroplasticity, impaired neurogenesis, and increased susceptibility to neurodegeneration, reflecting both age-dependent and age-independent mechanisms (Koen \u0026amp; Rugg, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Culig et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These observations highlight the importance of cellular models that can recapitulate aging-related changes and facilitate the identification of potential protective interventions.\u003c/p\u003e \u003cp\u003eIn this context, naturally derived compounds with antioxidant and anti-inflammatory properties have attracted increasing attention as potential modulators of aging-related cellular dysfunction. Cinnamon, obtained from the bark of the Cinnamomum genus, exhibits broad pharmacological activities, largely attributed to its active component cinnamaldehyde. Researchers have reported that trans-cinnamaldehyde (TCA) exerts antioxidant, anti-inflammatory, cardioprotective, anti-aging, and antitumor effects (Guo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), highlighting its potential relevance in oxidative stress-associated conditions.\u003c/p\u003e \u003cp\u003eHydrogen peroxide (H₂O₂) is commonly used to model aging-related oxidative stress in vitro (Campisi, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Expanding upon this methodology, the present study developed an H₂O₂-induced senescence model in SVGp12 astroglial cells to ascertain the protective effects of TCA. Based on these considerations, we hypothesized that TCA exerts senomorphic effects on astroglial cells by alleviating oxidative stress, modulating redox-sensitive signaling pathways, and attenuating senescence-associated inflammatory responses. The objective of this study was to assess the impact of TCA on oxidative stress-induced cellular senescence and its effect on HIF-1α, KEAP1/NRF2 signaling, and inflammatory cytokine expression (IL-1β, IL-6, and TNF-α) in SVGp12 cells. Researchers have studied TCA in many different experiments, and they have found that it has antioxidant and anti-inflammatory properties. However, scientists still don't know enough about how TCA might influence the process of astroglial senescence and the related pathways that respond to redox and stress. In particular, the integrated assessment of HIF-1α signaling, KEAP1/NRF2-mediated redox regulation, and senescence-associated inflammatory responses within an astroglial senescence model represents a distinctive aspect of the present study. By examining these pathways concurrently, this work contributes novel insight into the senomorphic potential of redox-modulating phytochemicals and their relevance to aging-related neuroinflammatory processes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell Culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human fetal astroglial cell line SVGp12 (ATCC\u0026reg; CRL-8621\u0026trade;, Manassas, VA, USA) was obtained from the American Type Culture Collection (ATCC). Cells at passages 2-4 were cultured in Dulbecco\u0026rsquo;s Modified Eagle (Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 11965-092) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen Life Technologies, Paisley, UK; Cat. No. 10270-106) and 1% penicillin/streptomycin (Multicell, Wisent Inc., St-Bruno, QC, Canada; Cat. No. 450-202-EL). Cells were maintained in a humidified incubator (MCO18AC-PE, Panasonic Healthcare Co., Ltd., Tokyo, Japan) at 37 \u0026deg;C with 5% CO₂ and were subcultured at approximately 80% confluence using 0.05% trypsin-EDTA (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. T3924).\u003c/p\u003e\n\u003cp\u003eFor cell viability assays, SVGp12 cells were seeded into 96-well plates (Thermo Scientific, Waltham, MA, USA) at a density of 5 \u0026times; 10\u0026sup3; cells per well. For senescence-associated \u0026beta;-galactosidase (SA-\u0026beta;-gal) staining, cells were plated into 6-well plates (Thermo Scientific, Waltham, MA, USA) at a density of 1 \u0026times; 10⁵ cells per well. Following senescence induction and determination of the appropriate trans-Cinnamaldehyde (TCA) (Aldrich, Sigma-Aldrich; Cat. No. C80687) concentration range, cells were seeded into 25 cm\u0026sup2; culture flasks (Thermo Scientific, Waltham, MA, USA) at a density of 1.5 \u0026times; 10⁶ cells per flask for subsequent ELISA analyses. For immunocytochemistry (ICC) experiments, cells were seeded onto sterile 13-mm glass coverslips (Marienfeld GmbH \u0026amp; Co. KG, Lauda-K\u0026ouml;nigshofen, Germany) placed in 24-well plates (NEST Biotechnology, Wuxi, China) at a density of 3 \u0026times; 10⁴ cells per well.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eViability Assessment of H₂O₂-Induced Senescence in SVGp12 Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo generate the senescence model, human SVGp12 astroglial cells were plated into 96-well culture plates (Thermo Scientific, Waltham, MA, USA) at a volume of 100 \u0026micro;L per well, with four replicate wells allocated to each experimental group, and allowed to adhere overnight. The cells were subsequently exposed to hydrogen peroxide (H₂O₂) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. 216763) at final concentrations of 50, 100, and 200 \u0026micro;M for a duration of 2 h. After treatment, the H₂O₂-containing medium was aspirated and replaced with fresh complete growth medium, followed by an additional incubation period of 48 h.