18-β-glycyrrhetinic acid facilitates nuclear-mitochondrial communications to alleviate oxidative stress through HMGB1-cGAS-Mul1 axis in tendinopathy.

18-β-glycyrrhetinic acid facilitates nuclear-mitochondrial communications to alleviate oxidative stress through HMGB1-cGAS-Mul1 axis in tendinopathy - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. 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Learn more: PMC Disclaimer | PMC Copyright Notice J Transl Med . 2026 Apr 17;24:539. doi: 10.1186/s12967-026-08091-4 Search in PMC Search in PubMed View in NLM Catalog Add to search 18-β-glycyrrhetinic acid facilitates nuclear-mitochondrial communications to alleviate oxidative stress through HMGB1-cGAS-Mul1 axis in tendinopathy Yuan-Yuan Gao Yuan-Yuan Gao 1 Department of Orthopaedics, Jinling Hospital, Nanjing University of Chinese Medicine, Nanjing, China 2 Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Media, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Yuan-Yuan Gao 1, 2, # , Wen-Shuang Sun Wen-Shuang Sun 1 Department of Orthopaedics, Jinling Hospital, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Wen-Shuang Sun 1, # , Zi-Ying Sun Zi-Ying Sun 1 Department of Orthopaedics, Jinling Hospital, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Zi-Ying Sun 1, # , Jun-Rui Wang Jun-Rui Wang 2 Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Media, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Jun-Rui Wang 2 , Zhong-Yang Lv Zhong-Yang Lv 1 Department of Orthopaedics, Jinling Hospital, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Zhong-Yang Lv 1 , Yu-Jia Li Yu-Jia Li 3 Department of Pharmacy, Nantong Third People’s Hospital, Nantong, China Find articles by Yu-Jia Li 3 , Hao-Yuan Tian Hao-Yuan Tian 2 Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Media, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Hao-Yuan Tian 2 , Zheng-Yang Bao Zheng-Yang Bao 2 Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Media, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Zheng-Yang Bao 2 , Xin-Ran Qiu Xin-Ran Qiu 2 Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Media, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Xin-Ran Qiu 2 , Zheng Wang Zheng Wang 4 State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Drum Tower Hospital, Nanjing University, Nanjing, China Find articles by Zheng Wang 4 , Shi-Zhong Zheng Shi-Zhong Zheng 2 Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Media, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Shi-Zhong Zheng 2 , Jiang-Juan Shao Jiang-Juan Shao 2 Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Media, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Jiang-Juan Shao 2, ✉ , Zi-Li Zhang Zi-Li Zhang 2 Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Media, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Zi-Li Zhang 2, ✉ , Jia Meng Jia Meng 1 Department of Orthopaedics, Jinling Hospital, Nanjing University of Chinese Medicine, Nanjing, China Find articles by Jia Meng 1, ✉ Author information Article notes Copyright and License information 1 Department of Orthopaedics, Jinling Hospital, Nanjing University of Chinese Medicine, Nanjing, China 2 Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Media, Nanjing University of Chinese Medicine, Nanjing, China 3 Department of Pharmacy, Nantong Third People’s Hospital, Nantong, China 4 State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Drum Tower Hospital, Nanjing University, Nanjing, China ✉ Corresponding author. # Contributed equally. Received 2025 Sep 22; Accepted 2026 Mar 29; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13091248  PMID: 41998635 Abstract Background Tendinopathy is a prevalent orthopaedic condition characterized by disrupted tendon homeostasis, with oxidative stress being a key contributing mechanism. Although the natural compound 18-β-Glycyrrhetinic acid (GA) exhibits antioxidant properties and is a therapeutic candidate for tendinopathy, its precise molecular mechanism remains unclear. This study aimed to elucidate how GA alleviates tendinopathy, with a focus on its role in regulating the HMGB1-cGAS-STING axis and NLRP3 inflammasome activation in the context of oxidative stress. Methods We employed single-cell RNA sequencing (scRNA-seq) of clinical samples, proteomics of animal tissues, and comprehensive pharmacological assays to investigate the mechanisms of tendinopathy. Furthermore, the rat tendinopathy model and H 2 O 2 -induced oxidative stress model of tendon stem cells (TSCs) were used to validate the protective effects of GA. Results We found that GA significantly reduced oxidative stress and subsequent inflammation, thereby mitigating collagen disruption in rats with tendinopathy. Notably, scRNA-seq revealed that the proportion of TSCs increased significantly during tendinopathy, which were particularly susceptible to reactive oxygen species (ROS). TSCs from oxidative damage and inhibited activation of the NLRP3 inflammasome by suppressing the cGAS-STING pathway. Mechanistically, GA promoted cGAS degradation by enhancing its interaction with the mitochondrial E3 ubiquitin ligase Mul1. This effect was mediated through high-mobility group box 1 (HMGB1), as GA disrupted the HMGB1-cGAS interaction. Specifically, GA induced methylation of HMGB1 at lysine 43, a modification essential for its activity. This methylation was catalyzed by the methyltransferase DOT1L, which was upregulated and directly bound by GA. Collectively, GA alleviates tendinopathy by targeting the DOT1L-HMGB1-cGAS axis to resolve oxidative stress and inflammation. Conclusion Collectively, our findings provide new insights into how oxidative stress accelerates tendinopathy progression. Moreover, they delineate the mechanism by which GA in mitigates oxidative damage and inflammation in TSCs by inhibiting the co-localization of HMGB1 and cGAS. Overall, this study offers scientific support for further developing GA as a promising therapeutic agent for tendinopathy treatment. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12967-026-08091-4. Keywords: 18-β-Glycyrrhetinic acid, Tendinopathy, Oxidative stress, cGAS-STING, NLRP3 inflammasome, HMGB1 methylation Introduction Tendinopathy is a complex musculoskeletal disorder characterized by disordered collagen arrangement. Clinically, it presents with symptoms such as pain, stiffness, tendon hypoplasia and impaired exercise tolerance, which seriously impair the quality of life of patients [ 1 ]. The onset of tendinopathy is generally related to aging, overuse, genetic factors and medication use [ 2 ]. Despite a variety of treatment options, including surgery, extracorporeal shock wave therapy and pharmacological interventions, the therapeutic outcomes remain unsatisfactory due to the hypocellularity and poor vascularity of the tendon tissues [ 3 , 4 ]. In this context, tendon stem cells (TSCs) have emerged as a key effector population. They are resident cell population with multi-differentiation potential, first isolated from mouse and human tendons [ 5 ]. A growing number of studies have demonstrated that TSCs play key roles in tendon homeostasis and repair owing to their self-renewal and tenogenic differentiation capacity [ 6 , 7 ]. Thus, understanding how external stimuli or therapeutic agents regulate TSC function represents a strategic approach to decoding tendinopathy mechanisms and identifying potential treatment targets. cGAS acts as a pattern recognition receptor (PRR) in response to DAMPs. It can bind to the DNA and generates 2′3′cyclic GMP-AMP (cGAMP) from ATP and GTP upon the release of double-stranded DNA (dsDNA) into the cytoplasm [ 8 ]. Our previous study revealed that oxidative stress induces mitochondrial DNA (mtDNA) release into the cytoplasm of TSCs, a key event that activates the cGAS-STING pathway [ 9 ]. This pathway, alongside the NLRP3 inflammasome, forms a critical axis linking oxidative stress to sustained inflammation [ 10 ]. Consequently, targeting the cGAS-STING-NLRP3 axis represents a promising therapeutic strategy. 