The impaired repair of intestinal mucosal damage is an important pathological feature of ulcerative colitis (UC). The critical role of intestinal epithelial cells (IECs) proliferation and migration in the repair of damaged mucosal epithelium has been well established. However, the molecular circuitry that decodes IECs sense intestinal mucosal damage signals to initiate and drive repair program remains elusive. Here, we identify a tryptophan (Trp) metabolic gatekeeping mechanism wherein G protein-coupled receptor 35 (GPR35) senses intestinal mucosal damage through monitoring Trp-kynurenine (KYN)-kynurenic acid (KA) axis metabolism with a unique “sandwich” structural binding mode. We delineate a GPR35-Kruppel-like factor 5 (KLF5) regulatory circuit in which KLF5 serves as the central effector, translating GPR35-mediated KA sensing into repair programming through PI3K-AKT-mTOR signaling cascade. This circuitry precisely orchestrates IECs proliferation and migration by regulating KLF5-dependent gene expression networks that essential for restoring damaged mucosa. Once this metabolic gatekeeping system is disrupted, either through impaired GPR35-mediated KA sensing or defective signal transduction, compromises damage signal decoding, leading to inadequate repair responses. Such dysregulation results in delayed intestinal mucosal repair and exacerbation of tissue damage. Our findings highlight GPR35 as a surveillant of abnormal Trp-KYN-KA axis metabolism, enabling IECs to detect intestinal mucosal damage and orchestrate repair through KLF5 response. This provides important implications for UC prevention and treatment by targeting GPR35-KLF5 circuit.

Chronic kidney disease (CKD) is a major global health burden, with renal fibrosis as the key pathological driver of disease progression. Fucoidan (FPS) is a sulfated polysaccharide from brown algae that has demonstrated renoprotective and anti-fibrotic properties. However, its metabolic and microbiota-related mechanisms remain unclear. Here, we evaluated the efficacy of FPS in a unilateral ureteral obstruction (UUO) mouse model, in which mice received oral FPS (100 or 200 mg kg-1 day-1) for 14 days. Kidney metabolomics and 16S rRNA sequencing of the gut microbiota were performed. As a result, UUO induced pronounced disturbances in tryptophan metabolism and marked gut microbial dysbiosis. FPS treatment attenuated renal fibrosis, normalized key tryptophan pathway intermediates and related metabolic enzymes in the kidneys, and partially restored the gut microbial composition. These results implicate the modulation of tryptophan metabolism and the gut microbiota in the anti-fibrotic effects of FPS, supporting its potential as a natural therapeutic candidate for renal fibrosis and CKD.

Acylcarnitines (ACs) are a diverse class of fatty acid esters of L-carnitine that serve as critical mediators in energy homeostasis and mitochondrial function. Beyond their classical role in newborn screening for inborn errors of metabolism, ACs have emerged as promising biomarkers for complex pathologies, including cardiovascular diseases, diabetes, and drug-induced toxicities. However, the accurate quantification and comprehensive profiling of ACs in biological matrices remain analytically challenging due to their broad polarity range, vast concentration disparities, and the presence of isomers. This review provides a comprehensive overview of the current bioanalytical strategies for ACs, covering sample preparation techniques and detection platforms, with a focus on liquid chromatography-mass spectrometry. Special attention is given to the differentiation of isomers. Finally, we discuss the clinical applications of ACs profiling and highlight future perspectives, including the integration of ion mobility spectrometry, automated high-throughput workflows, spatial and single-cell metabolomics, and AI-driven analytics, to pave the way for precision medicine.

