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. 2026 Mar 1;15:11. doi: 10.1186/s13619-026-00285-x

ACSS2-mediated lysine crotonylation attenuates senescence and enhances the therapeutic efficacy of adipose-derived stem cells in inflammatory bowel disease

Ming Yuan 1,2,3,#, Senmao Li 4,#, Shaopeng Chen 1,2,3,#, Minghui Zhu 5,#, Runfeng Yu 4, Junfeng Huang 4, Guanzhan Liang 1,2,3, Chi Zhang 6, Xiaowen He 1,2,3,✉, Ping Lan 1,2,3,7,✉, Xianrui Wu 4,✉
PMCID: PMC12950121  PMID: 41764109

Abstract

Background

Inflammatory bowel disease (IBD) remains a significant clinical challenge with limited curative options. Adipose-derived mesenchymal stem cells (ADSCs) hold therapeutic promise, but their anti-inflammatory efficacy is often compromised by cellular senescence. This study investigates the role of lysine crotonylation (Kcr) in ADSCs senescence and explores its therapeutic potential.

Methods

We analyzed Pan-Kcr levels in senescent ADSCs and evaluated the effects of sodium crotonate (NaCr), a crotonyl-CoA precursor, on senescence, proliferation, and anti-inflammatory function. A murine colitis model was used to assess therapeutic efficacy. Molecular mechanisms focusing on ACSS2-mediated Kcr regulation and H3K9 crotonylation (H3K9cr) at the ACSS2 promoter.

Results

Senescent ADSCs exhibited a marked decline in Pan-Kcr levels. NaCr treatment ameliorated senescence, enhanced proliferation, and improved anti-inflammatory capacity. ACSS2, a key regulator of Kcr, was downregulated in senescent ADSCs. Moreover, the anti-senescence effect of NaCr depended on ACSS2-mediated crotonylation. NaCr promoted H3K9cr modification at the ACSS2 promoter, forming a positive feedback loop that elevated Kcr levels. Mechanistically, ACSS2-mediated Kcr suppressed the NF-κB pathway to delay ADSCs senescence.

Conclusion

Our findings reveal an epigenetic pathway (ACSS2-Kcr-H3K9cr) regulating ADSCs senescence and propose Kcr modulation as a novel strategy to enhance ADSC-based therapy for IBD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13619-026-00285-x.

Keywords: Crotonylation, ACSS2, Cellular senescence, Adipose-derived stem cells, Inflammatory bowel disease

Background

Inflammatory bowel disease (IBD), which encompasses conditions such as Crohn’s disease and ulcerative colitis, manifests as persistent and recurrent intestinal inflammation (Chang 2020; Ng et al. 2020). Globally, IBD has shown an upward trend in both prevalence and incidence, becoming a major clinical and socioeconomic concern (Kaplan and Windsor 2021; Xu et al. 2023; Yu et al. 2024). Although the current treatments for IBD are constantly evolving, including novel small-molecule drugs and biological agents, there remains an absence of definitive therapeutic solutions (Baumgart and Le Berre 2021; Moutsoglou et al. 2025; Sun et al. 2026). Therefore, developing new therapeutic strategies for IBD is urgent.

Given the anti-inflammatory properties and biological safety, mesenchymal stem cells (MSCs) have been recognized as a potential therapy for IBD (Li et al. 2024b; Liu et al. 2020; Wu et al. 2023; Zhou et al. 2020), with numerous clinical trials underway (Garcia-Olmo et al. 2022; Hadizadeh et al. 2024; Panes et al. 2016). Recently, adipose-derived mesenchymal stem cells (ADSCs) have been identified as a highly advantageous MSC source due to their tissue accessibility, stronger anti-inflammation activity, and greater therapeutic efficacy in colitis models (Buscail et al. 2021; Dozois et al. 2023; Liang et al. 2023). Nevertheless, ADSCs’ therapeutic capacity is frequently constrained by cellular senescence occurring during in vitro expansion (Foti et al. 2024; Mullen et al. 2023; Zhang et al. 2023b), which compromises their anti-inflammatory functions in IBD. Therefore, understanding the mechanisms underlying ADSCs senescence and developing strategies to mitigate senescence process are significant.

