Objective:To investigate the impact of evaluating the alcohol intake on all-cause mortality in patients with metabolic-associated fatty liver disease（MAFLD）and metabolic dysfunction and alcohol-related liver disease（MetALD）.Method:The retrospective study included patients aged 20 to 74 years with hepatic steatosis diagnosed by ultrasound，with data from the Third National Health and Nutrition Examination Survey（NHANES III）between 1988 and 1994. Participants were categorized into light，moderate，and heavy drinking groups according to daily alcohol intake. Multivariable-adjusted hazard ratios（aHR）and their 95% confidence intervals（ CI）were calculated by Cox proportional risk regression modeling to assess the effect of alcohol intake on all-cause mortality. Results:A total of 2 322 patients were included in the study. Males accounted for 50.2%（1 166/2 322），with a age of 42.0（31.3，57.0）years，a median follow-up of 316.0（270.0，337.0）months，and an all-cause mortality rate of 1.48% per person-year. There were 1，763 cases in the light drinking group，333 in the moderate drinking group，and 226 in the heavy drinking group.The all-cause mortality rates for patients in the three drinking groups were 1.38%，1.67%，and 2.10% per person-year，respectively. The moderate（a HR=1.37，95% CI：1.12 to 1.67， P=0.002）and heavy（a HR=1.45，95% CI：1.17 to 1.80， P=0.001）drinking groups were independently associated with increased all-cause mortality following covariate adjustment. There was a difference in all-cause mortality for alcohol intake in non-type 2 diabetes mellitus（T2DM）patients under 60 years of age（ P<0.05），but the difference was not statistically significant between non-T2DM patients over 60 years of age and T2DM patients of all ages（ P>0.05）according to the analysis of diabetes status and age subgroups. Conclusion:Alcohol intake has a dose-dependent negative effect on patients with MAFLD and MetALD. The risk of all-cause mortality increased significantly with increasing alcohol intake.

Objective:To investigate the efficacy of fibrosis-4 index (FIB-4), NAFLD fibrosis score (NFS), aspartate aminotransferase to platelet count ratio (APRI), liver stiffness value (LSM), and Agile 3+ score and their combined model in predicting advanced-stage liver fibrosis in patients with chronic hepatitis B (CHB) combined with metabolic-associated fatty liver disease (MAFLD).Methods:A multicenter retrospective cohort study was conducted on the BMOVE population.Nine hundred twenty CHB cases combined with MAFLD who underwent liver biopsy at seven medical centers in China from April 2006 to December 2023 were included. The patients were divided into advanced-stage liver fibrosis (159 cases) and non-advanced-stage liver fibrosis (761 cases) according to the Scheuer's scoring system.The area under the receiver operating characteristic curve (AUROC), decision curve, and calibration curve analysis were used to evaluate the efficacy of the firbrosis-4 index (FIB-4) score, NFS score, APRI index, LSM, and Agile 3+ score and their combined model in predicting advanced-stage fibrosis. The liver fibrosis grade of all patients was diagnosed by liver biopsy. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each scoring model and combined model, as well as the proportion of correctly classified patients, were calculated based on different cutoff values.Results:AUROC analysis showed that Agile 3+ (0.814, 95% CI: 0.787-0.838) and LSM (0.805, 95% CI: 0.778-0.829) had similar accuracy and were superior to FIB-4 (0.721, 95% CI: 0.691-0.749), NFS (0.687, 95% CI: 0.656-0.716) and APRI ( 0.689, 95% CI: 0.658-0.718); however, HBV DNA level and HBV e antigen status had no effect on this outcome. Decision curve analysis showed that interventions based on LSM and Agile 3+ had provided higher net benefits compared with serological scores. Calibration curves showed that Agile 3+ had better predicitive accuracy than all other models. Agile 3+ had the highest PPV (0.54), minimal uncertainty interval (11.6%), and the highest proportion of correctly classified patients (76%); followed by LSM (PPV: 0.43, uncertainty interval: 15.5%, correct classification rate: 66%), and FIB-4 (PPV: 0.42, uncertainty interval: 26.1%, correct classification rate: 62.6%) in terms of identifying advanced-stage liver fibrosis. Combined model analysis demonstrated that FIB-4 combined with Agile 3+ had improved the correct classification rate and reduced the proportion of missed patients compared with FIB-4 combined with LSM. Conclusion:The Agile 3+ score is superior than LSM, FIB-4, NFS, and APRI index at identifying advanced-stage fibrosis in patients with CHB combined with MAFLD. This study supports the use of FIB-4 index combined with Agile 3+ for risk stratification in patients with CHB combined with MAFLD.