An-Gong-Niu-Huang Wan (AGNHW) is a traditional Chinese medicine preparation containing cinnabar (HgS). However, its clinical use has been controversial due to concerns regarding mercury nephrotoxicity. To evaluate the renal toxicity, mice were administered AGNHW, cinnabar, methylmercury (MeHg), and mercuric chloride (HgCl₂) for 28 days. Blood biochemistry, histopathology, and the expression of nephrotoxicity-sensitive genes were assessed. To assess the health risks associated with AGNHW, we established an innovative Physiologically Based Extraction Test (IPBET) /small intestinal organ-on-a-chip model for the first time to simulate gastrointestinal digestion and absorption of mercury from AGNHW. This organ-on-a-chip model with HPLC-ICP-MS enabled the first determination of bioavailable mercury species in AGNHW. Urinary creatinine (UCr), total protein (TP), kidney injury molecule-1 (Kim-1), α1-microglobulin (a1-MG), N-cadherin levels, and histological findings in the MeHg and HgCl₂ groups showed significant differences compared to the control, cinnabar, and AGNHW groups. The expression of F4/80, CD11b, and MMP7 was markedly altered in the MeHg group. Our organ-on-a-chip model revealed that bioavailable MeHg in the MeHg group was 298-fold and 27-fold higher compared to the control and AGNHW groups. Our refined risk assessment indicated that, when adjusted by bioavailability and speciation, the health risks associated with mercury exposure from AGNHW consumption were acceptable. Collectively, considering bioavailability and mercury species, our IPBET/small intestinal organ-on-a-chip provides a novel approach for assessing cinnabar exposure risks in AGNHW. Our study provides a scientific basis that AGNHW and cinnabar are much less nephrotoxic than HgCl2 and MeHg, and does not pose a significant threat to human health clinically.

The use of irinotecan (CPT-11), a first-line chemotherapeutic agent for colorectal cancer (CRC), is limited by acquired drug resistance and severe adverse effects. It has been found that activation of farnesoid X receptor (FXR) signaling reduced the growth of CRC. Here, we investigated the combination of obeticholic acid (OCA), a well-characterized FXR agonist, with CPT-11 as a potential therapeutic combination for CRC. In vitro experiments demonstrated that the OCA-CPT-11 synergistically inhibited proliferation and migration in FXR-high HT-29 and SW620 cells, but exhibited minimal synergy in FXR-low HCT116 and Caco-2 cells. Genetic knockdown of FXR in HT-29 cells attenuated the synergistic effects, whereas FXR overexpression in HCT116 cells enhanced them. These findings indicate that the synergistic effect of OCA-CPT-11 depends on basal FXR expression levels, highlighting the need to identify both FXR inducers and agonists. Through literature and database screening, silibinin (SB) and nigakinone (Nig) were identified to upregulate and activate FXR. Both SB-CPT-11 and Nig-CPT-11 exhibited strong synergistic anti-CRC effects in vitro, even under low FXR conditions. Nig achieved synergy with CPT‑11 at lower effective concentrations than SB, so it was prioritized as the lead candidate for in vivo evaluation. Consistently, the Nig-CPT‑11 combination synergistically inhibited the growth of HCT116 subcutaneous xenograft tumors in vivo. Notably. In summary, combining FXR inducers and agonists with CPT-11 offers a promising strategy for the treatment of CRC.

Cisplatin (DDP) is a widely used chemotherapeutic agent, but its clinical application is limited by dose-dependent nephrotoxicity. Although metabolic dysregulation is a hallmark of DDP-induced acute kidney injury (AKI), the specific changes in fatty acid oxidation (FAO)-associated metabolic programs and the enzymes linking metabolic disturbances to cell death remain incompletely defined. In this study, targeted metabolomic profiling of the kidney revealed a marked blockade of FAO, evidenced by the accumulation of fatty acids and a decrease in downstream acylcarnitines. Among FAO-related enzymes, acyl-CoA synthetase short-chain family member 2 (ACSS2) emerged as the most significantly downregulated enzyme, which was further confirmed in an ischemia/reperfusion AKI model. ACSS2 overexpression in HK-2 cells aggravated DDP-induced inflammation and apoptosis, whereas ACSS2 knockdown significantly reduced cytotoxicity and pro-inflammatory cytokine production. Furthermore, pharmacological inhibition of ACSS2 in vivo alleviated DDP-induced AKI characterized by reduced oxidative stress and improved renal function. Together, these findings indicate that targeting ACSS2 may represent a promising therapeutic strategy to mitigate DDP-induced renal injury.

Liver fibrosis can progress to cirrhosis and hepatocellular carcinoma, yet effective antifibrotic therapies remain limited. Fibroblast activation protein α (FAPα) is a promising biomarker and therapeutic target, but existing probes are constrained by limited tissue penetration and single-modality imaging, and effective FAPα-targeted antifibrotic agents remain lacking. Here we show that Cs-FAP, a hemicyanine-based near-infrared fluorescence/photoacoustic dual-modal probe, enables real-time and noninvasive visualization of FAPα activity in fibrotic liver tissue. Using a Cs-FAP-guided screening platform integrating high-content imaging and chromatographic purification, we screened 95 medicinal herbs and identified sappanchalcone (Sap) as a potent low-toxicity FAPα inhibitor. Sap markedly attenuated fibrosis progression and improved liver function in multiple liver fibrosis models using male mice. Mechanistically, Sap suppressed hepatic stellate cell activation through inhibition of the FAPα-PI3K-AKT signaling pathway. These findings establish Cs-FAP as a versatile platform for fibrosis imaging and drug discovery and identify Sap as a promising antifibrotic candidate.

