Redox Activity of APE1 Mediates SASP Suppression in Senescent Fibroblasts

Redox Activity of APE1 Mediates SASP Suppression in Senescent Fibroblasts | 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 Redox Activity of APE1 Mediates SASP Suppression in Senescent Fibroblasts Yi Duan, Xiao Yang, Xunjie Kuang, He Xiao, Xin Guo, Yang Peng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6841207/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 Cellular senescence, triggered by multiple stressors, was characterized by senescence-associated secretory phenotype (SASP) that drive age-related pathologies. Apurinic apyrimidinic endonuclease 1 (APE1), a bifunctional enzyme with redox and DNA repair activities, regulates inflammation and senescence, but its role in SASP expression remains unclear. This study aimed to elucidate how APE1 regulates SASP and to identify new target for senomorphic therapy. APE1-deficient senescence models were established in human fibroblasts, and senescence markers (SA-β-gal, SenTraGor, p16/p21) and SASP factors (IL-6, IL-8, TNF-α, CCL5) were assessed via RT-qPCR, RNA-seq, and Western blot. APE1 inhibitors and point mutation plasmids were used to further investigate the contributions of the dual functions of APE1 in SASP modulation. Contrary to classical senescence models, APE1 deficiency induced premature senescence but paradoxically suppressed SASP expression. Transcriptomic profiling revealed that APE1 knockdown attenuated cytokine signaling and NF-κB pathway activation, accompanied by downregulation of key SASP factors (IL-6, IL-8, CCL5, TNF-α, IFN-β). Mechanistically, inhibiting the redox activity of APE1 resulted in the inactivation of NF-κB/C/EBPβ signaling and suppression of SASP expression, while the impaired DNA repair activity of APE1 triggered cellular senescence. Furthermore, inhibiting the redox function of APE1 via E3330 attenuated radiation-induced SASP. In conclusion, APE1 governs SASP expression through NF-κB/C/EBPβ signaling, highlighting its dual role in senescence and SASP regulation. Targeting the redox function of APE1 represents a senomorphic strategy to alleviate SASP-related aging, and provides therapeutic potential for age-related and radiotherapy-associated conditions. APE1 SASP NF-κB/C/EBPβ pathway Radiotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Over the lifetime, a wide range of endogenous and exogenous stresses, like replicative exhaustion, oxidants, viral infection, malnutrition, and cancer therapies can induce cellular senescence, which is accompanied with changes in cell morphology, functions, and secretions. Among these changes, the soluble factors including pro-inflammatory cytokines, chemokines, growth factors, and proteases secreted by senescent cells are collectively known as the senescence-associated secretory phenotypes (SASP) (López-Otín C 2023; Calcinotto A and Kohli J 2019). Accumulating evidence demonstrates that the SASP acts as a central orchestrator of senescence programs. Specifically, certain components of the SASP can mediate immune cell recruitment to execute clearance functions and promote tissue repair and regenerative responses during the early senescence phase (Yin K 2022; Ritschka B 2017; Zhao S and Qiao Z 2024). Nevertheless, excessive SASP secretion from accumulated senescent cells exacerbates systemic chronic inflammation, thereby accelerating the progression of senescence-related pathologies such as diabetes, tumorigenesis, and cardiovascular disease (Zhang L 2022; Mehdizadeh M 2022). Senotherapeutics encompasses two primary therapeutic modalities: senolytics and senomorphics. Senolytics function by eliminating anti-apoptotic or pro-survival key enzymes, thereby inducing apoptosis in senescent cells (Zhang L 2021). In contrast, senomorphics modulate the SASP by targeting the NF-κB, mTOR, L1, and p38 MAPK signaling pathways, effectively mitigating the adverse impact of senescent cells on tissue homeostasis (Birch J 2020). Notably, senolytics face some limitations. The off-target effects of senolytic agents often give rise to undesirable side effects. Moreover, because senescent cells play indispensable roles in processes such as tissue renewal, wound healing, cancer prevention, and embryonic development, senolytics seem not to be the optimal senotherapeutic therapy. By selectively dampening the pro-inflammatory, pro-fibrotic, and pro-tumorigenic signals emitted by senescent cells, senomorphics serve as a non-cytotoxic complementary strategy to alleviate age-related pathologies without directly eliminating senescent cells (Zhang L 2023; Zheng L et al. 2024). This also makes senomorphics a research hotspot in the field of senotherapeutics. Apurinic apyrimidinic endonuclease 1 (APE1), a bifunctional enzyme with DNA repair and redox activities, is critically involved in maintaining cellular homeostasis, signal transduction, cellular senescence, and inflammation regulation (Oliveira TT 2022; Caston RA and Gampala S 2021; Zhao H 2024). Many studies have demonstrated that inhibition of APE1's redox activity effectively suppresses the production of pro-inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) in many cell lines, such as tumor cells, endothelial cells, and macrophages (Chu A 2025; Zhao CR et al. 2021; Tang Z and Wang Y 2021). Our previous research demonstrated that APE1 deficiency promoted cellular senescence both in human fibroblasts and in an APE1 knockout mouse model (Li M and Yang X 2018). However, the SASP expression profiles in APE1-deficient senescent fibroblasts and the regulatory mechanisms linking APE1's dual functions remain unclear. Methods Extraction primary foreskin fibroblasts Primary human foreskin fibroblasts (HFFs) were isolated from circumcised foreskins of 6–18-year-old healthy human donors at DaPing Hospital. Foreskin tissues were obtained with written informed consent under a protocol approved by the Institutional Review Board of the Army Medical Center, Chongqing DaPing Hospital. Dermal tissues were separated using dispase II (42613-33-2, KEHBIO) and digested with type I collagenase (2275GR001, BIoforxx). Cells culture and chemical reagents HFL-1 cells (Pricella, CL-0106) were purchased. HFF and HFL-1 cells were cultured in F12K medium (PMI150910, Pricella) containing 10% fetal bovine serum (164210, Pricella) and 1% penicillin-streptomycin at 37°C in a humidified 5% CO 2 incubator. E3330 (S7445, Selleck) and Inhibitor III (262017, Sigma) were used. Lentivirus transfection to construct APE1 overexpressing/interfering fibroblasts HFF and HFL-1 fibroblast lines were seeded into 6-well plates at 1×10 4 cells/well with 2 mL medium, incubated at 37°C in 5% CO 2 . For lentivirus transfection, cell density was adjusted to ~2×10 4 cells/well. The next day, medium was replaced with 2 mL fresh medium containing 2 μg/mL polybrene, followed by 10 μL of 1×10⁸ TU/mL APE1 overexpression/interference lentivirus (Genechem, Shanghai). After 48 h incubation, medium was changed to fresh 2 mL medium. APE1 expression was analyzed by western blot 72 h post-infection. Western blot For Western blot, treated cells were lysed in buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% Triton X-100, 1% SDS) with fresh protease (Roche, 11873580001) and phosphatase inhibitors (SPERIKON, SP002730001). Protein concentration was quantified by DC assay (Bio-Rad). Equal protein (15 µg/lane) was separated by 10–12% SDS-PAGE and transferred to PVDF membranes. After blocking with 10% skim milk, membranes were incubated overnight at 4°C with primary antibodies: anti-APE1 (ab137708, Abcam), anti-Tubulin (ab52866, Abcam), anti-p16INK4A (ab51243, Abcam), anti-p21 (A19094, ABclonal), anti-LamB1 (ab256380, Abcam), anti-p65 (ab32536, Abcam), anti-p-p65 (ab76302, Abcam), and anti-C/EBPβ (23431-1-AP, Proteintech). After washing, membranes were incubated with secondary antibodies in 5% skim milk for 1 h at RT, detected by ECL reagent (PK10001, Proteintech), and imaged via ChemiDoc (Bio-Rad). RT-qPCR Total RNA was isolated using Trizol (Invitrogen) or the RNeasy Micro kit. Complementary DNA was synthesized with the iScript cDNA synthesis kit (K16225, ThermoFisher). PCR was performed using SYBR Green qPCR Master Mix (Q311-01, Vazyme) and gene-specific primers (Supplementary Data Table 1) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher). Relative RNA levels were normalized to ACTB mRNA (β-Actin) and analyzed via the 2 -ΔΔCT method and specific normalization transcripts are noted in Figureure legends. SA-β-gal activity For senescence-associated β-galactosidase (SA-β-gal) staining, cells were processed using the Beyotime kit (C0602) according to the manufacturer’s protocol. Briefly, cells were fixed with SA-β-gal fixation solution for 15 min at RT and washed twice with PBS, followed by staining with fresh SA-β-gal detection solution (pH 6.0). Incubation was performed overnight at 37°C in a CO 2 free incubator. SA-β-gal-positive cells were quantified by calculating the percentage of stained cells across three fields per replicate using Olympus microscopy. SenTraGor stainning Cellular senescence was detected using SenTraGor™ 40 mg (Cat no: AR8850040, Arriani Pharmaceuticals, Attika, Greece) following the manufacturer’s protocol. For staining, cells were fixed with 4% polyformaldehyde for 15 min, washed sequentially with 50% and 70% EtOH (5 min each), and incubated with SenTraGor reagent at 37°C for 10 min. After washing with 50% EtOH (2×5 min) and TBS, cells were permeabilized with 0.3% Triton X-100 for 5 min, then incubated overnight at 4°C with anti-biotin primary antibody (Abcam, ab201341, 1:300). The next day, samples were washed with TBS, treated with Primary Antibody Amplifier Quanto for 10 min, and incubated with HRP Polymer Quanto at RT for 13 min. DAB Quanto Chromogen (1:150) was applied for 30–60 sec under light microscopy until dark brown signals