Objective:To investigate the impact of evaluating the alcohol intake on all-cause mortality in patients with metabolic-associated fatty liver disease（MAFLD）and metabolic dysfunction and alcohol-related liver disease（MetALD）.Method:The retrospective study included patients aged 20 to 74 years with hepatic steatosis diagnosed by ultrasound，with data from the Third National Health and Nutrition Examination Survey（NHANES III）between 1988 and 1994. Participants were categorized into light，moderate，and heavy drinking groups according to daily alcohol intake. Multivariable-adjusted hazard ratios（aHR）and their 95% confidence intervals（ CI）were calculated by Cox proportional risk regression modeling to assess the effect of alcohol intake on all-cause mortality. Results:A total of 2 322 patients were included in the study. Males accounted for 50.2%（1 166/2 322），with a age of 42.0（31.3，57.0）years，a median follow-up of 316.0（270.0，337.0）months，and an all-cause mortality rate of 1.48% per person-year. There were 1，763 cases in the light drinking group，333 in the moderate drinking group，and 226 in the heavy drinking group.The all-cause mortality rates for patients in the three drinking groups were 1.38%，1.67%，and 2.10% per person-year，respectively. The moderate（a HR=1.37，95% CI：1.12 to 1.67， P=0.002）and heavy（a HR=1.45，95% CI：1.17 to 1.80， P=0.001）drinking groups were independently associated with increased all-cause mortality following covariate adjustment. There was a difference in all-cause mortality for alcohol intake in non-type 2 diabetes mellitus（T2DM）patients under 60 years of age（ P<0.05），but the difference was not statistically significant between non-T2DM patients over 60 years of age and T2DM patients of all ages（ P>0.05）according to the analysis of diabetes status and age subgroups. Conclusion:Alcohol intake has a dose-dependent negative effect on patients with MAFLD and MetALD. The risk of all-cause mortality increased significantly with increasing alcohol intake.

Objective:To investigate the efficacy of fibrosis-4 index (FIB-4), NAFLD fibrosis score (NFS), aspartate aminotransferase to platelet count ratio (APRI), liver stiffness value (LSM), and Agile 3+ score and their combined model in predicting advanced-stage liver fibrosis in patients with chronic hepatitis B (CHB) combined with metabolic-associated fatty liver disease (MAFLD).Methods:A multicenter retrospective cohort study was conducted on the BMOVE population.Nine hundred twenty CHB cases combined with MAFLD who underwent liver biopsy at seven medical centers in China from April 2006 to December 2023 were included. The patients were divided into advanced-stage liver fibrosis (159 cases) and non-advanced-stage liver fibrosis (761 cases) according to the Scheuer's scoring system.The area under the receiver operating characteristic curve (AUROC), decision curve, and calibration curve analysis were used to evaluate the efficacy of the firbrosis-4 index (FIB-4) score, NFS score, APRI index, LSM, and Agile 3+ score and their combined model in predicting advanced-stage fibrosis. The liver fibrosis grade of all patients was diagnosed by liver biopsy. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each scoring model and combined model, as well as the proportion of correctly classified patients, were calculated based on different cutoff values.Results:AUROC analysis showed that Agile 3+ (0.814, 95% CI: 0.787-0.838) and LSM (0.805, 95% CI: 0.778-0.829) had similar accuracy and were superior to FIB-4 (0.721, 95% CI: 0.691-0.749), NFS (0.687, 95% CI: 0.656-0.716) and APRI ( 0.689, 95% CI: 0.658-0.718); however, HBV DNA level and HBV e antigen status had no effect on this outcome. Decision curve analysis showed that interventions based on LSM and Agile 3+ had provided higher net benefits compared with serological scores. Calibration curves showed that Agile 3+ had better predicitive accuracy than all other models. Agile 3+ had the highest PPV (0.54), minimal uncertainty interval (11.6%), and the highest proportion of correctly classified patients (76%); followed by LSM (PPV: 0.43, uncertainty interval: 15.5%, correct classification rate: 66%), and FIB-4 (PPV: 0.42, uncertainty interval: 26.1%, correct classification rate: 62.6%) in terms of identifying advanced-stage liver fibrosis. Combined model analysis demonstrated that FIB-4 combined with Agile 3+ had improved the correct classification rate and reduced the proportion of missed patients compared with FIB-4 combined with LSM. Conclusion:The Agile 3+ score is superior than LSM, FIB-4, NFS, and APRI index at identifying advanced-stage fibrosis in patients with CHB combined with MAFLD. This study supports the use of FIB-4 index combined with Agile 3+ for risk stratification in patients with CHB combined with MAFLD.

Tumor microenvironment(TME)is a complex cellular environment where tumor cells reside,along with various types of cells and extracellular components surrounding the tumor cells.Immune cells are key components of TME,including tumor-associated macrophages(TAMs),myeloid-derived suppressor cells(MDSCs),lymphocytes,regulatory T cells(Tregs),natural killer cells(NK cells),dendritic cells(DCs),and many others.It is worth noting that drug resistance is currently a major factor limiting the efficacy of cancer treatment methods such as chemotherapy,radiotherapy,targeted therapy,and immunotherapy,and a leading cause of treatment failure.Research has found that the development of drug resistance in tumor cells is the result of interactions between tumor cells and TME.Consequently,overcoming drug resistance in tumors caused by TME is considered a significant challenge in cancer treatment.In recent years,with in-depth research into immune cells within TME,significant progress has been made in understanding the specific mechanisms by which immune cells regulate drug resistance in tumor cells.Furthermore,therapeutic strategies that target these immune cells,signaling pathways,or cytokines have been shown to effectively combat tumor drug resistance and enhance the therapeutic outcomes of cancer treatment.This article reviews the research advancements regarding the roles of TAMs,MDSCs,Tregs,and NK cells in tumor drug resistance within TME and discusses the development of targeting strategies to overcome this resistance.Additionally,we explore the relationship of tumor-associated neutrophils(TANs)and B regulatory cells(Bregs)with tumor drug resistance.It is hoped that this review will offer insights and serve as reference for reducing tumor drug resistance and improving the efficacy of anti-tumor therapies.