\u003c/p\u003e\n\u003cp\u003eCell viability was then evaluated using the Water-Soluble Tetrazolium Salt-8 (WST-8) assay. Briefly, 10 \u0026micro;L of CCK-8 reagent (Cell Counting Kit-8; Abbkine Scientific Co., Ltd., Wuhan, Hubei, China; Cat. No. KTA1020) was added to each well, and the plates were incubated for 2 h at 37 \u0026deg;C. Absorbance values were recorded at 450 nm using a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA). Cell viability was expressed as a percentage relative to untreated control cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSenescence-Associated \u0026beta;-Galactosidase (SA-\u0026beta;-gal) Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSenescence-associated \u0026beta;-galactosidase (SA-\u0026beta;-gal) activity was evaluated using a commercially available staining kit (Cell Senescence \u0026beta;-Galactosidase Staining Kit, MedChemExpress, Monmouth Junction, NJ, USA; Cat. No. HY-K1089-100T) in accordance with the supplier\u0026rsquo;s guidelines. Briefly, cells were gently washed twice with phosphate-buffered saline (PBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 10010-023) and subsequently fixed using the kit-provided fixative for 10\u0026ndash;15 min at room temperature. Following fixation, the cells were rinsed again with PBS. A freshly prepared X-gal staining solution adjusted to pH 6.0 was then added to fully cover the cell monolayer, and the plates were incubated overnight at 37 \u0026deg;C in a dry, non-CO₂ incubator (Panasonic Healthcare Co., Ltd., Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003eAfter the incubation period, SA-\u0026beta;-gal-positive cells were identified by the presence of blue cytoplasmic staining and examined using an inverted light microscope (Primovert, Carl Zeiss Microscopy GmbH, Jena, Germany) fitted with a 20\u0026times; Plan-Achromat objective. Representative images were acquired at 200\u0026times; total magnification. For quantitative analysis, five randomly selected fields were analyzed per well, and a minimum of 500 cells per well were manually counted. In parallel, characteristic senescence-associated morphological changes, such as increased cell size and a flattened cellular appearance, were qualitatively documented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunocytochemistry and H-score analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunocytochemical analysis was performed to evaluate the expression of HIF-1\u0026alpha; in human SVGp12 cells across Negative Control, Control, Senescence, and Senescence + TCA groups. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. 158127) for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. T8787), and blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. A2153). Samples were incubated overnight at 4 \u0026deg;C with a primary antibody against HIF-1\u0026alpha; (Santa Cruz Biotechnology, Dallas, TX, USA; sc-13515), followed by incubation with an HRP-conjugated secondary antibody (goat anti-mouse IgG-HRP; Santa Cruz Biotechnology, Dallas, TX, USA; Cat. No. sc-2005) for 1 hour at room temperature. Immunoreactivity was visualized using DAB chromogen (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. D5637), and nuclei were counterstained with hematoxylin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; Cat. No. H9627).\u003c/p\u003e\n\u003cp\u003eFor each experimental group, H-score values were determined by counting the number of cells exhibiting positive immunostaining in five randomly selected microscopic fields. Staining intensity was scored on a scale from 0 to 3, corresponding to no staining (0), weak (1), moderate (2), and strong staining (3), and the percentage of cells at each intensity level was estimated. The H-score was calculated by multiplying the percentage of positively stained cells by the corresponding intensity score and summing these values across all intensity categories, yielding a final score ranging from 0 to 300, with higher values indicating greater immunoreactivity, as previously described (Detre et al., 1995).\u003c/p\u003e\n\u003cp\u003eThe sections were examined using a light microscope (Eclipse 80i; Nikon Instruments Inc., Melville, NY, USA) equipped with a DS-Fi1 digital camera (5-megapixel CCD sensor, maximum resolution 2560 \u0026times; 1920 pixels; Nikon Instruments Inc.) and a Digital Sight DS-L2 control unit (Embedded System Software version 450.1032.3220.100531). Images were captured and analyzed using the integrated Nikon image analysis system with a 12-bit depth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were washed twice with phosphate-buffered saline (PBS; used for washing) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), collected, and homogenized in five volumes of ice-cold buffer consisting of 20 mM potassium phosphate (pH 7.3), 2 mM EDTA, 150 mM NaCl, and 0.2% Triton X-100. Homogenization was carried out on ice using a Pro-200 homogenizer (Pro Scientific, Oxford, CT, USA) at 25,000 rpm for two cycles of 20 s each. The lysates were centrifuged, and supernatants were aliquoted and stored at -80 \u0026deg;C for subsequent assays.\u003c/p\u003e\n\u003cp\u003eCell lysates were analyzed for IL-1\u0026beta;, IL-6, TNF-\u0026alpha;, Keap1, and Nrf2 using human-specific ELISA kits (BT-Laboratory, Shanghai, China) in accordance with the manufacturer\u0026rsquo;s protocols. Briefly, standards and samples were loaded into 96-well plates, incubated with specific antibodies and streptavidin-HRP at 37 \u0026deg;C, and washed repeatedly. Following substrate reaction and termination, absorbance was measured at 450 nm using a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA). Concentrations were calculated from standard curves.