appeared, followed by tap water washing and counterstaining with ¼-diluted Hematoxylin Harris for 1 min. Images were captured using Olympus microscopy. EDU assay EdU assay was performed using Beyotime's EdU kit (C0071S) to assess cell viability. Briefly, cells were incubated with EdU for 2 h, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 15 min. Afterward, cells were incubated with Click Reaction Mixture in the dark for 30 min at RT, stained with DAPI for 10 min, and imaged using Olympus fluorescence microscopy. Immunofluorescence Cells were seeded on coverslips and fixed with 4% paraformaldehyde for 15 min at RT, washed twice with PBS, and permeabilized with 0.3% Triton X-100 for 20 min. After blocking with 5% goat serum for 1 h at RT, cells were incubated overnight at 4°C with primary antibodies: anti-p65 (ab32536, Abcam) and anti-C/EBPβ (23431-1-AP, Proteintech). The next day, cells were treated with Alexa Fluor secondary antibody for 1 h and DAPI for 5 min, then imaged using an Olympus fluorescence microscope. Fluorescence intensity was analyzed via Image J software 2.1. Cell cycle profile The cell cycle was analyzed using the Cell Cycle Analysis Kit (C1052; Beyotime). Cells were fixed in 70% ethanol at 4°C for 2 h, stained with propidium iodide (0.05 mg/ml), RNase A (1 mg/ml), and 0.3% Triton X-100 in the dark for 30 min. DNA content was analyzed by flow cytometry, and the percentages of cells in G1, S, and G2/M phases were determined using ModFIT software. RNA sequencing and data analysis Total RNA was extracted using the TRIzol kit (TIANGEN, DP424), quality-checked via 0.8% agarose gel electrophoresis and spectrophotometry. mRNA libraries were constructed by ApexBio using the Hieff NGS® Ultima Dual-mode mRNA Library Prep Kit (12310ES), followed by second-strand cDNA synthesis and AMPure XP purification (Beckman Coulter). Library quality was assessed on an Agilent 2100 Bioanalyzer, and paired-end 150 bp sequencing was performed on the Illumina NovaSeq 6000. Differentially expressed genes (DEGs) were identified using DESeq2 (replicated samples) or edgeR (non-replicated) with thresholds of log₂|FC| > 1 and adjusted p < 0.05. Functional enrichment analysis of significant DEGs was performed using clusterProfiler for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (p < 0.05). Gene set enrichment analysis (GSEA) was conducted with gene lists ranked by log2 FC. Statistical Analysis Data are presented as mean values ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for multiple group comparisons. Statistical significance was defined as a P value < 0.05. All statistical analyses were performed using GraphPad Prism v9.0 softwareStatistical significance was indicated as follows: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Graphs were generated using GraphPad Prism 9. Results APE1 deficiency induced cellular senescence while suppressed SASP expression To explore the SASP expression in APE1-deficiency induced senescence model, we isolated primary human foreskin fibroblasts (HFF) from the foreskin tissues of healthy donors from Army Medical Center of PLA and identified them with fibroblast marker Vimentin by immunofluorescence (Supplementary Figure. 1A). We employed lentiviral particles loaded with APE1 shRNAs [TRCN0000007958 (#58) ] to deplete APE1 protein in HFF and human fetal lung fibroblast1(HFL-1) , both of which are well-established cell models for cellular senescence (Nadalutti CA 2020; Marthandan S 2016). After 14 days, the cells manifested senescence phenotypes. The percentages of cells with positive staining for SA-β-gal and SenTraGor were increased (Figure. 1A-B), and the EUD assay showed the Cell proliferation activity was decreased (Figure. 1C-D). Western blot analysis revealed elevated protein levels of the senescence markers p16 and p21, which coincided with loss of LamB1, a nuclear envelope integrity marker (Figure. 1E). These data demonstrate the successful establishment of an APE1 depletion-induced senescence model. Next, we examined the mRNA levels of classical SASP factors, including IL-6, IL-8, CCL5, TNF-α, and IFN-β by RT-qPCR analysis. To our surprise, the SASP was reduced compared with the control group both in HFF and HFL-1 cell lines (Figure. 1F-G). This was contray to other classic senescence models we established, including the replication and radiotherapy-induced senescence model (Figure. 1H-I, Supplementary Figure 1B). Collectively, our findings identified an intriguing senescence phenotype characterized by reduced SASP, which is induced by APE1 deficiency. Transcriptomic alterations in APE1 deficiency fibroblasts To investigate the transcriptional alterations caused by APE1 loss, we performed RNA-seq on scramble control and shAPE1 HFF cells from two different donors (HFF1 and HFF4). Compared with scramble control cells, APE1 knockdown resulted in 1081 upregulated differentially expressed genes (up-DEGs) and 1407 downregulated differentially expressed genes (down-DEGs) (Figure. 2A). We respectively utilized GO enrichment analysis on the up and down DEGs. The up-DEGs were significantly enriched in the gene sets related to the cell cycle and chromosome segregation (Figure. 2B). The down-DEGs were enriched in the cell chemotaxis and cytokine-mediated signaling pathway (Figure. 2C). We employed cell cycle detection and found the cell cycle was significantly arrested at G1 phase after knocking down APE1 (Figure. 2D). The GSEA analysis also revealed that cytokine activity, TNF signaling, growth factor activity, and NF-κB signaling pathway, were significantly inhibited (Figure. 2E). Further analysis of SASP genes identified 37 downregulated SASP factors, including IL-6, IL-8, CCL5, TNF-α, and IFN-β, consistent with our prior RT-qPCR findings Additionally, other reduced SASP components were detected, such as IL-7, IL-11, IL-1α, CSF2, CXCL2 and VEGFC (Figure. 2F). Taken together, our transcriptome data comprehensively depicted a downregulated SASP profile induced by APE1 deficiency. APE1 mediated SASP expression through NF-κΒ/C/EBPβ pathway Transcription factors critically regulate SASP transcription by binding to specific cis-elements in target gene promoters. To explore the TFs for APE1 to modulate the SASP. We used a web-based tool TRRUST for identification of candidate TFs involved in regulation of SASP gene expression. Using the TRRUST transcriptional regulatory network database, RELA (NF-κB p65) and C/EBPβ were identified as potential downstream transcription factors of APE1 responsible for mediating SASP gene transcription (Figure. 3A). Concurrently, NF-κB and C/EBPβ have been extensively documented to play critical roles in SASP regulation across multiple studies (Wang B 2024; Salotti J 2019). To validate this hypothesis, we performed APE1 knockdown and overexpression in HFF and HFL1 cells, respectively. Western blotting showed that APE1 depletion inhibited the expression of p65, phosphorylated p65 (p-p65) and C/EBPβ, whereas APE1 overexpression exerted the opposite effect (Figure. 3B-C). The same finding was also confirmed by immunofluorescence in HFF cells (Figure. 3D-E). We also conducted a time course experiment to detect the relationship between SASP and NF-κΒ/C/EBPβ during senescence induction by APE1 deficiency. In the APE1-depleted senescence model, persistent inactivation of NF-κB/C/EBPβ signaling axis was found to underlie the prolonged suppression of SASP, highlighting a novel regulatory node in SASP control(Figure. 3F-G). In summary, the SASP attenuation in the APE1 deficiency induced senescence model is attributed to the sustained inactivation of the NF-κB/C/EBPβ signaling axis. APE1 regulated NF-κΒ/C/EBPβ pathway via its redox activity To explore the bifunctional nature of APE1 in cellular senescence and SASP regulation, we respectively treated HFF cells with its redox activity inhibitor E3330 20 μM and DNA repair function inhibitor Inhibitor3 5 μM continuously cultured for 72 h. Then,we harvested the cells for subsequent experiments. Following treatment with Inhibitor3, cells displayed senescence hallmarks, including increased positivity for SA-β-gal and SenTraGor staining, reduced cell proliferation capacity, G1 phase cell cycle arrest, and upregulated protein levels of p16 and p21. Concurrently, this treatment triggered SASP expression. By contrast, treatment with E3330 significantly inhibited the NF-κB/C/EBPβ pathway and suppressed SASP without affecting cellular senescence (Figure. 4A-E). In order to more accurately elucidate the bifunctional nature of APE1 on the NF-κB/C/EBPβ signaling pathway and SASP. We separately transfected point mutation plasmids to overexpress C65S (APE1 redox function mutant) and D210 (APE1 DNA repair function mutant) in APE1-knockdown cells. Results showed that the NF-κB/C/EBPβ pathway and SASP were significantly attenuated in cells expressing APE1 C65S(Figure. 4F-G). Collectively, these findings confirm that loss of APE1 redox function inhibits the NF-κB/C/EBPβ pathway and SASP, whereas impairment of its DNA damage repair activity drives cellular senescence. Inhibiting the redox function of APE1 attenuated radiation-induced SASP APE1 has been reported to increase in liver tissue during radiotherapy and can serve as a biomarker to predict the occurrence of radioactive liver injury (Yue R and Li Z 2024). Our above results strongly suggest that targeting APE1, particularly its redox function, holds significant potential for reducing SASP during radiotherapy. To validate this hypothesis, we exposed HFFs to 10 Gy X-ray irradiation to induce cellular senescence. 10 days post-irradiation, we assessed cellular senescence and SASP expression. Ionizing radiation led to abundant SA-β-gal and SenTraGor positive staining, demonstrating efficient senescence induction. Western blot analysis confirmed significant upregulation of p16 and p21. Notably, both APE1-knockdown and control cells exhibited enhanced senescence after irradiation, with no significant difference between groups. Although APE1 did not increase after radiation, APE1 depletion impaired radiation-induced activation of the NF-κB/C/EBPβ signaling pathway and reduced SASP levels (Figure. 