ABSTRACT Unidentified pathogen intestinal infections (UPIIs) represent a severe clinical dilemma, characterised by clear signs of intestinal infection yet no identifiable causative pathogen, often leading to prolonged, antibiotic‐refractory illness. A nationwide retrospective study based on the prospective cohorts from September 2015 to February 2025 was conducted in China to evaluate washed microbiota transplantation (WMT) on this challenging condition. Patients diagnosed with UPIIs and then underwent WMT were included. The primary outcome was the clinical response rate one month post‐WMT. Finally, among the 81 included patients, 71.6% were bedridden, 46.9% required ICU admission and 51.9% developed multiple organ dysfunction syndrome. Diarrhoea was the primary symptom, and over half received ≥ 3 empirical antibiotics. Despite the challenges, WMT achieved a one‐month clinical response rate of 63.0% and a cure rate of 43.2%. Multivariate analysis identified several baseline risk factors affecting WMT efficacy, including adverse events (AEs) related to WMT (β = 1.545, p = 0.026, OR = 4.690, 95% CI 1.208–18.206), total abdominal symptom scores (TASS) before WMT (β = 0.292, p = 0.047, OR = 1.340, 95% CI 1.004–1.788) and WHO performance status score ≥ 4 (β = 1.583, p = 0.031, OR = 4.867, 95% CI 1.160–20.423). The overall AEs rate was only 8.3% (18/216). A nomogram based on logistic regression [akaike information criterion (AIC) = 93.75] was developed to predict the clinical non‐response at one month after WMT. The favourable clinical outcomes observed in this study provide cohort‐based evidence on using WMT for treating refractory UPIIs. These findings implied that if WMT is available, earlier WMT may be beneficial for UPIIs.

The incidence of vulvar cancer has been increasing, particularly among younger women, yet prognostic tools specifically designed for early-onset vulvar cancer (EOVC) remain limited. Using the Surveillance, Epidemiology, and End Results (SEER) database, we identified 1386 patients with EOVC and developed prognostic models for overall survival (OS) and cancer-specific survival (CSS). Independent prognostic factors were identified using multivariable Cox regression, and nomograms were subsequently constructed and compared with five machine learning (ML) survival models, including random survival forest, gradient boosting machine, CoxBoost, LASSO-Cox, and survival support vector machine. Model performance was evaluated using the concordance index (C-index), time-dependent area under the curve (AUC), Brier score, calibration slope and intercept, and decision curve analysis (DCA). Age, marital status, histological type, tumor differentiation, lymph node status, SEER stage, primary tumor site, surgery, radiotherapy, and chemotherapy were independently associated with prognosis. The Cox-based nomograms demonstrated favorable discrimination, calibration, and greater net benefit than TNM staging. Although RSF and GBM showed competitive discriminative ability, no statistically significant improvement over the Cox-based nomogram was observed. In validation analyses, most ML models demonstrated reduced generalizability and less stable calibration, whereas the Cox-based model maintained more consistent predictive performance with lower Brier scores. In conclusion, we developed and validated clinically applicable nomograms for individualized survival prediction in EOVC. Compared with more complex ML approaches, the Cox-based nomogram showed robust performance, stability, and interpretability, supporting its potential clinical utility.

Circular RNAs (circRNAs) are stable, widespread regulatory molecules produced primarily by back-splicing of pre-mRNAs, with critical roles in gene regulation and disease pathogenesis. While significant progress has been made in circRNA identification, their targets remain largely unexplored. To address this, we present CircTarget, the first comprehensive database of experimentally supported circRNA-target RNA interactions mapped at single-nucleotide resolution. By analyzing RNA-RNA interactome data from multiple crosslinking methods across 13 cell lines and hippocampal tissues from human and mouse, we identified 132 517 high-confidence circRNA-target RNA interactions. Each entry in CircTarget is richly annotated, including circRNA basic information, disease associations, interaction regions, supporting chimeric read counts, predicted hybrid duplex structures, and disease-linked variants from GWAS Catalog, ClinVar, and ICGC databases. CircTarget provides a valuable resource for deciphering circRNA functions and is freely accessible at https://circtarget.cn.