Lactylation is a widespread protein modification with important regulatory roles in health and disease. Emerging evidence also links metabolite lactylation to disease onset, progression, and treatment response. It is estimated that an uncharacterized pool of lactoyl-metabolites exists in vivo, according to its formation mechanism. To mine this pool, we combined knowledge-driven prediction with stable isotopic labeling-based high-resolution mass spectrometry (SIL-HRMS). A predictive library of 151 lactoyl-metabolites was generated by expanding 20 basic amino acids (AAs) to their upstream and downstream metabolites. To improve detection, we developed a pair of new hydroxyl-reactive labeling reagents, 3,5-(dimethylamino)-2,4,6-triazine benzene-1-chlorine (Tmt-aycl-Cl) and its deuterium form d12-Tmt-aycl-Cl. Using HRMS, 95 lactoyl-metabolites were putatively identified across 14 tissue types, 64 structures of which are reported for the first time. Representative lactoyl-metabolites were chemically synthesized and characterized to verify some of the annotations. We then developed a pseudotargeted metabolomics workflow for semiquantitative profiling in a diabetic kidney disease (DKD) mouse model. DKD mice showed elevated lactate in serum and the kidney and widespread dysregulations of lactoyl-metabolites. Many lactoyl-metabolites correlated strongly with the urinary albumin-to-creatinine ratio, suggesting biomarker potential for DKD. This integrated strategy expands the catalog of lactoyl-metabolites and provides a platform for investigating their biological significance.

ETHNOPHARMACOLOGICAL RELEVANCE:Dachaihu Decoction (DCHD) is a traditional Chinese medicine formula from the Shanghan Lun. It has been used for over 1800 years to treat "Shaoyang and Yangming concurrent syndromes", which are closely related to modern liver disorders, including metabolic dysfunction-associated steatohepatitis (MASH). While DCHD has demonstrated hepatoprotective effects in preclinical studies, the specific bioactive components and mechanism of action against MASH remain elusive. AIM OF THE STUDY:To systematically identify the key active components of DCHD mediating its anti-MASH effects and to elucidate their molecular mechanisms. MATERIALS AND METHODS:The components in DCHD were characterized using high resolution-mass spectrometry (HRMS). The potential bioactive components were screened using network pharmacology. The therapeutic efficacy of candidate compounds was evaluated both in free fatty acid (FFA)-induced hepatocyte injury and activated hepatic stellate cells (HSCs) and in a high-fat diet/CCl4-induced MASH mouse model. The molecular mechanism was investigated via transcriptomic profiling, drug-target interaction validation, and gene knockdown assays. RESULTS:A total of 128 components were identified from DCHD using HRMS. Lonicerin (LON), a flavonoid glycoside predominantly derived from Citrus trifoliata L., was identified as the key active component. LON significantly ameliorated steatosis, inflammation and fibrosis both in vitro and in vivo. Mechanistically, LON bound to lymphocyte antigen 6 family member D (LY6D), which was significantly upregulated during MASH progression, and dose-dependently suppressed its expression. Importantly, LY6D knockdown abolished the protective effect of LON in AML12 hepatocytes and activated HSCs, confirming LY6D as a functional target. CONCLUSIONS:This study identified LON as a key bioactive component mediating the anti-MASH effects of DCHD and revealed that it alleviated MASH progression by targeting LY6D.

Cisplatin (DDP) is a widely used chemotherapeutic agent, but its clinical application is limited by dose-dependent nephrotoxicity. Although metabolic dysregulation is a hallmark of DDP-induced acute kidney injury (AKI), the specific changes in fatty acid oxidation (FAO)-associated metabolic programs and the enzymes linking metabolic disturbances to cell death remain incompletely defined. In this study, targeted metabolomic profiling of the kidney revealed a marked blockade of FAO, evidenced by the accumulation of fatty acids and a decrease in downstream acylcarnitines. Among FAO-related enzymes, acyl-CoA synthetase short-chain family member 2 (ACSS2) emerged as the most significantly downregulated enzyme, which was further confirmed in an ischemia/reperfusion AKI model. ACSS2 overexpression in HK-2 cells aggravated DDP-induced inflammation and apoptosis, whereas ACSS2 knockdown significantly reduced cytotoxicity and pro-inflammatory cytokine production. Furthermore, pharmacological inhibition of ACSS2 in vivo alleviated DDP-induced AKI characterized by reduced oxidative stress and improved renal function. Together, these findings indicate that targeting ACSS2 may represent a promising therapeutic strategy to mitigate DDP-induced renal injury.