5A-C). We previously confirmed that APE1 regulates the NF-κB/C/EBPβ pathway and SASP via its redox function. In order to explore whether E3330 also has the potential to reduce the SASP induced by radiotherapy. Cells were exposed to 10 Gy X-ray irradiation and cultured for 7 days to allow senescence induction. Subsequently, they were treated with 20 μM E3330 for 72 hours. We found that E3330 treatment blocked radiation-induced upregulation of the NF-κB/C/EBPβ signaling axis and SASP without affecting cellular senescence (Figure. 5D-F). In this part, Our results demonstrate that both knocking down APE1 and using its redox function inhibitor E3330 can reduce the radiation-induced SASP via the NF-κB/C/EBPβ pathway. Discussion The SASP secreted by senescent cells is detrimental to the tissue microenvironment, accelerating tumorigenesis and promoting the progression of aging-related diseases (Li X et al. 2023; Wang X 2024). Therefore, elucidating the regulatory mechanisms of SASP and developing potential therapies to alleviate SASP-mediated pathological processes are of critical importance. To investigate the expression and regulatory mechanisms of the SASP in APE1-deficient senescent cells, we isolated primary fibroblasts from foreskin tissue and successfully established an APE1-deficient senescence model. Through RNA-seq and RT-qPCR, we identified a distinct phenotype of SASP downregulation in APE1-deficient senescent cells, which contrasted with our established classical replication and radiation induced senescence models. The bifunctional characteristic of APE1 plays distinct roles in regulating cellular senescence and the SASP. Specifically, the impairment of its DNA repair function accelerates cellular senescence and SASP, while the loss of its redox activity suppresses SASP expression. Mechanistic investigations further revealed that the down-regulation of SASP is mainly mediated by APE1's redox function via the inhibition of the NF-κB/C/EBPβ signaling pathways. Additionally, we found that both targeting APE1 and using its redox activity inhibitor E3330 could reduce radiation-induced SASP. Due to inherent variations in organs, tissues, cell types, and senescence-inducing stimuli, distinct senescence models exhibit characteristic differences (Cohn RL and Gasek NS 2023). The SENNET Consortium has confirmed that the SASP is the most prevalent senescence marker, observed in 80% of senescent cell populations across diverse tissues. However, their findings also emphasized that cellular senescence should not be defined by a single marker, calling for multimethodological approaches to characterize this state (Suryadevara V et al. 2024). To address this, we comprehensively characterized the senescence state of APE1-deficient cells using multi approaches. Our analyses revealed that APE1 deficiency induced canonical senescence hallmarks: reduced proliferative capacity, cell cycle arrest, enlarged morphology, upregulated expression of senescence markers p21 and p16, and downregulated LamB1 expression. Additionally, increased positive staining for SA-β-gal and SenTraGor was observed, consistent with phenotypes in other senescence models. Notably, despite these shared features, the APE1-deficiency model exhibited a unique divergence from classical paradigms: SASP factor expression was significantly downregulated, in stark contrast to the upregulated pro-inflammatory SASP typically observed in most senescence models. To elucidate the mechanism by which APE1 regulates the SASP, we queried upstream TFs of key SASP factors (including IL-6, IL-8, TNF-α, CCL2, etc.) and downstream TFs of APE1 using the TRRUST databases. This analysis revealed that APE1 mightily regulates SASP through modulation of two transcription factors: NF-κBand C/EBPβ. Functional assays demonstrated that APE1 knockdown significantly suppressed protein expression of both NF-κB and C/EBPβ, whereas APE1 overexpression specifically enhanced their expression. These results indicate that APE1 is involved in the NF-κB/C/EBPβ signaling pathway. Further, the time course experiment showed that in APE1-deficient senescent cells, sustained inhibition of NF-κB/C/EBPβ pathway led to a marked reduction of SASP factors. Mechanistic validation using APE1 bifunctional inhibitors and point-mutated plasmids confirmed that APE1 loss-mediated SASP downregulation depends on its redox activity to modulate the NF-κB/C/EBPβ pathway. Collectively, these results uncover a novel mechanism whereby APE1 regulates the NF-κB/C/EBPβ pathway through its redox function, thereby specifically suppressing SASP. To explore whether targeting APE1 serves as a senomorphic strategy to alleviate the SASP during aging process, we conducted preliminary investigations in a classical radiation-induced senescence model. Our results showed that APE1 knockdown and E3330 treatment both inhibited the NF-κB/C/EBPβ pathway and reduced radiation-induced SASP expression. The redox function of APE1 in inflammation regulation has been extensively studied. Knocking down APE1 can reduce the expression of the myocardial inflammatory factors IL-6, TNF-α and IL-1β induced by Ang II, thereby reducing the occurrence of myocardial infarction and fibrosis (Lu F 2024). Similarly, in APE1 knockout mice and mice treated with E3330, NF-κB activity and the expression of its downstream target CXCL1 are suppressed. This suppression reduces neutrophil infiltration and decreases IL-1β expression in injured tissues, which exerts protective effects against DSS-induced colitis (Chen Q and Chen T 2024). The anti-inflammatory effect of E3330 has been verified in a mouse model of alcoholic hepatitis. Treatment with E3330 improves alcoholic liver injury and hepatitis in mice, and the expression levels of TNF-α, AST and ALT in the plasma of mice are reduced after E3330 treatment (Nagakawa J 1992). Our results are consistent with these previous studies, indicating that the redox function of APE1 indeed plays a critical role in SASP. There are some limitations in our study. Although we demonstrated that targeting APE1 can suppress the SASP in a radiation induced senescence model, its regulatory effects on SASP in other senescence models, such as those induced by oncogenes, chemotherapy, or nutrient deficiency, remain unclear. Additionally, as our findings predominantly stem from in vitro experiments, the in vivo effects of APE1 on SASP, along with determining whether this mechanism is broadly applicable or context-dependent, necessitate further investigation. These remaining issues will also be further elucidated in our future research. In summary, APE1 deletion in fibroblasts elicits a distinct senescent phenotype with suppressed SASP. This phenotype arises from APE1's dual functions: DNA repair impairment drives cellular senescence, while blocking its redox activity inhibits NF-κB/C/EBPβ and SASP expression. The most significant highlight of our study is the discovery that the APE1 redox activity inhibitor E3330 has the potential to serve as a senomorphics therapy for improving age-related diseases. Declarations Ethics approval and consent to participate All human foreskin tissue experimental procedures were reviewed and approved by the ethics committee of Army Medical Center of PLA. We certify that the study was performed in accordance with the 1964 declaration of HELSINKI and later amendments. Written informed consent was obtained from all the participants prior to the enrollment of his study. Consent for publication All authors approved the final manuscript and the submission to this journal. Availability of data and materials The datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing financial interests exist. Fundings This research was supported by grants from the National Natural Science Foundation of China (No. 82002444 to X.Y.) and (No. 82173203 to JW.Z.), the Natural Science Foundation of Chongqing Municipality (cstc2021jcyj-msxmX0446 to J.Z.), and the Talent Innovation Ability Training Plan of the Army Medical Center of PLA [ No. ZXYZZKY07 to MX.L]. Authors' contributions Conceptualization: Mx L, XY; Data Curation:YD; Formal Analysis: Xj K, HX, XG , YP; Funding Acquisition: XY, Mx L, JW Z; Writing - Original Draft Preparation: YD, Jw Z. Acknowledgements We are grateful to Zhengwei Hu from Urology department of Army Medical Center of PLA for providing foreskin tissue;GuiHong Yang from dermatology department of Army Medical Center of PLA for experimental technical support. References López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G (2023) Hallmarks of aging: An expanding universe. Cell 186(2):243-278. Calcinotto A, Kohli J, Zagato E, Pellegrini L, Demaria M, Alimonti A (2019) Cellular Senescence: Aging, Cancer, and Injury. Physiol Rev 99(2):1047-1078. Yin K, Patten D, Gough (2022) Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance. Genes Dev 36(9-10):533-549. Ritschka B, Storer M, Mas A, Heinzmann F, Ortells MC (2017) The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration. Genes Dev 31(2):172-183. Zhao S, Qiao Z, Meng B, Chen S, Liu H (2024) Modulation of fracture healing by senescence-associated secretory phenotype (SASP): a narrative review of the current literature. Eur J Med Res 29(1):38. Zhang L, Pitcher LE, Yousefzadeh MJ, Niedernhofer LJ, Robbins PD, Zhu Y (2022) Cellular senescence: a key therapeutic target in aging and diseases. J Clin Invest 132(15):e158450. Mehdizadeh M, Aguilar M, Thorin E, Ferbeyre G, Nattel S (2022) The role of cellular senescence in cardiac disease: basic biology and clinical relevance. Nat Rev Cardiol 19(4):250-264. Zhang L, Pitcher