Background: For most colorectal cancer (CRC) patients, expanding the benefits of immunotherapy, particularly through blocking programmed cell death-1 (PD-1) and its ligand (PD-L1), is crucial, especially in cases with limited response to neoadjuvant therapy. This study investigates the role of Myoferlin (MYOF) as a novel target in CRC immunotherapy. Methods: Human CRC cell lines (RKO, HCT116), normal intestinal epithelial cells (HIEC-6), and the murine CRC cell line MC38 were used to study the effects of apatinib and MYOF in CRC cells. RNA sequencing, the CPTAC and TCGA databases, and other molecular and cellular methods were applied to disclose the mechanisms involved. A series of mouse models were established to assess the effects of apatinib and MYOF knockdown on tumor progression, immune cell infiltration, and immune checkpoint protein response. Results: We found that MYOF is overexpressed in CRC and linked to immune cell infiltration and checkpoint expression. Suppression of MYOF expression significantly inhibited CRC cell proliferation and migration, as well as reduced PD-L1 protein levels. Integrative analysis showed that apatinib modulates MYOF expression via VEGFR2, resulting in decreased PD-L1 expression, increased CD8+ T cell infiltration, and reduced pro-tumor M2 macrophages. Animal experiments further revealed that apatinib treatment or MYOF knockdown enhanced the efficacy of immune checkpoint blockade (ICB) in CRC. Conclusions: These findings highlight novel antitumor mechanisms of MYOF and suggest that combining apatinib with ICB therapy may improve CRC treatment outcomes, offering a promising strategy to enhance immune responses.

Background: Finding effective strategies and novel targets for reversing drug resistance is one of the major frontiers in hepatocellular carcinoma (HCC) research. Ferroptosis is participate in the malignant progression and drug resistance of HCC. However, the underlying molecular mechanisms remail largely uninvestigated. Methods: HCC cell lines and xenografted nude mice were used as experimental models. Biological functions were investigated by various molecular biology experiments. An HCC population was used to reveal clinical significance. Results: In our study, HCG18 and RRM2 was found to be associated with unfavorable prognosis. HCG18 regulates RRM2 expression through competitively binding to miR-30a-5p, consequently impacting ferroptosis. RRM2 directly regulated GSS to increase GSH synthesis. The colony formation assay demonstrated that overexpression of HCG18 inhibited erastin-induced cell death. In addition, in vivo experiments have also confirmed that HCG18 can inhibit ferroptosis by regulating the expression of RRM2, thereby promoting HCC proliferation. Conclusion: Our study discovered a novel lncRNA HCG18, as a "switch-like" molecule of the axis of miR-30a-5p/RRM2/GSS, confers resistance to ferroptosis and holds promise as a potential target for ferroptosis-dependent therapy.

Receptor-Interacting Protein Kinase 2 (RIPK2) is a critical component of the signaling pathways downstream of Nucleotide-binding oligomerization domain-like receptor (NOD-like receptor), playing a vital role in the immune response, particularly in the context of cellular transport, adaptive immunity, and tumorigenesis. Recent advances have further clarified the complex roles of RIPK2, offering insights into its structural and functional characteristics. In this review, we provide a comprehensive overview of RIPK2’s involvement in signaling, examine the development of RIPK2 inhibitors, and discuss novel strategies for targeting RIPK2 in therapeutic applications. Additionally, we highlight the dynamic interactions between RIPK2 and NOD-like receptors and explore future directions for improving RIPK2-targeted therapies.

Early gastric cancer (EGC) represents a critical stage in preventing and controlling the progression from gastritis to advanced gastric cancer (AGC). Therefore, identifying the single-cell characteristics of EGC, particularly the cellular composition of the tumor microenvironment (TME), as well as identifying potential predictive markers and therapeutic targets, could significantly enhance the monitoring of gastric cancer and improve clinical cure rates. We constructed a comprehensive single-cell RNA sequencing atlas for 184,426 high-quality gastric cancer cells from various stages, utilizing clinical biopsies and surgical samples. Our single-cell atlas highlights the cellular and molecular characteristics of EGC. Eight distinct cell lineage states were identified, and it was observed that the number of epithelial cell meta-clusters gradually decreased, while the number of T&NK, B, plasma, fibroblast, myeloid, and endothelial cells increased with disease progression. Certain epithelial subclusters (metaplastic stem-like cells (MSCs), pit mucous-like cells (PMC-like), proliferating cells), T-cell subclusters (Treg, CCR7 + naive, CH25H + CD4+, TEM CD8+, and GFPT2 + CD8+ T cells), and endothelial subclusters (IL-33 + Venous-1 and AMAMTSL2 + Artery-2) were found to be increased in EGC. The Venous-1 subcluster was found to express high levels of IL-33. Mechanistically, it was revealed that IL-33 enhances the survival and angiogenesis of endothelial cells by upregulating the expression of adhesion proteins CD34 and PECAM1. Patient-derived EGC and AGC organoids were subsequently generated, and it was demonstrated that endothelial-derived IL-33 promoted the growth of both EGC and AGC organoids ex vitro and in vivo. Furthermore, IL-33 was found to increase the expression of KRT17 in EGC organoids. Notably, we also found that high expression of IL-33 was positively correlated with the depth of invasion and malignancy of EGC. This study provides novel insights into the single-cell components involved in EGC and reveals the role of the IL-33 + endothelial subcluster in EGC progression.

