ETHNOPHARMACOLOGICAL RELEVANCE:The modified Zuojin formula (SQQT) has been clinically prescribed for gastric metaplasia (GM) for several decades in China. The therapeutic efficacy of SQQT and potential mechanisms have been demonstrated in our previous studies. This research will further investigate its mechanism in the immune microenvironment. AIM OF THE STUDY:We aimed to determine the influence of SQQT on the ILC2-mediated JAK-2/STAT5/c-Myc pathway during GM. METHODS:The mechanism of GM in patients was measured through single-cell RNA sequencing. The constituents of SQQT have been examined before. The role of SQQT targets JAK-2/STAT5/c-Myc pathway was verified by network pharmacology and molecular docking. The model of GM was induced by tamoxifen (5 mg/20 g), CD90.2 protein (200 μg) or SQQT (1.69, 3.38, 6.76 g/kg) was given to treat GM mice. The SQQT mechanism was confirmed by both in vitro and in vivo studies. Histological analysis, serum cytokines, and protein levels were assessed. RESULTS:Single-cell RNA sequencing analysis indicated that ILC2 increased in the GM patients, the goblet cells in GM were probably transferred from endocrine cells. Compounds in SQQT are related to cell proliferation and can bind to the JAK-2/STAT5/c-Myc pathway proteins. The main components of SQQT, can spontaneously bind to the JAK-2 protein in 9 sites. Tamoxifen caused body weight decrease, spleen weight increase, stomach injury, ILC2 increase, and cytokines increase in the GM group. After examining the cytokines, IL-5 was the only one significantly increased in the GM group. CD90.2 and SQQT can alleviate histological changes of the stomach corpus, inflammation cytokines, and other GM-related indicators. Moreover, cell proliferation and JAK-2 pathway markers were depressed in GM mice. Besides, SQQT protects GES-1 cells from IL-5 injury related to upregulating JAK-2/STAT5/c-Myc proteins in 24h, 48h and 72h. CONCLUSION:The mechanism of SQQT protected the stomach from metaplasia associated to ILC2 activation and the subsequent cell proliferation through IL-5/JAK-2/STAT5/c-Myc pathway.

Patients were divided into three groups based on the percentages of CD8+ T cells

Spatial phenotypes of CD8+ T cells

INTRODUCTION:High-risk chronic atrophic gastritis (CAG; OLGA/OLGIM Ⅲ-Ⅳ) carries significant gastric cancer (GC) risk yet lacks reliable gastric stem cell (GSC)-based biomarkers. We evaluated GSC markers LGR5 (proliferative) and TFF2 (protective) for risk stratification. METHODS:TCGA/GEO bioinformatics analysis preceded immunohistochemical validation in 60 clinical samples. Protein co-expression (Wnt/β-catenin, Ki67, Bax) was assessed. Diagnostic/prognostic power was tested via ROC and Kaplan-Meier analyses. Functional networks were deciphered through GO/KEGG enrichment. RESULTS:High-risk CAG and GC tissues showed LGR5 upregulation and TFF2 downregulation (p < 0.001). IHC confirmed these patterns, with concurrent Wnt activation (β-catenin↑, cyclin D1↑) and proliferation-apoptosis imbalance (Ki67↑, Bax↓). TFF2 outperformed LGR5 in diagnosing high-risk CAG (AUC: 0.842 vs. 0.681). Poor GC prognosis correlated with high LGR5/low TFF2 (p < 0.05). Co-expression networks linked LGR5 to metabolic genes (CPS1, ADH6) and TFF2 to mucosal defense (GKN1, PGC). CONCLUSION:The coordinated assessment of LGR5 and TFF2 offers a promising approach to identifying high-risk CAG. This biomarker pair captures a homeostatic imbalance in GSCs linked to Wnt/β-catenin signaling, establishing a novel molecular framework for early detection and future targeted strategies.