\u003c/p\u003e\n\u003cp\u003eTotal protein content was determined by the bicinchoninic acid (BCA) method using a commercial assay kit (Pierce\u0026trade; BCA Protein Assay Kit; Thermo Scientific, Waltham, MA, USA; Cat. No. 23225). Cell lysates and bovine serum albumin (BSA) standards were mixed with the prepared working reagent and incubated at 37 \u0026deg;C for 30 min. Following incubation, absorbance values were recorded at 562 nm using a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA). Protein concentrations were subsequently calculated based on the corresponding standard curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in three independent replicates. Results are expressed as mean values with corresponding standard deviations (SD). Data distribution was first examined for normality using the Shapiro\u0026ndash;Wilk test, while variance homogeneity was evaluated with Levene\u0026rsquo;s test. Intergroup comparisons were conducted using one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s multiple comparison test when appropriate. Statistical analyses were carried out using SPSS software version 27 (IBM Corp., Armonk, NY, USA). In cases where the assumption of equal variances was not met, Welch\u0026rsquo;s ANOVA was applied, with subsequent post hoc evaluation using the Games\u0026ndash;Howell test. For biochemical parameters, nonparametric comparisons were performed using the Kruskal\u0026ndash;Wallis test, followed by Dunn\u0026rsquo;s correction for multiple comparisons. All statistical analyses were two-sided, and a p value of less than 0.05 was considered indicative of statistical significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eImmunocytochemical Evaluation of HIF-1\u0026alpha; Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunocytochemical analysis revealed low basal HIF-1\u0026alpha; immunoreactivity in control human SVGp12 astroglial cells, whereas no specific staining was observed in the negative control group (Figure 2A,B). Exposure to H₂O₂ (100 \u0026micro;M) resulted in a marked increase in HIF-1\u0026alpha; immunopositivity, accompanied by enlarged, senescence-associated cellular morphology (Figure 2C). In contrast, cells treated with trans-cinnamaldehyde (TCA) in the presence of H₂O₂ exhibited visibly reduced HIF-1\u0026alpha; staining intensity compared with the senescence group (Figure 2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative H-Score Analysis of HIF-1\u0026alpha; Immunoreactivity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH-score values obtained from the Control, Senescence, and TCA groups were first assessed for normality and variance homogeneity. The Shapiro-Wilk test indicated that the data were normally distributed (W = 0.978, p = 0.782), whereas Levene\u0026rsquo;s test revealed a violation of the homogeneity of variances assumption (F = 6.42, p = 0.005). Accordingly, Welch\u0026rsquo;s one-way ANOVA, which is robust to unequal variances, was employed for group comparisons.\u003c/p\u003e\n\u003cp\u003eWelch\u0026rsquo;s ANOVA demonstrated a statistically significant difference in mean H-score values among the groups (F(2, 17.1) = 25.3, p \u0026lt; 0.001). Descriptive analysis showed that the Control group exhibited a mean H-score of 145 \u0026plusmn; 4.82, the Senescence group 158 \u0026plusmn; 4.97, and the TCA group 139 \u0026plusmn; 9.29 (Table 1).\u003c/p\u003e\n\u003cp\u003ePost hoc analysis using the Games\u0026ndash;Howell test revealed that the Senescence group displayed significantly higher H-score values compared with the Control group (mean difference = +13.4, t = 6.12, p \u0026lt; 0.001). In contrast, the TCA-treated group exhibited significantly lower H-score values compared with the Senescence group (mean difference = \u0026minus;19.06, t = 5.72, p \u0026lt; 0.001). No statistically significant difference was observed between the Control and TCA groups (p = 0.234).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: The mean levels of HIF-1\u0026alpha;\u003c/strong\u003e \u003cstrong\u003egroups\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 100%;\"\u003e\n \u003cp\u003eH score\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003e145\u0026plusmn;4.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eSenescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003e158\u0026plusmn;4.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eTCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003e139\u0026plusmn;9.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eData are presented as arithmetic mean \u0026plusmn; standard deviation (SD).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduction of Cellular Senescence Assessed by SA-\u0026beta;-gal Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe control human SVGp12 astroglial cells exhibited normal cell morphology and minimal SA-\u0026beta;-gal positivity, as revealed by senescence-associated \u0026beta;-galactosidase (SA-\u0026beta;-gal) analysis (Figure 3A). Treatment with 50 \u0026micro;M H₂O₂ did not result in a noticeable change in the proportion of SA-\u0026beta;-gal-positive cells or cellular morphology compared with the control group (Figure 3B). In contrast, exposure to 100 \u0026micro;M H₂O₂ led to a pronounced increase in SA-\u0026beta;-gal-positive cells, accompanied by characteristic senescence-associated morphological changes, including enlarged and flattened cell shapes (Figure 3C). These findings were consistently observed across multiple microscopic fields. Notably, treatment with 200 \u0026micro;M H₂O₂ resulted in a marked reduction in cell density, indicating reduced cell viability rather than a senescence-dominant phenotype (Figure 3D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Optimal H₂O₂ Concentration for Senescence Induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe combined WST-8 viability data (Figure 1) and