LE, Prahalad V, Niedernhofer LJ, Robbins PD (2021) Recent advances in the discovery of senolytics. Mech Ageing Dev 200:111587. Birch J, Gil J(2021) Senescence and the SASP: many therapeutic avenues. Genes Dev 34(23-24):1565-1576. Zhang L, Pitcher LE, Prahalad V, Niedernhofer LJ, Robbins P (2023) Targeting cellular senescence with senotherapeutics: senolytics and senomorphics. FEBS J 290(5):1362-1383. Zheng L, He S, Wang H, Li J, Liu Y, Liu S (2024) Targeting Cellular Senescence in Aging and Age-Related Diseases: Challenges, Considerations, and the Emerging Role of Senolytic and Senomorphic Therapies. Aging Dis 15(6):2554-2594. Oliveira TT, Coutinho LG, de Oliveira LOA (2022) APE1/Ref-1 Role in Inflammation and Immune Response. Front Immunol 13:793096. Caston RA, Gampala S, Armstrong L, Messmann RA, Fishel ML, Kelley MR (2021) The multifunctional APE1 DNA repair-redox signaling protein as a drug target in human disease. Drug Discov Today 26(1):218-228. Zhao H, Richardson C, Marriott I, Yang IH, Yan S (2024) APE1 is a master regulator of the ATR-/ATM-mediated DNA damage response. DNA Repair (Amst) 144:103776. Chu A, Liu X, Liu S, Li M, Song R, Gan L, Wang Y, Liu Z, Sun C (2025) RNA-seq analysis reveals key genes associated with downregulation of APE1 in esophageal squamous cell carcinoma. Front Genet 16:1549371. Zhao CR, Yang FF, Cui Q, Wang D (2021) Vitexin inhibits APEX1 to counteract the flow-induced endothelial inflammation. Proc Natl Acad Sci U S A 118(48):e2115158118. Tang Z, Wang Y, Wan Y, Xie Y, Li S, Tao D, Wang C, Wu YZ, Sui JD (2021) Apurinic/apyrimidinic endonuclease 1/reduction-oxidation effector factor-1 (APE1) regulates the expression of NLR family pyrin domain containing 3 (NLRP3) inflammasome through modulating transcription factor NF-κB and promoting the secretion of inflammatory mediators in macrophages. Ann Transl Med 9(2):145. Li M, Yang X,Wang D, Wilson DM 3rd (2018) APE1 deficiency promotes cellular senescence and premature aging features. Nucleic Acids Res 46(11):5664-5677. Nadalutti CA, Wilson SH (2020) Using Human Primary Foreskin Fibroblasts to Study Cellular Damage and Mitochondrial Dysfunction. Curr Protoc Toxicol 86(1):e99. Marthandan S, Menzel U, Diekmann S (2016) Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence. Biol Res 49(1):34. Wang B, Han J, Elisseeff JH, Demaria M (2024) The senescence-associated secretory phenotype and its physiological and pathological implications. Nat Rev Mol Cell Biol 25(12):958-978. Salotti J, Johnson PF (2019) Regulation of senescence and the SASP by the transcription factor C/EBPβ. Exp Gerontol 128:110752. Yue R, Li Z, Zhang X, Song G (2024) Imaging-guided companion diagnostics in radiotherapy by monitoring APE1 activity with afterglow and MRI imaging. Nat Commun 15(1):6349. Li X, Li C, Zhang W, Wang Y, Qian P, Huang H (2023) Inflammation and aging: signaling pathways and intervention therapies. Signal Transduct Target Ther 8(1):239. Wang X, Fukumoto T, Noma KI (2024) Therapeutic strategies targeting cellular senescence for cancer and other diseases. J Biochem 175(5):525-537. Cohn RL, Gasek NS, Kuchel GA, Xu M (2023) The heterogeneity of cellular senescence: insights at the single-cell level. Trends Cell Biol 33(1):9-17. Suryadevara V, Hudgins AD, Dou Z, Neretti N (2024) SenNet recommendations for detecting senescent cells in different tissues. Nat Rev Mol Cell Biol. Lu F, Ding L, Qiao Y (2024) APEX1 Knockdown Alleviates Inflammation and Fibrosis in Myocardial Infarction Through Promoting ZCCHC9 Expression and Blocking the p38 MAPK Signaling. Biochem Genet. Chen Q, Chen T, Wang D, Li MX. APEX1 in intestinal epithelium triggers neutrophil infiltration and intestinal barrier damage in ulcerative colitis. Free Radic Biol Med. 2024 Nov 20;225:359-373. Nagakawa J, Yamatsu I (1992) Protective effects of (2E)-3- [5 - (2,3-dimethoxy-6- methyl -1, 4-benzoquinoyl)] -2-nonyl -2- propenoic acid on endotoxin-mediated hepatitis in mice. J Pharmacol Exp Ther. 262(1):145-50. Additional Declarations No competing interests reported. Supplementary Files Table1Theprimersequencesusedinthestudy.docx Supplementaryfigure.tif Supplementary Figure 1 Micrographs and Immunostaining for Vimentin in HFF cells (A). SA-β-gal staining (B) in replication and radiation induced senescence modle. 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-6841207","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":468768939,"identity":"58830c32-b6a7-421b-81a0-0e82dafca6c8","order_by":0,"name":"Yi Duan","email":"","orcid":"","institution":"Army Medical Center of PLA","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Duan","suffix":""},{"id":468768940,"identity":"38f242d8-7f10-4d7b-b01e-ddf55b5659e9","order_by":1,"name":"Xiao Yang","email":"","orcid":"","institution":"Army Medical Center of PLA","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Yang","suffix":""},{"id":468768941,"identity":"0c0f288e-e38b-4af1-ab5d-f41cecff34ae","order_by":2,"name":"Xunjie Kuang","email":"","orcid":"","institution":"Army Medical Center of PLA","correspondingAuthor":false,"prefix":"","firstName":"Xunjie","middleName":"","lastName":"Kuang","suffix":""},{"id":468768942,"identity":"f0b1cb71-3e26-4918-b669-2e3b7b30dc34","order_by":3,"name":"He Xiao","email":"","orcid":"","institution":"Army Medical Center of PLA","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Xiao","suffix":""},{"id":468768943,"identity":"53feef65-b59a-4d77-9a5b-ba25e668dd0d","order_by":4,"name":"Xin Guo","email":"","orcid":"","institution":"Army Medical Center of PLA","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Guo","suffix":""},{"id":468768944,"identity":"6172ceb7-d505-4cef-8ce5-9cf03a3bb122","order_by":5,"name":"Yang Peng","email":"","orcid":"","institution":"Army Medical Center of PLA","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Peng","suffix":""},{"id":468768945,"identity":"c74ff1e7-e8b2-4f57-a666-7008de2a868d","order_by":6,"name":"JianWu Zhu","email":"","orcid":"","institution":"Army Medical Center of PLA","correspondingAuthor":false,"prefix":"","firstName":"JianWu","middleName":"","lastName":"Zhu","suffix":""},{"id":468768946,"identity":"1003b846-74cf-464f-8ed6-c71024fb7b78","order_by":7,"name":"Mengxia Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBACPmYIzcPA3gAVOkBACxtcC89hYrXAWRLJxGph5058XPDrsAz/zPdHN91sY5Dju5HA+LkAr8N4NxvP7EvjkbidzHY7t43BWPJGArP0DPxatknz9tjwMEC1JG64kcDGzINfy/bfvD0SPPI3D4O11BOjZRszzw8bHoMbzGAtCQZEaNkszduQxmN4Jtnsds45CcOZZx42S+PTws9/duNnnj+H7eWOH3x2O6fMRp7vePLBz/i0gAFjG5wpAeI2ENIABH+IUDMKRsEoGAUjFwAAuL5E1FcBGB8AAAAASUVORK5CYII=","orcid":"","institution":"Army Medical Center of PLA","correspondingAuthor":true,"prefix":"","firstName":"Mengxia","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-06-07 07:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6841207/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6841207/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84420617,"identity":"9358a948-96c7-4f02-9790-d89a71fb456e","added_by":"auto","created_at":"2025-06-11 18:02:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":798145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of APE1 blockades the SASP in senescent fibroblast\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrographs of SA-β-Gal and SenTraGor stainning (A-B) in senescent cells(shAPE1) relative to corresponding proliferating controls(scr) in HFF and HFL-1 cells. The EDU test (C-D) in APE1-deficiency HFF and HFL-1 cells. Western blot analysis (E) of the levels of p16, p21, LamB1, APE1 and control Tubulin in HFF and HFL-1 cells . RT-qPCR analysis (F-G) of the levels of IL-6, IL-8, CCL5, TNF-α, and IFN-β mRNA, normalized to the levels of ACTIN mRNA in HFF and HFL-1 cells . Western blot (H) of indicated proteins in replication and radiation-induced senescent HFF cells. Heatmap of SASP(I) in APE1-deficient and other senescent HFF cells detected by RT-qPCR.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6841207/v1/8c0abb6e71d142c3d9ea1c3f.png"},{"id":84421089,"identity":"2427c548-5338-43ad-909b-f7072b230ac2","added_by":"auto","created_at":"2025-06-11 18:10:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":602592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA sequencing of senescent cells induced by APE1 deficiency reveals decreased activation of cytokine and inflammatory signaling pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe volcano plot (A) showed the profile of differentially expressed transcripts. Heatmap of the GO enrichment analysis for the upregulated DEGs (B) and downregulated DEGs (C). The cell cycle distribution (D) after kocking down APE1 in HFFs. GSEA was performed on the the DEGs(E). Heatmap depicting the variation of SASP on transcriptomic expression profile in APE1 deficiency HFFs (F).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6841207/v1/08c7008969a06bf9554f1afc.png"},{"id":84420616,"identity":"7298a520-6910-4403-ab37-c104b434e505","added_by":"auto","created_at":"2025-06-11 18:02:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":680073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNF-κB/C/EBPβ is a critical pathway for APE1 to regulate the SASP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTRRUST website and APE1-related TF indicates RELA(NF-κB, p65) and C/EBPβ as two of the key transcriptional regulators in APE1 induced SASP expression (A). Western blot analysis (B) of the indicated proteins in APE1-overexpressing and Scr HFF and HFL-1 cells. Western blot analysis (C) of the indicated proteins in APE1-deleted and Scr HFF and HFL-1 cells. Immunostaining (D-E)for NF-κB and C/EBPβ in HFF cells with different APE1 expression including shAPE1, scr and APE1-overexpressed in HFF cells. Western blot (F) of the indicated proteins in APE1-deleted HFF cells in a time course experiment. Time Course Analysis of SASP (G) Dynamics in APE1-Deleted HFF Cells.