Chronic atrophic gastritis (CAG) severity risk assessment remains challenging due to limitations of current static histologic/endoscopic tools. We evaluated the leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)/trefoil factor 2 (TFF2) ratio — quantifying gastric stem cell niche imbalance— as a dynamic biomarker for high-risk CAG (OLGA/OLGIM III-IV). In a retrospective cohort (n = 60 CAG patients), we constructed four prediction models (Baseline: demographics; Clinical: +H. pylori/family history; Endoscopic; Full: +LGR5/TFF2 ratio) using Firth penalized regression. Model performance was assessed via area under the curve (AUC), Akaike (AIC), and Bayesian (BIC) information criteria, and likelihood ratio tests. Restricted cubic splines and subgroup analyses validated threshold effects. The LGR5/TFF2 ratio > 1.066 (AUC = 0.93) identified high-risk CAG with 48.5-fold increased odds (95

Clinical characteristics of training and validation cohorts

ETHNOPHARMACOLOGICAL RELEVANCE:Zuojin pill is a well-known traditional Chinese medicine (TCM) for treating gastric disorders. The modified Zuojin pill (SQQT) has been used in the treatment of gastric metaplasia (GM) in China for decades. However, the mechanisms of SQQT treat GM remain unclear. AIM OF THE STUDY:Our goals are to evaluate the effect of SQQT on GM and to investigate its potential mechanisms. METHODS:An animal model of metaplasia was established to study the mechanism of SQQT. RNA-seq was employed to analyze the pathogenesis of GM. Network pharmacological approaches and molecular docking were used to elucidate the mechanisms of SQQT. Common targets of the SQQT and GM mechanism pathways are defined as the key mechanisms of SQQT's treatment in GM. The key mechanisms were validated through in vivo and in vitro experiments. RESULTS:RNA-seq analysis of GM animals and network pharmacology of SQQT indicated that SQQT might treat GM via 20 pathways, including the PPAR pathway. Among the 3 core targets of the PPAR pathway, only PPARG is related to GM progression. Besides, the core components of SQQT have a lower affinity for binding to PPARG. The main mechanism of SQQT ameliorated GM is related to PPARG. In animal experiments, SQQT ameliorated GM through ROS decreasing, mitochondrial damage repairing, and protein marker rectification. In cell experiments, SQQT notably decreased the levels of ferroptosis and metaplasia markers including GPX4, PPARG, MUC6, and ACSL4. CONCLUSION:SQQT ameliorated gastric metaplasia by inhibiting the PPARG/ferroptosis pathway.

Clinical characteristics of patients from TMA study

Pearson correlation of screened factors

Immune cell status changes after αPD-1 + αTIM-3 treatment

Analysis of immune cell statuses across different tumor locations in training and validation cohorts.

Background: Gastric cancer (GC) is the third leading cause of cancer-related death and is associated with high mortality and morbidity. Helicobacter pylori (HP) infection is the most important cause of GC. We aimed to identify the core genes of HP caused GC and further elucidate the underlying mechanisms. Methods: GC and HP associated gastritis (HPAG) gene expression data were sourced from Gene Expression Omnibus. Key genes affecting GC prognosis were identified using Cytoscape software. Patient groups were formed based on key gene expression, and the immune analyses were performed with R. MNU, derived from nitrite by HP, was given to GC mice (240ppm) for histology and fluorescence assays. For in vitro experiments, cells received MNU (20 μM) stimulation for 24 hours. Results: CD14 was the only key gene identified. A total of 412 GC patients were divided into CD14-high and CD14-low groups. The two groups showed significant differences in immune cell populations and immune checkpoints. In particular, there was a notable increase in M2 macrophages in GC patients with high CD14 expression (P <0.001). GC Patients with high CD14 expression exhibited a more pronounced immune response than those with low CD14 expression, and elevated CD14 expression positively correlated with the efficacy of CTLA4 therapy (P <0.05). These results indicated that CD14 expression was strongly correlated with the GC immune response. A noticeable increase in CD14 levels was observed in MNU-induced GC animals, cell models, and GC patients. In addition, the number of M2 macrophages was increased in MNU-induced GC mice. Conclusion: Reducing CD14 expression may increase the survival rate of GC patients through the modulation of immune responses. The complex mechanism of CD14's influence on prognosis deserves further investigation.