Background:Gastric ulcers are a form of peptic ulcers that present as ruptures of the mucosal lining of the stomach or the proximal intestinal lining extending beyond the muscularis mucosae, and Helicobacter pylori infection is one of the main causative factors of gastric ulcers. However, the growing incidence of Helicobacter pylori drug resistance and the emergence of specialised ulcers has necessitated continued research on gastric ulcers. This study surveyed global gastric ulcer research over the past two decades with the aim of identifying the major findings and emerging trends in the field. Methods:Bibliometric analysis was performed using the search terms 'Gastric ulcer', 'Gastric ulcer disease', 'Gastrohelcoma', and 'Stomach ulcers'. Data were extracted from the Web of Science Core Database (WoSCC) and visualised using CiteSpace software. Results:The Journal of Gastroenterology had the most cited papers. The largest number of papers was from the United States. The most frequently cited keywords were 'Helicobacter pylori', 'peptic ulcer', and 'gastric ulcer'. Conclusion:The field of gastric ulcer research is rapidly expanding, and the existing research is focused on preventing the occurrence of gastric ulcers, exploring the pathogenesis of gastric ulcers, and identifying new methods of treating gastric ulcers.

Atherosclerosis is a chronic and progressive vascular disease involving the gradual accumulation of lipids, cholesterol, cellular debris, and fibrous elements within the arterial wall. This process leads to the thickening and hardening of arteries, resulting in restricted blood flow and reduced oxygen delivery to tissues. Over time, these pathological changes significantly elevate the risk of life‑threatening cardiovascular events, including myocardial infarction and ischemic stroke. Recent studies emphasize the significant role of epigenetic modifications and non‑coding RNAs (ncRNAs) in regulating the progression of atherosclerosis. Histone modifications, DNA methylation, and ncRNAs interact to modulate gene expression, influencing endothelial dysfunction, lipid metabolism, and inflammatory processes. Epigenetic regulators, such as DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), control key vascular genes, while ncRNAs like microRNAs (miRNAs), long non‑coding RNAs (LncRNAs), and circular RNAs (circRNAs) contribute to the modulation of cholesterol efflux and foam cell formation. Understanding the complex interplay between these molecular pathways offers new therapeutic insights for managing atherosclerosis and its complications. The reversible nature of epigenetic changes, alongside ncRNA‑based therapies, holds promising potential for future clinical applications, though challenges such as delivery mechanisms and specificity remain.

Helicobacter pylori (H. pylori) is carcinogenic and has the potential to cause progressive gastric lesions and gastric cancer (GC), which also represents one of the essential constituents of the tumor microenvironment (TME) of GC. The infiltration and functional status of tumor-infiltrating lymphocytes (TILs) in the TME affect the anti-tumor function of the body. However, the impact of H. pylori on anti-tumor immunity and prognosis of GC is still unclear. In this study, we constructed a tissue microarray (TMA) consisting of GC tissues from patients with or without H. pylori infection. We evaluated the status of TILs and the expression of CD3, CD8 and PD-L1 by Hematoxylin-eosin (H E) staining and immunohistochemical (IHC) staining, respectively. Correlation, Cox regression, and survival analyses were performed. We found that TIL, CD3, and CD8 were negatively correlated with H. pylori infection. In addition, TILhigh and CD8+TILs status were positively associated with better survival. Simultaneously, patients with H. pylori-positive status had decreased survival compared to those in the H. pylori-negative group. Our study supports the hypothesis that H. pylori infection was positively correlated with less TILs and CD8+TILs, which may contribute to anti-tumor immune escape, thus lead to a poor prognosis of GC.