18-β-Glycyrrhetinic acid (GA) is the major bioactive compound derived from licorice and possesses numerous biological properties, including anticancer [ 11 ], antioxidative [ 12 ], anti-inflammatory and hepatoprotective effects [ 13 ]. This specific property provides a strong rationale for investigating GA’s potential mechanism of action in this disease context, although how it alleviates oxidative damage and inflammatory responses in TSCs remains unclear. We initially confirmed that GA alleviates tendinopathy phenotypes and inhibits cGAS-STING-NLRP3 activation, in part by promoting cGAS ubiquitination and degradation. However, a critical question remained: how does GA achieve this specific inhibition? Cellular thermal shift assays indicated that GA does not directly bind to cGAS, ruling out a straightforward interaction. This prompted us to search for an upstream mediator. HMGB1 emerged as a prime candidate. It is a key damage-associated molecular pattern (DAMP) protein implicated in tendinopathy progression [ 14 ], capable of forming complexes with mtDNA and cGAS to potentiate pathway activation. Notably, GA is a recognized pharmacological inhibitor of HMGB1 [ 15 ]. We therefore hypothesized that GA exerts its effects by targeting HMGB1 to indirectly suppress the cGAS-STING axis. In the present study, we elucidate the mechanism by which GA alleviates tendinopathy, with a focus on the HMGB1-cGAS–NLRP3 axis. To establish a coherent mechanism linking GA to pathway inhibition, we followed a sequential investigative strategy. Firstly, we validated the therapeutic efficacy of GA against tendinopathy and its antioxidative and anti-inflammatory effects. We then determined that its protection is mediated through suppressing the cGAS-STING-NLRP3 axis and promoting cGAS ubiquitination. Since GA does not directly bind cGAS, we investigated the upstream regulator HMGB1, testing the hypothesis that GA disrupts the HMGB1-cGAS interaction by promoting HMGB1 methylation. Finally, to complete the mechanistic circuit, we identified and validated the methyltransferase DOT1L as the direct target through which GA initiates this modification. Our findings reveal that GA ameliorates tendinopathy by promoting DOT1L-dependent methylation of HMGB1, which inhibits its interaction with cGAS and leads to its degradation. This work not only delineates a novel molecular pathway for GA but also highlights the therapeutic potential of precisely modulating the HMGB1-cGAS interface in tendinopathy. Materials and methods Clinical samples All participants enrolled in this clinical investigation received thorough preoperative briefings regarding the specimen acquisition protocols (Table S1 ), with written informed consent forms duly completed and signed. The analysed tendon tissue samples represented pathological tissue exhibiting inflammatory changes that required surgical excision during standard therapeutic procedures. Similarly, amputation specimens were exclusively obtained from nonsalvageable limbs requiring resection due to severe traumatic deformities or oncological indications, with all surgical interventions maintaining equivalent risk profiles equivalent to those of conventional operations without introducing additional patient morbidity. This research protocol was conducted in compliance with the ethical standards established by the Institutional Review Board of Nanjing Jinling Hospital (Ethical Approval No.2024DZKY-035-01). Single cell sequencing and analysis Tendon tissues from clinical samples were extracted and dissociated into single cells. Following lysis for RNA extraction, cDNA was synthesized by reverse transcription using microbeads labeled with unique barcodes. Sequencing libraries were then prepared through amplification and subjected to high-throughput sequencing. The sequencing depth was approximately 100 Gb per sample. Cell quality control was performed by excluding cells with mitochondrial gene content exceeding 10%, as well as cells with feature counts below 200 or above 5000. Potential doublets were identified using DoubletFinder 2.0.6 removed during data processing. Bioinformatics analysis, including functional enrichment analysis performed with the clusterProfiler 4.14.6, was subsequently carried out on the filtered dataset. The single-cell RNA sequencing experiment and initial data processing were conducted by Beijing Xunyin Biotechnology. Animal experiments Male Wistar rats (4 weeks old, 90–110 g) were obtained from the Animal Experimental Center, Nanjing University of Chinese Medicine and were randomly divided into 8 groups of 5 rats each after a 1-week acclimatization period. The following groups were identified: control group, ciprofloxacin group, GA (25 mg/kg) group, GA (50 mg/kg) group, GA (100 mg/kg) group, Lip-VA-control-Plasmid + GA group, Lip-VA-cGAS-Plasmid + GA group and positive control group respectively. Ciprofloxacin hydrochloride (Shanghai Yuanye Bio-Technology; # B24370 ) was used as an intragastric dose of 800 mg/kg once daily for two weeks to induce tendinopathy in a rat model. The plasmid was constructed and aseptically administered via tail vein injection to Wistar rats. Prior to injection, the animals were acclimatized to a temperature-controlled environment (22 ± 1 °C) and briefly anaesthetized with 2% isoflurane to minimize stress. The injection volume was precisely calibrated at 0.5 ml per 100 g body weight with a 27-gauge insulin syringe. Postprocedural monitoring confirmed normal locomotor activity within 10 min, indicating successful administration without acute complications. After two weeks of drug treatment, the administration group was given the corresponding concentration of GA once a day by gavage for 2 weeks. The patellar tendons and serum of the rats in each group were collected at the end of treatment. The protocols of the institutional and local animal care and use committees at Nanjing University of Chinese Medicine (Nanjing, China) were followed for all in vivo experimental procedures (Animal ethical code: 202211A011, November 7, 2022). Proteomic analysis of clinical and animal samples DIA (data independent acquisition) proteomic detection was performed on the tendons of clinical samples and rats in the healthy and injured groups. Protein was first extracted from the tissue samples, and then digested into peptides by trypsin. After the peptide segment was desalted, DIA was detected on the Orbitrap Astral mass spectrometer. Finally, all the original DIA data detected by LC‒MS/MS were imported into DIA-NN for analysis. Histological analysis The spatial distribution and ultrastructural characteristics of the collagen matrices in the tendon tissues were histologically assessed through combined H&E and Masson staining, and Sirius red staining further distinguished the different types of collagens. The expression of collagen Ⅰ and Ⅲ, together with the colocalization of HMGB1 and cGAS was assessed by immunofluorescence staining. The spatial distributions of PTGS2, cGAS, and HMGB1 in tendon matrices were determined through standardized IHC protocols. A microscope (Zeiss Axio Vert. A1, Germany), was used to obtain random photos of these stained sections. Isolation and culture of primary rat tendon stem cells The tendon stem cells (TSCs) were isolation from three-week-old SD rats. The specific procedures were as follows. First, the rats were euthanized and soaked in 75% ethanol for 10 min, then the Achilles tendon tissues were exposed and washed for 2–3 times with PBS. Finally, the Sterile 0.2% collagenase type I was used to digest the tendon tissues overnight. The next day, the cells were filtered with a 100 μm filter, and the precipitate was centrifuged to obtain P0 TSCs. TSCs were cultured in Duchenne’s Modified Eagle’s Medium (DMEM/F-12, #8122437, Gibco) supplemented with 10% FBS (Sempervirens Biotechnology, # BC-SE-FB07) and 1% penicillin/streptomycin (Solarbio, #P1400) to prevent cell contamination. To determine stem cell characteristics, cells from P2 ~ 6 were used for the experimental studies. Cell transfection The cGAS overexpression plasmid and HMGB1 mutant plasmid were constructed by Nanjing Yike Biology. HMGB1-WT, HMGB1 K43G and HMGB1 K112G were confirmed by DNA sequencing. We