TIM-3 and PD-1 dual blockade reverses T-cell exhaustion and enhances the antitumor immune response. A, Tumor volume. A measure of 2.5 × 105 CT26 cells were injected subcutaneously into BALB/C mice, followed by treatment with isotype or αPD-1 (RMP1-14) and αTIM-3 (RMT3-23) antibodies (10 mg/kg) every 4 days (isotype: n = 6 mice per arm; αPD-1 + αTIM-3: n = 7 mice per arm). Data are presented as the mean ± SEM and determined by two-way ANOVA. B, Representative images of multiplex immunofluorescence staining (CD8, red; Ki67, white; PD-1, cyan; TIM-3, yellow; and TOX, green) in isotype or αPD-1 + αTIM-3 treatment tumor. C, mIHC quantification of PD-1+CD8+ T cells, TIM-3+CD8+ T cells, TOX+CD8+ T cells, PD-1+TIM-3+CD8+ T cells, PD-1+TIM-3+TOX+CD8+ T cells, and PD-1−TIM-3−TOX−CD8+ T cells as a percentage of CD8+ cells. Data are presented as the mean ± SEM and determined by a two-tailed unpaired t test. D, T-cell panel gating strategy. E, FACS quantification of PD-1+CD8+ T cells, TIM-3+CD8+ T cells, and PD-1+TIM-3+CD8+ T cells as a percentage of CD8+ T cells. Data are presented as the mean ± SEM and determined by a two-tailed unpaired t test. F, FACS quantification of PD-1+CD4+ T cells, TIM-3+CD4+ T cells, and PD-1+TIM-3+CD4+ T cells as a percentage of CD4+ cells. Data are presented as the mean ± SEM and determined by a two-tailed unpaired t test. G, Myeloid cell panel gating strategy. H, FACS quantification of dendritic cells, macrophages, and myeloid-derived suppressor cells as a percentage of CD45+ cells in the αPD-1 + αTIM-3 experiment. Data are presented as the mean ± SEM and determined by a two-tailed unpaired t test. DC, dendritic cell; MDSC, myeloid-derived suppressor cell.

The clinical advantage staging and underlying mechanisms of Wangbi Tablets against knee osteoarthritis (KOA) were studied based on the " disease-formula" interaction network. Firstly,the clinical symptoms and related genes corresponding to Wangbi Tablets and KOA in the acute,remission,and recovery phases were collected from clinical guidelines/consensus and SoFDA database,and the putative targets of Wangbi Tablets were obtained from ETCM 2. 0. Then,Jaccard similarity and cosine similarity were employed to assess the similarities of clinical symptoms,genes,and enriched pathways between Wangbi Tablets and KOA in different phases.The " disease-formula" interaction network of the drug targets and disease genes was constructed,and the key targets were screened by topological feature calculation. KEGG and Reactome database were used for the functional enrichment of the key targets,on the basis of which the functional characteristics of Wangbi Tablets against KOA in the acute,remission,and recovery phases were predicted. Final-ly,the SW1353 cells exposed to lipopolysaccharide were used to decipher the mechanism of Wangbi Tablets against KOA. The results showed that 92/3 921,138/3 708,139/3 800,and 196/3 946 clinical symptoms and the related genes corresponded to KOA in the acute,remission,and recovery phases and Wangbi Tablets were collected from SoFDA,and 260 putative targets of Wangbi Tablets were obtained from ETCM 2. 0. Wangbi Tablets had highest similarity of clinical symptoms,genes,and enriched pathways with KOA in the remission phase and the secondary highest similarity with KOA in the recovery phase. The key targets of Wangbi Tablets mainly participated in the regulation of immunity-inflammation imbalance and exerted pain-relieving and bone-protecting effects to alleviate symptoms such as knee joint pain,joint swelling,soreness,fatigue,and dysfunction. Intriguingly,the key targets of Wangbi Tablets possessed antioxidant effects during KOA in the acute and remission phases,while they maintained material and energy metabolism ho-meostasis and protected vessels during KOA in the recovery phase. The cell experiment indicated that Wangbi Tablets down-regulated the expression of interleukin ( IL)-6,IL-1β,tumor necrosis factor-α ( TNF-α),and Bcl-2-associated X protein ( Bax)/B-cell lympho-ma 2 ( Bcl-2) via regulating the phosphatidylinositol 3-kinase ( PI3K)-protein kinase B ( Akt) signaling pathway. The findings lay a theoretical foundation for further clarifying the clinical advantage stage and precise clinical application of Wangbi Tablets in treating KOA.