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6841207/v1/5a16b73569d0ef652ab9632f.png"},{"id":84420619,"identity":"d91886a6-17e8-44dd-91ce-f36002c23fda","added_by":"auto","created_at":"2025-06-11 18:02:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":758792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe NF-κB/C/EBPβ pathway is modulated by the redox function of APE1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSA-β-gal and SenTraGor staining (A) with inhibitors treatment in HFF cells. EDU Assay (B), the cell cycle distribution (C) in HFF cells treated with E3330 and Inhibitor3. Western blot of indicated proteins (D) and the expression of SASP (E) with Inhibitors treatment. Western blot of indicated proteins (F) and the expression of SASP (G) with Point mutant plasmids.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6841207/v1/d1776ae5210914f9d0f46758.png"},{"id":84420623,"identity":"895ff0d9-2d08-4ea9-bd82-28a675166945","added_by":"auto","created_at":"2025-06-11 18:02:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1029317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibiting the redox activity of APE1 can mitigate radiation-induced SASP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSA-β-gal and SenTraGor staining (A) in radiation-induced senescent HFF cells with or without APE1 knockdown. Western blot (B) and RT-qpcr analysis of SASP (C) in radiation-induced senescent HFF cells with or without APE1 knockdown. SA-β-gal and SenTraGor staining (D) in radiation-induced senescent HFF cells with or without E3330 treatment.Western blot of indicated proteins (E) and RT-qpcr analysis of SASP (F) in radiation-induced senescent HFF cells with or without E3330 treatment.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6841207/v1/4dce9ad74903abbee09edaf6.png"},{"id":84434953,"identity":"3e04d5a0-f825-42e2-9061-259add60dd25","added_by":"auto","created_at":"2025-06-12 02:01:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4565881,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6841207/v1/f87b5330-e8a3-4690-bc33-9b5e696f9141.pdf"},{"id":84420621,"identity":"b504fe1f-44af-486e-8612-f164a26fd72b","added_by":"auto","created_at":"2025-06-11 18:02:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12326,"visible":true,"origin":"","legend":"","description":"","filename":"Table1Theprimersequencesusedinthestudy.docx","url":"https://assets-eu.researchsquare.com/files/rs-6841207/v1/3e6f6af0fd4684af032ebe4e.docx"},{"id":84421090,"identity":"4c46fcb7-a557-4bfb-a9bd-b75c4179986b","added_by":"auto","created_at":"2025-06-11 18:10:52","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1245147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrographs and Immunostaining for Vimentin in HFF cells (A). SA-β-gal staining (B) in replication and radiation induced senescence modle.\u003c/p\u003e","description":"","filename":"Supplementaryfigure.tif","url":"https://assets-eu.researchsquare.com/files/rs-6841207/v1/1f760b70a77a89440222a9a1.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Redox Activity of APE1 Mediates SASP Suppression in Senescent Fibroblasts","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOver the lifetime, a wide range of endogenous and exogenous stresses, like replicative exhaustion, oxidants, viral infection, malnutrition, and cancer therapies can induce cellular senescence, which is accompanied with changes in cell morphology, functions, and secretions. Among these changes, the soluble factors including pro-inflammatory cytokines, chemokines, growth factors, and proteases secreted by senescent cells are collectively known as the senescence-associated secretory phenotypes (SASP) (López-Otín C 2023; Calcinotto A and Kohli J 2019). Accumulating evidence demonstrates that the SASP acts as a central orchestrator of senescence programs. Specifically, certain components of the SASP can mediate immune cell recruitment to execute clearance functions and promote tissue repair and regenerative responses during the early senescence phase (Yin K 2022; Ritschka B 2017; Zhao S and Qiao Z 2024). Nevertheless, excessive SASP secretion from accumulated senescent cells exacerbates systemic chronic inflammation, thereby accelerating the progression of senescence-related pathologies such as diabetes, tumorigenesis, and cardiovascular disease (Zhang L 2022; Mehdizadeh M 2022).\u003c/p\u003e\n\u003cp\u003eSenotherapeutics encompasses two primary therapeutic modalities: senolytics and senomorphics. Senolytics function by eliminating anti-apoptotic or pro-survival key enzymes, thereby inducing apoptosis in senescent cells (Zhang L 2021). In contrast, senomorphics modulate the SASP by targeting the NF-κB, mTOR, L1, and p38 MAPK signaling pathways, effectively mitigating the adverse impact of senescent cells on tissue homeostasis (Birch J 2020). Notably, senolytics face some limitations. The off-target effects of senolytic agents often give rise to undesirable side effects. Moreover, because senescent cells play indispensable roles in processes such as tissue renewal, wound healing, cancer prevention, and embryonic development, senolytics seem not to be the optimal senotherapeutic therapy. By selectively dampening the pro-inflammatory, pro-fibrotic, and pro-tumorigenic signals emitted by senescent cells, senomorphics serve as a non-cytotoxic complementary strategy to alleviate age-related pathologies without directly eliminating senescent cells (Zhang L 2023; Zheng L et al. 2024). This also makes senomorphics a research hotspot in the field of senotherapeutics.\u003c/p\u003e\n\u003cp\u003eApurinic apyrimidinic endonuclease 1 (APE1), a bifunctional enzyme with DNA repair and redox activities, is critically involved in maintaining cellular homeostasis, signal transduction, cellular senescence, and inflammation regulation (Oliveira TT 2022; Caston RA and Gampala S 2021; Zhao H 2024). Many studies have demonstrated that inhibition of APE1's redox activity effectively suppresses the production of pro-inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) in many cell lines, such as tumor cells, endothelial cells, and macrophages (Chu A 2025; Zhao CR et al. 2021; Tang Z and Wang Y 2021). Our previous research demonstrated that APE1 deficiency promoted cellular senescence both in human fibroblasts and in an APE1 knockout mouse model (Li M and Yang X 2018). However, the SASP expression profiles in APE1-deficient senescent fibroblasts and the regulatory mechanisms linking APE1's dual functions remain unclear.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eExtraction primary foreskin fibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary human foreskin fibroblasts (HFFs) were isolated from circumcised foreskins of 6–18-year-old healthy human donors at DaPing Hospital. Foreskin tissues were obtained with written informed consent under a protocol approved by the Institutional Review Board of the Army Medical Center, Chongqing DaPing Hospital. Dermal tissues were separated using dispase II (42613-33-2, KEHBIO) and digested with type I collagenase (2275GR001, BIoforxx).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCells culture and chemical reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHFL-1 cells (Pricella, CL-0106) were purchased. HFF and HFL-1 cells were cultured in F12K medium (PMI150910, Pricella) containing 10% fetal bovine serum (164210, Pricella) and 1% penicillin-streptomycin at 37°C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. E3330 (S7445, Selleck) and Inhibitor III (262017, Sigma) were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLentivirus transfection to construct APE1 overexpressing/interfering fibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHFF and HFL-1 fibroblast lines were seeded into 6-well plates at 1×10\u003csup\u003e4\u003c/sup\u003e cells/well with 2 mL medium, incubated at 37°C in 5% CO\u003csub\u003e2\u003c/sub\u003e. For lentivirus transfection, cell density was adjusted to ~2×10\u003csup\u003e4\u003c/sup\u003e cells/well. The next day, medium was replaced with 2 mL fresh medium containing 2 μg/mL polybrene, followed by 10 μL of 1×10⁸ TU/mL APE1 overexpression/interference lentivirus (Genechem, Shanghai). After 48 h incubation, medium was changed to fresh 2 mL medium. APE1 expression was analyzed by western blot 72 h post-infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor Western blot, treated cells were lysed in buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% Triton X-100, 1% SDS) with fresh protease (Roche, 11873580001) and phosphatase inhibitors (SPERIKON, SP002730001). Protein concentration was quantified by DC assay (Bio-Rad). Equal protein (15 µg/lane) was separated by 10–12% SDS-PAGE and transferred to PVDF membranes. After blocking with 10% skim milk, membranes were incubated overnight at 4°C with primary antibodies: anti-APE1 (ab137708, Abcam), anti-Tubulin (ab52866, Abcam), anti-p16INK4A (ab51243, Abcam), anti-p21 (A19094, ABclonal), anti-LamB1 (ab256380, Abcam), anti-p65 (ab32536, Abcam), anti-p-p65 (ab76302, Abcam), and anti-C/EBPβ (23431-1-AP, Proteintech). After washing, membranes were incubated with secondary antibodies in 5% skim milk for 1 h at RT, detected by ECL reagent (PK10001, Proteintech), and imaged via ChemiDoc (Bio-Rad).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using Trizol (Invitrogen) or the RNeasy Micro kit. Complementary DNA was synthesized with the iScript cDNA synthesis kit (K16225, ThermoFisher). PCR was performed using SYBR Green qPCR Master Mix (Q311-01, Vazyme) and gene-specific primers (Supplementary Data Table 1) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher). Relative RNA levels were normalized to ACTB mRNA (β-Actin) and analyzed via the 2\u003csup\u003e-ΔΔCT\u003c/sup\u003e method and specific normalization transcripts are noted in Figureure legends.