Objective·To investigate the causal relationship between plasma zinc-alpha-2-glycoprotein 1(AZGP1)and heart failure(HF)by using Mendelian randomization(MR)analysis and experimental validation.Methods·A two-sample MR analysis was performed to assess the causal relationship between AZGP1 and HF by integrating large-scale genome-wide association study(GWAS)data on plasma proteins and HF.The inverse-variance weighted(IVW)method was employed as the primary analytical approach,supplemented by MR-Egger regression,weighted median,and simple median methods.Horizontal pleiotropy was tested by using MR-PRESSO global test and MR-Egger intercept analysis.Colocalization analysis was conducted to validate genetic locus overlap.Additionally,a clinical cohort(84 HF patients and 68 healthy controls)was analyzed,with plasma AZGP1 levels quantified by enzyme-linked immunosorbent assay(ELISA).Results·MR analysis showed that elevated plasma AZGP1 levels were significantly associated with reduced HF risk(OR=0.82,95%CI 0.75?0.90,P=1.70×10-5).Colocalization analysis confirmed that AZGP1 expression and HF shared causal genetic variants(posterior probability for H4=0.69).Sensitivity and reverse MR analyses supported the robustness of the results.ELISA confirmed that plasma AZGP1 levels were significantly lower in HF patients compared to healthy controls,reinforcing its protective role in HF.Conclusion·This study demonstrates AZGP1 exerts a protective causal effect on HF and may serve as a potential biomarker for HF treatment.

Non-small cell lung cancer(NSCLC)is one of leading causes of cancer-related mortality worldwide.In recent years,immune checkpoint inhibitors(ICIs)have become the first-line treatment option for advanced NSCLC.Although ICIs have significantly improved the prognosis of some patients,not all individuals benefit from these therapies.The immunosuppressive tumor microenvironment(TME)is considered a key factor limiting the efficacy of ICIs.The adenosinergic axis refers to pathway in which extracellular ATP is sequentially degraded into adenosine(ADO)by enzymes and subsequently regulates immune responses through binding to ADO receptors.In TME of NSCLC,pathological conditions such as hypoxia,cell necrosis,and inflammation can lead to abnormal accumulation of ADO,thereby suppressing immune cell function and driving expansion of immunosuppressive cells.Due to its central role in immunosuppression,adenosinergic axis has become a major focus of tumor immunotherapy research.This article further explores complex interactions among ADO,its receptors and TME,proposes potential strategies for targeting adenosinergic axis to enhance antitumor immunity,evaluates the progress of early clinical studies of related pharmacological inhibitors in NSCLC,and discusses future research directions.

Objective·To investigate the causal relationship between plasma zinc-alpha-2-glycoprotein 1(AZGP1)and heart failure(HF)by using Mendelian randomization(MR)analysis and experimental validation.Methods·A two-sample MR analysis was performed to assess the causal relationship between AZGP1 and HF by integrating large-scale genome-wide association study(GWAS)data on plasma proteins and HF.The inverse-variance weighted(IVW)method was employed as the primary analytical approach,supplemented by MR-Egger regression,weighted median,and simple median methods.Horizontal pleiotropy was tested by using MR-PRESSO global test and MR-Egger intercept analysis.Colocalization analysis was conducted to validate genetic locus overlap.Additionally,a clinical cohort(84 HF patients and 68 healthy controls)was analyzed,with plasma AZGP1 levels quantified by enzyme-linked immunosorbent assay(ELISA).Results·MR analysis showed that elevated plasma AZGP1 levels were significantly associated with reduced HF risk(OR=0.82,95%CI 0.75?0.90,P=1.70×10-5).Colocalization analysis confirmed that AZGP1 expression and HF shared causal genetic variants(posterior probability for H4=0.69).Sensitivity and reverse MR analyses supported the robustness of the results.ELISA confirmed that plasma AZGP1 levels were significantly lower in HF patients compared to healthy controls,reinforcing its protective role in HF.Conclusion·This study demonstrates AZGP1 exerts a protective causal effect on HF and may serve as a potential biomarker for HF treatment.

