The widespread use of aspartame has raised increasing health concerns, particularly regarding its potential endocrine-disrupting effects and a possible association with polycystic ovary syndrome (PCOS), a condition marked by endocrine and metabolic abnormalities. This study systematically investigated the molecular mechanisms by which aspartame may contribute to PCOS pathogenesis using a network toxicology approach. A total of 281 common targets between aspartame and PCOS were identified. Protein-protein interaction network analysis and five topological algorithms revealed 20 hub targets significantly enriched in pathways such as endocrine resistance, TNF signaling, and progesterone-mediated oocyte maturation. Eight of these targets showed significant differential expression in PCOS patients. Weighted gene co-expression network analysis (WGCNA) further demonstrated that several of these hub targets are embedded in co-expression modules positively associated with PCOS phenotypes. Transcription factor prediction indicated that these targets are primarily regulated by FOS and JUN. Molecular docking showed favorable binding affinities between aspartame and both transcription factors. Subsequent molecular dynamics simulations supported the dynamic stability of aspartame binding to FOS and JUN. These findings suggest that aspartame may contribute to PCOS by modulating FOS- and JUN-mediated transcriptional programs, thereby disrupting biological pathways related to endocrine regulation and inflammation. Collectively, this study reveals a potential mechanism by which aspartame mediates PCOS and offers novel insights for toxicological assessment of food additives.

Oestrogen receptor (ER) activation leads to the formation of DNA double strand breaks (DSB), promoting genomic instability and tumour heterogeneity. The single-stranded DNA cytosine deaminase APOBEC3B (A3B) serves as a co-activator of ER and is implicated in inducing DSBs at transcriptional enhancers regulated by ER. Using whole-genome sequencing in an engineered cell model lacking base excision repair (BER) function, we demonstrate that A3B preferentially targets transcriptionally active regulatory regions in an R-loop-dependent manner. Strand-specific DNA:RNA immunoprecipitation sequencing (ssDRIP-seq) and ssDNA-associated protein immunoprecipitation sequencing (SPI-seq) confirm that A3B binds to and deaminates ssDNA within R-loops, a process facilitated by ER transactivation. Furthermore, BER-mediated processing of A3B-induced uracil bases contributes to the formation of R-loop-associated DSBs, which are essential for ER-regulated gene activation. These findings establish a role for A3B in R-loop homeostasis and transcriptional regulation, with implications for understanding ER-driven genomic instability and potential therapeutic targeting of A3B.

Rationale:Inflammatory bowel disease (IBD), known for its complexity and frequent relapses, urgently demands novel therapeutics due to the limited efficacy of current treatments. Cinnamaldehyde (CMA), a bioactive compound derived from Cinnamomum cassia Presl, has exhibited therapeutic potential for IBD. However, the therapeutic mechanism of CMA remains incompletely elucidated, and clinical translation is hampered by its poor oral pharmacokinetics. Methods:Using RAW 264.7 cells stimulated with either LPS or IL-4, we evaluated the effects of CMA on macrophage polarization. Subsequently, the impact of CMA on glucose metabolism in M1 macrophages was analyzed. RNA sequencing identified the signaling pathways through which CMA inhibits M1 macrophage polarization, and this was further validated through genetic or pharmacological blockade. To overcome the pharmacokinetic challenges of CMA, macrophage membrane-biomimetic CMA-loaded nanoparticles (MM@CMANP) were designed, and their pharmacokinetics and targeting to intestinal inflammation sites were evaluated. Finally, the efficacy of MM@CMANP was assessed in DSS-induced IBD mice. Results:CMA suppresses M1 macrophage polarization in vitro. Notably, CMA disrupted M1 macrophage glucose metabolic reprogramming, characterized by glycolysis suppression and enhanced oxidative phosphorylation. RNA sequencing demonstrated a clear association with mitophagy pathway following CMA treatment, and mechanistic studies verified that CMA promotes BCL2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3)-mediated mitophagy activation. Crucially, CMA-induced inhibition of M1 macrophages was mitigated by BNIP3 knockdown or autophagy inhibitors. MM@CMANP enhanced CMA accumulation in inflamed colonic tissues. In IBD mice, MM@CMANP significantly alleviated epithelial barrier disruption and mucosal inflammation. Consistent with in vitro findings, CMA modulated macrophage polarization and autophagy in vivo. Conclusions:These results establish mitophagy as a central mechanism underlying anti-IBD effects of CMA and position MM@CMANP as a clinically translatable nanotherapeutic platform for IBD.