generated the transfection solution by combining the overexpression plasmid, Lip3000, and MEM at specific ratios according to the instructions. After standing at room temperature for 15 min, the transfection reagents were thoroughly combined with the cell media. The western blotting was utilized to confirm that the cell transfection was effective. Test for cell viability TSCs were cultured in 96-well plates (1 × 10 4 cells per well) and following drug treatment in accordance with the group settings, the viability of the cells was assessed with the MTT (Aladdin, #T100806) and CCK8 (YEASEN, #40203ES76) assay. In brief, 20 µl of MTT or CCK8 solution was added to each well, and the 96-well plates were incubated at 37 °C for 4 h or 1 h. The absorbance of each well was measured at 490 nm (MTT assay) or 450 nm (CCK-8 assay) with a SpectraMaxTM microtiter spectrophotometer (Molecular Devices, Sunnyvale, CA). GSH, MDA, IL-1β and IFN-β Measurements The glutathione (GSH) content of TSCs and tendon tissues was determined with a reduced GSH Assay Kit (Best Bio; #BB-4711). The concentration of malondialdehyde (MDA) in tendon tissues and cells was determined with an MDA detection kit (Best Bio; # BB-4709). An IL-1β ELSIA kit (yfxbio Biotech. Co., Ltd; #YFXER00022) was used to detect the content of IL-1β in TSCs and tendon serums. An IFN-β ELSIA kit (yfxbio Biotech. Co., Ltd; #YFXER00719) was used to assess the content of IFN-β in TSCs. Each experiment was carried out in compliance with the instructions included in the corresponding kit. Western blotting Precooled PBS was used to remove dead and suspended cells from a six-well plate. The cells were then thoroughly ground and 200 µl of lysis solution (RIPA: PMSF: protease inhibitors 100:1:1, Beyotime, #P0013B) was added to each well. Nanodrop was used to measure the concentration of the supernatant. The proteins were incubated for 15 min at 95 °C in a metal bath after the loading buffer was added and thoroughly mixed. The extracted protein was subsequently subjected to electrophoresis on a polyacrylamide gel, transferred to a PVDF membrane, blocked with 5% skim milk for a 2 h, and subsequently incubated with secondary antibody for 2 h. ROS Measurement First, TSCs were seeded on slides (2 × 10 4 cells/well) that had been inserted in 24-well plates. Following the group dosing treatments, each well of the culture plate received 1 mL of 1000-fold diluted DCFH-DA probe (Beyotime; #S0033) in serum-free media. The plates were then incubated at 37 °C for 20 min. Ultimately, the fluorescence microscope and flow cytometry were used to detect the level of ROS in TSCs. Quantitative real-time PCR Trizol Regent (Life, # 15596018) was used to extract RNA from TSCs, and a designated kit (ABclonal, #RK20429) was used to reverse transcribe the extracted RNA into cDNA. Finally, real-time PCR was carried out with the SYBR Green PCR Kit (ABclonal, #RK21206) in accordance with the manufacturer’s instructions. GAPDH was used as the internal reference. The sequences of primers used are displayed in Table S2 . Immunofluorescence assay The immunofluorescence tests were conducted in 24-well plates. First, the plates were filled with cell slides, after which TSCs were added at a density of 2 × 10 4 cells/ml onto the slides. Following exposure to the experimental compounds, the cellular samples were subjected to sequential processing: fixation in 4% paraformaldehyde, permeabilization with 0.5% Triton X-100, and overnight primary antibody incubation at 4°C. Subsequent immunolabelling involved incubating with fluorophore-conjugated secondary antibodies, nuclear counterstaining with DAPI, and fluorescence microscopic imaging. For double immunofluorescence staining, after fixation with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100, the cells were incubated with the primary antibody against the first target protein overnight at 4°C and then with the corresponding secondary antibody for 30 min at 37 °C in the dark. The incubation was subsequently repeated with the corresponding primary and secondary antibodies for the other target protein. Finally, the nuclei were restained with DAPI. Co-immunoprecipitation After drug treatment, the cells in the dish were washed with precooled PBS and then lysed on ice for 30 min by adding an appropriate amount of lysis solution (RIPA: PMSF: protease inhibitors 100:1:1) (Beyotime, #P0013B) and grinding thoroughly. After centrifugation, the appropriate antibody or IgG was added to the collected supernatant and incubated overnight in an ice box with shaking to fully combine the antigen with the antibody. The antigen-antibody complex was mixed with pretreated rProtein A/G Plus MaqPoly Beads (ABclonal, #RM09008) and incubated overnight in an ice box with shaking. After centrifugation, the supernatant was collected for subsequent use, and the precipitate contained the antigen-antibody-magnetic bead complex. After sufficient washing, the precipitate was mixed well with 2x SDS-PAGE Loading Buffer. The system was heated in a metal bath for 15 min before centrifugation and the collected supernatant was used for protein blotting analysis. Cellular thermal shift assay (CETSA) TSCs were treated with GA or vehicle control for 2 h prior to CETSA analysis. After treatment, the harvested cells were aliquoted into 10 thermal challenge groups (43–70 °C, 3 °C increments), each heated for 3 min. Thermocycling involved sequential incubation at 80 °C (12 h) and ambient temperature (5 min), which was repeated twice. Lysates obtained via high-speed centrifugation (20,000 ×g, 20 min) were analysed for cGAS stabilization by immunoblotting. Bioinformatics prediction and database analysis Ubibrowser ( http://ubibrowser.ncpsb.org/ ) was used to predict potential E3 ubiquitin ligases of target proteins. PhosphoSitePlus ( http://www.phosphosite.org/ ) and GPS-MSP ( http://msp.biocuckoo.org/download.php ) were used to determine the methylation sites of the target proteins. Molecular docking The 2D structure of the small-molecule ligand was retrieved from the PubChem database ( http://pubchem.ncbi.nlm.nih.gov/ ) and imported into ChemOffice to generate its 3D conformation. Potential protein targets were screened using the RCSB PDB database ( http://www.rcsb.org/ ), from which a high-resolution crystal structure was selected as the docking receptor. The protein structure was prepared with PyMOL 2.6 by removing water molecules and phosphate groups. Subsequent preparation in AutoDock 1.5.6 included adding hydrogen atoms to the protein, as well as optimizing the ligand by adding hydrogens and defining its rotatable bonds. The docking grid coordinates were then assigned. Molecular docking was performed using AutoDock Vina to evaluate protein–ligand interactions. The conformation with the most favorable docking score was selected as the optimal binding pose. Results were visualized using Discovery Studio 2019 and PyMOL 2.6 to produce 2D interaction diagrams and 3D plots highlighting key binding residues. A binding energy below − 7.0 kcal/mol was considered indicative of strong binding activity. Reagents and antibodies 18-β-Glycyrrhetinic acid (#HY-N0180, Purity: 99.88%) and 3-MA (#HY-19312 S) were obtained from MedChemExpress. The 3% hydrogen peroxide solution (#323381) was supplied by Sigma-Aldrich. Cycloheximide (CHX) (#C7698) and MG-132 (#M8699) were supplied by Merck KGaA. The anti-collagen-Ⅰ antibody (#WL0088) and anti-IRF3 antibody (#WL02431) were purchased from Wanlei Biotechnology. The anti-collagen-Ⅲ antibody (#22734-1-AP), anti-Ub antibody (#10201-2-AP), anti-TOM20 antibody (#66777-1-Ig), anti-HMGB1 antibody (#10829-1-AP), anti-CD68 antibody (#28058-1-AP) and anti-Flag antibody (#66008-4-Ig) were purchased from Proteintech. The anti-TNMD antibody (#A17753), anti-IL-6 antibody (#A22222), anti-IL-1β antibody (#A16288), anti-TBK1 antibody (#A3458), anti-P-IRF3-S386 antibody (#AP0857) and anti-P-TBK1-S172 antibody (#AP1026) were purchased from ABclonal Technology. The anti-STING antibody (#ab302617), anti-cGAS antibody (#ab288157) and anti-Methyl K antibody (#ab23366) were purchased from Abcam. The anti-NLRP3 antibody (#BA3677) and anti-Collagen Type III antibody (#M00788-1) were obtained from Boster, and the anti-ASC antibody (#HA721306) was obtained from Hangzhou Huaan Biotechnology. The anti-Scleraxis antibody (#sc-518082) was bought from Santa Cruz. Statistical analyses Three separate cell tests were performed and the data were subsequently normalized to those of the control group. All experimental data are presented as means ± SEM. Statistical analyses were performed with unpaired Student’s t-test or one-way ANOVA, depending on the number of groups. All the statistical analyses were conducted with GraphPad prism 9.0 (San Diego, California, USA). Results Oxidative stress levels are increased in patients with tendinopathy To define the pathological basis of tendinopathy and identify key cellular targets for intervention, we first analyzed clinical tendon samples. Proteomic analysis was performed on tendon tissues obtained from clinical samples. The results revealed a significant increase in antioxidant activity during the progression of tendinopathy (Fig. 1 A). These findings suggested that oxidative stress may play a pivotal role in the pathogenesis of tendinopathy. Given the established role of PTGS2/COX2 in oxidative stress-related pathological processes, we assessed the expression of PTGS2 in tendon tissues by immunohistochemical staining. The results revealed a marked increase in PTGS2 expression in injured tendon tissues (Fig. 1 B). Mononuclear cell libraries were subsequently constructed from three healthy and three injured tendon tissues, and single-cell RNA sequencing was performed. Based on lineage-specific marker expression (Table S3 ), seven distinct cell types were identified: tendon cells, endothelial cells, smooth muscle cells, TSCs, macrophages, T cells, and Mast Cells (Fig. 1 C). Notably, the relative abundance of several cell subtypes changed significantly in tendinopathy, with a particularly pronounced alteration observed in TSCs (Fig. 1 D-F). To verify this result, we performed immunofluorescence staining (SCX + CD146 + ) on TSCs, confirming an increase in the number of TSCs in patients with tendinopathy (Fig. 1 G). In addition to TSCs, the proportions of other cell types also underwent significant changes. Compared to cells whose numbers decreased in the diseased state (smooth muscle cells, endothelial cells, and tendon cells) and cells with very small proportions (such as T cells), the significant increase in macrophage numbers also caught our attention. To rule out the influence of macrophage oxidative stress in the disease, we used dihydroethidium (DHE) to stain ROS in the tissue and labelled TSCs and macrophages separately (Fig. 1 H). The results showed that ROS production was primarily present in TSCs. Therefore, we hypothesized that TSCs are key mediators of the response to oxidative damage in tendinopathy. This established TSCs as the primary cellular model for subsequent investigations into the therapeutic mechanism of GA. Fig. 1. Open in a new tab Oxidative stress levels are increased in patients with tendinopathy. The tendons of normal clinical samples and tendinopathy patients were collected and tested. ( A ) Proteomics was used for detection, and the results were subjected to GO analysis for Biological Process, Cellular Component and Molecular Function of proteins. ( B ) The protein expression of PTGS2 in tendon tissues was assessed by immunohistochemical staining (Scale bars: 20×, 50 μm). ( C-D ) Uniform manifold approximation and projection (UMAP) was used to visualize nuclear clusters, which are coloured and labelled according to cell identities, in normal (left panel) and injured (right panel) tendon tissues. ( E-F ) Proportion of cell types in normal and injured tendon tissues. Each cell type is colour-coded. ( G ) Immunofluorescence staining identifying TSCs (Red: SCX + ; Green: CD146 + ). ( H ) The superoxide levels in TSCs (SCX + ) and macrophages (CD68 + ) were detected by DHE staining GA attenuates oxidative stress by inhibiting inflammation to alleviate tendinopathy Having identified TSCs as a key cellular compartment under oxidative stress in tendinopathy, we next sought to evaluate the therapeutic potential of GA in vivo. To investigate this, a tendon injury model under oxidative stress was established [ 16 ]. Considering that compared to mechanical overload and collagenase-induced models, ciprofloxacin hydrochloride (a fluoroquinolone antibiotic) is clinically known to cause tendinitis and tendon rupture, and that oxidative stress has been clearly established as the key initiating and driving factor of this model [ 17 ], this approach was selected for tendinopathy modeling. Fluoroquinolone-induced tendinopathy is characterized by a disorganized collagen architecture, reduced type I collagen, and increased type III collagen in tendon tissues [ 18 ]. H&E, Masson and Sirius red staining revealed that GA promoted the repair of collagen arrangement and ameliorated the pathological disorganization of collagen fibres in rat tendon tissues (Fig. 2 A). Immunofluorescence staining analysis further confirmed alterations in the expression of type I and type III collagen in the tendon tissues (Fig. 2 B). Additionally, the expression of PTGS2 in tendon tissues was assessed and found to be downregulated in the GA-treated group (Fig. 2 C). For comparative purposes, we included aspirin, a commonly used positive control in tendinopathy. Both aspirin and high concentrations of GA administration resulted in significant histological improvement compared to the untreated control group in the tendinopathy model. Furthermore, tendon tissues from four rats in both the control and tendinopathy model groups were selected for proteomic analysis. Gene Ontology (GO) enrichment analysis of biological processes revealed that the tendinopathy model group presented significantly upregulated responses to changes in oxygen levels compared with the control group (Fig. 2 D). Molecular function enrichment analysis furtherly revealed a significant enrichment and downregulation of oxidoreductase activity, whereas protein binding remained unaffected (Fig. 2 E). Collectively, these results indicated that oxidative stress played a critical role in the progression of tendinopathy. Therefore, oxidative stress levels were quantified by GSH and MDA assays. In addition, we extracted the proteins of primary TSCs from different groups of rats for kit detection and found that the oxidative stress level of TSCs increased in tendinopathy rats, while GA administration inhibited its oxidative stress level (Figure. S6 ). The results demonstrated that GA treatment effectively alleviated oxidative stress during tendinopathy (Fig. 2 F-G). As ROS in tendinopathy can trigger a cascade of inflammatory responses [ 4 ], we further evaluated the level of inflammation by detecting the content of serum pro-inflammatory cytokine IL-1β. The results confirmed that GA treatment attenuated inflammatory responses in tendinopathy rats (Fig. 2 H). In summary, our findings confirmed that GA can alleviate tendon injury in vivo in a concentration-dependent manner. The established therapeutic efficacy prompted us to investigate the underlying molecular mechanisms in TSCs. Fig. 2. Open in a new tab GA attenuates oxidative stress by inhibiting inflammation to alleviate tendinopathy. In vivo, Wistar rats were divided into 6 groups: vehicle, ciprofloxacin, ciprofloxacin + 25 mg/kg GA, ciprofloxacin + 50 mg/kg GA, ciprofloxacin + 100 mg/kg GA and positive control (five rats in each group). ( A ) H&E and Masson staining were used to observe collagen organization (blue: collagen fibres; red: muscle fibres); Sirius red staining was used to distinguish between type I and type III collagen (scale bars: 10×, 100 μm); ( B ) The expression of collagen I/III was determined by immunofluorescence staining (scale bars: 10×, 100 μm); ( C ) The protein expression of PTGS2 in rat tendon tissues was assessed by immunohistochemical staining (scale bars: 10×, 100 μm). ( D-E ) Biological process and protein function enrichment analysis of the TMT proteomics data. ( F-H ) The serum levels of