SA-\u0026beta;-gal staining results (Figure 3) demonstrated that 100 \u0026micro;M H₂O₂ effectively induced a senescence phenotype in human SVGp12 astroglial cells without causing overt cytotoxicity. Therefore, this concentration was selected for subsequent experiments involving TCA administration under senescent conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of the Therapeutic TCA Dose in Senescent Human SVGp12 Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the therapeutic dose range of TCA, human SVGp12 cells were treated with various concentrations of TCA (1 \u0026micro;M, 5 \u0026micro;M, 10 \u0026micro;M, 50 \u0026micro;M, and 100 \u0026micro;M). Cell viability was then assessed using the WST-8 assay (see Figure 4). The analysis revealed that 1 \u0026micro;M and 5 \u0026micro;M TCA were the only concentrations that did not produce a statistically significant decrease in cell viability compared with control cells (p \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eFollowing senescence induction with 100 \u0026micro;M H₂O₂, the therapeutic window of TCA was further evaluated. Among the tested concentrations, only the Senescence and Senescence + 1 \u0026micro;M TCA groups exhibited no statistically significant difference in cell viability (p \u0026gt; 0.05). Accordingly, 1 \u0026micro;M TCA was selected as the optimal therapeutic concentration for subsequent experiments (Figure 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of TCA on Proinflammatory Cytokines and KEAP1-NRF2 Signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of proinflammatory cytokines demonstrated that IL-1\u0026beta;, IL-6, and TNF-\u0026alpha; levels were markedly elevated in the senescence group compared with control cells (Figure 6). TCA treatment mitigated the senescence-associated inflammatory response, decreasing the levels of all three cytokines relative to the senescence group, with a statistically significant reduction observed exclusively in TNF-\u0026alpha; (p \u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, expression levels of the redox-regulatory proteins KEAP1 and NRF2 were increased under senescent conditions compared with controls. Notably, TCA administration resulted in a substantial reduction in the expression of both KEAP1 and NRF2 (Figure 7).\u003c/p\u003e\n\u003cp\u003eCollectively, these findings indicate that oxidative stress-induced senescence in astroglial cells is associated with pronounced activation of inflammatory signaling and redox-responsive pathways, whereas TCA treatment partially mitigates these senescence-associated alterations.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe establishment of an oxidative stress-induced senescence model in human SVGp12 astroglial cells using hydrogen peroxide was successful in the present study. Exposure to 100 \u0026micro;M H₂O₂ induced characteristic senescent morphological alterations and a marked increase in SA-\u0026beta;-gal positivity without a significant reduction in cell viability, indicating the induction of premature senescence rather than overt cytotoxicity. These findings align with those of other studies that have identified oxidative stress as a significant factor in the aging of neural and glial cells. In particular, Shang et al. (2020) demonstrated that H₂O₂-induced oxidative stress promotes astrocytic senescence, as evidenced by increased SA-\u0026beta;-gal activity, senescence-associated molecular markers, and characteristic morphological changes, closely resembling the phenotype observed in our model. Similarly, Gao et al. (2021) reported that H₂O₂-induced oxidative stress elicits senescence-like features in astrocytes, supporting the use of H₂O₂-based models for investigating astroglial senescence and aging-related mechanisms in vitro. At a broader level, these observations align with the concept that cellular senescence represents a stress-responsive program frequently driven by oxidative damage and contributing to cellular aging and age-associated pathological conditions (Campisi, 2013). These reports and our results show that human SVGp12 astroglial cells change a lot when they are exposed to 100 \u0026micro;M H2O2. This change is called \u0026quot;senescence,\u0026quot; and it is shown by increased SA-\u0026beta;-gal positivity and changes in cell shape. The cells don\u0026apos;t die a lot when this happens.\u003c/p\u003e\n\u003cp\u003eFollowing the induction of cellular senescence, TCA treatment markedly improved cell viability and alleviated senescence-associated phenotypic alterations, indicating a cytoprotective effect against oxidative stress\u0026ndash;mediated injury. These findings are consistent with accumulating evidence demonstrating that cinnamaldehyde possesses potent antioxidant and anti-inflammatory properties and effectively mitigates oxidative damage across a broad range of neural and non-neural cellular models (Karimirad et al., 2025). In line with this evidence, TCA has been shown to exert robust neuroprotective effects in multiple neural injury models. In PC12 cells, cinnamaldehyde attenuates oxidative and metabolic stress by reducing intracellular ROS levels, suppressing apoptotic signaling, and improving cellular viability under toxic conditions (Rashidi et al., 2021). Similarly, in models of dopaminergic degeneration, cinnamaldehyde mitigates oxidative stress-mediated neuronal damage and preserves neuronal integrity, as demonstrated in 6-hydroxydopamine\u0026ndash;induced dopaminergic injury models (Pyo et al., 2013). Collectively, these studies highlight the broader neuroprotective potential of cinnamaldehyde against oxidative stress-driven cellular injury. Consistent with this framework, our findings demonstrate that TCA significantly improves cell viability in senescent human SVGp12 cells, supporting its protective role against oxidative stress\u0026ndash;induced cellular damage within an astroglial senescence model.