Osteoarthritis (OA) is a degenerative joint disorder characterized by cartilage degradation and synovial inflammation, with its pathogenesis involving intricate interactions between cellular metabolic processes and inflammatory pathways. In this study, we established OA models in both wild-type and histone deacetylase 6 (HDAC6) knockout mice via anterior cruciate ligament transection (ACLT) to investigate the role of HDAC6 in OA progression. Histological and immunohistochemical analyses revealed that the absence of HDAC6 significantly mitigated ACLT-induced cartilage damage and synovitis in mice, while also restoring imbalances in cartilage metabolic markers. In vitro experiments demonstrated that, upon stimulation with inflammatory factors IL-1β or TNF-α, neither HDAC6 overexpression nor knockdown significantly altered the metabolic processes of chondrocytes, suggesting that HDAC6 may not directly regulate chondrocyte metabolism in an inflammatory context. However, conditioned medium from macrophages overexpressing HDAC6 promoted catabolic processes in chondrocytes and inhibited anabolic processes, whereas the knockdown of HDAC6 reversed this effect. These findings imply that HDAC6 contributes to the progression of OA by amplifying macrophage-mediated synovial inflammation, thereby indirectly disrupting the metabolic homeostasis of chondrocytes. In conclusion, these results suggest that targeting HDAC6 may provide a novel strategy for alleviating OA pathology by modulating macrophage-chondrocyte interactions.

Flavonoid glycosides exhibit compromised bioavailability due to low membrane permeability. To address this limitation, we acetylated flavonoids through enzymatic reactions to increase bioavailability. This study first reported that Hesperetin-7-O-glucoside (Hes-7-G) was acetylated by galactoside acetyltransferase (GAT), and the apparent permeability (Papp) of the Caco-2 monolayer was increased by 69

Positron emission tomography (PET) is a common imaging technique and can provide accurate information about the size, shape, and location of tumors. Recent evidence has shown that G-quadruplex structures (G4s) are identified in human oncogenes, and these special structures are recognized as diagnostic cancer markers and drug targets for anticancer therapies. Although a number of techniques for in vivo imaging of G4s have been developed, achieving sufficient sensitivity and selectivity in vivo remains challenging. Herein, we have engineered and developed a radiolabeled peptide probe [18F]AlF-NOTA-RHAU18 targeting mitochondrial DNA G4s for in vivo PET imaging. The results of the study indicate that this probe is able to visualize and detect solid tumors in living homozygous mice. In addition, the distribution of the probe in cancer cells was investigated using FITC-RHAU18. This work may offer new insights into the development of cancer diagnostic tools by targeting in vivo G4s.

BACKGROUND AND PURPOSE:Regulation of mitochondrial calcium overload and ferroptosis with mitochondria-targeting ligands is an attractive anticancer strategy but it remains a challenge. The aim of the present study was to demonstrate that a mitochondria-targeting and mtDNA G-quadruplex-binding ligand, BYB, induced mitochondrial calcium overload and ferroptosis in HeLa cells and showed potent in vitro and in vivo anticancer activity. EXPERIMENTAL APPROACH:Cellular functions and molecular mechanism were studied using cell viability assay, live-cell imaging, western blotting, immunofluorescence, cell uptake, cell cycle arrest and apoptosis analysis, mitochondrial metabolism analysis, Comet assay, and wound-healing analysis. Pharmacokinetic studies were conducted in rat. In vivo antitumor activity was studied in a cervical cancer HeLa cell xenograft mouse model. KEY RESULTS:Cellular results showed that BYB induced mitochondrial calcium overload, attributed to ligand-induced mitochondrial dysfunction via the mechanism of inhibiting mitochondrial DNA replication and transcription. The expression of respiratory chain complexes was markedly downregulated in BYB-treated HeLa cells. The respiratory chain function was also dysregulated. Mitophagy and mitochondrial calcium overload were induced in BYB-treated HeLa cells. Mitochondrial calcium overload markedly induced mtROS production. The induced mtDNA stress activated cGAS-STING pathway, leading to autophagy-dependent ferroptosis. The antitumour efficacy of BYB, evaluated in a HeLa tumour xenograft mouse model, achieved over 60% tumour weight reduction. CONCLUSION AND IMPLICATIONS:BYB, via targeting mitochondria and mtDNA G-quadruplexes, induced mitochondrial calcium overload and ferroptosis, exhibited high in vivo antitumour efficacy and low toxicity. It shows high potential to be a mitochondria-targeting lead compound for chemical biology and drug discovery.