Lysine crotonylation (Kcr) represents a new type of post-translational modification (PTM) that participates in diverse biological functions, notably in the regulation of cellular senescence (Dai et al. 2021; Han et al. 2024; Tan et al. 2011; Yang et al. 2023; Yu et al. 2021). Kcr is known to play roles in stimulating proliferation and directing differentiation in embryonic stem cells (Fang et al. 2021; Zhang et al. 2023a), periodontal ligament stem cells (Han et al. 2024), neural stem cells (Dai et al. 2021) and so on. However, its role in stem cell senescence, especially in ADSCs, remains largely unexplored.

This research explored how Kcr modification is connected to senescence in ADSCs. We observed a decline in Pan-Kcr levels and intracellular crotonyl-CoA during ADSCs senescence. Treatment with sodium crotonate (NaCr), a crotonyl-CoA precursor, ameliorated senescence and improved proliferation capacity. NaCr-treated ADSCs exhibited enhanced anti-inflammatory effects in a murine colitis model. Furthermore, ACSS2, an enzyme within the acyl-CoA short-chain synthetase family, was identified as an essential regulator of Kcr in ADSCs, with its expression reduced during senescence. We confirmed that the anti-senescence effects of NaCr were dependent on ACSS2-mediated Kcr. Moreover, NaCr increases H3K9 crotonylation (H3K9cr) at the ACSS2 promoter, thereby enhancing its transcription and forming a positive feedback loop that elevates Kcr levels. Mechanistically, ACSS2-mediated Kcr inhibits NF-κB pathway to alleviate ADSCs senescence. Overall, our findings reveal a novel epigenetic mechanism regulating ADSCs senescence and propose an alternative strategy aimed at boosting the therapeutic performance of ADSCs for IBD treatment by modulating lysine crotonylation.

Results

Cellular senescence is associated with a decline in Pan-Kcr levels in ADSCs

To investigate whether crotonylation modification plays a role in the senescence of ADSCs, ADSCs at different passages were analyzed. Data indicated a notable increase in senescence marker levels in ADSCs at passage seven (P7) compared with passage three (P3; Fig. 1A). As senescence progressed, the Pan-Kcr level in ADSCs gradually decreased (Fig. 1B), along with a reduction in intracellular crotonyl-CoA levels (Fig. 1C), suggesting a significant attenuation of crotonylation during cellular senescence. Based on these findings, we hypothesized that improving lysine crotonylation level could alleviate ADSCs senescence. Given that crotonate has been shown to increase crotonyl-CoA and Pan-Kcr levels (Cai et al. 2022; Liao et al. 2023; Yuan et al. 2023), we treated both P3 and P7 ADSCs with sodium crotonate (NaCr) and assessed senescence levels and proliferation capacity. NaCr treatment significantly reduced senescence markers (Fig. 1D), SA-β-gal staining level (Fig. 1E) and the senescence-associated secretory phenotype (SASP) in ADSCs (Fig. 1F). Moreover, NaCr treatment markedly enhanced the proliferation capacity of ADSCs (Fig. 1G). These results indicate that NaCr treatment can delay the senescence process in ADSCs.

Fig. 1.

Fig. 1

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Reduced Pan-Kcr in senescent ADSCs. A Immunoblotting of indicated senescence markers in P3 and P7 ADSCs. B Immunoblotting of Pan-Kcr levels in ADSCs at different passages. C The intracellular crotonyl-CoA levels in ADSCs at different passages. D Immunoblotting of indicated senescence markers in P3 and P7 ADSCs with or without NaCr supplement. E The SA-β-gal staining level in P3 and P7 ADSCs with or without NaCr supplement. F The mRNA expression of SASPs in P3 and P7 ADSCs with or without NaCr supplement. G The growth curves of P3 and P7 ADSCs with or without NaCr supplement. The concentration of NaCr used is 10 mmol/L. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, determined by two-tailed Student’s t-test (A), one-way ANOVA with Bonferroni’s post hoc test (B-F) and repeated measures ANOVA with Bonferroni’s post hoc test (G)

Pre-treated with NaCr enhances therapeutic efficacy of ADSCs in murine colitis model

To determine the in vivo anti-inflammatory capacity of NaCr-treated ADSCs, a DSS-triggered colitis mouse model was generated, and ADSCs pre-treated with NaCr were administered. The results demonstrated that, compared with control ADSCs, NaCr-treated ADSCs significantly alleviated intestinal inflammation, as evidenced through reduced weight loss (Fig. 2A), increased colon length, lower disease activity index (DAI), and improved histopathological scores (Fig. 2B-F). These findings indicate that NaCr pre-treatment can enhance the anti-colitic efficacy of ADSCs in vivo.