Non-small cell lung cancer(NSCLC)is one of leading causes of cancer-related mortality worldwide.In recent years,immune checkpoint inhibitors(ICIs)have become the first-line treatment option for advanced NSCLC.Although ICIs have significantly improved the prognosis of some patients,not all individuals benefit from these therapies.The immunosuppressive tumor microenvironment(TME)is considered a key factor limiting the efficacy of ICIs.The adenosinergic axis refers to pathway in which extracellular ATP is sequentially degraded into adenosine(ADO)by enzymes and subsequently regulates immune responses through binding to ADO receptors.In TME of NSCLC,pathological conditions such as hypoxia,cell necrosis,and inflammation can lead to abnormal accumulation of ADO,thereby suppressing immune cell function and driving expansion of immunosuppressive cells.Due to its central role in immunosuppression,adenosinergic axis has become a major focus of tumor immunotherapy research.This article further explores complex interactions among ADO,its receptors and TME,proposes potential strategies for targeting adenosinergic axis to enhance antitumor immunity,evaluates the progress of early clinical studies of related pharmacological inhibitors in NSCLC,and discusses future research directions.

Objective:To investigate the effect of bupivacaine on Apelin/APJ pathway and subsequent gene expression in Sprague-Dawley (SD) rat cardiomyocytes.Methods:H9c2 cardiomyocytes were cultured in vitro and treated with 0-1 mmol/L bupivacaine for 6 h. The concentration of Apelin was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of APJ was detected by Western blot; Fifteen adult male SD rats were randomly divided into sham group (sham group), bupivacaine 30 mg/kg group (Bupi group), bupivacaine 30 mg/kg+ [Pyr 1] apelin-13 0.15 mg/kg (Bupi-A) group. After corresponding treatment, myocardial tissue was taken to detect the expression of Apelin and APJ, and high-throughput sequencing was performed. Results:Western blot and ELISA results showed that bupivacaine down regulated the expression of Apelin and APJ in H9c2 cardiomyocytes (all P<0.05). The expression levels of Apelin and APJ in the myocardial tissue of SD rats in Bupi group were lower than those in sham group (all P<0.05). The expression of Apelin in myocardial tissue of Bupi-A group was higher than that of Bupi group ( P=0.006), but the expression of APJ had no significant difference ( P>0.05). High throughput sequencing revealed that compared with sham group, the top five differentially expressed genes that were significantly upregulated in Bupi group but significantly downregulated in Bupi-A group were ubiquinone NADH dehydrogenase Fe-S protein 3, enolase 1, aquaporin 1, ATP synthase F0 complex C subunit 1 lipid binding protein, and peroxidase 2 (all P<0.001); The top five genes that were significantly down regulated in Bupi group but significantly up-regulated in Bupi-A group were mesothelin, Rho GTPase activating protein 29, sten20 like kinase, carbonic anhydrase, and paraplatelet lysin (all P<0.001). These genes were associated with increased cardiomyocyte apoptosis, energy metabolism disorders and exercise attenuation. Conclusions:bupivacaine can reduce the expression of Apelin, leading to the changes of gene expression related to increased apoptosis, energy metabolism disorder and exercise attenuation in rat cardiomyocytes.