MDA, GSH and IL-1β were determined with the relevant kits; ( n = 5, data are presented as mean ± SD, ** p < 0.01, *** p < 0.001 vs. vehicle group; ## p < 0.01, ### p < 0.001 vs. Ciprofloxacin group) GA increases TSCs viability by inhibiting oxidative stress and inflammatory responses The tendon lineage differentiation of TSCs contributes to the repair of tendon tissue, whereas sustained oxidative stress leads to the aberrant differentiation of stem cells, thereby exacerbating tendon injury [ 19 ]. Therefore, we investigated whether GA could alleviate oxidative damage of TSCs. Firstly, TSCs were incubated with different concentrations of H 2 O 2 for 24 h to establish an in vitro model of oxidative stress. According to our previous research [ 9 ], 100 µM H 2 O 2 was used for subsequent modelling of oxidative stress in TSCs, and we found that the survival rate of TSCs decreased significantly with increasing concentrations of H 2 O 2 . Next, we pretreated TSCs with different concentrations of GA for 12 h before treatment with H 2 O 2 . The MTT results revealed that GA was not obviously cytotoxic to TSCs and increased the cell viability when the drug concentration was 2.5, 5, and 10 µM, indicating that GA could increase the level of resistance to oxidative stress in TSCs within a certain range (Fig. 3 A-B). Furtherly, we compared the cell viability of TSCs in each GA concentration group with and without H₂O₂ treatment conditions. When the GA concentration was higher than 20 µM, the antioxidant stress level of GA weakened, which might be the result of enhanced cytotoxicity (Fig. 3 C). We then explored the alleviation of oxidative stress and inflammation in TSCs by GA. As expected, pretreatment with GA significantly suppressed GSH depletion in TSCs (Fig. 3 D), together with the production of the lipid peroxidation products MDA and ROS (Fig. 3 E-F, S4 ). Additionally, the protein and gene expression levels of inflammatory factors IL-1β, IL-6 and IL-8 were also detected by western blotting and qPCR (Fig. 3 G-H). The imbalance between type I and type III collagen is a core pathological feature of tendinopathy [ 1 ]. We found that under oxidative stress, the ability of TSCs to generate type I collagen was weakened, while the ability to generate type III collagen was increased. However, GA could reverse these effects. Similarly, H₂O₂ inhibited the expression of tenomodulin, a marker of tendon-lineage differentiation in stem cells, which may potentially exacerbate disease progression. (Fig. 3 I). Collectively, these results revealed that GA can inhibit oxidative damage and inflammation to attenuate H 2 O 2 -induced damage to TSCs. This furtherly promoted us to investigate the specific signaling pathway involved. Fig. 3. Open in a new tab GA increases TSC viability by inhibiting oxidative stress and inflammatory responses. In vitro, TSCs were pretreated with different concentrations of GA for 12 h and then subjected to the prescribed concentration of H 2 O 2 (100 µM) for 24 h. ( A-B ) Cell viability was assessed by MTT assay; ( C ) Cell viability was assessed by CCK-8 assay; ( D-E ) The levels of GSH and MDA were determined with the relevant kits; ( F ) The content of ROS in TSCs was measured by flow cytometry using DCFH-DA probe; ( G-H ) Western blotting and qPCR were used to assess the protein and gene expression of inflammatory cytokines, including IL-1β, IL-6, and IL-8; ( I ) Tenomodulin, Collagen I and Collagen III protein levels were assessed via western blotting. ( n = 3, data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. H₂O₂ group) GA inhibits the cGAS-STING-NLRP3 axis to alleviate H 2 O 2 -induced TSCs damage To elucidate the specific molecular mechanism underlying GA’s protection, we investigated its effect on the cGAS-STING pathway, a key sensor of cytosolic DNA known to be activated under oxidative stress and implicated in inflammatory responses. We hypothesized that H₂O₂ activates the cGAS‒STING pathway, subsequently inducing oxidative damage in TSCs. This hypothesis was tested by assessing levels of cGAS-STING pathway proteins under different concentrations of GA (Fig. 4 A-B). H₂O₂ stimulation upregulated the expression of cGAS, STING, and phosphorylated forms of IRF3 and TBK1, all of which were inhibited by GA treatment in a concentration-dependent manner. IFN-β was used as an indicator of cGAS-STING pathway activation. Both qPCR and ELISA confirmed that H₂O₂ induced IFN-β expression, and this induction was suppressed by GA in a dose-dependent manner (Fig. 4 C-D). Under oxidative stress, mitochondrial DNA (mtDNA) release can activate both the cGAS-STING pathway and the intracellular NLRP3 inflammasome [ 20 ]. However, the regulatory relationship between cGAS-STING and NLRP3 activation remained unclear. Therefore, we conducted further investigations. First, we examined the expression of NLRP3 and ASC, two key components of the NLRP3 inflammasome pathway. H 2 O 2 administration promoted the activation of the NLRP3 inflammasome, which was blocked by GA (Fig. 4 E). To verify whether the protective effect of GA against oxidative damage in TSCs was dependent on cGAS inhibition, we constructed a cGAS overexpression plasmid and validated its efficiency via western blotting (Fig. 4 F). As expected, overexpression of cGAS reversed the GA-mediated suppression of IFN-β expression, as demonstrated by ELISA (Fig. 4 G). GA significantly reduced H 2 O 2 -induced inflammatory responses in TSCs via the cGAS-STING pathway, as indicated by reduced levels of activated IL-1β and IL-6 (Fig. 4 H, S3A ). Furthermore, we investigated the regulatory relationship between cGAS and NLRP3. The overexpression of cGAS diminished the GA-mediated inhibition of NLRP3 (Fig. 4 I, S3B ), indicating that cGAS signalling contributed to the regulation of NLRP3 inflammasome activation. We next measured intracellular MDA accumulation in TSCs. GA treatment reduced MDA levels, whereas cGAS overexpression increased MDA accumulation (Fig. 4 J). Consistently, the GSH levels also supported the conclusion that inhibition of the cGAS‒STING pathway is essential for the antioxidative effect of GA in TSCs (Figure. 4 K). Since collagen expression imbalance and impaired tenogenic differentiation in TSCs are among the main features of tendon lesions, we detected the expression of collagen I, collagen III, and Tnmd in TSCs (Figure. S3C ). The results showed that overexpression of cGAS exacerbated the disease progression. In summary, our findings suggested that GA alleviated H₂O₂-induced oxidative stress and inflammation in TSCs via modulation of the cGAS‒STING‒NLRP3 axis. Having identified the pathway, we next sought to determine how GA specifically induces cGAS downregulation. Fig. 4. Open in a new tab GA inhibits the cGAS-STING-NLRP3 axis to alleviate H 2 O 2 -induced TSC damage. TSCs were pretreated with the cGAS plasmid or GA at different concentrations for 12 h, and then exposed to 100 µM H 2 O 2 (freshly diluted in media) for 24 h. ( A-B ) Protein levels of the cGAS-STING axis were evaluated by western blotting; ( C ) The mRNA levels of IFN-β were measured by qPCR; ( D ) An IFN-β ELISA kit was used to detect the content of IFN-β in TSCs; ( E ) The NLRP3 and ASC protein levels were evaluated by western blotting; ( F ) Western blotting was used to verify the overexpression efficiency of the cGAS plasmid; ( G ) The IFN-β ELISA kit was used to detect the content of IFN-β in TSCs; ( H ) Western blotting was used to detect the protein expression of inflammatory cytokines, including IL-1β and IL-6. ( I ) NLRP3 and ASC protein levels were evaluated by western blotting; ( J-K ) The corresponding kits were used to measure the levels of oxidative stress in TSCs; ( n = 3, data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. H₂O₂ group, & p < 0.05, && p < 0.01 vs. H₂O₂ + GA group) GA inhibits the expression of cGAS by promoting its ubiquitination-mediated degradation Having established that GA suppresses the cGAS-STING-NLRP3 axis, we next sought to determine the precise mechanism by which GA downregulates cGAS protein. cGAS is a nucleotide transferase that plays a critical role