\u003c/p\u003e\n\u003cp\u003eOne of the key mechanistic insights emerging from this study is the suppression of HIF-1\u0026alpha; expression following TCA treatment in senescent astroglial cells. HIF-1\u0026alpha; functions as a critical regulator of cellular responses to hypoxic and oxidative stress conditions, and sustained activation of stress-adaptive signaling pathways has been closely associated with metabolic imbalance, mitochondrial perturbation, and stabilization of the senescent state. Within this framework, the reduction of HIF-1\u0026alpha; observed in our model suggests that TCA may facilitate a partial reversal of stress-imposed transcriptional and metabolic constraints, thereby promoting the re-establishment of cellular homeostasis. This interpretation is supported by prior evidence indicating that chronic metabolic and mitochondrial stress signaling reinforces senescence-associated metabolic remodeling, whereas attenuation of these stress-responsive pathways mitigates oxidative stress-related cellular dysfunction (Wiley et al., 2016). Consistently, Bellot et al. (2009) demonstrated that prolonged activation of HIF-1\u0026alpha;\u0026ndash;dependent metabolic pathways under sustained stress conditions supports short-term cellular survival while imposing long-term constraints on cellular homeostasis.\u003c/p\u003e\n\u003cp\u003eSenescence-associated inflammatory signaling is regulated through complex and partially independent pathways. Previous studies have demonstrated that SASP components are not uniformly regulated during senescence but are instead controlled at different levels through distinct regulatory networks (Copp\u0026eacute; et al., 2010). In particular, IL-1 dependent signaling functions as a pivotal upstream regulator of the senescence-associated secretory phenotype, enabling sustained inflammatory signaling and stabilization of the senescent program, even in contexts where downstream cytokines such as IL-6 or TNF-\u0026alpha; are partially modulated (Acosta et al., 2013). Accordingly, the relatively preserved IL-1\u0026beta; expression observed in our model may reflect its central regulatory role in maintaining senescence-associated inflammatory responses in aging-related neural environments. In line with this regulatory hierarchy, our experimental data indicate that TCA treatment significantly reduced TNF-\u0026alpha; levels and partially attenuated IL-6 expression, whereas IL-1\u0026beta; levels remained comparatively preserved in senescent human SVGp12 cells.\u003c/p\u003e\n\u003cp\u003eThe KEAP1-NRF2 pathway operates as a tightly regulated redox-responsive system. Elevated KEAP1 and NRF2 levels in senescent cells may reflect prolonged oxidative stress driving continuous, yet insufficient, compensatory signaling. In contrast, the reduction of both proteins following TCA treatment likely indicates a decreased requirement for antioxidant activation due to improved redox balance, consistent with adaptive regulation of NRF2 signaling (Baird \u0026amp; Yamamoto, 2020). This coordinated reduction is more consistent with alleviation of oxidative stress rather than impairment of antioxidant defense mechanisms, as discussed in the context of dynamic NRF2 regulation (Cuadrado et al., 2019). In our model, senescence induction was accompanied by elevated KEAP1 and NRF2 expression, whereas TCA treatment resulted in concurrent reductions in both proteins, supporting an overall attenuation of oxidative burden.\u003c/p\u003e\n\u003cp\u003eConsistent with our in vitro observations, in vivo studies have identified senescent astrocytes as active contributors to neuroinflammatory environments in the aging brain. Clearance of these cells has been shown to mitigate neuroinflammation and functional decline, underscoring the pathological relevance of astroglial senescence beyond cell culture models (Bussian et al., 2018). Similarly, antioxidant-deficient Sod1⁻/⁻ mouse models demonstrate that persistent oxidative stress promotes cellular senescence and inflammatory activation, which can be attenuated by interventions that reduce oxidative burden (Zhang et al., 2017). These observations further support a central role for redox imbalance in senescence-associated tissue dysfunction.\u003c/p\u003e\n\u003cp\u003eTaken together, our findings demonstrate that oxidative stress\u0026ndash;induced senescence in astroglial cells is accompanied by coordinated alterations in inflammatory signaling, redox regulation, and stress-responsive transcriptional pathways. The ability of TCA to attenuate these senescence-associated changes supports its potential role as a senomorphic agent that restores cellular homeostasis by reducing oxidative and inflammatory burden rather than eliminating senescent cells. Accordingly, the present study provides experimental evidence that TCA exerts senomorphic effects in astroglial cells through modulation of oxidative stress, inflammatory signaling, and HIF-1\u0026alpha; associated pathways, highlighting redox-modulating phytochemicals as promising candidates for therapeutic strategies targeting age-related neurodegenerative processes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the present study demonstrates that trans-cinnamaldehyde (TCA) improves cell viability and attenuates oxidative stress-induced senescence in human SVGp12 astroglial cells. These effects are accompanied by a reduction in HIF-1α expression, partial suppression of proinflammatory cytokine production, and modulation of KEAP1-NRF2 redox signaling, indicating an overall restoration of cellular homeostasis under senescent conditions. Importantly, TCA appears to exert senomorphic rather than senolytic effects by alleviating oxidative and inflammatory burden without eliminating senescent cells. Collectively, these results underscore the promise of TCA as a redox-modifying phytochemical that may be significant for addressing astroglial senescence and age-related neuroinflammatory processes. To validate these observations and better define the therapeutic applicability of TCA in age-related neurodegenerative conditions, more studies using in vivo models and expanded molecular analyses are needed.