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSA-β-gal activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor senescence-associated β-galactosidase (SA-β-gal) staining, cells were processed using the Beyotime kit (C0602) according to the manufacturer’s protocol. Briefly, cells were fixed with SA-β-gal fixation solution for 15 min at RT and washed twice with PBS, followed by staining with fresh SA-β-gal detection solution (pH 6.0). Incubation was performed overnight at 37°C in a CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003efree incubator. SA-β-gal-positive cells were quantified by calculating the percentage of stained cells across three fields per replicate using Olympus microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSenTraGor stainning\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCellular senescence was detected using SenTraGor™ 40 mg (Cat no: AR8850040, Arriani Pharmaceuticals, Attika, Greece) following the manufacturer’s protocol. For staining, cells were fixed with 4% polyformaldehyde for 15 min, washed sequentially with 50% and 70% EtOH (5 min each), and incubated with SenTraGor reagent at 37°C for 10 min. After washing with 50% EtOH (2×5 min) and TBS, cells were permeabilized with 0.3% Triton X-100 for 5 min, then incubated overnight at 4°C with anti-biotin primary antibody (Abcam, ab201341, 1:300). The next day, samples were washed with TBS, treated with Primary Antibody Amplifier Quanto for 10 min, and incubated with HRP Polymer Quanto at RT for 13 min. DAB Quanto Chromogen (1:150) was applied for 30–60 sec under light microscopy until dark brown signals appeared, followed by tap water washing and counterstaining with ¼-diluted Hematoxylin Harris for 1 min. Images were captured using Olympus microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEDU assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEdU assay was performed using Beyotime's EdU kit (C0071S) to assess cell viability. Briefly, cells were incubated with EdU for 2 h, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 15 min. Afterward, cells were incubated with Click Reaction Mixture in the dark for 30 min at RT, stained with DAPI for 10 min, and imaged using Olympus fluorescence microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded on coverslips and fixed with 4% paraformaldehyde for 15 min at RT, washed twice with PBS, and permeabilized with 0.3% Triton X-100 for 20 min. After blocking with 5% goat serum for 1 h at RT, cells were incubated overnight at 4°C with primary antibodies: anti-p65 (ab32536, Abcam) and anti-C/EBPβ (23431-1-AP, Proteintech). The next day, cells were treated with Alexa Fluor secondary antibody for 1 h and DAPI for 5 min, then imaged using an Olympus fluorescence microscope. Fluorescence intensity was analyzed via Image J software 2.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell cycle was analyzed using the Cell Cycle Analysis Kit (C1052; Beyotime). Cells were fixed in 70% ethanol at 4°C for 2 h, stained with propidium iodide (0.05 mg/ml), RNase A (1 mg/ml), and 0.3% Triton X-100 in the dark for 30 min. DNA content was analyzed by flow cytometry, and the percentages of cells in G1, S, and G2/M phases were determined using ModFIT software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using the TRIzol kit (TIANGEN, DP424), quality-checked via 0.8% agarose gel electrophoresis and spectrophotometry. mRNA libraries were constructed by ApexBio using the Hieff NGS® Ultima Dual-mode mRNA Library Prep Kit (12310ES), followed by second-strand cDNA synthesis and AMPure XP purification (Beckman Coulter). Library quality was assessed on an Agilent 2100 Bioanalyzer, and paired-end 150 bp sequencing was performed on the Illumina NovaSeq 6000.\u003c/p\u003e\n\u003cp\u003eDifferentially expressed genes (DEGs) were identified using DESeq2 (replicated samples) or edgeR (non-replicated) with thresholds of log₂|FC| \u0026gt; 1 and adjusted p \u0026lt; 0.05. Functional enrichment analysis of significant DEGs was performed using clusterProfiler for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (p \u0026lt; 0.05). Gene set enrichment analysis (GSEA) was conducted with gene lists ranked by log2 FC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean values ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for multiple group comparisons. Statistical significance was defined as a P value \u0026lt; 0.05. All statistical analyses were performed using GraphPad Prism v9.0 softwareStatistical significance was indicated as follows: *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001; ****, p\u0026lt;0.0001. Graphs were generated using GraphPad Prism 9.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAPE1 deficiency induced cellular senescence while suppressed SASP expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the SASP expression in APE1-deficiency induced senescence model, we isolated primary human foreskin fibroblasts (HFF) from the foreskin tissues of healthy donors from Army Medical Center of PLA and identified them with fibroblast marker Vimentin by immunofluorescence (Supplementary Figure. 1A). We employed lentiviral particles loaded with APE1 shRNAs [TRCN0000007958 (#58) ] to deplete APE1 protein in HFF and human fetal lung fibroblast1(HFL-1) , both of which are well-established cell models for cellular senescence (Nadalutti CA 2020; Marthandan S 2016). After 14 days, the cells manifested senescence phenotypes. The percentages of cells with positive staining for SA-β-gal and SenTraGor were increased (Figure. 1A-B), and the EUD assay showed the Cell proliferation activity was decreased (Figure. 1C-D). Western blot analysis revealed elevated protein levels of the senescence markers p16 and p21, which coincided with loss of LamB1, a nuclear envelope integrity marker (Figure. 1E). These data demonstrate the successful establishment of an APE1 depletion-induced senescence model. Next, we examined the mRNA levels of classical SASP factors, including IL-6, IL-8, CCL5, TNF-α, and IFN-β by RT-qPCR analysis. To our surprise, the SASP was reduced compared with the control group both in HFF and HFL-1 cell lines (Figure. 1F-G). This was contray to other classic senescence models we established, including the replication and radiotherapy-induced senescence model (Figure. 1H-I, Supplementary Figure 1B). Collectively, our findings identified an intriguing senescence phenotype characterized by reduced SASP, which is induced by APE1 deficiency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic alterations in APE1 deficiency fibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the transcriptional alterations caused by APE1 loss, we performed RNA-seq on scramble control and shAPE1 HFF cells from two different donors (HFF1 and HFF4). Compared with scramble control cells, APE1 knockdown resulted in 1081 upregulated differentially expressed genes (up-DEGs) and 1407 downregulated differentially expressed genes (down-DEGs) (Figure. 2A). We respectively utilized GO enrichment analysis on the up and down DEGs. The up-DEGs were significantly enriched in the gene sets related to the cell cycle and chromosome segregation (Figure. 2B). The down-DEGs were enriched in the cell chemotaxis and cytokine-mediated signaling pathway (Figure. 2C). We employed cell cycle detection and found the cell cycle was significantly arrested at G1 phase after knocking down APE1 (Figure. 2D). The GSEA analysis also revealed that cytokine activity, TNF signaling, growth factor activity, and NF-κB signaling pathway, were significantly inhibited (Figure. 2E). Further analysis of SASP genes identified 37 downregulated SASP factors, including IL-6, IL-8, CCL5, TNF-α, and IFN-β, consistent with our prior RT-qPCR findings Additionally, other reduced SASP components were detected, such as IL-7, IL-11, IL-1α, CSF2, CXCL2 and VEGFC (Figure. 2F). Taken together, our transcriptome data comprehensively depicted a downregulated SASP profile induced by APE1 deficiency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAPE1 mediated SASP expression through NF-κΒ/C/EBPβ pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranscription factors critically regulate SASP transcription by binding to specific cis-elements in target gene promoters. To explore the TFs for APE1 to modulate the SASP. We used a web-based tool TRRUST for identification of candidate TFs involved in regulation of SASP gene expression. Using the TRRUST transcriptional regulatory network database, RELA (NF-κB p65) and C/EBPβ were identified as potential downstream transcription factors of APE1 responsible for mediating SASP gene transcription (Figure. 3A). Concurrently, NF-κB and C/EBPβ have been extensively documented to play critical roles in SASP regulation across multiple studies (Wang B 2024; Salotti J 2019). To validate this hypothesis, we performed APE1 knockdown and overexpression in HFF and HFL1 cells, respectively. Western blotting showed that APE1 depletion inhibited the expression of p65, phosphorylated p65 (p-p65) and C/EBPβ, whereas APE1 overexpression exerted the opposite effect (Figure. 3B-C). The same finding was also confirmed by immunofluorescence in HFF cells (Figure. 3D-E). We also conducted a time course experiment to detect the relationship between SASP and NF-κΒ/C/EBPβ during senescence induction by APE1 deficiency. In the APE1-depleted senescence model, persistent inactivation of NF-κB/C/EBPβ signaling axis was found to underlie the prolonged suppression of SASP, highlighting a novel regulatory node in SASP control(Figure. 