Objective:To screen the differentially expressed immune-related genes(DEIRGs)in in-stent restenosis(ISR),and to analyze the immune cell infiltration in ISR,and to clarify the mechanism of occurrence and development of ISR.Methods:The mRNA gene expression data of GSE46560 dataset samples were downloaded from the Gene Expression Omnibus(GEO),and divided into ISR group and non-ISR group.The"Limma"package in R software was used to identify the differentially expressed genes(DEGs)which were then intersected with immune-related genes(IRGs)to identify the DEIRGs in ISR;R software was used for Gene Ontology(GO)functional enrichment andalysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis on DEIRGs;the STRING database was used to construct the protein-protein interaction(PPI)network,which was visualized and analyzed for Hub genes by Cytoscape software;the receiver operating characteristic(ROC)curve of the Hub genes were plotted,and the area under the curve(AUC)was calculated and the diagnostic value was evaluated;CIBERSORT software was used to analyze the immune cell infiltration in ISR;Pearson correlation analysis was used to analyze the relationships between the immune cells and the relationships between the immune cells and key genes.Results:A total of 331 DEGs were identified(P<0.05,|log2FC|>1),including 176 upregulated genes and 155 downregulated genes,and 38 DEIRGs were obstained.The GO functional enrichment analysis results showed that the DEIRGs were mainly enriched in biological processes(BP)such as defense response,immune response,and immune system;in cellular components(CC),the DEIRGs were located primarily in the extracellular region and cytoplasmic membrane;and in molecular functions(MF),the DEIRGs were mainly involved in regulating signaling receptor binding and cytokine receptor activity.The KEGG signaling pathway enrichment analysis results indicated that the DEIRGs in ISR were primarily enriched in the phosphatidylinositol 3-kinase/protein kinase B(PI3K-AKT)and transforming growth factor-β(TGF-β)signaling pathways.In the PPI network,CD19 had the highest node among the top 10 Hub genes.Compared with non-ISR group,the expression level of the CD19 gene in the samples in ISR group was increased(P<0.05).The AUC value in the ROC curve of CD19 gene expression was 0.92(P<0.05).The immune cell infiltration analysis results showed that compared with non-ISR group,the infiltration level of T lymphocyte follicular helper(Tfh)cells in the patients in ISR group were increased(P<0.05),the infiltration levels of immature B lymphocytes,CD8+T lymphocytes,naive CD4+T lymphocytes,and M0 macrophages were increased,but the differences were not statistically significant(P>0.05),while the infiltration levels of memory B lymphocytes,activated memory CD4+T lymphocytes,regulatory T cells,resting natural killer(NK)cells,activated NK cells,monocytes,resting mast cells,and neutrophils were decreased,but the differences were not statistically significant(P>0.05).There were positive correlations between Tfh cells and M0 macrophages and resting mast cells(r=0.88,P<0.05;r=0.68,P<0.05),and there were negative correlations between Tfh cells and monocytes and neutrophils(r=-0.49,P<0.05;r=-0.42,P<0.05).Conclusion:CD19 may influence the occurrence and development of ISR by regulating the activation of the PI3K-AKT signaling pathway to affect the Tfh and B lymphocytes.CD19 can serve as a biomarker for the diagnosis of ISR.