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA \u0026nbsp; \u0026nbsp; \u0026nbsp;Analysis of Variance\u003c/p\u003e\n\u003cp\u003eATCC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; American Type Culture Collection\u003c/p\u003e\n\u003cp\u003eBCA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bicinchoninic Acid\u003c/p\u003e\n\u003cp\u003eBSA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bovine Serum Albumin\u003c/p\u003e\n\u003cp\u003eCCK-8 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Cell Counting Kit-8\u003c/p\u003e\n\u003cp\u003eDMEM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Dulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e\n\u003cp\u003eFBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eHIF-1\u0026alpha; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Hypoxia-Inducible Factor 1 AlphaİC\u003c/p\u003e\n\u003cp\u003eICC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Immunocytochemistry\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Phospate Buffered Saline\u003c/p\u003e\n\u003cp\u003eIL-1\u0026beta; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Interleukin-1 Beta\u003c/p\u003e\n\u003cp\u003eIL-6 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Interleukin-6\u003c/p\u003e\n\u003cp\u003eKEAP1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Kelch-like ECH-associated protein 1\u003c/p\u003e\n\u003cp\u003eNRF2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Nuclear factor erythroid 2\u0026ndash;related factor 2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Phosphate-Buffered Saline\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Reactive Oxygen Species\u003c/p\u003e\n\u003cp\u003eSA-\u0026beta;-gal \u0026nbsp; \u0026nbsp; Senescence-Associated \u0026beta;-galactosidase\u003c/p\u003e\n\u003cp\u003eSASP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Senescence-Associated Secretory Phenotype\u003c/p\u003e\n\u003cp\u003eSD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Standard Deviation\u003c/p\u003e\n\u003cp\u003eSOD1 \u0026nbsp; \u0026nbsp; \u0026nbsp;Superoxide dismutase\u003c/p\u003e\n\u003cp\u003eTCA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Trans-Cinnamaldehyde\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha; \u0026nbsp; \u0026nbsp; \u0026nbsp; Tumor Necrosis Factor Alpha\u003c/p\u003e\n\u003cp\u003eWST-8 \u0026nbsp; \u0026nbsp; \u0026nbsp; Water-Soluble Tetrazolium Salt-8\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy design: RNG, HEB, VY.\u003c/p\u003e\n\u003cp\u003eData collection and analysis: RNG, HEB, VY, MG.\u003c/p\u003e\n\u003cp\u003eWriting: RNG, HEB, VY, MG.\u003c/p\u003e\n\u003cp\u003eSupervision: HE.\u003c/p\u003e\n\u003cp\u003eAll authors (RNG, HEB, VY, MG, HE) revised the manuscript and approved the submitted version.\u003c/p\u003e\n\u003cp\u003eCorresponding Author:\u0026nbsp;Rana Nur Gursu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\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\n\u003cp\u003e\u003cstrong\u003eEthical approvements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs this study was conducted using a commercially available cell line, ethical approval was not necessary.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript has neither been previously published nor is under consideration by any other journal. The authors have all approved the content of the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcosta, J. C., Banito, A., Wuestefeld, T., Georgilis, A., Janich, P., Morton, J. P., Athineos, D., Kang, T. W., Lasitschka, F., Andrulis, M., Pascual, G., Morris, K. J., Khan, S., Jin, H., Dharmalingam, G., Snijders, A. P., Carroll, T., Capper, D., Pritchard, C., \u0026hellip; Gil, J. (2013). A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nature Cell Biology, 15(8), 978\u0026ndash;990. https://doi.org/10.1038/ncb2784\u003c/li\u003e\n\u003cli\u003eATCC. (n.d.). SVGp12 (SV40 transformed human fetal glial cells). Retrieved September 12, 2025, from https://www.atcc.org/products/crl-8621\u003c/li\u003e\n\u003cli\u003eBaird, L., \u0026amp; Yamamoto, M. (2020). The molecular mechanisms regulating the KEAP1-NRF2 pathway. Molecular and Cellular Biology, 40(13), e00099-20. https://doi.org/10.1128/MCB.00099-20\u003c/li\u003e\n\u003cli\u003eBellot, G., Garcia-Medina, R., Gounon, P., Chiche, J., Roux, D., Pouyss\u0026eacute;gur, J., \u0026amp; Mazure, N. M. (2009). Hypoxia-induced autophagy is mediated through HIF-1 induction of BNIP3 and BNIP3L. Cell Metabolism, 9(5), 419\u0026ndash;431. https://doi.org/10.1016/j.cmet.2009.03.009\u003c/li\u003e\n\u003cli\u003eBussian, T. J., Aziz, A., Meyer, C. F., Swenson, B. L., van Deursen, J. M., \u0026amp; Baker, D. J. (2018). Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline. Nature, 562(7728), 578\u0026ndash;582. https://doi.org/10.1038/s41586-018-0543-y\u003c/li\u003e\n\u003cli\u003eCampisi, J. (2013). Aging, cellular senescence, and cancer. Annual Review of Physiology, 75, 685\u0026ndash;705. https://doi.org/10.1146/annurev-physiol-030212-183653\u003c/li\u003e\n\u003cli\u003eCopp\u0026eacute;, J. P., Desprez, P. Y., Krtolica, A., \u0026amp; Campisi, J. (2010). The senescence-associated secretory phenotype: The dark side of tumor suppression. Annual Review of Pathology, 5, 99\u0026ndash;118. https://doi.org/10.1146/annurev-pathol-121808-102144\u003c/li\u003e\n\u003cli\u003eCsekes, E., \u0026amp; Račkov\u0026aacute;, L. (2021). Skin aging, cellular senescence and natural polyphenols. International Journal of Molecular Sciences, 22(23), 12641. https://doi.org/10.3390/ijms222312641\u003c/li\u003e\n\u003cli\u003eCuadrado, A., Rojo, A. I., Wells, G., Hayes, J. D., Cousin, S. P., Rumsey, W. L., Attucks, O. C., Franklin, S., Levonen, A. L., Kensler, T. W., \u0026amp; Dinkova-Kostova, A. T. (2019). Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases. Nature Reviews Drug Discovery, 18(4), 295\u0026ndash;317. https://doi.org/10.1038/s41573-018-0008-x\u003c/li\u003e\n\u003cli\u003eCulig, L., Chu, X., \u0026amp; Bohr, V. A. (2022). Neurogenesis in aging and age-related neurodegenerative diseases. Ageing Research Reviews, 79, 101636. https://doi.org/10.1016/j.arr.2022.101636\u003c/li\u003e\n\u003cli\u003eForman, H. J., \u0026amp; Zhang, H. (2021). Targeting oxidative stress in disease: Promise and limitations of antioxidant therapy. Nature Reviews Drug Discovery, 20(9), 689\u0026ndash;709. https://doi.org/10.1038/s41573-021-00233-1\u003c/li\u003e\n\u003cli\u003eFreeman, M. R. (2010). Specification and morphogenesis of astrocytes. Science, 330(6005), 774\u0026ndash;778. https://doi.org/10.1126/science.1190928\u003c/li\u003e\n\u003cli\u003eGao, L., Zhang, Y., Wang, L., Li, C., Yu, J., \u0026amp; Liu, Y. (2021). Baicalein delays H₂O₂-induced astrocytic senescence through inhibition of SASP and JAK2/STAT1/NF-\u0026kappa;B signaling. ACS Chemical Neuroscience, 12(13), 2320\u0026ndash;2335. https://doi.org/10.1021/acschemneuro.1c00024\u003c/li\u003e\n\u003cli\u003eGuo, J., Yan, S., Jiang, X., Su, Z., Zhang, F., Xie, J., Hao, E., \u0026amp; Yao, C. (2024). Advances in pharmacological effects and mechanism of action of cinnamaldehyde. Frontiers in Pharmacology, 15, 1365949. https://doi.org/10.3389/fphar.2024.1365949\u003c/li\u003e\n\u003cli\u003eHayflick, L., \u0026amp; Moorhead, P. S. (1961). The serial cultivation of human diploid cell strains. Experimental Cell Research, 25, 585\u0026ndash;621.\u003c/li\u003e\n\u003cli\u003eHenriksen, S., Tylden, G. D., Dumoulin, A., Sharma, B. N., Hirsch, H. H., \u0026amp; Rinaldo, C. H. (2014). The human fetal glial cell line SVG p12 contains infectious BK polyomavirus. Journal of Virology, 88(9), 5355\u0026ndash;5365. https://doi.org/10.1128/JVI.00696-14\u003c/li\u003e\n\u003cli\u003eHuang, D., Ou, B., \u0026amp; Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53(6), 1841\u0026ndash;1856. https://doi.org/10.1021/jf030723c\u003c/li\u003e\n\u003cli\u003eIdriss, H. T., \u0026amp; Naismith, J. H. (2000). TNF alpha and the TNF receptor superfamily: Structure\u0026ndash;function relationship(s). Microscopy Research and Technique, 50(3), 184\u0026ndash;195. https://doi.org/10.1002/1097-0029(20000801)50:3\u0026lt;184::AID-JEMT2\u0026gt;3.0.CO;2-H\u003c/li\u003e\n\u003cli\u003eKarimirad, R., Inbaraj, B. S., \u0026amp; Chen, B.-H. (2025). Recent Advances on the Analysis and Biological Functions of Cinnamaldehyde and Its Derivatives. Antioxidants, 14(7), 765. https://doi.org/10.3390/antiox14070765\u003c/li\u003e\n\u003cli\u003eKishimoto, T. (2006). Interleukin-6: Discovery of a pleiotropic cytokine. Arthritis Research \u0026amp; Therapy, 8(Suppl 2), S2. https://doi.org/10.1186/ar1916\u003c/li\u003e\n\u003cli\u003eKoen, J. D., \u0026amp; Rugg, M. D. (2019). Neural dedifferentiation in the aging brain. Trends in Cognitive Sciences, 23(7), 547\u0026ndash;559. https://doi.org/10.1016/j.tics.2019.04.012\u003c/li\u003e\n\u003cli\u003eLee, B. Y., Han, J. A., Im, J. S., Morrone, A., Johung, K., Goodwin, E. C., Kleijer, W. J., DiMaio, D., \u0026amp; Hwang, E. S. (2006). Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell, 5(2), 187\u0026ndash;195. https://doi.org/10.1111/j.1474-9726.2006.00199.x\u003c/li\u003e\n\u003cli\u003eLopez-Castejon, G., \u0026amp; Brough, D. (2011). Understanding the mechanism of IL-1\u0026beta; secretion. Cytokine \u0026amp; Growth Factor Reviews, 22(4), 189\u0026ndash;195. https://doi.org/10.1016/j.cytogfr.2011.10.001\u003c/li\u003e\n\u003cli\u003eNowakowski, P., Markiewicz-Żukowska, R., Gromkowska-Kępka, K., Naliwajko, S. K., Moskwa, J., Bielecka, J., Grabia, M., Borawska, M., \u0026amp; Socha, K. (2021). Mushrooms as potential therapeutic agents in the treatment of cancer: Evaluation of anti-glioma effects of Coprinus comatus, Cantharellus cibarius, Lycoperdon perlatum and Lactarius deliciosus extracts. Biomedicine \u0026amp; Pharmacotherapy, 133, 111090. https://doi.org/10.1016/j.biopha.2020.111090\u003c/li\u003e\n\u003cli\u003eOsemwegie, O., Butler, L., Subbiah, S., \u0026amp; Smith, E. (2021). Effects of in vitro exposure of perfluorooctanoic acid and monocrotophos on astroglia SVG p12 cells. Journal of Applied Toxicology, 41(6), 1010\u0026ndash;1021. https://doi.org/10.1002/jat.4129\u003c/li\u003e\n\u003cli\u003ePyo JH, Jeong YK, Yeo S, Lee JH, Jeong MY, Kim SH, Choi YG, Lim S. Neuroprotective effect of trans-cinnamaldehyde on the 6-hydroxydopamine-induced dopaminergic injury. Biol Pharm Bull. 2013;36(12):1928-35. doi: 10.1248/bpb.b13-00537. PMID: 24292051.