Fig. 2.

Fig. 2

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Pre-treated with NaCr enhances therapeutic efficacy of ADSCs in murine colitis model. A Body weight trajectory of mice across the various experimental cohorts. B, C Representative colon photographs accompanied by a comparative analysis of colon length among groups. D The DAI scores obtained from the specified experimental categories. E, F Quantitative histological scoring alongside typical H&E-stained colon tissue images from every treatment group. Scale bar = 50 μm. ADSCs were pre-treated with 10 mmol/L NaCr for 48 h before administration. Data are expressed as mean ± SD. n.s. (not significant, p > 0.05), **p < 0.01, ***p < 0.001, determined by repeated measures ANOVA with Bonferroni’s post hoc test (A), one-way ANOVA with Bonferroni’s post hoc test (C, D, E)

ACSS2 is downregulated in senescent ADSCs and its overexpression alleviates senescence

To explore the mechanism underlying the decline in Pan-Kcr levels in senescent ADSCs, we performed RNA-seq on P3 and P7 ADSCs and examined key regulatory factors of Kcr homeostasis, namely crotonyl-transferases (writers), decrotonylases (erasers), and enzymes for crotonyl-CoA synthesis (Liu et al. 2017; Sabari et al. 2015; Wei et al. 2017). The results revealed a significant downregulation of ACSS2 expression in senescent ADSCs (Fig. 3A). We further confirmed the reduction of ACSS2 in senescent ADSCs using qPCR and immunoblotting (Fig. 3B-C). ACSS2 functions as an essential enzyme in crotonyl-CoA biosynthesis and plays a pivotal role in regulating Kcr modifications across various biological pathways. We therefore hypothesized that modulating ACSS2 expression could influence the senescence process in ADSCs. ADSCs with ACSS2 stable overexpression were constructed (Fig. 3D-E), showing that ACSS2 overexpression increased Pan-Kcr levels in ADSCs. ACSS2 overexpression significantly diminished senescence markers (Fig. 3E), SA-β-gal staining level (Fig. 3F) and SASP expression (Fig. 3G), and enhanced cell proliferation capacity (Fig. 3H). These results suggest that senescent ADSCs downregulate ACSS2, thereby impairing crotonylation modification levels. Overexpression of ACSS2 can delay ADSCs senescence and promote proliferation.

Fig. 3.

Fig. 3

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ACSS2 is downregulated in senescent ADSCs and its overexpression alleviates senescence. A Heatmap illustrating gene expression patterns associated with crotonylation. Red star denotes genes markedly reduced in P7 ADSCs (adj. P value < 0.01 and Log2Fold Change < −0.5). B The ACSS2 mRNA level in ADSCs at different passages. C The protein level of ACSS2 in ADSCs at different passages. D The ACSS2 mRNA level in ADSCs with or without ACSS2 overexpression. E Immunoblotting of indicated senescence markers and Pan-Kcr levels in ADSCs with or without ACSS2 overexpression. F The SA-β-gal staining level in ADSCs with or without ACSS2 overexpression. G The mRNA expression of SASPs in ADSCs with or without ACSS2 overexpression. H The growth curves of ADSCs with or without ACSS2 overexpression. Values are mean ± SD. **p < 0.01, ***p < 0.001, determined by one-way ANOVA with Bonferroni’s post hoc test (B, C), two-tailed Student’s t-test (D-G), and repeated measures ANOVA with Bonferroni’s post hoc test (H)

The anti-senescence effect of NaCr is dependent on ACSS2-mediated Kcr

We next sought to determine whether the anti-senescence effect of NaCr depends on enhancing cellular Kcr level. We generated ADSCs with stable ACSS2 knockdown (Fig. 4A-B), showing that ACSS2 knockdown decreased Pan-Kcr levels in ADSCs. ACSS2 knockdown significantly increased senescence markers (Fig. 4C), SA-β-gal staining level (Fig. 4D) and SASP expression (Fig. 4E), while impairing proliferation capacity (Fig. 4F), which could not be rescued by NaCr treatment (Fig. 4C-F). Thus, the anti-senescence effect of NaCr depends on ACSS2-mediated Kcr.