Cisplatin is widely used in anti-tumor therapy, but the ototoxicity caused by high-dose cisplatin often limits its efficacy, and the specific mechanism of cisplatin-induced cochlear damage is still not perfect. The Wnt/β-catenin signaling pathway is closely related to aging, embryonic development, and apoptosis. Meanwhile, B lymphoma Moloney murine leukemia virus insertion region 1 (BMI1) plays a certain role in the evolution and development of the inner ear and the occurrence and development of inner ear-related diseases. Our study intends to explore the role and specific mechanism of the Wnt/β-catenin signaling pathway and BMI1 in improving cisplatin ototoxicity. The appropriate experimental concentrations for each drug were selected by CCK-8 cell proliferation assay and Western Blot to detect apoptosis. The lentivirus transfection of HEI-OC1 cochlear hair cells was used to overexpress BMI1. Western Blot, qPCR, and immunofluorescence detected the activation of each component of BMI1 and Wnt/β-catenin signaling pathway in each experimental model. Wnt/β-catenin signaling pathway and BMI1 are jointly involved in cisplatin-induced cell injury. Low lithium chloride (LiCl) concentrations activated the Wnt/β-catenin pathway, increased BMI1 expression, and reduced cisplatin-induced hair cell injury. In contrast, overexpression of BMI1 inhibited the Wnt/β-catenin pathway and reduced hair cell injury. Meanwhile, the increased cisplatin-induced damage to hair cells by inhibiting BMI1 could not be rescued by LiCl. In conclusion, LiCl can ameliorate cisplatin ototoxicity by elevating BMI1 expression through activation of the Wnt/β-catenin pathway. Overexpression of BMI1 inhibits the Wnt/β-catenin pathway and reduces cisplatin-induced hair cell damage.

Colorectal cancer (CRC) ranks as the third most prevalent cancer globally and is the second leading cause of cancer mortality. FAM49B, a member of the FAM49 gene family, is a recently identified, evolutionarily conserved gene. Emerging studies indicate that FAM49B plays a role in various cancers, though its specific mechanism in CRC remains largely unexplored. In this study, we observed that FAM49B was abnormally expressed in CRC tissues and cell lines, with elevated expression correlating with poor patient prognosis. FAM49B knockdown markedly suppressed CRC cell proliferation by arresting the cell cycle and reducing cell migration and invasion. Single-cell RNA-seq (ScRNA-seq) analysis revealed that high FAM49B expression in malignant epithelial cell clusters was strongly linked to c-Myc oncogene activation. Further, FAM49B knockdown significantly reduced c-Myc expression by enhancing its K48 ubiquitination. We identified NEK9 as a direct interacting partner of FAM49B, with FAM49B knockdown inhibiting NEK9-Thr210 phosphorylation. Similarly, high NEK9 expression was linked to unfavorable prognosis in CRC. In FAM49B-overexpressing CRC cells, NEK9 knockdown significantly suppressed c-Myc expression, c-Myc-ser62 phosphorylation, and reduced cell proliferation, migration, and invasion. Thus, directly targeting the FAM49B/NEK9/c-Myc pathway presents a promising therapeutic approach for c-Myc positive CRC patients.

Background: Lipid reprogramming represents a pivotal stage in tumor progression. N6-methyladenosine (m6A), the most prevalent RNA modification in eukaryotic cells, plays a significant role in colorectal cancer (CRC) development, though its specific involvement in lipid reprogramming remains unclear. Methods: Bioinformatics analysis of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases revealed differential expression of METTL16 (M16), which was further validated through qRT-PCR and Western blotting in CRC tissues and cell lines. The impact of M16 on CRC proliferation, metastasis, invasion, and lipid reprogramming was evaluated using both in vivo and in vitro approaches. Regulatory mechanisms underlying M16's role in CRC progression were explored using immunofluorescence (IF) staining, RNA immunoprecipitation (RIP), MERIP assay, RNA pull-down assay, total m6A measurement, RNA stability assay, protein stability analysis, and luciferase reporter assays. Results: Analysis results demonstrated a significant upregulation of the m6A methyltransferase METTL16 in CRC, closely associated with poor prognosis and abnormal lipid droplet accumulation. Functional assays revealed that M16 overexpression markedly promotes CRC cell proliferation, migration, and invasion both in vitro and in vivo, primarily by enhancing lipid reprogramming. Mechanistically, M16 induces m6A modification of TM7SF2 mRNA, stabilizing it via an IGF2BP1- and IGF2BP2-dependent pathway, thereby upregulating TM7SF2 expression and driving lipid reprogramming in CRC. Conclusion: In conclusion, these findings highlight the critical role of the M16/m6A/TM7SF2 axis in lipid metabolic reprogramming in CRC, offering potential therapeutic targets for its treatment.