\u003c/li\u003e\n\u003cli\u003eRashidi, R., Moallem, S. A., Moshiri, M., Hadizadeh, F., \u0026amp; Etemad, L. (2021). Protective Effect of Cinnamaldehyde on METH-induced Neurotoxicity in PC12 Cells via Inhibition of Apoptotic Response and Oxidative Stress. Iranian journal of pharmaceutical research : IJPR, 20(2), 135-143. https://doi.org/10.22037/ijpr.2020.111891.13411.\u003c/li\u003e\n\u003cli\u003eRuan, Y., Luo, H., Tang, J., Ji, M., Yu, D., Yu, Q., Cao, Z., Mai, Y., Zhang, B., Chen, Y., Liu, J., \u0026amp; Liao, W. (2024). Curcumin inhibits oxidative stress and autophagy in C17.2 neural stem cells through ERK1/2 signaling pathways. Aging Medicine, 7(5), 559\u0026ndash;570. https://doi.org/10.1002/agm2.12361\u003c/li\u003e\n\u003cli\u003eSemenza, G. L. (2001). HIF-1 and mechanisms of hypoxia sensing. Current Opinion in Cell Biology, 13(2), 167\u0026ndash;171. https://doi.org/10.1016/S0955-0674(00)00194-0\u003c/li\u003e\n\u003cli\u003eShang, D., Hong, Y., Xie, W., Tu, Z., \u0026amp; Xu, J. (2020). Interleukin-1\u0026beta; drives cellular senescence of rat astrocytes induced by oligomerized amyloid \u0026beta; peptide and oxidative stress. \u003cstrong\u003eFrontiers in Neurology, 11\u003c/strong\u003e, Article 929. https://doi.org/10.3389/fneur.2020.00929\u003c/li\u003e\n\u003cli\u003eTsikas, D., \u0026amp; Theodoridis, G. (2016). Analytical tools and protocols in oxidative stress. Journal of Chromatography B, 1019, 1\u0026ndash;3. https://doi.org/10.1016/j.jchromb.2016.03.015\u003c/li\u003e\n\u003cli\u003eVerkhratsky, A., \u0026amp; Nedergaard, M. (2018). Physiology of astroglia. Physiological Reviews, 98(1), 239\u0026ndash;389. https://doi.org/10.1152/physrev.00042.2016\u003c/li\u003e\n\u003cli\u003eWiley, C. D., Velarde, M. C., Lecot, P., Liu, S., Sarnoski, E. A., Freund, A., Shirakawa, K., Lim, H. W., Davis, S. S., Ramanathan, A., Gerencser, A. A., Verdin, E., \u0026amp; Campisi, J. (2016). Mitochondrial dysfunction induces senescence with a distinct secretory phenotype. Cell Metabolism, 23(2), 303\u0026ndash;314. https://doi.org/10.1016/j.cmet.2015.11.011\u003c/li\u003e\n\u003cli\u003eZhang Y, Unnikrishnan A, Deepa SS, Liu Y, Li Y, Ikeno Y, Sosnowska D, Van Remmen H, Richardson A. A new role for oxidative stress in aging: The accelerated aging phenotype in Sod1\u003csup\u003e-/-\u003c/sup\u003e mice is correlated to increased cellular senescence. Redox Biol. 2017 Apr;11:30-37. doi: 10.1016/j.redox.2016.10.014. Epub 2016 Nov 2. PMID: 27846439; PMCID: PMC5109248.\u003c/li\u003e\n\u003cli\u003eZhong, Y., Zhang, B., Huang, Y., Du, J., Liang, B., Li, Z., Ye, R., Wang, B., Xian, H., Yang, X., Rong, W., Guo, X., Yang, X., \u0026amp; Huang, Z. (2023). CircBCL11B acts as a ceRNA to facilitate 1,2-dichloroethane-induced astrocyte swelling via miR-29b-3p/AQP4 axis in SVG p12 cells. Toxicology Letters, 373, 88\u0026ndash;97. https://doi.org/10.1016/j.toxlet.2023.04.003\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aging, Cellular senescence, Oxidative stress, Astrocytes, SVGp12 cells, Trans-cinnamaldehyde, Neuroinflammation, Antioxidant activity","lastPublishedDoi":"10.21203/rs.3.rs-8457575/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8457575/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAging is characterized by progressive functional decline driven by oxidative stress, inflammation, and cellular senescence. Astrocytes are particularly vulnerable to oxidative injury, making them a relevant cellular model for investigating aging-related neurodegenerative mechanisms. Trans-cinnamaldehyde (TCA), a major bioactive component of cinnamon, exhibits antioxidant and anti-inflammatory properties; however, its role in astroglial senescence remains poorly understood.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOxidative stress-induced senescence was established in SVGp12 astroglial cells100 µM hydrogen peroxide (H2O2) was used to achieve this. Senescence was confirmed by morphological alterations, reduced cell viability (WST-8 assay), and senescence-associated β-galactosidase (SA-β-gal) staining. Following cytotoxicity screening, TCA was applied at a non-toxic concentration. HIF-1α expression was evaluated by immunocytochemistry (ICC) and semi-quantified using H-score analysis. Levels of KEAP1, TNF-α, IL-1β, IL-6 and NRF2 were measured by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH₂O₂ exposure significantly induced astroglial senescence, increased SA-β-gal positivity, elevated HIF-1α immunoreactivity, and upregulated proinflammatory cytokines and KEAP1/NRF2 signaling. TCA treatment made a big difference in cell viability, reduced the changes to cells that happen as they age, and decreased the expression of HIF-1α. Moreover, TCA markedlyattenuated the H₂O₂-induced increases in IL-6, KEAP1, TNF-α, NRF2 and IL-1β levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTCA effectively mitigates oxidative stress-induced senescence in astroglial cells by reducing HIF-1α expression, inflammatory cytokine production, and redox stress signaling. TCA may be a natural compound that can target astrocyte-associated ageing and neuroinflammation, according to these findings.\u003c/p\u003e","manuscriptTitle":"Trans-cinnamaldehyde Attenuates H₂o₂-induced Cellular Senescence in Human Svgp12 Astroglial Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-08 09:34:15","doi":"10.21203/rs.3.rs-8457575/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"057e8a0f-db3a-4c11-9222-56449c8499d8","owner":[],"postedDate":"January 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-13T13:54:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-08 09:34:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8457575","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8457575","identity":"rs-8457575","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}