in innate immunity by catalysing the synthesis of cGAMP from ATP and GTP [ 21 ]. Therefore, we aimed to determine whether GA inhibits the cGAS‒STING pathway by affecting the enzymatic activity of cGAS. The synthesis of cGAMP was used to reflect the enzyme activity of cGAS. The results showed that GA administration had no effect on cGAS enzyme activity (Fig. 5 A). Our previous experiments demonstrated that GA suppresses cGAS protein expression (Fig. 4 A). To further investigate the mechanism of cGAS inhibition by GA, we assessed cGAS mRNA expression levels. Surprisingly, GA had no effect on the cGAS mRNA level (Fig. 5 B), suggesting that GA may regulate cGAS expression by promoting its protein degradation. First, TSCs were treated with cycloheximide (CHX), a protein synthesis inhibitor, with or without GA. Co-administration of GA and CHX significantly reduced the half-life of cGAS protein (Fig. 5 C). Given that autophagy and the ubiquitin-proteasome system are the two primary pathways of protein degradation, we further investigated the specific mechanism involved. Treatment with the autophagy inhibitor 3-MA did not prevent the GA-induced reduction in the cGAS protein (Fig. 5 D). Therefore, we hypothesized that GA inhibits the protein expression of cGAS by promoting its ubiquitination-mediated degradation. Ubiquitination plays an important role in protein stability [ 22 ]. To test this hypothesis, CHX was co-administered with the proteasome inhibitor MG-132. Western blotting analysis revealed that MG-132 reversed the decrease of cGAS levels, whereas CHX cotreatment exacerbated this effect (Fig. 5 E). In summary, these results indicated that GA suppressed cGAS expression by promoting its degradation via the ubiquitin-proteasome pathway. Fig. 5. Open in a new tab GA inhibits the expression of cGAS by promoting its ubiquitination-mediated degradation. ( A ) The cGAMP ELISA kit was used to assess the enzyme activity of cGAS; ( B ) qPCR was used to detect the mRNA expression of cGAS under different GA concentrations; ( C ) TSCs were treated with cycloheximide (CHX, 20 µg/ml) in the presence or absence of GA (5 µM), and cGAS levels were analyzed by western blot at the indicated time points over 8 h; ( D-E ) TSCs were pretreated with 3-MA (5 µM), CHX (20 µg/ml) or MG-132 (1.25 µM) for 4 h and then exposed to 100 µM H 2 O 2 (freshly diluted in media) for 24 h, the protein expression of cGAS was detected by western blotting; ( F ) Ubiquitination of cGAS was analyzed by immunoprecipitation (IP) followed by immunoblotting (IB); ( G ) Ubibrowser ( http://ubibrowser.ncpsb.org/ ) was used to predict the E3 ubiquitin ligase of cGAS in rats; ( H ) The binding of cGAS and predicted E3 ubiquitin ligase was detected by co-immunoprecipitation; ( I ) The colocalization of cGAS and TOMM20 was detected by immunofluorescence staining. ( n = 3, data are presented as mean ± SD, ** p < 0.01, *** p < 0.001 vs. control group; ## p < 0.01, ### p < 0.001 vs. H₂O₂ group; && p < 0.01, &&& p < 0.001 vs. H₂O₂+GA group) Ubiquitination assays revealed that GA treatment increased the ubiquitination level of cGAS in TSCs (Fig. 5 F). This finding raised the question of which E3 ubiquitin ligase mediates the ubiquitination and degradation of cGAS. Using Ubibrowser ( http://ubibrowser.ncpsb.org/ ), a computational tool for predicting substrate-E3 ligase interactions, we identified Pml, mitogen-activated protein kinase kinase 1 (Map3k1), and the E3 ligase RING-type E3 ubiquitin transferase (Mul1) as the top candidates predicted to interact with cGAS at comparable levels (Fig. 5 G). Accordingly, immunoprecipitation assays were conducted to assess the interaction between cGAS and the predicted E3 ligases. Notably, only the interaction between cGAS and Mul1 was increased following GA treatment, whereas the interactions with Map3k1 and Pml remained unchanged (Fig. 5 H). Furthermore, the HEK293T cells were used to prove enhanced binding of cGAS to Mul1 after GA administration (Figure. S2A ). According to previous studies, Mul1 was a multifunctional protein located on the mitochondrial membrane, and one of its key roles depended on its E3 ubiquitin ligase activity [ 23 ]. We therefore hypothesized that GA promotes the translocation of cGAS to the mitochondrial membrane, where it was ubiquitinated by Mul1 and subsequently degraded. Immunofluorescence staining revealed increased colocalization of cGAS with the mitochondrial outer membrane protein TOMM20 following GA treatment (Fig. 5 I). In summary, GA downregulates cGAS not by inhibiting its transcription or enzyme activity, but by promoting its ubiquitination and proteasomal degradation. Our data demonstrate that GA enhances the interaction between cGAS and the mitochondrial E3 ubiquitin ligase Mul1, suggesting Mul1 plays a key role in mediating this process. This detailed mechanism, however, left a fundamental question unanswered: how does GA initiate this process, given that it does not directly bind cGAS? This prompted us to search for an upstream mediator linking GA to cGAS regulation. GA promotes the cGAS degradation by promoting K43 methylation of HMGB1 Furthermore, we wanted to explore the interaction mode between GA and cGAS, which was evaluated by Cellular thermal shift assay. GA treatment did not affect the thermal stability of cGAS, suggesting that GA did not exert its effects through direct binding to cGAS (Figure. S1 ). Hence, we sought to identify the upstream mediator through which GA regulates cGAS stability. As a known pharmacological inhibitor of HMGB1, GA was investigated for its potential to modulate the cGAS pathway via this key DAMP protein. We constructed an overexpression plasmid of HMGB1 and verified its overexpression efficiency (Fig. 6 A). Through western blotting and immunofluorescence, we found that the HMGB1 overexpression reversed the inhibitory effect of GA on cGAS to a certain extent (Fig. 6 B, S5 ). Furthermore, we wondered whether HMGB1 overexpression would affect the therapeutic effect of GA. By measuring collagen synthesis, oxidative stress, and inflammation-related indicators in TSCs, we confirmed our hypothesis that HMGB1 overexpression reversed the efficacy of GA (Figure. S7 ). It has been reported that HMGB1 can bind to nucleic acids in host cells to strongly stimulate DNA-sensing pathways, including the cGAS-STING axis [ 24 , 25 ]. Therefore, we tested the combination of cGAS and HMGB1 in TSCs after GA administration. The results revealed that the binding of HMGB1 and cGAS was increased by H 2 O 2 stimulation and inhibited by GA administration (Fig. 6 C), which was also confirmed by the immunofluorescence staining results (Fig. 6 D). HMGB1, a non-histone chromatin-associated protein widely distributed in eukaryotic cells, undergoes nucleoplasmic translocation in response to cell stress, and methylation at the protein level is involved in the translocation of HMGB1 [ 26 , 27 ].Our results revealed that the methylation level increased significantly (Fig. 6 E). Therefore, we hypothesized that the methylation of HMGB1 inhibits its binding to cGAS and activation of downstream signalling pathways. Similarly, we verified that H 2 O 2 stimulation promoted the binding of cGAS and HMGB1 in tool cells, whereas GA could inhibit this process by promoting the methylation process of HMGB1 (Figure. S2B ). Methylation sites of HMGB1 were predicted through two databases (Fig. 6 F), PhosphoSitePlus and GPS-MSP. In addition, we investigated the methylation sites related to HMGB1 nucleoplasmic translocation described in existing reports, focusing on the K43 and K112 sites [ 26 ], and synthesized a locus mutation plasmid for validation. Interestingly, mutation of K43 rather than K112 inhibited HMGB1 methylation and its binding to cGAS (Fig. 6 G, S2C ). After the overexpression of different mutant HMGB1 plasmids, the ubiquitination level of cGAS was restored in the K43 mutant group (Fig. 6 H). To elucidate how GA promotes HMGB1 methylation, we screened the expression of potential lysine methyltransferases and demethylases in TSCs following GA treatment. Among the candidates, the methyltransferase DOT1L was