Colorectal liver metastasis (CRLM) remains lethal, and the convergence of cellular senescence with metabolic reprogramming via epigenomic rewiring is poorly understood. We integrated genome-wide DNA methylation and RNA-seq data from 10 paired primary tumors and liver metastases (GSE213402). After calling differentially methylated genes (3,399 hyper- and 9,519 hypomethylated) and differentially expressed genes (406 DEGs), we intersected them with curated senescence (n = 866) and metabolic reprogramming (n = 948) gene sets, yielding 28 differentially expressed cellular-senescence-related genes (DE-CSRGs) and 24 metabolic-reprogramming-related genes (DE-MRRGs). Machine-learning pipelines (LASSO + SVM-RFE) converged on a five-gene signature: CXCL1, SERPINE1, NDRG1, SRM and GATM, most of which are hypomethylated and over-expressed in metastases. Gene-set enrichment analysis revealed that these genes are involved in pathways such as oxidative phosphorylation, focal adhesion, complement-coagulation cascades, and PPAR signaling. Immune de-convolution revealed strong positive correlations between signature genes and immunosuppressive subsets (MDSCs, Tregs, type-1 T-helper cells; p < 0.05). Elevated IC50 values for oxaliplatin and 5-fluorouracil in metastatic samples were positively associated with NDRG1 and negatively with SRM, indicating chemo-resistance modulation. This five-gene epigenetic-transcriptomic hub identifies a molecular signature that warrants prospective validation as a potential biomarker for patient stratification and combination therapy in CRLM.

Introduction:Esophageal cancer (ESCA) is a highly aggressive malignancy with poor prognosis. Small nuclear ribonucleoprotein polypeptide B2 (SNRPB2) is a core component of the spliceosome involved in pre-mRNA splicing. However, its role in tumor development and progression remains largely unclear. This study aimed to evaluate the clinical relevance and prognostic value of SNRPB2 in ESCA. Methods:SNRPB2 mRNA expression and genetic alterations were analyzed using GEPIA2 and cBioPortal. Protein expression was assessed by immunohistochemistry in paraffin-embedded esophageal squamous cell carcinoma (ESCC) tissues. Functional assays in ESCC cell lines were conducted to determine the biological role of SNRPB2. Immune-related and functional analyses were performed using TIMER, TISIDB, TISCH, Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA). Cycloheximide (CHX) chase assays were used to assess protein stability. Results:SNRPB2 mRNA was upregulated in ESCA and associated with tumor progression and poor prognosis. Immunohistochemistry confirmed high SNRPB2 protein expression in ESCC, correlating with vessel carcinoma embolus, lymph node metastasis, clinical stage, and tumor grade. SNRPB2 knockdown significantly inhibited ESCC cell proliferation, migration, and invasion in vitro and in vivo. GSEA indicated that SNRPB2 suppresses the Rb/E2F pathway. Mechanistically, SNRPB2 stabilized E2F4 protein by preventing its proteasomal degradation, and E2F4 overexpression reversed the tumor-suppressive effects of SNRPB2 silencing. Immune analyses showed that SNRPB2 expression correlated with increased infiltration of activated CD8+ T cells, γδ T cells, dendritic cells, and monocytes, as well as immune-related genes including PDCD1, CD274, CTLA4, HLA-DRA, and B2M. These findings suggest a dual role for SNRPB2 in promoting tumor progression and modulating the immune microenvironment in ESCA. Conclusion:SNRPB2 promotes ESCC progression by stabilizing E2F4 and regulating cell cycle genes. It is also associated with immune infiltration and gene expression in ESCA. SNRPB2 may serve as a prognostic biomarker and potential therapeutic target in esophageal cancer.