Oocyte maturation-coupled mRNA post-transcriptional regulation is essential for the establishment of developmental potential. Previously, oocyte mRNA translation efficiencies focused on the trans-regulation of key RNA-binding protein (RBPs), rarely related to RNA structure. RNA G-quadruplexes (rG4s) are four-stranded RNA secondary structures involved in many different aspects of RNA metabolism. In this study, we have developed a low-input technique for rG4 detection (G4-LACE-seq) in mouse oocytes and found that rG4s were widely distributed in maternal transcripts, with enrichment in untranslated regions, and they underwent transcriptome-wide removal during meiotic maturation. The rG4-selective small-molecule ligand BYBX stabilized rG4s in the oocyte transcriptome and impaired spindle assembly and meiotic cell cycle progression. The proteomic spectrum results revealed that rG4 accumulation weakened the binding of a large number of RBPs to mRNAs, especially those associated with translational initiation. Ribosomal immunoprecipitation and translational reporter assays further proved that rG4s in the untranslated regions negatively affected the translational efficiency of key maternal mRNAs. Overexpression DEAH/RHA family helicase-36 partially reverses BYBX-induced oocyte developmental defects, suggesting its importance in rG4 regulation. Collectively, this study describes the distribution, dynamic changes, and regulation of rG4s in the mouse maternal transcriptome. Before meiosis resumption, a large number of rG4s in oocytes are necessary to maintain the translatome at a low level, and DHX36-mediated rG4 removal promotes a translational switch and is required for successful maternal-to-zygotic transition.

Sclerostin (SOST) serves as a pivotal negative regulator of bone formation, and its dysregulated expression is implicated in the pathogenesis of skeletal disorders, including osteoporosis. While substantial progress has been made in elucidating the intracellular signaling pathways activated by SOST, the molecular mechanisms governing its expression remain less well understood. In this study, we identify an RNA G-quadruplex (RG4) secondary structure within the 3' untranslated region (3' UTR) of SOST. Utilizing biophysical and fluorescent probe analyses, we demonstrate that the SOST RG4 can form a typical parallel RG4 folding topology structure. Functional analyses reveal that the SOST RG4 acts as a crucial positive regulator of cellular SOST expression. Mechanistically, we show that the RG4 structure interferes with the binding of miR-4648 to the SOST 3' UTR, thereby enhancing SOST expression. Furthermore, this regulatory function is potentiated by the RG4-stabilizing ligand, Pyridostatin. Collectively, our findings highlight the biological significance of the RG4 structure in regulating SOST expression and suggest novel strategies for the post-transcriptional modulation of this essential gene in bone biology.