consistently upregulated at the mRNA level, whereas no significant changes were observed for other tested enzymes ( Figure. S8A-F ). Molecular docking analysis indicated a strong binding affinity between GA and DOT1L, with a calculated binding energy of − 7.6 kcal/mol (Figure. S8G ). Cellular thermal shift assay further demonstrated that GA treatment enhanced the thermal stability of DOT1L, confirming their direct interaction (Figure. S8H ). In summary, this series of experiments delineates a mechanism wherein GA, by directly binding to and upregulating the methyltransferase DOT1L, promotes methylation of HMGB1 at K43. This modification inhibits the HMGB1-cGAS interaction, leading to the subsequent Mul1-mediated ubiquitination and degradation of cGAS. These findings collectively map the upstream molecular events through which GA achieves precise inhibition of the cGAS-STING-NLRP3 axis in tendinopathy. Fig. 6. Open in a new tab GA promotes cGAS degradation by promoting the K43 methylation of HMGB1. ( A ) Western blotting was used to verify the overexpression efficiency of the HMGB1 plasmid; ( B ) TSCs were pretreated with GA (5 µM) with and without the HMGB1 plasmid for 12 h and then exposed to 100 µM H 2 O 2 (freshly diluted in media) for 24 h, then western blotting was used to assess the expression of cGAS; ( C-D ) The binding of cGAS and HMGB1 was detected by co-immunoprecipitation and immunofluorescence; ( E ) Immunoprecipitation was used to detect methylation levels of HMGB1; ( F ) PhosphoSitePlus ( www.phosphosite.org ) and GPS-MSP ( http://msp.biocuckoo.org/download.php ) were used to predict the methylation sites in HMGB1; ( G ) An HMGB1 site mutation plasmid was constructed, after which TSCs were pretreated with the HMGB1 plasmid for 12 h, and the binding of cGAS and HMGB1 was detected by coimmunoprecipitation; ( H ) Ubiquitination of cGAS was detected by Co-IP. ( n = 3, data are presented as mean ± SD, * p < 0.05, *** p < 0.001 vs. control group; ### p < 0.001 vs. H₂O₂ group; &&& p < 0.001 vs. H₂O₂+GA group) GA protects TSCs against oxidative stress via the HMGB1-cGAS-NLRP3 axis in tendinopathy To validate the physiological relevance of the molecular pathway delineated in vitro, we next investigated whether GA protects against tendinopathy in vivo through the HMGB1-cGAS axis. In addition, we investigated whether GA could protect tendons from oxidative stress via the HMGB1/cGAS pathway in vivo. The expression of HMGB1 and cGAS in tendon tissues were assessed by immunohistochemical staining, which revealed increased levels of HMGB1 and cGAS in injured tendons, and these levels were significantly reduced following GA treatment (Fig. 7 A). GA treatment also reduced the expression of NLRP3 and ASC in tendon tissues (Fig. 7 B). To determine whether cGAS overexpression affects oxidative stress and inflammation in TSCs in vivo, a VA-Lip-cGAS-Plasmid was constructed to increase cGAS expression. Macroscopic observation of tendon morphology revealed pathological differences between the tendinopathy and control groups, while GA treatment mitigated ciprofloxacin-induced tendinopathy. Notably, pretreatment with the VA-Lip-cGAS Plasmid completely abolished the protective effect of GA against tendon injury (Fig. 7 C). Tendinopathy is also characterized by the replacement of type I collagen with type III collagen [ 1 ]. Immunofluorescence staining analysis further confirmed that the GA-mediated suppression of collagen III expression was completely reversed by the VA-Lip-cGAS Plasmid (Fig. 7 D). The expression of NLRP3 and ASC in tendon tissue was also inhibited after GA administration (Fig. 7 E). Furthermore, oxidative stress and inflammation markers in rat serum were assessed with commercial assay kits. Serum GSH depletion and MDA accumulation observed during tendinopathy were mitigated by GA, but these effects were abolished by the VA-Lip-cGAS Plasmid (Fig. 7 F-G). Similarly, IL-1β levels in the serum showed the same trend (Fig. 7 H). Mechanistically, immunofluorescence staining further confirmed the interaction between HMGB1 and cGAS in tendon tissues with elevated cGAS expression in the tendinopathy group compared with the control group. In parallel, GA treatment reduced the co-localization of HMGB1 and cGAS. The VA-Lip-cGAS-Plasmid significantly reversed the GA-induced suppression of HMGB1-cGAS colocalization and increased cGAS pathway activation (Fig. 7 I). Finally, colocalization of cGAS and HMGB1 was also observed in tendon tissues from patients with tendinopathy (Fig. 7 J). In conclusion, these findings supported the hypothesis that GA mitigated oxidative stress and inflammation during tendinopathy by suppressing the HMGB1-cGAS axis. Fig. 7. Open in a new tab GA protects TSCs against oxidative stress via the HMGB1-cGAS-NLRP3 axis in tendinopathy. Wistar rats were divided into five groups (five rats per group): vehicle, ciprofloxacin, ciprofloxacin + 50 mg/kg GA, ciprofloxacin + 50 mg/kg GA + VA-Lip-Ctrl-Plasmid, and ciprofloxacin + 50 mg/kg GA + VA-Lip-cGAS-Plasmid. ( A-B ) Immunohistochemical detection for cGAS, HMGB1, NLRP3 and ASC in tendon tissues (scale bars: 10×, 100 μm); ( C ) H&E and Masson staining were used to observe the collagen organization of tendon tissues (blue indicates collagen fibres and red indicates muscle fibres); Sirius red staining was used to distinguish between type I and type III collagen (Scale bars: 10×, 100 μm); ( D ) The expression of collagen I/III were assessed by immunofluorescence staining (scale bars: 10×, 100 μm); ( E ) Immunohistochemical detection of the expression of NLRP3 and ASC (scale bars: 10×, 100 μm); ( F-H ) Serum levels of MDA and IL-1β were measured by commercial ELISA kits; ( I ) Immunofluorescence staining was used to detect the expression and colocalization of cGAS and HMGB1 in the tendon tissues of the rats (scale bars: 20×, 50 μm). ( J ) Immunofluorescence staining was used to detect the colocalization of cGAS and HMGB1 in the tendon tissue of clinical sample 1. ( n = 5, data are presented as mean ± SD, ** p < 0.01, *** p < 0.001 vs. vehicle group; ## p < 0.01, ### p < 0.001 vs. Ciprofloxacin group; && p < 0.01, &&& p < 0.001 vs. Ciprofloxacin + GA group) Discussion Our study establishes that TSCs are a major target of oxidative damage in tendinopathy and identifies GA as an effective agent that alleviates the condition by disrupting the pathogenic cycle between oxidative stress and inflammation. The consistent beneficial effects of GA across both models strongly reinforce that targeting oxidative stress is a central mechanism through which GA alleviates tendinopathy. To comprehensively evaluate this therapeutic potential, we made a deliberate choice of complementary experimental models. For in vivo validation, we employed a clinically relevant ciprofloxacin-induced rat model, which recapitulates complex pathology including inflammation and extracellular matrix disruption, and in which oxidative stress is a well-established key driver [ 28 , 29 ]. This allowed us to assess the integrative effects of GA on tissue repair. In contrast, the pro-oxidant effect of fluoroquinolones in vivo involves metabolic conversion, which is not reliably modeled in isolated cells. The H₂O₂ model [ 30 ] thus provided a reductionist and controllable system to precisely isolate and interrogate the core mechanistic link between oxidative damage and the subsequent inflammatory cascade without in vivo confounding factors. This complementary approach confirmed that targeting oxidative stress is central to GA’s therapeutic action and prompted us to define the underlying molecular pathway. Building on our previous finding that oxidative stress triggers mtDNA release in TSCs [ 9 ]. As a cytosolic DNA sensor, cGAS is pivotal in innate immune defence [ 31 ]. Given that the released mtDNA can activate the cGAS-STING pathway, we demonstrated that this activation within TSCs leads to NLRP3 inflammasome assembly and the consequent release of inflammatory factors in this paper. Based on this established pathway, we sought to determine whether GA’s protective effect against oxidative stress in tendinopathy