Fig. 4.

Fig. 4

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The anti-senescence effect of NaCr is dependent on ACSS2-mediated Kcr. A The mRNA expression of ACSS2 in ADSCs with or without ACSS2 knockdown. B The protein level of ACSS2 and Pan-Kcr level in ADSCs with or without ACSS2 knockdown. C Immunoblotting of senescence markers in ADSCs following the designated treatment regimen. D The SA-β-gal staining level following the designated treatment regimen. E SASP mRNA levels in ADSCs following the designated treatment regimen. F Growth curve analysis of ADSCs following the designated treatment regimen. NaCr was applied at 10 mmol/L. Data are presented as mean ± SD. n.s. (not significant, p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, determined by one-way ANOVA with Bonferroni’s post hoc test (A-E) and repeated measures ANOVA with Bonferroni’s post hoc test (F)

ACSS2 knockdown abrogates the ability of NaCr to enhance the anti-colitis activity of ADSCs

Furthermore, to demonstrate whether ACSS2 knockdown would also compromise the beneficial efficacy of NaCr-pretreated ADSCs in alleviating colitis. ACSS2 knockdown or control ADSCs were pretreated with NaCr and then administered to colitis model mice. ACSS2 knockdown not only significantly attenuated the inherent anti-colitis activity of ADSCs but also abolished the additional benefit conferred by NaCr pretreatment (Fig. 5). This was reflected in more severe disease phenotypes, including increased weight loss (Fig. 5A), reduced colon length (Fig. 5B-C), higher disease activity index (Fig. 5D), and worse histopathological damage (Fig. 5E-F). Together, these results suggest that ACSS2 is required for NaCr pretreatment to enhance the therapeutic potential of ADSCs against colitis.

Fig. 5.

Fig. 5

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ACSS2 knockdown abrogates the anti-Colitis activity of ADSCs. A Body weight trajectory of mice across the various experimental cohorts. B, C Representative colon photographs accompanied by a comparative analysis of colon length among groups. D The DAI levels from indicated groups. E, F Quantitative histological scoring alongside typical H&E-stained colon tissue images from every treatment group. Scale bar = 50 μm. ADSCs were pre-treated with 10 mmol/L NaCr for 48 h before administration. Data are expressed as mean ± SD. n.s. (not significant, p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, determined by repeated measures ANOVA with Bonferroni’s post hoc test (A), one-way ANOVA with Bonferroni’s post hoc test (C, D, E)

NaCr upregulates ACSS2 expression via enhancing H3K9 crotonylation

Previous studies have suggested that NaCr not only serves as a substrate for crotonylation to enhance this modification but may also upregulate ACSS2 expression in certain cell types, forming a positive feedback loop (Jiang et al. 2018). However, whether NaCr directly upregulates ACSS2 in ADSCs remained unknown. Treatment of ADSCs with NaCr significantly increased ACSS2 levels (Fig. 6A-B).

Fig. 6.

Fig. 6

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NaCr upregulates ACSS2 expression via enhancing H3K9cr modification. A The ACSS2 mRNA level in ADSCs treated with or without NaCr. B The protein level of ACSS2 in ADSCs treated with or without NaCr. C The H3K9cr modification level in ADSCs at different passages. D IGV track display of ACSS2 derived from the specified ChIP-seq data analysis. E Results from ChIP-qPCR assessing H3K9cr presence on ACSS2 in the specified ADSC samples. F ChIP-qPCR analysis showing H3K9cr occupancy on ACSS2 in ADSCs administered with or without NaCr. The concentration of NaCr used is 10 mmol/L. Values are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, determined by two-tailed Student’s t-test (A, B) and one-way ANOVA with Bonferroni’s post hoc test (C, E, F)

Based on this, we hypothesized that NaCr treatment might promote ACSS2 gene transcription through histone Kcr. H3K9 crotonylation (H3K9cr), a Kcr modification on histone H3, is known to facilitate gene transcription (Li et al. 2024a). We found that H3K9cr levels decreased with ADSCs senescence (Fig. 6C). Subsequent ChIP-seq analysis revealed that H3K9cr modification level at the ACSS2 promoter region was significantly reduced in senescent ADSCs (Fig. 6D), which was further validated by ChIP-qPCR (Fig. 6E). Moreover, ChIP-qPCR analysis also revealed that NaCr treatment increased the enrichment of H3K9cr at the ACSS2 gene locus (Fig. 6F). These results demonstrate that NaCr upregulates ACSS2 gene expression via H3K9cr in ADSCs.