Multidrug resistance (MDR) is a significant challenge in cancer treatment, with limited effective strategies available. Neuropilin-1 (NRP1) is emerging as a potential therapeutic target for overcoming drug resistance, but its role in MDR and the identification of potential inhibitors require further exploration. In this study, we investigated the role of NRP1 in MDR and identifies potential inhibitors targeting NRP1. Elevated NRP1 expression was observed in oxaliplatin (OXP)-resistant HCT116 (HCT116/L) and cisplatin (DDP)-resistant A549 cells (A549/DDP). Virtual screening and biological assays identified pitavastatin (Ptv) as a potent NRP1 inhibitor that restored chemosensitivity in resistant cells both in vitro and in vivo. Mechanistic studies revealed that Ptv directly binds to NRP1, promotes degradation of Zinc finger X-chromosomal protein (ZFX), and disrupts the NRP1-ZFX axis to reverse MDR. This study provides promising prospects for targeting the NRP1-ZFX axis as a therapeutic strategy for MDR and highlights the potential clinical application of Ptv in diseases involving NRP1.

Pulmonary hypertension (PH) is a deadly disease with limited treatment options and poor long-term survival, necessitating the discovery of novel therapeutics. Our previous study has revealed that dimeric proanthocyanidins (PACs) mainly existed in the ethyl acetate extract from the roots of Ephedra sinica Stapf (ERE), however, its therapeutic effects on SU5416/hypoxia-induced pulmonary hypertension (PH) rats remain elusive. In this study, column chromatography combined with UPLC-LTQ-Orbitrap-HRMS analysis was performed to comprehensively characterize polyphenols in ERE. The therapeutic effects of ERE were investigated using the SU5416/hypoxia rat model, in which the rats were injected with SU5416 (20 mg/kg), followed by a three-week hypoxia exposure (10% O2). Hemodynamic indicators determined by right heart catheterization, pulmonary arterial morphological changes assessed by histopathological analysis, cardiac function and pulmonary hemodynamics using echocardiography, as well as oxidative stress markers measured by corresponding kits were used to test the therapeutic effects of ERE. Moreover, 16S rRNA sequencing combined with untargeted metabolomics was employed to capture changes in gut microbiota and serum metabolites after ERE treatment. Comprehensive chemical analysis of polyphenols in ERE revealed various levels of proanthocyanidin monomers, dimers and trimers, especially A-type dimers. In vivo experiments showed that ERE decreased pulmonary arterial pressure, right ventricular hypertrophy, right ventricular free wall (RVFW) thickness and oxidative stress levels, increased pulmonary acceleration time (PAT) and alleviated pulmonary vascular remodeling in rats exposed to SU5416/hypoxia treatment. Meanwhile, ERE improved gut microbial dysbiosis and the disturbed glycerophospholipid metabolism. Collectively, this study presents the first report on the efficacy of A-type PACs from Ephedra sinica for the treatment of PH through regulating gut microbiota and host metabolism.

Cisplatin (DDP) is widely utilized in the clinical treatment of malignant tumors, but its effectiveness is significantly compromised by the adverse effects of acute kidney injury (AKI). Renal tubular cells are primarily responsible for DDP-induced AKI (DDP-AKI); however, the responses of heterogeneous renal tubular cells to DDP exposure have not been thoroughly explored. In this study, we employed a targeted metabolomics approach to investigate the metabolic responses of renal tubular cells in DDP-AKI rats. Tubular cells were isolated from the renal cortex and outer medulla, and a chemical derivatization-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method was applied. Our findings revealed distinct metabolic profiles in tubular cells from the renal cortex and outer medulla, with outer medullary cells exhibiting greater sensitivity to DDP exposure. Further analyses identified the tryptophan pathway as a critical factor contributing to these regional differences. Additional functional investigations showed that intermediate metabolites of the tryptophan pathway alleviated DDP cytotoxicity in both cortical and outer medullary tubular cells primarily through modulation of the Bcl2/Bax and Caspase-3 pathway. This study enhances our understanding of the metabolic characteristics of tubular cells across heterogeneous renal regions in DDP-AKI and facilitates further exploration of the underlying mechanisms of DDP-induced nephrotoxicity.