OBJECTIVE: To investigate the association between the glucose-to-lymphocyte ratio (GLR) and prognosis of patients with sepsis-associated acute kidney injury (SA-AKI). METHODS: Based on the Medical Information Mart for Intensive Care-IV (MIMIC-IV), SA-AKI patients aged ≥ 18 years were selected. According to the tertiles of GLR, the patients were divided into GLR1 group (GLR ≤ 4.97×10^-9 mmol), GLR2 group (4.97×10^-9 mmol < GLR < 9.75×10^-9 mmol) and GLR3 group (GLR ≥ 9.75×10^-9 mmol). Patients with SA-AKI were divided into survival group and death group according to whether they survived 28 days after admission. The patient's gender, age, vital signs, laboratory test results, comorbidities, sequential organ failure assessment (SOFA), acute physiology score III (APS III) score and treatment measures were extracted from the database. Kaplan-Meier survival analysis was used to make the survival curves of patients with SA-AKI at 28 days, 90 days, 180 days and 1 year. Multivariate Logistic regression analysis model was used to explore the independent risk factors of 28-day mortality in patients with SA-AKI. Receiver operator characteristic curve (ROC curve) was used to analyze the predictive efficacy of GLR for the prognosis of patients with SA-AKI. RESULTS: A total of 1 524 patients with SA-AKI were included, with a median age of 68.28 (58.96, 77.24) years old, including 612 females (40.16%) and 912 males (59.84%). There were 507 patients in the GLR1 group, 509 patients in the GLR2 group and 508 patients in the GLR3 group. There were 1 181 patients in the 28-day survival group and 343 patients in the death group. Grouping according to GLR tertiles showed that with the increase of GLR, the 28-day, 90-day, 180-day and 1-year mortality of SA-AKI patients gradually increased (28-day mortality were 11.64%, 22.00%, 33.86%, respectively; 90-day mortality were 15.98%, 26.72%, 40.55%, respectively; 180-day mortality were 17.16%, 28.29% and 41.73%, and the 1-year mortality were 17.95%, 29.27% and 42.72%, respectively, all P < 0.01). According to 28-day survival status, the GLR of the death group was significantly higher than that of the survival group [×10^-9 mmol: 9.81 (5.75, 20.01) vs. 6.44 (3.64, 10.78), P < 0.01]. Multivariate Logistic regression analysis showed that GLR was an independent risk factor for 28-day mortality in patients with SA-AKI [when GLR was used as a continuous variable: odds ratio (OR) = 1.065, 95% confidence interval (95%CI) was 1.045-1.085, P < 0.001; when GLR was used as a categorical variable, compared with GLR1 group: GLR2 group OR = 1.782, 95%CI was 1.200-2.647, P = 0.004; GLR3 group OR = 2.727, 95%CI was 1.857-4.005, P < 0.001]. ROC curve analysis showed that the area under the ROC curve (AUC) of GLR for predicting 28-day mortality in patients with SA-AKI was 0.674, when the optimal cut-off value was 8.769×10^-9 mmol, the sensitivity was 57.1% and the specificity was 67.1%. The predictive performance was improved when GLR was combined with APS III score and SOFA score, and the AUC was 0.806, the sensitivity was 74.6% and the specificity was 71.4%. CONCLUSIONS GLR is an independent risk factor of 28-day mortality in patients with SA-AKI, and high GLR is associated with poor prognosis in patients with SA-AKI.
Male ; Female ; Humans ; Blood Glucose ; Glucose ; ROC Curve ; Prognosis ; Sepsis/diagnosis* ; Acute Kidney Injury ; Retrospective Studies ; Intensive Care Units

Cytosine methylation is one of the major types of DNA epigenetic modifications and plays an important role in maintaining normal cell function and regulating gene expression. Bisulfite sequencing PCR (BSP) based cloning and sequencing is a general method for detecting DNA methylation at specific sites, which can clarify the methylation status of each CpG site in the target fragment. However, this method requires large amounts of single-clonal sequencing, which is complicated to operate, time consuming and expensive. Therefore, the development of an accurate, efficient and convenient DNA methylation detection technology is of great significance to improve the efficiency of epigenetic research. Based on the high-throughput mutation detection platform Hi-TOM (high-throughput tracking of mutations) developed by our group, we further established a site-specific DNA methylation high-throughput detection platform Hi-Meth (High-throughput Detection of DNA Methylation). After bisulfite treatment of DNA samples, the specific site-specific DNA methylation analysis results could be obtained through the Hi-Meth platform by performing only one round of PCR amplification. Using the Hi-Meth platform, the DNA methylation status of two promoter regions of rice were detected. The DNA methylation results from Hi-Meth were consistent with the results from BSP-based method. Thus, site-specific DNA methylation analysis results could be obtained accurately and conveniently through the Hi-Meth platform. In conclusion, Hi-Meth provides an important methylation detection platform for specific DNA regions, which has important significance for epigenetic research.
DNA Methylation ; Epigenesis, Genetic ; Epigenomics ; High-Throughput Nucleotide Sequencing/methods* ; Polymerase Chain Reaction ; Sequence Analysis, DNA/methods*