ACSS2-mediated Kcr inhibits NF-κB pathway to alleviate ADSCs senescence

The mechanism by which ACSS2 inhibits cellular senescence in ADSCs remained unclear. Given the pivotal involvement of the NF-κB pathway in modulating cellular senescence (Chen et al. 2019; Hu et al. 2022; Wang et al. 2022), we investigated whether ACSS2 affects this pathway. The results revealed that overexpression of ACSS2 suppressed the phosphorylation and nuclear translocation of p65 (Fig. 7A-B). Similarly, treatment with NaCr also inhibited p65 phosphorylation and nuclear import (Fig. 7C-D). However, in ACSS2-knockdown ADSCs, the effects of NaCr were abolished (Fig. 7C-D). These findings suggest that ACSS2-mediated Kcr attenuates ADSCs senescence by inhibiting the NF-κB pathway.

Fig. 7.

Fig. 7

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ACSS2-mediated Kcr inhibits NF-κB pathway to alleviate ADSCs senescence. A Protein expression of p65 and p-p65 in ADSCs with or without ACSS2 overexpression. B Immunoblotting results showing p65 distribution in both the nucleus and cytoplasm of ADSCs with or without ACSS2 overexpression. C Protein expression of p65 and p-p65 in ADSCs with indicated treatment. D Immunoblotting results showing p65 distribution in both the nucleus and cytoplasm of ADSCs with indicated treatment. The concentration of NaCr used is 10 mmol/L. E Schematic diagram of the ACSS2-H3K9cr axis in delaying senescence and improving the function of ADSCs. Values are mean ± SD. n.s. (not significant, p > 0.05), **p < 0.01, ***p < 0.001, determined by two-tailed Student’s t-test (A, B) and one-way ANOVA with Bonferroni’s post hoc test (C, D)

Discussion

ADSCs have been recognized as a promising candidate for IBD therapy due to their accessible tissue source and anti-inflammatory effects (Buscail et al. 2021; Panes et al. 2016). However, ADSCs often undergo senescence during in vitro expansion (Foti et al. 2024), which compromises their therapeutic efficacy in IBD treatment. Crotonylation, a newly identified post-translational modification, has roles in the senescence process of ADSCs that are still not well understood. This study revealed that the Pan-Kcr level decreased in senescent ADSCs. Enhancing Pan-Kcr levels through NaCr treatment could delay senescence, boost proliferation and anti-inflammatory capacity of ADSCs in a murine colitis model. Furthermore, NaCr’s effect on mitigating senescence depends on ACSS2-mediated crotonylation. NaCr increased H3K9cr modification at the ACSS2 promoter, forming a positive feedback loop to enhance Kcr level in ADSCs. Mechanistically, ACSS2-mediated Kcr inhibits NF-κB pathway to delay ADSCs senescence. This study reveals the important role of Kcr in ADSCs senescence and identifies it as a potential therapeutic target (Fig. 7E).

The senescence of ADSCs constitutes a major obstacle to their clinical translation. The mechanisms underlying ADSCs senescence are complex and have not been fully elucidated (Foti et al. 2024). Protein lysine crotonylation (Kcr) has been previously reported to regulate the differentiation and proliferation in multiple stem cell types, such as human embryonic stem cells (Dai et al. 2021; Fang et al. 2021; Han et al. 2024). Our research revealed a marked decline in ACSS2-mediated Kcr during ADSC senescence. Supplementation with NaCr markedly delayed the senescent phenotype of ADSCs. Furthermore, we demonstrated that ACSS2-mediated Kcr suppressed the NF-κB pathway activation, a classical pathway promoting cellular senescence. However, our study did not further explore the specific mechanism by which Kcr regulates the NF-κB pathway and future research should investigate this process in greater depth.