Astragali Radix (AR), a traditional Chinese medicine (TCM), has demonstrated therapeutic efficacy against various diseases, including cardiovascular conditions, over centuries of use. While doxorubicin serves as an effective chemotherapeutic agent against multiple cancers, its clinical application remains constrained by significant cardiotoxicity. Research has indicated that AR exhibits protective properties against doxorubicin-induced cardiomyopathy (DIC); however, the specific bioactive components and underlying mechanisms responsible for this therapeutic effect remain incompletely understood. This investigation seeks to identify the protective bioactive components in AR against DIC and elucidate their mechanisms of action. Through network medicine analysis, astragaloside IV (AsIV) and formononetin (FMT) were identified as potential cardioprotective agents from 129 AR components. In vitro experiments using H9c2 rat cardiomyocytes revealed that the AsIV-FMT combination (AFC) effectively reduced doxorubicin-induced cell death in a dose-dependent manner, with optimal efficacy at a 1∶2 ratio. In vivo, AFC enhanced survival rates and improved cardiac function in both acute and chronic DIC mouse models. Additionally, AFC demonstrated cardiac protection while maintaining doxorubicin's anti-cancer efficacy in a breast cancer mouse model. Lipidomic and metabolomics analyses revealed that AFC normalized doxorubicin-induced lipid profile alterations, particularly by reducing fatty acid accumulation. Gene knockdown studies and inhibitor experiments in H9c2 cells demonstrated that AsIV and FMT upregulated peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) and PPARα, respectively, two key proteins involved in fatty acid metabolism. This research establishes AFC as a promising therapeutic approach for DIC, highlighting the significance of multi-target therapies derived from natural herbals in contemporary medicine.

Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder with an increasing incidence worldwide. Due to the complex and unclear therapeutic targets, unmet UC therapeutic drugs still exist. Recently, acylcarnitine metabolism disorder has been linked to intestinal inflammation, but its role in UC remains elusive. According to our preliminary non-targeted metabolomics data, acylcarnitines (ACs) was screened as the disturbed metabolites in the different intestinal inflammation-related diseases. Here we quantified 26 ACs within liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the dextran sulfate sodium (DSS)-induced UC rat model, and found that long-chain acylcarnitines (LCACs) were increased to varying degrees. As the key metabolites of fatty acid β-oxidation (FAO), the upstream metabolites long-chain fatty acids (LCFAs) and the related metabolic enzymes were further characterized, the results showed that the rate-limiting enzyme carnitine palmitoyltransferase 1A (CPT1A)-mediated LCFAs-LCACs metabolic axis was activated sharply. Next in vitro experiments exhibited that CPT1A was significantly upregulated in both inflammatory macrophages and colonic epithelial cells, and inhibition or knockdown of CPT1A could reduce the inflammation level remarkably. Thus, we screened the pharmacologic inhibitors of CPT1A from US Food and Drug Administration (FDA) approved drugs, within molecular docking, Western blot and cell membrane chromatography (CMC) technology, gliquidone was found to inhibit CPT1A in a dose-dependent manner and exert anti-inflammatory effects in vitro. Animal experiments also showed that gliquidone alleviated DSS-induced UC significantly. In summary, our study presents that within metabolomics analysis, inhibiting CPT1A is focused to be a potential therapeutic strategy against UC, and gliquidone represents an alternative treatment.

Acylated polyamines (acyl-PAs) are gaining significant attention due to their involvement in various diseases. However, their annotation and quantification remain challenging, as robust analytical methods are lacking, with only a few acyl-PAs characterized to date. In this study, we integrated prior-knowledge-guided prediction with chemical isotope labeling-based metabolomics to identify novel acyl-PAs. An in silico library of 267 predicted acyl-PAs was constructed. Using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) and paired labeling reagents (dansyl chloride and d6-dansyl chloride), we successfully annotated 41 acyl-PAs across diverse biological samples, 38 of which were novel. Representative acyl-PAs were synthesized for the validation of annotation. Furthermore, we developed a pseudotargeted metabolomic approach for the semiquantification of acyl-PAs in a mouse model of ulcerative colitis (UC), revealing significant changes in 13 acyl-PAs in the feces or colon samples from UC mice. Notably, an antibiotic-treated mouse model revealed that gut microbiota significantly influenced the abundance of acyl-PAs. This study introduces a comprehensive workflow for discovering novel metabolites and provides valuable insights into the roles of acyl-PAs in health and disease.