is mediated through the cGAS-STING axis. As anticipated, GA treatment enhanced the expression of tenogenic markers and reduced inflammatory responses in TSCs in a concentration-dependent manner. This effect relied on the inhibition of cGAS-STING pathway activation, as overexpressing cGAS in TSCs reversed the antioxidative and anti-inflammatory actions of GA. Furthermore, while GA did not affect cGAS mRNA levels or enzymatic activity, it significantly downregulated cGAS protein expression—an effect attributable to increased ubiquitination of cGAS, in which the E3 ubiquitin ligase MUL1 appeared to play an important role. A critical question arose from the observation that GA does not directly bind cGAS. This led us to investigate the upstream damage-associated molecular pattern protein HMGB1, a known GA target that facilitates cGAS activation [ 32 ]. In our study, we found that oxidative stress triggers the cytoplasmic translocation of HMGB1, initiating a cascade that leads to cGAS-STING pathway activation and NLRP3 inflammasome-mediated inflammation. GA treatment was shown to inhibit this cascade at its origin by blocking the formation of the mtDNA-HMGB1-cGAS complex. Mechanistically, this blockade is achieved through a novel epigenetic regulation: GA directly binds to and stabilizes the methyltransferase DOT1L, which in turn catalyzes methylation of HMGB1 at the K43 residue. This specific post-translational modification disrupts the HMGB1-cGAS interaction, thereby uncoupling the damage signal from the inflammatory cascade. The functional centrality of this mechanism was confirmed by the loss of GA‘s protection upon HMGB1 K43 mutation. In summary, we delineate a coherent signaling pathway through which GA alleviates tendinopathy: by targeting DOT1L, GA promotes HMGB1 K43 methylation, which inhibits the HMGB1-cGAS interaction and subsequently leads to the downregulation of the cGAS-STING-NLRP3 axis, ultimately resolving oxidative stress and inflammation. This work reveals a previously unrecognized regulatory node at the interface of epigenetic modification and innate immune signaling in tendinopathy. Beyond elucidating this mechanism, our study prompts consideration of GA’s translational potential and future research directions. The promising preclinical data are tempered by GA’s poor water solubility and off-target effects upon systemic delivery, which could be addressed by developing local delivery systems, such as nanoparticles or hydrogels, to enhance its bioavailability and specificity. Furthermore, the identified HMGB1-cGAS-STING-NLRP3 axis likely interacts with other signaling networks in tendinopathy. For instance, in later repair phases driven by TGF-β, persistent inflammation via this axis might suppress TGF-β signaling, thereby impairing organized collagen regeneration and promoting pathological fibrosis. Apart from its role in inflammation, the chronically activated cGAS-STING pathway has been reported to induce cellular senescence [ 33 ]. This link raises the question of whether this axis also promotes the senescence of TSCs in tendinopathy, consequently depleting their regenerative capacity. These interconnected questions outline meaningful avenues for further investigation. Conclusion In conclusion, this study demonstrates that GA ameliorates tendinopathy by suppressing the cGAS-STING-NLRP3 inflammatory axis (Fig. 8 ). The therapeutic action of GA is achieved through a defined molecular pathway: it binds to and stabilizes the methyltransferase DOT1L, which catalyzes methylation of HMGB1 at K43. This modification disrupts the HMGB1-cGAS interaction, leading to the degradation of cGAS and subsequent suppression of downstream inflammatory signaling. These findings not only establish a mechanistic foundation for GA but also highlight the DOT1L-HMGB1-cGAS axis as a promising target for the development of new therapeutic strategies against tendinopathy. Fig. 8. Open in a new tab GA plays a role in the treatment of tendinopathy by mitigating oxidative stress and the inflammatory response. During tendinopathy, oxidative stress triggers HMGB1 translocation from the nucleus to the cytoplasm, where it facilitates the binding of cGAS to cytosolic DNA. This interaction activates the cGAS-STING signalling pathway, subsequently driving NLRP3 inflammasome activation and perpetuating inflammation. GA counteracts this process by inducing methylation at the K43 residue of HMGB1, which disrupts its binding to cGAS. Concurrently, GA increases the recruitment of cGAS to the mitochondrial E3 ubiquitin ligase Mul1, promoting cGAS ubiquitination and subsequent proteasomal degradation. Together, these mechanisms suppress inflammatory signalling, positioning GA as a therapeutic candidate for tendinopathy through targeted modulation of the HMGB1-cGAS-NLRP3 axis Supplementary Information Below is the link to the electronic supplementary material. Supplementary Material 1 (1.8MB, docx) Supplementary Material 2 (10.7MB, docx) Abbreviations cGAMP 2′3′cyclic GMP-AMP cGAS GMP-AMP synthase GA Glycyrrhetinic acid GSH Glutathione HMGB1 High mobility group box 1 protein Map3k1 Mitogen-activated protein kinase kinase kinase 1 MDA Malondialdehyde mtDNA mitochondrial DNA Mul1 E3 ligase RING-type E3 ubiquitin transferase NLRP3 NLR Family, Pyrin Domain Containing Protein 3‌ IFN-β Interferon-β IL-1β Interleukin-1β IL-6 Interleukin-6 IL-8 Interleukin-8 IRF3 Interferon regulatory Factor 3 p-IRF3 phospho-Interferon regulatory Factor 3 p-TBK1 phospho-TANK Binding Kinase 1 PTGS2 prostaglandin peroxidase synthase 2 ROS Reactive oxygen species STING Stimulator of interferon genes TBK1 TANK Binding Kinase 1 TSCs Tendon stem cells Author contributions Yuanyuan Gao: Validation, Investigation, Writing-Original Draft. Wenshuang Sun: Validation, Investigation. Ziying Sun: Methodology. Junrui Wang: Methodology, Validation, Investigation. Zhongyang Lv: Investigation. Yujia Li: Validation, Investigation. Haoyuan Tian: Validation. Zhengyang Bao: Writing-Original Draft. Xinran Qiu: Validation. Zheng wang: Validation. Shizhong Zheng: Writing - Review & Editing, Supervision, Funding acquisition. Jiangjuan Shao: Methodology, Validation, Writing - Review & Editing, Funding acquisition. Zili Zhang: Methodology, Validation, Investigation. Jia Meng: Resources, Writing - Review & Editing, Supervision, Funding acquisition. All authors read and approved the final manuscript. Funding The work was fund by the National Natural Science Foundation of China (82274185, 882374124, 82474164), Leading Program for First Class Disciplines at Nanjing University of Traditional Chinese Medicine (ZYXYL2024-008), National Key Laboratory of Traditional Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacturing Technology Research Innovation Project (NZYSKL240102), Traditional Chinese Medicine Cancer Poison Disclosure and Leading Project (AD202403), National Ten-Thousand Talents Program for Top-notch Talents, Key Project of Jiangsu Provincial Science and Technology Development Plan for Traditional Chinese Medicine (ZD202402), the Natural Science Foundation of Jiangsu Province (BK20230458), Program of Jinling hospital (2024JCYJQN096, 2023JCYJYB100). Data availability The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate The clinical research protocol in this manuscript was conducted in compliance with the ethical standards established by the Institutional Review Board of Nanjing Jinling Hospital (Ethical Approval No.2024DZKY-035-01). The protocols of the institutional and local animal care and use committees at Nanjing University of Chinese Medicine (Nanjing, China) were followed for all in vivo experimental procedures (Animal ethical code: 202211A011, November 7, 2022). Consent for publication All authors agree to this submission. Competing interests The authors have declared that no competing interests exist. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Yuan-Yuan Gao, Wen-Shuang Sun and Zi-Ying Sun contributed equally to this work. Contributor Information Jiang-Juan Shao, Email: [email protected]. Zi-Li Zhang, Email: [email protected]. 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