Numerous strategies have been documented to boost the anti-inflammatory function and survival of ADSCs through pre-treatment approaches (Hwang et al. 2020; Li et al. 2023; Li et al. 2024c; Mahajan et al. 2023; Suvakov et al. 2021). For example, preconditioning ADSCs with inflammatory cytokines can augment their anti-inflammatory efficacy (Huang et al. 2024; Li et al. 2022). Additionally, pre-culturing ADSCs under hypoxic conditions can improve anti-inflammatory functions of ADSC-derived exosomes in vivo (Zhao et al. 2023). We observed that NaCr administration could improve the anti-colitis efficacy of ADSCs in a murine model, highlighting its translational clinical potential. Pre-treatment with NaCr represents a simple yet effective strategy to precondition ADSCs, thereby overcoming senescence during ex vivo expansion in their therapeutic application. This metabolic priming strategy could be readily integrated into existing cell manufacturing protocols.

Nevertheless, this research is subject to certain limitations. Firstly, our investigation concentrated solely on the ACSS2-H3K9cr axis, and it is feasible that other histone lysine crotonylation marks may also contribute to the regulation of ADSCs senescence. Furthermore, whether NaCr exerts similar effects in other stem cell types warrants further investigation. Additionally, the safety profile and long-term effects of NaCr-preconditioned ADSCs also need to be thoroughly evaluated in future preclinical studies.

In conclusion, our work elucidates an essential link between metabolic regulation, epigenetic modification, and cellular senescence in ADSCs. By revealing the role of the ACSS2-Kcr axis, we not only provide insights into the biology of ADSCs senescence but also offer a novel and practical approach to improve the efficacy of ADSC-based therapeutic strategies for IBD.

Materials and methods

ADSCs isolation and culture

Adipose tissue specimens were collected from the greater omentum of IBD patients during surgical procedures. Samples were rinsed with phosphate-buffered saline (PBS) containing 1% streptomycin–penicillin (Gibco, USA) and then minced into small pieces. Later, samples were enzymatically digested by 0.25% collagenase type I for 30 min at 37 °C. Then DMEM (Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA) was mixed to stop digestion. After filtering out the debris, cells were plated into DMEM with 1 g/L glucose containing 1% streptomycin–penicillin and 10% FBS. After the third passage, ADSCs were authenticated by cytometry according to the previously described methods (Liang et al. 2023). The ADSCs administered to colitis model mice were at passage 4 (P4), and those used for in vitro experiments were at passage 5 (P5), unless otherwise specified for particular experiments. To prepare sodium crotonate (NaCr) solution for cell treatment, crotonate solution was mixed with sodium hydroxide solution, and the pH was adjusted to 7.

Immunoblotting

Indicated cells were washed twice with cold PBS before lysis. RIPA lysis buffer (Beyotime, China) was added to ADSCs for protein extraction. Protein samples were separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, USA). To prevent nonspecific binding, the PVDF membranes were blocked using skim milk and then incubated with the specified antibodies against p53 (CST, #2527, 1:1000), p21 (CST, #2947, 1:1000), p16 (CST, #23,200, 1:1000), β-actin (Servicebio, #GB15003, 1:3000), Pan-Kcr (PTMBIO, #PTM-501, 1:1000), ACSS2 (CST, #3658, 1:1000), H3K9cr (PTMBIO, #PTM-539, 1:1000), H3 (Proteintech, #17,168–1-AP, 1:1000), p65 (CST, #8242, 1:1000), phospho-p65 (CST, #3033, 1:1000), GAPDH (Servicebio, #GB15002, 1:3000), PCNA (CST, #13,110, 1:2000). The next day, membranes were treated with the corresponding secondary antibody and incubated for 1 h at room temperature. Subsequently, the protein bands were detected using ECL reagents.

Crotonyl-CoA detection

Ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was employed to determine the intracellular levels of crotonyl-CoA. Briefly, ADSCs at different passages were placed in liquid nitrogen for 10 min, and then added with pre-chilled methanol. Later, samples were added with chloroform and ultra-pure water, and the mixtures were maintained on ice for an additional 10 min before centrifugation to collect the supernatants. The collected samples were dried and processed for UPLC-MS/MS. Quantification was carried out by comparing the obtained signals with a crotonyl-CoA (Sigma-Aldrich, #28,007) standard calibration curve.