Ischemia-reperfusion injury (IRI) remains an unavoidable challenge in liver surgery, with macrophages playing a critical role in its pathogenesis. However, the mechanisms by which macrophages regulate the pathogenesis of IRI are not well understood. Through a target-guided screening approach, we identified a small 3 kDa peptide (SjDX5-271) from various schistosome egg-derived peptides that induced M2 macrophage polarization. SjDX5-271 treatment protected mice against liver IRI by promoting M2 macrophage polarization, and this protective effect was abrogated when the macrophages were depleted. Transcriptomic sequencing showed that the TLR signaling pathway was significantly inhibited in macrophages from the SjDX5-271 treatment group. We further identified that SjDX5-271 promoted M2 macrophage polarization by inhibiting the TLR4/MyD88/NF-κB signaling pathway and alleviated hepatic inflammation in liver IRI. Collectively, SjDX5-271 exhibited some promising therapeutic effects in IRI and represented a novel therapeutic approach, potentially applicable to other immune-related diseases. The current study demonstrates the potential of new biologics from the parasite, enhances our understanding of host-parasite interplay, and provides a blueprint for future therapies for immune-related diseases.

To investigate the utility of combining clinical and contrasted-enhanced tomography (CECT) parameters for the preoperative evaluation of perineural invasion (PNI) in gallbladder carcinoma (GBC). A total of 134 patients with GBC (male/female, 52/82; age, 64.4 ± 9.7 years) were divided into PNI-positive (n = 63) and PNI-negative groups (n = 71). Clinical characteristics (demographic information, liver function indicators and tumor markers) and CECT parameters (tumor type, tumor size, gallbladder stone, invasion of gallbladder neck/cystic duct, clinical T stage and N stage) were collected and compared between two groups. Binary logistic regression analysis, receiver operating characteristic curves analyses and Delong test were used in further statistical analyses in clinical T3-4 stage (cT3-4) GBC patients. Overall survival (OS) rates after surgery were compared between PNI-negative group and PNI-positive group of cT3-4 GBC patients. The majority of GBC patients with PNI were classified as cT3-4 (61/63, 96.8

Metabolic studies at the single cell level can directly define the cellular phenotype closest to physiological or disease states. However, the current single cell metabolome (SCM) study using mass spectroscopy has difficulty giving a complete view of the metabolic activity in the cell, and the prediction of the metabolism-phenotype relationship is limited by the potential inconsistency between transcriptomic and metabolic levels. Here, the single-cell simultaneous metabolome and transcriptome profiling method (scMeT-seq) is developed at one single cell, based on sub-picoliter sampling from the cell for the initial metabolome profiling followed by single cell transcriptome sequencing. This design not only provides sufficient cytoplasm for SCM but also nicely keeps the cellular viability for the accurate transcriptomic analysis in the same cell. Integrative analysis of scMeT-seq reveals both dynamical and cell state-specific associations between metabolome and transcriptome in the macrophages with defined metabolic perturbations. Moreover, metabolite signatures are mapped to the single-cell trajectory and gene correlation network of macrophage transition, which allows the unsupervised functional interpretation of metabolome. Thus, the established scMeT-seq should lead to a new perspective in metabolic research by transforming metabolomics from a metabolite snapshot to a functional approach.

Autophagic dysfunction-induced deterioration of the retinal microenvironment drives the progression of wet age-related macular degeneration (wAMD). The efficacy of single-target anti-VEGF antibodies in treating wAMD has long been suboptimal due to the intricate interplay between autophagy dysfunction, oxidative stress, and angiogenesis. Here, we introduce an intravitreal hydrogel depot, named Rab&Rapa-M@G, consisting of rapamycin-loaded microemulsion (Rapa-M, an mTOR inhibitor), ranibizumab (anti-VEGF antibody), and a thermosensitive hydrogel matrix. A single intravitreal injection of Rab&Rapa-M@G can sustainably deliver Rapa-M and ranibizumab to the retinal pigment epithelium for at least 14 days. This formulation significantly improves retinal autophagic flux homeostasis and reduces oxidative stress injury in wAMD mice by modulating the AMPK/mTOR/HIF-1α/VEGF and AMPK/ROS/HO-1/VEGF pathways. Consequently, it synergistically disrupts the “autophagic dysfunction-oxidative stress-angiogenesis” loop, leading to a remarkable reduction in choroidal neovascularization area and retinal damage compared to ranibizumab alone. Notably, the sequential administration of ranibizumab and Rab&Rapa-M@G further enhances the overall anti-wAMD efficacy, achieved through sequential delivery of Rab and Rapa, allowing for a more precise grasp of the treatment window. In conclusion, this hydrogel depot design, with its sequential and sustained delivery of mTOR inhibitors and anti-VEGF antibodies, offers a promising strategy for multi-target synergistic therapy in wAMD.