ETHNOPHARMACOLOGICAL RELEVANCE:Osteoporosis is a chronic metabolic bone disorder characterized by excessive bone resorption. The NuanXin Formula (NX) is a classical traditional Chinese medicine formula that can warm and tonify kidney Yang, as well as replenish Qi and blood, which are essential for maintaining bone health and regulating bone metabolism. Nevertheless, the functions and mechanisms of NX in osteoporosis therapy remain unclear. AIM OF THE STUDY:This study aims to evaluate the effects and mechanisms of NX on osteoclastogenesis and to investigate its potential in combating osteoporosis. MATERIALS AND METHODS:The inhibitory effects of NX on RANKL-induced osteoclastogenesis were evaluated using Western blotting, quantitative PCR (Q-PCR), TRAP staining, and pit-formation assays. Bone mass and structure were assessed through micro-CT, biomechanical testing, TRAP staining, IHC staining, and H&E staining. The mechanism of action of NX on osteoclasts was investigated using RNA sequencing, ROS staining, ATP measurement, and mitochondrial membrane potential assays. RESULTS:The in vitro findings demonstrated that NX treatment significantly inhibited osteoclast differentiation and bone resorption activity. Q-PCR and WB analyses indicated that NX substantially downregulates the expression levels of key osteoclast markers, including Nfatc1, Ctsk, Mmp9, and Trap. In vivo experiments revealed that intragastric administration of NX effectively suppressed bone loss and bone resorption, while enhancing the biomechanical properties of bone in ovariectomized (OVX) mice. Mechanistically, NX inhibits oxidative phosphorylation (OXPHOS), decreases mitochondrial membrane potential, and reduces ATP production and reactive oxygen species generation, thereby impeding osteoclast differentiation and activity. CONCLUSION:NX mitigates osteoporosis by modulating OXPHOS to inhibit osteoclast differentiation and activity, thus offering a potential therapeutic approach for osteoporosis management. However, the study has limitations that require further investigation. NX did not show a clear dose-dependent effect in animal tests, suggesting a need for improved dosing designs. Although we emphasize NX's therapeutic potential, more research is necessary to clarify its mechanism. Variability in plant materials and ingredient ratios might influence NX's pharmacological effects, with specific bioactive components potentially playing a major role. Future research should integrate network pharmacology with experimental validation for a more thorough understanding.

Inflammation is an important predisposing factor for many chronic diseases. The dietary flavonoid silibinin (SB) has excellent anti-inflammatory properties in cells, but its low bioavailability in the blood compromises its therapeutic potential. This study aims to investigate the potential of dibenzoylmethane (DBM) to synergistically enhance the anti-inflammatory benefits of SB. The synergistic effects of DBM and SB in combination were evaluated in lipopolysaccharide (LPS)-induced RAW264.7 cells and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mice. In addition, a network pharmacology approach and molecular docking were used to explore the key targets and signaling pathways of DBM and SB in combination. The results showed that DBM and SB synergistically inhibited the production of nitric oxide (NO), reactive oxygen species (ROS), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in a 1:1 concentration ratio. These two compounds may exert their synergistic effects by modulating the nuclear factor kappa-B (NF-κB) and HIF-1 signaling pathways, among others. Molecular docking revealed that both compounds exhibited high binding affinities to inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Compared with single-compound use, the two compounds in combination significantly reduced ear edema and inflammatory cell infiltration and inhibited the protein expression of iNOS and COX-2 in TPA-induced mice. This research provides a rationale for the combination of DBM and SB as an effective anti-inflammatory agent.

G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures formed by guanine-rich DNA or RNA sequences. These structures play pivotal roles in cellular processes, including DNA replication, transcription, RNA splicing, and protein translation. High-throughput sequencing has significantly advanced the study of G4s by enabling genome-wide mapping and detailed characterization. This review provides a comprehensive overview of current methods for G4 identification using high-throughput sequencing, focusing on key techniques such as G4-seq, G4-ChIP-seq, G4-CUT&Tag, LiveG4ID-seq, G4assess, HepG4-seq, rG4-seq, RT-stop profiling with DMS-m7G footprinting, G4RP-seq, Keth-seq, and SHALIPE-seq. We discuss the principles, advantages, limitations, and applications of these methods, highlighting their contribution to our understanding of G4 biology. The review also emphasizes the need for improved tools to explore the dynamic behavior of G4s, particularly in living organisms.

Semiconductor-mediated photodegradation (SMPD) is a tempting photocatalytic technique that utilizes visible light to decompose toxic organic pollutants but has often hit a bottleneck of poor mineralization. In this study, a triphasic interface is created by fabricating hydrophobic hollow TiO2 nanospheres (HB-HTS) to promote the generation of reactive oxygen species, such as •O2- and •OH, and thereby the kinetics of photo-oxidation reactions. Mechanistically, improving the surface hydrophobicity of hollow catalysts not only facilitates gas storage and augments the O2 supply, but also enhances surface adsorption of dye molecules via hydrophobic interactions, contributing concertedly to the faster photodegradation rate and higher degree of mineralization. Consequently, the optimal photocatalytic system based on HB-HTS, compared to the unmodified and uncarved controls, exhibits much faster kinetics of dye degradation up to 15-fold with almost complete mineralization. This study underlines the regulation of the reaction interfacial microenvironment to leverage the geometrical nanostructure and surface property of semiconductor nanoparticles for enhanced photocatalytic performance.