Senescence-associated β-galactosidase (SA-β-gal) staining

Senescence-associated β-galactosidase (SA-β-gal) activity was detected using a commercial staining kit (Beyotime, #C0602) according to the manufacturer’s instructions. Briefly, ADSCs under different treatment conditions were seeded in 6-well plates. After removal of the culture medium and washing with PBS, the cells were fixed with the fixative solution provided in the kit at room temperature for 15 min. Following fixation, the cells were incubated with the SA-β-gal staining working solution overnight at 37 °C in a humidified chamber without CO₂. The next day, SA-β-gal-positive cells were observed and counted under a light microscope.

RNA isolation and qRT-PCR

TRIzol Reagent (Invitrogen, USA) was used to isolate total RNA, which was subsequently converted into cDNA with the qPCR RT Kit (Toyobo, Japan). Quantitative real-time PCR (qRT-PCR) was carried out on the ABI QuantStudio™ 7 RealTime PCR Systems, with primer sequences detailed in Table S1. Relative mRNA expression was calculated and normalized by β-actin.

Cell viability assay

Briefly, 1000 ADSCs with different gene transfection or treatment were cultured in 96-well plates. At a specific time point, the existing medium was substituted with fresh culture medium supplemented with 10% CCK-8 reagent (Solarbio, China, #CA1210). The plates were maintained at 37 °C for 2 h, after which optical density was recorded at 450 nm.

Dextran sulfate sodium salt (DSS)-induced colitis

All experimental mice were housed at the Sixth Affiliated Hospital of Sun Yat-Sen University, with procedures authorized by the Institutional Animal Care and Use Committee (IACUC) at Sun Yat-Sen University (IACUC-2024111102). Male C57BL/6 J mice, 6–8 weeks old and weighing 20–28 g, were purchased from Gempharmatech Co. Ltd. (Guangdong, China) and kept in specific pathogen-free facilities with a 12-h light/dark schedule.

To establish experimental colitis, mice were randomly assigned to groups and treated with 3% DSS solution (MP Biomedicals, USA) in drinking water for 7 days, while the control groups received normal water. All procedures followed the ARRIVE reporting guidelines. Body weight, rectal bleeding, and stool consistency were monitored daily to calculate disease activity index (DAI) score. For ADSCs treatment, indicated cells (1×106/mouse) were injected intraperitoneally on the first day. On day 8, mice were euthanized and intestinal tissues were collected for histopathological examination. Protocols for calculating the DAI score and conducting histopathological assessments were reported in earlier studies (Zheng et al. 2019).

RNA sequencing

ADSCs at different passages were processed to extract total RNA. After being purified and fragmented, the cDNA libraries were constructed and sequenced. Then the resulting sequencing reads were mapped to reference genome using Bowtie2, and the differential gene expression was evaluated by DESeq2 packages.

Lentiviral transduction

The plasmids for short hairpin RNA (shRNA) targeted ACSS2 and ACSS2 overexpression were purchased from MiaoLingBio (Wuhan, China). The target plasmids were combined with packaging plasmids (psPAX2 and pMD2.G; Addgene) and polyethylenimine (#24765–100, Polysciences) prior to transfection into HEK293T cells. ADSCs were then exposed to lentiviral supernatant from HEK293T cultures for 2 days, followed by puromycin selection to establish stably transfected cells.

Chromatin immunoprecipitation (ChIP)-seq and ChIP-qPCR

The ChIP assay was carried out using the ChIP assay kit (#17–10085, Sigma-Aldrich, USA). ADSCs at different passages were crosslinked, quenched and sonicated for chromatin fragmentation. Then the chromatin-containing lysates were treated overnight at 4 °C with H3K9cr antibodies (PTMBIO, #PTM-539) or mouse IgG controls (Proteintech, #B900620), together with protein A/G magnetic beads. On the following day, H3K9cr-modified chromatin was eluted and purified for ChIP-seq and ChIP-qPCR analysis. For ChIP-seq, raw reads were processed using fastp, aligned to the human reference genome via Bowtie2, and subjected to PCR duplicate removal and peak identification. IGV software was used to visualize peaks within the ACSS2 locus. ChIP-qPCR results were reported as fold enrichment relative to input samples, with ACSS2 promoter primers as: Forward 5′-AGACTTCAGTGGCTCTGCAC-3′ and Reverse 5′-AGAGGGATTTGCGGTCAGTG-3′.