Objective:To investigate the effect of extracellular vesicles derived from lung tissue on intrapulmonary inflammation and the formation of neutrophil extracellular traps (NETs) in sepsis rats.Methods:Sepsis rat model was established by cecal ligation and puncture (CLP). Collagenase D and DNase I were used to dissociate the lung tissue, the impurities were removed by centrifugation, and finally, the extracellular vesicles (Ti-EVs) derived from lung tissue were separated and extracted by differential ultracentrifugation. Eighteen male SD rats were randomly divided into the sham group, sepsis group and Ti-EVs group: in the Ti-sEV group, a sepsis model was established by CLP, and Ti-EVs suspension was instilled through the airway; rats in the CLP group received CLP, and an equal volume of PBS was instilled through the airway; and rats in the sham group was treated with sham operation. The pathological changes of lung tissue were detected by hematoxylin-eosin (HE) staining after 24 h. The content of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the NETs content in lung tissue.Results:The isolated extracellular vesicles derived from rat lung tissue were observed by transmission electron microscopy as double-layer circular cystic vesicles with particle diameter mainly distributed at 150 nm. Western blot showed positive expression of EVs markers CD9, CD63, and Tsg101. HE staining of lung tissue showed alveolar integrity damage and a large number of inflammatory cells infiltrated in the lung of sepsis rats. Compared with the CLP group, the degree of lung tissue damage was more serious in the Ti-EVs group and the levels of IL-1β, TNF-α and IL-6 in the BALF of rats were significantly increased ( P<0.01). The formation of NETs in the lungs of the rats in the sepsis group and the Ti-EVs group was observed under the laser confocal microscope. Compared with the sepsis group, the fluorescence intensity of NETs in the lung tissues of the Ti-EVs group increased significantly. Conclusions:After enzymatic digestion, differential ultracentrifugation and other treatments, the extracellular vesicles derived from rat lung tissue with high purity can be successfully isolated and extracted. In the process of septic lung injury, extracellular vesicles in lung tissue can aggravate the inflammatory response in the lung and promote the formation of NETs.

OBJECTIVE：To improve the quality standard of M ongolian med icine Artemisia sacrorum ，and to provide scientific basis for comprehensive quality evaluation. METHODS ：The appearance and microscopic characteristics of A. sacrorum were identified；scopoletin，chlorogenic acid ，caffeic acid ，scopoletin and 3，5-dicaffeoylquinic acid were identified quantitatively by TLC；the contents of above 5 components were determined by HPLC. The water content ，total ash and extract were examined. RESULTS：The stem of A. sacrorum was cylindrical ，and its surface was purple or purple-brown or cyan-brown ；the leaves were ovate or oblong-ovate ，fragrant；the flowers were yellow ，head-shaped，subglobose or hemispherical. The powder was green or yellow-green，its pollen grain had three germination ；the parenchymal cell clusters with sharp edges and numerous threaded ducts ， occasionally having marginal pitted ducts ；its wood fibers were in bundles mostly. Results of TLC showed that the spots of the same color were found in the corresponding positions of chromatogram for 5 substance control and samples. The linear range of scopoletin， chlorogenic acid ， caffeic acid ， scopolactone and 3，5-dicaffeoylquinic acid were 85.60-428.00， 10.16-101.60， 10.20-102.00，40.84-408.40 and 40.80-408.00 μg/mL（all r＞0.999 0）. RSDs of precision ，stability，repeatability tests were all less than 3.00％（n＝6）. The average recoveries were 103.07％，99.66％，98.37％，97.78％，98.40％（all RSDs ＜3.00％，n＝6）. The contents of the above-mentioned 5 compounds in 10 batches of samples were 0.36％-1.23％，0.09％-0.51％，0.04％-0.13％， 0.61％ -1.13％ ，0.12％ -1.11％ ，respectively；the average com contents of water ，total ash and water soluble extract were 6.25％，5.86％，26.50％，respectively. CONCLU SIONS：O the basis of the original quality standard of A. sacrorum ， microscopic identification，TLC identification ，content determination and examination items of water ，total ash and extract are added. The method shows good precision ，accuracy and stability ，which can provide reference for more scientific and standardized evaluation of the quality of this medicinal material.