3F-G). In summary, the SASP attenuation in the APE1 deficiency induced senescence model is attributed to the sustained inactivation of the NF-κB/C/EBPβ signaling axis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAPE1 regulated NF-κΒ/C/EBPβ pathway via its redox activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the bifunctional nature of APE1 in cellular senescence and SASP regulation, we respectively treated HFF cells with its redox activity inhibitor E3330 20 μM and DNA repair function inhibitor Inhibitor3 5 μM continuously cultured for 72 h. Then,we harvested the cells for subsequent experiments. Following treatment with Inhibitor3, cells displayed senescence hallmarks, including increased positivity for SA-β-gal and SenTraGor staining, reduced cell proliferation capacity, G1 phase cell cycle arrest, and upregulated protein levels of p16 and p21. Concurrently, this treatment triggered SASP expression. By contrast, treatment with E3330 significantly inhibited the NF-κB/C/EBPβ pathway and suppressed SASP without affecting cellular senescence (Figure. 4A-E). In order to more accurately elucidate the bifunctional nature of APE1 on the NF-κB/C/EBPβ signaling pathway and SASP. We separately transfected point mutation plasmids to overexpress C65S (APE1 redox function mutant) and D210 (APE1 DNA repair function mutant) in APE1-knockdown cells. Results showed that the NF-κB/C/EBPβ pathway and SASP were significantly attenuated in cells expressing APE1 C65S(Figure. 4F-G). Collectively, these findings confirm that loss of APE1 redox function inhibits the NF-κB/C/EBPβ pathway and SASP, whereas impairment of its DNA damage repair activity drives cellular senescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibiting the redox function of APE1 attenuated radiation-induced SASP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAPE1 has been reported to increase in liver tissue during radiotherapy and can serve as a biomarker to predict the occurrence of radioactive liver injury (Yue R and Li Z 2024). Our above results strongly suggest that targeting APE1, particularly its redox function, holds significant potential for reducing SASP during radiotherapy. To validate this hypothesis, we exposed HFFs to 10 Gy X-ray irradiation to induce cellular senescence. 10 days post-irradiation, we assessed cellular senescence and SASP expression. Ionizing radiation led to abundant SA-β-gal and SenTraGor positive staining, demonstrating efficient senescence induction. Western blot analysis confirmed significant upregulation of p16 and p21. Notably, both APE1-knockdown and control cells exhibited enhanced senescence after irradiation, with no significant difference between groups. Although APE1 did not increase after radiation, APE1 depletion impaired radiation-induced activation of the NF-κB/C/EBPβ signaling pathway and reduced SASP levels (Figure. 5A-C). We previously confirmed that APE1 regulates the NF-κB/C/EBPβ pathway and SASP via its redox function. In order to explore whether E3330 also has the potential to reduce the SASP induced by radiotherapy. Cells were exposed to 10 Gy X-ray irradiation and cultured for 7 days to allow senescence induction. Subsequently, they were treated with 20 μM E3330 for 72 hours. We found that E3330 treatment blocked radiation-induced upregulation of the NF-κB/C/EBPβ signaling axis and SASP without affecting cellular senescence (Figure. 5D-F). In this part, Our results demonstrate that both knocking down APE1 and using its redox function inhibitor E3330 can reduce the radiation-induced SASP via the NF-κB/C/EBPβ pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe SASP secreted by senescent cells is detrimental to the tissue microenvironment, accelerating tumorigenesis and promoting the progression of aging-related diseases (Li X et al. 2023; Wang X 2024). Therefore, elucidating the regulatory mechanisms of SASP and developing potential therapies to alleviate SASP-mediated pathological processes are of critical importance. To investigate the expression and regulatory mechanisms of the SASP in APE1-deficient senescent cells, we isolated primary fibroblasts from foreskin tissue and successfully established an APE1-deficient senescence model. Through RNA-seq and RT-qPCR, we identified a distinct phenotype of SASP downregulation in APE1-deficient senescent cells, which contrasted with our established classical replication and radiation induced senescence models. The bifunctional characteristic of APE1 plays distinct roles in regulating cellular senescence and the SASP. Specifically, the impairment of its DNA repair function accelerates cellular senescence and SASP, while the loss of its redox activity suppresses SASP expression. Mechanistic investigations further revealed that the down-regulation of SASP is mainly mediated by APE1's redox function via the inhibition of the NF-κB/C/EBPβ signaling pathways. Additionally, we found that both targeting APE1 and using its redox activity inhibitor E3330 could reduce radiation-induced SASP.\u003c/p\u003e\n\u003cp\u003eDue to inherent variations in organs, tissues, cell types, and senescence-inducing stimuli, distinct senescence models exhibit characteristic differences (Cohn RL and Gasek NS 2023). The SENNET Consortium has confirmed that the SASP is the most prevalent senescence marker, observed in 80% of senescent cell populations across diverse tissues. However, their findings also emphasized that cellular senescence should not be defined by a single marker, calling for multimethodological approaches to characterize this state (Suryadevara V et al. 2024). To address this, we comprehensively characterized the senescence state of APE1-deficient cells using multi approaches. Our analyses revealed that APE1 deficiency induced canonical senescence hallmarks: reduced proliferative capacity, cell cycle arrest, enlarged morphology, upregulated expression of senescence markers p21 and p16, and downregulated LamB1 expression. Additionally, increased positive staining for SA-β-gal and SenTraGor was observed, consistent with phenotypes in other senescence models. Notably, despite these shared features, the APE1-deficiency model exhibited a unique divergence from classical paradigms: SASP factor expression was significantly downregulated, in stark contrast to the upregulated pro-inflammatory SASP typically observed in most senescence models.\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanism by which APE1 regulates the SASP, we queried upstream TFs of key SASP factors (including IL-6, IL-8, TNF-α, CCL2, etc.) and downstream TFs of APE1 using the TRRUST databases. This analysis revealed that APE1 mightily regulates SASP through modulation of two transcription factors: NF-κBand C/EBPβ. Functional assays demonstrated that APE1 knockdown significantly suppressed protein expression of both NF-κB and C/EBPβ, whereas APE1 overexpression specifically enhanced their expression. These results indicate that APE1 is involved in the NF-κB/C/EBPβ signaling pathway. Further, the time course experiment showed that in APE1-deficient senescent cells, sustained inhibition of NF-κB/C/EBPβ pathway led to a marked reduction of SASP factors. Mechanistic validation using APE1 bifunctional inhibitors and point-mutated plasmids confirmed that APE1 loss-mediated SASP downregulation depends on its redox activity to modulate the NF-κB/C/EBPβ pathway. Collectively, these results uncover a novel mechanism whereby APE1 regulates the NF-κB/C/EBPβ pathway through its redox function, thereby specifically suppressing SASP.\u003c/p\u003e\n\u003cp\u003eTo explore whether targeting APE1 serves as a senomorphic strategy to alleviate the SASP during aging process, we conducted preliminary investigations in a classical radiation-induced senescence model. Our results showed that APE1 knockdown and E3330 treatment both inhibited the NF-κB/C/EBPβ pathway and reduced radiation-induced SASP expression. The redox function of APE1 in inflammation regulation has been extensively studied. Knocking down APE1 can reduce the expression of the myocardial inflammatory factors IL-6, TNF-α and IL-1β induced by Ang II, thereby reducing the occurrence of myocardial infarction and fibrosis (Lu F 2024). Similarly, in APE1 knockout mice and mice treated with E3330, NF-κB activity and the expression of its downstream target CXCL1 are suppressed. This suppression reduces neutrophil infiltration and decreases IL-1β expression in injured tissues, which exerts protective effects against DSS-induced colitis (Chen Q and Chen T 2024). The anti-inflammatory effect of E3330 has been verified in a mouse model of alcoholic hepatitis. Treatment with E3330 improves alcoholic liver injury and hepatitis in mice, and the expression levels of TNF-α, AST and ALT in the plasma of mice are reduced after E3330 treatment (Nagakawa J 1992). Our results are consistent with these previous studies, indicating that the redox function of APE1 indeed plays a critical role in SASP.\u003c/p\u003e\n\u003cp\u003eThere are some limitations in our study. Although we demonstrated that targeting APE1 can suppress the SASP in a radiation induced senescence model, its regulatory effects on SASP in other senescence models, such as those induced by oncogenes, chemotherapy, or nutrient deficiency, remain unclear. Additionally, as our findings predominantly stem from in vitro experiments, the in vivo effects of APE1 on SASP, along with determining whether this mechanism is broadly applicable or context-dependent, necessitate further investigation. These remaining issues will also be further elucidated in our future research. In summary, APE1 deletion in fibroblasts elicits a distinct senescent phenotype with suppressed SASP. This phenotype arises from APE1's dual functions: DNA repair impairment drives cellular senescence, while blocking its redox activity inhibits NF-κB/C/EBPβ and SASP expression. The most significant highlight of our study is the discovery that the APE1 redox activity inhibitor E3330 has the potential to serve as a senomorphics therapy for improving age-related diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll human foreskin tissue experimental procedures were reviewed and approved by the ethics committee of Army Medical Center of PLA. We certify that the study was performed in accordance with the 1964 declaration of HELSINKI and later amendments. Written informed consent was obtained from all the participants prior to the enrollment of his study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved the final manuscript and the submission to this journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the National Natural Science Foundation of China (No. 82002444 to X.Y.) and (No. 82173203 to JW.Z.), the Natural Science Foundation of Chongqing Municipality (cstc2021jcyj-msxmX0446 to J.Z.), and the Talent Innovation Ability Training Plan of the Army Medical Center of PLA [ No. ZXYZZKY07 to MX.L].