Environmental pollutants can induce multiorgan damage, with the digestive tract particularly susceptible. Diabetic enteropathy is a significant complication of type 2 diabetes mellitus (T2D). However, the relationship between environmental pollutant exposure and T2D-associated intestinal injury has not been previously explored. In this study, T2D mice were subjected to polystyrene microplastics (PS-MPs, 100 μg/day, 3 weeks) and bisphenol A (BPA, 100 μg/kg/day, 2 weeks). Metabolomics and 16S rRNA sequencing were used to detect changes in colonic metabolites and gut microbial composition. Caco-2 cells were utilized to investigate the functions of the altered metabolites. Compared to the T2D group, mice exposed to PS-MPs and BPA exhibited shorter colon length and reduced levels of gut barrier proteins ZO-1 and Occludin. Metabolomics analysis revealed that PS-MPs primarily affected colonic long-chain fatty acids (LCFAs) and adenosine metabolism, while BPA disrupted α-ketoisovaleric acid (KIVA) and pyruvic acid (PyrA) homeostasis. Moreover, PS-MPs exposure altered the abundance of Duncaniella and Olsenella, while BPA primarily affected Phocaeicola, Olsenella, and Variovorax. In vitro experiments showed that palmitoleic acid (C16:1), γ-linolenic acid (C18:3), adenosine (Ado), and KIVA promoted the expression of ZO-1 in Caco-2 cells. Our findings provide valuable insights into the impact of environmental pollutants on intestinal injury in T2D, underscoring the importance of environmental contaminant management, particularly in susceptible populations.

BACKGROUND:Irinotecan (CPT-11) is a standard first-line chemotherapy treatment for colorectal cancer (CRC). However, its clinical application is often comprised by gastrointestinal toxicity and limited therapeutic efficacy. Our previous study has revealed that the combination of Vardenafil (Vard) and Linagliptin (Linag) significantly alleviated CPT-11-induced intestinal toxicity in both in vitro and in vivo models. It remains unclear whether this combination can synergistically enhance the anticancer activity of CPT-11. METHODS:The in vitro synergism of Vard, Linag, and CPT-11 was assessed using cell viability assays on CRC cell lines, including HCT116, SW620, and HT29. An in vivo xenograft mouse model was established to evaluate the drug efficacy of both the original and liposomal forms of Vard and Linag combined with CPT-11. Additionally, untargeted metabolomics was utilized to explore the potential mechanisms underlying the observed synergistic effects. RESULTS:In vitro, the combination of Vard and Linag synergistically enhanced the anticancer activity of CPT-11 in CRC cell lines. In vivo, liposomal formulations of Vard and Linag tended to accumulate at the tumor site, improving drug targeting and synergistically enhancing the anticancer efficacy of CPT-11. Untargeted metabolomics analysis revealed that this synergistic effect was probably mediated through the regulation of lysophospholipid metabolism. CONCLUSION:Liposomal Vard and Linag combined with CPT-11 demonstrated a synergistic anti-CRC effect, offering valuable insights into novel combination therapies in CRC treatment.

Lung adenocarcinoma (LUAD), the predominant subtype of non-small cell lung cancer (NSCLC), poses significant therapeutic challenges due to its aggressive nature and limited treatment options. Cisplatin is a commonly used chemotherapeutic agent for advanced LUAD, often encounters PDR, leading to rapid disease progression and tumor recurrence. In this study, we demonstrate that LDHA expression is elevated in cisplatin-resistant LUAD cell lines and correlates with poor patient prognosis. Notably, inhibiting the expression of LDHA significantly enhances cisplatin sensitivity. Mechanistically, LDHA functions through non-metabolic enzymatic activity to promote the ubiquitination and degradation of AMBRA1 by facilitating its interaction with RNF2, thereby activating Cyclin D1 and BCL2, which drives cisplatin resistance. Importantly, we identified two LDHA inhibitors, triflupromazine (TRI) and tranylcypromine (TRA), that significantly improve cisplatin efficacy both in vitro and in vivo. These findings highlight a critical role of LDHA in promoting cisplatin resistance, and suggest that targeting LDHA may represent a promising therapeutic strategy for patients with advanced LUAD.