Statistical analysis

Data analysis was conducted using GraphPad Prism (La Jolla, USA). Results were obtained from a minimum of three independent experimental repeats and expressed as mean ± SD. Comprehensive statistical details are supplied in the relevant figure legends.

Supplementary Information

13619_2026_285_MOESM1_ESM.docx (27.5KB, docx)

Supplementary Material 1. Table S1. The primers used for qRT-PCR.

Acknowledgements

Not applicable.

Abbreviations

ACSS2

Acyl-CoA short-chain synthetase 2

ADSCs

Adipose-derived mesenchymal stem cells

ChIP-seq

Chromatin immunoprecipitation sequencing

DAI

Disease activity index

DSS

Dextran sulfate sodium salt

H3K9cr

Histone 3 lysine 9 crotonylation

IBD

Inflammatory bowel disease

Kcr

Lysine crotonylation

MSCs

Mesenchymal stem cells

NaCr

Sodium crotonate

PTM

Post-translational modification

qRT-PCR

Quantitative real-time polymerase chain reaction

SASP

Senescence-associated secretory phenotype

UPLC-MS/MS

Ultra-performance liquid chromatography coupled with tandem mass spectrometry

Authors' contributions

X.W.H., P.L., and X.R.W. contributed equally to this work as co-corresponding authors. M.Y., S.M.L., S.P.C., and M.H.Z. were co-first authors and performed most of the experiments. R.F.Y., J.F.H., G.Z.L., and C.Z. collected the clinical samples and provided technical assistance. M.Y., S.M.L., S.P.C., and M.H.Z. conceived the project, analyzed experimental results and wrote the original manuscript. X.W.H., P.L., and X.R.W. reviewed and edited the manuscript. All authors approved the submitted manuscripts.

Funding

This work is supported by the National Natural Science Foundation of China (No. 82370675), Fundamental Research Funds for the Central Universities, Sun Yat-sen University (No. 23xkjc023), Guangdong Basic and Applied Basic Research Foundation (No. 2024A1515012813) to Xianrui Wu; National Key R&D Program of China (No. 2022YFA1304000), the program of Guangdong Provincial Clinical Research Center for Digestive Diseases (2020B1111170004), the “Jie Bang Gua Shuai” Program from The Sixth Affiliated Hospital, Sun Yat-sen University (No. 2022JBGS01) to Dr. Ping Lan; Key Research and Development Program of Guangzhou (No. 2024B03J0211) to Dr. Xiaowen He. This work is supported by the National Key Clinical Discipline.

Data availability

Sequencing data have been deposited in Gene Expression Omnibus with accession codes GSE315663 (RNA-seq) and GSE315662 (ChIP-seq). Data supporting this study's results can be accessed from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Human sample collection procedures were approved by the Institutional Review Board of the Sixth Affiliated Hospital of Sun Yat-sen University (2023ZSLYEC-653). All experimental mice were housed at the Sixth Affiliated Hospital of Sun Yat-Sen University, with procedures authorized by the Institutional Animal Care and Use Committee (IACUC) at Sun Yat-Sen University (IACUC-2024111102).

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest.

Footnotes

Ming Yuan, Senmao Li, Shaopeng Chen and Minghui Zhu these authors were co-first authors.

Contributor Information

Xiaowen He, Email: hexiaow3@mail.sysu.edu.cn.

Ping Lan, Email: lanping@mail.sysu.edu.cn.

Xianrui Wu, Email: wuxianr5@mail.sysu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

13619_2026_285_MOESM1_ESM.docx (27.5KB, docx)

Supplementary Material 1. Table S1. The primers used for qRT-PCR.

Data Availability Statement

Sequencing data have been deposited in Gene Expression Omnibus with accession codes GSE315663 (RNA-seq) and GSE315662 (ChIP-seq). Data supporting this study's results can be accessed from the corresponding author upon reasonable request.

Articles from Cell Regeneration are provided here courtesy of Chinese Society for Cell Biology

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