DHX36 plays a crucial role in regulating transcriptional and post-transcriptional processes through its interaction with G-quadruplexes(G4s).The mechanisms by which DHX36 regulates G4s vary across dif-ferent cell types and physiological conditions.Oocyte-specific conditional knockout(CKO)mice were uti-lized to study the impact of DHX36 deficiency on female fertility.The results show that the CKO mice exhibit severely impaired hormone response,ovulation,and complete infertility.The CKO germinal vesi-cle(GV)oocytes display large nucleoli,aberrant chromatin configuration,decreased chromatin accessibil-ity,disturbed transcriptome,and inhibited meiosis progression.Following fertilization,the embryos derived from the CKO oocytes arrest at the zygote or 2-cell stage.Notably,we observed inadequate rRNA transcription in growing GV oocytes,as well as insufficient pre-rRNA processing and translation activity in fully-grown GV oocytes.Using a G4 probe and antibody,we found increased G4s formation at the chromatin and cytoplasm of CKO GV oocytes;these G4s mainly originate from the rDNA and pre-rRNA.Furthermore,the distribution of DHX36 was found to be spatiotemporally synchronized with that of pre-rRNA and G4s in early mouse embryos.In vitro experiments confirmed that DHX36 directly binds with pre-rRNA through the RHAU-specific motif(RSM).Overexpression of DHX36 could partially alleviate the pre-rRNA accumulation in fully-grown CKO oocytes.In conclusion,this study highlights the physiological significance of DHX36 in maintaining female fertility,underscoring its critical role in rRNA homeostasis and chromatin configuration through G4-unwinding mechanism in mouse oocytes.

Polycystic ovary syndrome (PCOS) can lead to increased abortion rates. Quercetin treats PCOS, but its specific mechanism has not been fully clarified. PCOS was induced in mice by dehydroepiandrosterone, and decidualization was induced by corn oil. Mice were treated with quercetin, autophagy inhibitor 3-MA, autophagy inducer rapamycin, PI3K inhibitor LY294002, and PI3K inducer 740Y-P. Pathological damage in the ovary and uterus was observed by HE staining. The levels of sex hormones, metabolism, and inflammatory factors were detected using ELISA. The survival and decidualization of endometrial stromal cells were identified by immunohistochemistry, immunofluorescence, qRT-PCR, and Western blot. Autophagy and the PI3K/Akt/FoxO1 pathway-related protein levels were detected by Western blot. The theca cell layer and endometrium of PCOS mice were significantly thinner. The levels of sex hormone, pro-inflammatory factors, COX-2, integrin ανβ3, and autophagy-related proteins were obviously raised, while Vimentin, IGFBP-1, PRL, and PI3K/Akt/FoxO1 pathway expression were significantly decreased. The above indices were reversed considerably after quercetin treatment. 3-MA could reduce the level of autophagy, LY294002 could reduce the levels of PI3K/Akt/FoxO1 pathway, Vimentin, and PRL, and increase the level of autophagy. In conclusion, quercetin enhanced autophagy through the PI3K/Akt/FoxO1 pathway; thereby protecting endometrial stromal cells and improving decidualization disorders.