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Mx L, XY; Data Curation:YD; Formal Analysis: Xj K, HX, XG , YP; Funding Acquisition: XY, Mx L, JW Z; Writing - Original Draft Preparation: YD, Jw Z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Zhengwei Hu from Urology department of Army Medical Center of PLA for providing foreskin tissue;GuiHong Yang from dermatology department of Army Medical Center of PLA for experimental technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eL\u0026oacute;pez-Ot\u0026iacute;n C, Blasco MA, Partridge L, Serrano M, Kroemer G (2023) Hallmarks of aging: An expanding universe. Cell 186(2):243-278.\u003c/li\u003e\n\u003cli\u003eCalcinotto A, Kohli J, Zagato E, Pellegrini L, Demaria M, Alimonti A (2019) Cellular Senescence: Aging, Cancer, and Injury. Physiol Rev 99(2):1047-1078.\u003c/li\u003e\n\u003cli\u003eYin K, Patten D, Gough (2022) Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance. Genes Dev 36(9-10):533-549.\u003c/li\u003e\n\u003cli\u003eRitschka B, Storer M, Mas A, Heinzmann F, Ortells MC (2017) The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration. Genes Dev 31(2):172-183.\u003c/li\u003e\n\u003cli\u003eZhao S, Qiao Z, Meng B, Chen S, Liu H (2024) Modulation of fracture healing by senescence-associated secretory phenotype (SASP): a narrative review of the current literature. Eur J Med Res 29(1):38.\u003c/li\u003e\n\u003cli\u003eZhang L, Pitcher LE, Yousefzadeh MJ, Niedernhofer LJ, Robbins PD, Zhu Y (2022) Cellular senescence: a key therapeutic target in aging and diseases. J Clin Invest 132(15):e158450.\u003c/li\u003e\n\u003cli\u003eMehdizadeh M, Aguilar M, Thorin E, Ferbeyre G, Nattel S (2022) The role of cellular senescence in cardiac disease: basic biology and clinical relevance. Nat Rev Cardiol 19(4):250-264.\u003c/li\u003e\n\u003cli\u003eZhang L, Pitcher LE, Prahalad V, Niedernhofer LJ, Robbins PD (2021) Recent advances in the discovery of senolytics. Mech Ageing Dev 200:111587.\u003c/li\u003e\n\u003cli\u003eBirch J, Gil J(2021) Senescence and the SASP: many therapeutic avenues. Genes Dev 34(23-24):1565-1576.\u003c/li\u003e\n\u003cli\u003eZhang L, Pitcher LE, Prahalad V, Niedernhofer LJ, Robbins P (2023) Targeting cellular senescence with senotherapeutics: senolytics and senomorphics. FEBS J 290(5):1362-1383.\u003c/li\u003e\n\u003cli\u003eZheng L, He S, Wang H, Li J, Liu Y, Liu S (2024) Targeting Cellular Senescence in Aging and Age-Related Diseases: Challenges, Considerations, and the Emerging Role of Senolytic and Senomorphic Therapies. Aging Dis 15(6):2554-2594.\u003c/li\u003e\n\u003cli\u003eOliveira TT, Coutinho LG, de Oliveira LOA (2022) APE1/Ref-1 Role in Inflammation and Immune Response. Front Immunol 13:793096.\u003c/li\u003e\n\u003cli\u003eCaston RA, Gampala S, Armstrong L, Messmann RA, Fishel ML, Kelley MR (2021) The multifunctional APE1 DNA repair-redox signaling protein as a drug target in human disease. Drug Discov Today 26(1):218-228.\u003c/li\u003e\n\u003cli\u003eZhao H, Richardson C, Marriott I, Yang IH, Yan S (2024) APE1 is a master regulator of the ATR-/ATM-mediated DNA damage response. DNA Repair (Amst) 144:103776.\u003c/li\u003e\n\u003cli\u003eChu A, Liu X, Liu S, Li M, Song R, Gan L, Wang Y, Liu Z, Sun C (2025) RNA-seq analysis reveals key genes associated with downregulation of APE1 in esophageal squamous cell carcinoma. Front Genet 16:1549371.\u003c/li\u003e\n\u003cli\u003eZhao CR, Yang FF, Cui Q, Wang D (2021) Vitexin inhibits APEX1 to counteract the flow-induced endothelial inflammation. Proc Natl Acad Sci U S A 118(48):e2115158118.\u003c/li\u003e\n\u003cli\u003eTang Z, Wang Y, Wan Y, Xie Y, Li S, Tao D, Wang C, Wu YZ, Sui JD (2021) Apurinic/apyrimidinic endonuclease 1/reduction-oxidation effector factor-1 (APE1) regulates the expression of NLR family pyrin domain containing 3 (NLRP3) inflammasome through modulating transcription factor NF-\u0026kappa;B and promoting the secretion of inflammatory mediators in macrophages. Ann Transl Med 9(2):145.\u003c/li\u003e\n\u003cli\u003eLi M, Yang X,Wang D, Wilson DM 3rd (2018) APE1 deficiency promotes cellular senescence and premature aging features. Nucleic Acids Res 46(11):5664-5677.\u003c/li\u003e\n\u003cli\u003eNadalutti CA, Wilson SH (2020) Using Human Primary Foreskin Fibroblasts to Study Cellular Damage and Mitochondrial Dysfunction. Curr Protoc Toxicol 86(1):e99.\u003c/li\u003e\n\u003cli\u003eMarthandan S, Menzel U, Diekmann S (2016) Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence. Biol Res 49(1):34.\u003c/li\u003e\n\u003cli\u003eWang B, Han J, Elisseeff JH, Demaria M (2024) The senescence-associated secretory phenotype and its physiological and pathological implications. Nat Rev Mol Cell Biol 25(12):958-978.\u003c/li\u003e\n\u003cli\u003eSalotti J, Johnson PF (2019) Regulation of senescence and the SASP by the transcription factor C/EBP\u0026beta;. Exp Gerontol 128:110752.\u003c/li\u003e\n\u003cli\u003eYue R, Li Z, Zhang X, Song G (2024) Imaging-guided companion diagnostics in radiotherapy by monitoring APE1 activity with afterglow and MRI imaging. Nat Commun 15(1):6349.\u003c/li\u003e\n\u003cli\u003eLi X, Li C, Zhang W, Wang Y, Qian P, Huang H (2023) Inflammation and aging: signaling pathways and intervention therapies. Signal Transduct Target Ther 8(1):239.\u003c/li\u003e\n\u003cli\u003eWang X, Fukumoto T, Noma KI (2024) Therapeutic strategies targeting cellular senescence for cancer and other diseases. J Biochem 175(5):525-537.\u003c/li\u003e\n\u003cli\u003eCohn RL, Gasek NS, Kuchel GA, Xu M (2023) The heterogeneity of cellular senescence: insights at the single-cell level. Trends Cell Biol 33(1):9-17.\u003c/li\u003e\n\u003cli\u003eSuryadevara V, Hudgins AD, Dou Z, Neretti N (2024) SenNet recommendations for detecting senescent cells in different tissues. Nat Rev Mol Cell Biol.\u003c/li\u003e\n\u003cli\u003eLu F, Ding L, Qiao Y (2024) APEX1 Knockdown Alleviates Inflammation and Fibrosis in Myocardial Infarction Through Promoting ZCCHC9 Expression and Blocking the p38 MAPK Signaling. Biochem Genet.\u003c/li\u003e\n\u003cli\u003eChen Q, Chen T, Wang D, Li MX. APEX1 in intestinal epithelium triggers neutrophil infiltration and intestinal barrier damage in ulcerative colitis. Free Radic Biol Med. 2024 Nov 20;225:359-373.\u003c/li\u003e\n\u003cli\u003eNagakawa J, Yamatsu I (1992) Protective effects of (2E)-3- [5 - (2,3-dimethoxy-6- methyl -1, 4-benzoquinoyl)] -2-nonyl -2- propenoic acid on endotoxin-mediated hepatitis in mice. J Pharmacol Exp Ther. 262(1):145-50.\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":"APE1, SASP, NF-κB/C/EBPβ pathway, Radiotherapy","lastPublishedDoi":"10.21203/rs.3.rs-6841207/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6841207/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cellular senescence, triggered by multiple stressors, was characterized by senescence-associated secretory phenotype (SASP) that drive age-related pathologies. Apurinic apyrimidinic endonuclease 1 (APE1), a bifunctional enzyme with redox and DNA repair activities, regulates inflammation and senescence, but its role in SASP expression remains unclear. This study aimed to elucidate how APE1 regulates SASP and to identify new target for senomorphic therapy. APE1-deficient senescence models were established in human fibroblasts, and senescence markers (SA-β-gal, SenTraGor, p16/p21) and SASP factors (IL-6, IL-8, TNF-α, CCL5) were assessed via RT-qPCR, RNA-seq, and Western blot. APE1 inhibitors and point mutation plasmids were used to further investigate the contributions of the dual functions of APE1 in SASP modulation. Contrary to classical senescence models, APE1 deficiency induced premature senescence but paradoxically suppressed SASP expression. Transcriptomic profiling revealed that APE1 knockdown attenuated cytokine signaling and NF-κB pathway activation, accompanied by downregulation of key SASP factors (IL-6, IL-8, CCL5, TNF-α, IFN-β). Mechanistically, inhibiting the redox activity of APE1 resulted in the inactivation of NF-κB/C/EBPβ signaling and suppression of SASP expression, while the impaired DNA repair activity of APE1 triggered cellular senescence. Furthermore, inhibiting the redox function of APE1 via E3330 attenuated radiation-induced SASP. In conclusion, APE1 governs SASP expression through NF-κB/C/EBPβ signaling, highlighting its dual role in senescence and SASP regulation. Targeting the redox function of APE1 represents a senomorphic strategy to alleviate SASP-related aging, and provides therapeutic potential for age-related and radiotherapy-associated conditions.","manuscriptTitle":"Redox Activity of APE1 Mediates SASP Suppression in Senescent Fibroblasts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 18:02:48","doi":"10.21203/rs.3.rs-6841207/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":"0e9be7f9-0403-4f8d-bafe-e8c26c289579","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-12T01:53:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 18:02:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6841207","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6841207","identity":"rs-6841207","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}