Spatial heterogeneity of the immune microenvironment correlates with patient prognosis in human colorectal cancer. A, Representative images of defined seven cell types (CD8+PD-1+TIM-3+, CD8+PD-1+TIM-3−, CD8+PD-1−TIM-3−, CD8−PD-1+TIM-3+, CD8+PD-1−TIM-3+, CD8−PD-1+TIM-3−, and CD8−PD-1−TIM-3+). B, Combination of the distance map and cell subtype map showed spatial infiltration patterns. Criteria of spatial division of the tumor: i.t. (within 10 μm to the tumor cell), proximal (10–30 μm to the tumor cell), and distal (more than 30 μm to the tumor cell). C, Representative image of microenvironment regions and different infiltration patterns of three-marker–defined CD8+ T cells. D, Hierarchical clustering of all quantified immune cells and their spatial phenotypes. Heatmap represents z-score–normalized data; red color represents expression above the mean, blue color represents expression below the mean, and white color represents the mean. E, Representative image and spatial state of three subtypes: cluster 1, high-infiltrated; cluster 2, medium-infiltrated; and cluster 3, low-infiltrated, characterized by infiltration of CTLs; blue color represents cells at a distance of 0 μm from the tumor cells, and yellow color represents cells at a distance of 30 μm from the tumor cells. F, K–M curve (log-rank test) survival plots depict the OS in the unsupervised clusters. DAPI,x; mid, medium.

Although immune checkpoint blockade (ICB) therapy has shown promise in colorectal cancer patients with high microsatellite instability (MSI-H), response rates remain low for the majority of patients. Key challenges include the exhaustion of CD8+ T cells and the presence of immunosuppressive cells within the tumor microenvironment. By integrating digital spatial profiling and scRNA sequencing, we found that the tumor-intrinsic activation of focal adhesion kinase (FAK) was strongly correlated with the heterogeneity and exhaustion of CD8+ T cells within the spatial context of the tumor microenvironment and correlated with patient survival. Pharmacological inhibition or genetic loss of FAK enhanced antitumor immune responses and synergistically potentiated αPD-1 and αTIM-3 immunotherapy by augmenting CD8+ T cell cytotoxicity and restoring exhaustion in preclinical models of colorectal cancer. Mechanistically, nuclear FAK was found to regulate SP1/IL-6-mediated T cell exhaustion and the transcription of proinflammatory cytokines/chemokines. These findings underscore FAK’s pivotal role in CD8+ T cell exhaustion within the immunosuppressive tumor microenvironment, suggesting that FAK is a promising target for advancing cancer immunotherapy. ### Competing Interest Statement The authors have declared no competing interest.

Background Spasmolytic polypeptide expression metaplasia (SPEM) occurs in the corpus of the stomach and is closely related to inflammations caused by H. pylori infection. Recently, SPEM was suggested as one of the dubious precancerous lesions of gastric cancer (GC). Thus, further research on SPEM cell transdifferentiation and its underlying mechanisms could facilitate the development of new molecular targets improving the therapeutics of GC. Using bibliometrics, we analyzed publications, summarized the research hotspots and provided references for scientific researchers engaged in related research fields. Methods We searched the Web of Science Core Collection (WoSCC) for publications related to SPEM-GC from 2002 to 2022. The VOSviewer, SCImago, CiteSpace and R software were used to visualize and analyze the data. Gene targets identified in the keyword list were analyzed for functional enrichment using the KEGG and GO databases. Results Of the 292 articles identified in the initial search, we observed a stable trend in SPEM-GC research but rapid growth in the number of citations. The United States was the leader in terms of quality publications and international cooperation among them. The total number of articles published by Chinese scholars was second to the United States. Additionally, despite its low centrality and average citation frequency, China has become one of the world’s most dynamic countries in academics. In terms of productivity, Vanderbilt University was identified as the most productive institution. Further, we also observed that Gastroenterology was the highest co-cited journal, and Goldenring Jr. was the most prolific author with the largest centrality. Conclusion SPEM could serve as an initial step in diagnosing gastric precancerous lesions. Current hotspots and frontiers of research include SPEM cell lineage differentiation, interaction with H. pylori , disturbances of the mucosal microenvironment, biomarkers, clinical diagnosis and outcomes of SPEM, as well as the development of proliferative SPEM animal models. However, further research and collaboration are still required. The findings presented in this study can be used as reference for the research status of SPEM-GC and determine new directions for future studies.