Cryptolepine, one important active ingredient of Cryptolepis sanguinolenta (Lour), exhibits various beneficial pharmacological activities. But its anti-alpha-glucosidase and hypoglycemic activities are still not cleared. Thence, in this study, we investigated its inhibitory effects on alpha-glucosidase by a multi-spectroscopic method and in vivo hypoglycemic activity by the oral glucose tolerance test (OGTT). The results showed that cryptolepine is a reversible mixed-type alpha-glucosidase inhibitor with IC50 value of 0.44 +/- 0.05 mM, similar to 2-fold stronger than positive control acarbose (IC50: 0.72 +/- 0.02 mM). Fluorescence quenching confirmed cryptolepine could quench the endogenous fluorescence of alpha-glucosidase in a static process. Fluorescence quenching, synchronous fluorescence, CD spectra, and 3D fluorescence results showed that the binding of cryptolepine with alpha-glucosidase caused chromophore microenvironment and conformation changes of alpha-glucosidase, consequently inhibiting its catalytic activity. Molecular docking revealed the detailed binding interactions between cryptolepine and alpha-glucosidase. Furthermore, oral administration of cryptolepine could improve the glucose tolerance in mice to reduce the postprandial hyperglycemia. These findings provide a solid foundation for understanding the inhibitory effects on alpha-glucosidase and hypoglycemic activity of cryptolepine.

Human telomerase reverse transcriptase (hTERT) may have noncanonical functions in transcriptional regulation and metabolic reprogramming in cancer cells, but it is a challenging target. We thus developed small-molecule ligands targeting hTERT promoter G-quadruplex DNA structures (hTERT G4) to downregulate hTERT expression. Ligand 5 showed high affinity toward hTERT G4 (K d = 1.1 μM) and potent activity against triple-negative breast cancer cells (MDA-MB-231, IC50 = 1 μM). In cell-based assays, 5 not only exerts markedly inhibitory activity on classical telomere functions including decreased telomerase activity, shortened telomere length, and cellular senescence but also induces DNA damage, acute cellular senescence, and apoptosis. This study reveals that hTERT G4-targeting ligand may cause mitochondrial dysfunction, disrupt iron metabolism and activate ferroptosis in cancer cells. The in vivo antitumor efficacy of 5 was also evaluated in an MDA-MB-231 xenograft mouse model and approximately 78.7% tumor weight reduction was achieved. No observable toxicity against the major organs was observed.

rRNAs are prevalent in living organisms. They are produced in nucleolus and mitochondria and play essential cellular functions. In addition to the primary biofunction in protein synthesis, rRNAs have been recognized as the emerging signaling molecule and drug target for studies on nucleolus morphology, mitochondrial autophagy, and tumor cell malignancy. Currently, only a few rRNA-selective probes have been developed, and most of them encounter the drawbacks of low water solubility, poor nuclear membrane permeability, short emission wavelength, low stability against photobleaching, and high cytotoxicity. These unfavorable properties of rRNA probes limit their potential applications. In the present study, we reported a new rRNA-selective and near-infrared fluorescent turn-on probe, 4MPS-TO, capable of tracking rRNA in live human cancer cells. The real-time monitoring performance in nucleolus morphology and mitochondrial autophagy is demonstrated in HeLa cells. The probe shows great application potential for being used as a rRNA-selective, sensitive, and photostable imaging tool in chemical biology study and drug screening.

Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 μM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

Gastrointestinal cancer is among the most common cancers worldwide. Immune checkpoint inhibitor-based cancer immunotherapy has become an innovative approach in cancer treatment; however, its efficacy in gastrointestinal cancer is limited by the absence of infiltration of immune cells within the tumor microenvironment. Therefore, it is therefore urgent to develop a novel therapeutic drug to enhance immunotherapy. In this study, we describe a previously unreported potentiating effect of Icariside I (ICA I, GH01), the main bioactive compound isolated from the Epimedium species, on anti-tumor immune responses. Mechanistically, molecular docking and SPR assay result show that ICA I binding with TRPV4. ICA I induced intracellular Ca2+ increasing and mitochondrial DNA release by targeting TRPV4, which triggered cytosolic ox-mitoDNA release. Importantly, these intracellular ox-mitoDNA fragments were taken up by immune cells in the tumor microenvironment, which amplified the immune response. Moreover, our study shows the remarkable efficacy of sequential administration of ICA I and anti-α-PD-1 mAb in advanced tumors and provides a strong scientific rationale for recommending such a combination therapy for clinical trials. ICA I enhanced the anti-tumor effects with PD-1 inhibitors by regulating the TRPV4/Ca2+/Ox-mitoDNA/cGAS/STING axis. We expect that these findings will be translated into clinical therapies, which will benefit more patients with cancer in the near future.