OBJECTIVES: To determine the potential molecular mechanisms underlying the therapeutic effect of curcumin on hepatocellular carcinoma (HCC) by network pharmacology and experimental in vitro validation. METHODS: The predictive targets of curcumin or HCC were collected from several databases. the identified overlapping targets were crossed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform. Two of the candidate pathways were selected to conduct an experimental verification. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay was used to determine the effect of curcumin on the viability of HepG2 and LO2 cells. The apoptosis and autophagy of HepG2 cells were respectively detected by flow cytometry and transmission electron microscopy. Besides, western blot and real-time polymerase chain reaction (PCR) were employed to verify the p53 apoptotic pathway and adenosine 5'-monophosphate (AMP)‍-activated protein kinase (AMPK) autophagy pathway. HepG2 cells were pretreated with pifithrin-‍α (PFT-‍α) and GSK690693 for further investigation. RESULTS: The 167 pathways analyzed by KEGG included apoptosis, autophagy, p53, and AMPK pathways. The GO enrichment analysis demonstrated that curcumin was involved in cellular response to drug, regulation of apoptotic pathway, and so on. The in vitro experiments also confirmed that curcumin can inhibit the growth of HepG2 cells by promoting the apoptosis of p53 pathway and autophagy through the AMPK pathway. Furthermore, the protein and messenger RNA (mRNA) of the two pathways were downregulated in the inhibitor-pretreated group compared with the experimental group. The damage-regulated autophagy modulator (DRAM) in the PFT-‍α-pretreated group was downregulated, and p62 in the GSK690693-pretreated group was upregulated. CONCLUSIONS Curcumin can treat HCC through the p53 apoptotic pathway and the AMPK/Unc-51-like kinase 1 (ULK1) autophagy pathway, in which the mutual transformation of autophagy and apoptosis may occur through DRAM and p62.
AMP-Activated Protein Kinases/pharmacology* ; Apoptosis ; Carcinoma, Hepatocellular/pathology* ; Curcumin/pharmacology* ; Humans ; Liver Neoplasms/pathology* ; Network Pharmacology ; Tumor Suppressor Protein p53/metabolism*

Objective The objective of this study was to utilize proton magnetic resonance spectroscopy (1H-MRS) to assess metabolites in cerebellar nuclei in unmedicated patients with insomnia disorder. Methods 1H-MRS was performed on cerebellar nuclei in 23 unmedicated patients with insomnia disorder (insomnia group) and 18 normal sleepers (control group). N-acetylaspartate (NAA), choline-containing compound (Cho) and creatine (Cr) were measured and the ratios of NAA/Cr and Cho/Cr were determined.Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI) were used to assess the subjective sleep quality and insomnia severity of all subjects, while State-Trait Anxiety Inventory (STAI) and Beck Depression Inventory (BDI) were used to assess the levels of anxiety and depression of all subjects. Sleep parameters of all subjects were measured by polysomnography (PSG). Results Mean NAA/Cr ratio of right cerebellar nuclei in insomnia group was significantly lower than that in control group (1.72±0.37 vs. 2.03±0.50, t=2.280, P=0.028). Mean NAA/Cr ratio of right cerebellar nuclei was significantly higher than that of left cerebellar nuclei within control group (2.03±0.50 vs. 1.68±0.21, t=3.386, P=0.004). There was no significant difference with regard to NAA/Cr ratio between bilateral cerebellar nuclei within insomnia group (t=1.416, P=0.171). Across all subjects, PSQI global scores (r=-0.369, P=0.018), and sleep latency (r=-0.437, P=0.004) and number of awakenings after sleep onset (r=-0.432, P=0.005) measured by PSG were negatively correlated with NAA/Cr ratios of right cerebellar nuclei, while percentages of stage 3 sleep (r=0.377,P=0.015) measured by PSG were positively correlated with NAA/Cr ratios of right cerebellar nuclei,respectively. Conclusion Patients with insomnia disorder have a hemispherically lateralized metabolic disturbance of NAA/Cr in right cerebellar nuclei,indicating that patients with insomnia disorder have neuronal damage in right cerebellar nuclei.

Objective To investigate the effects and mechanisms of shRNA interfered with expression of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) on the malignant behaviors of colorectal cancer stem cells (CSCs).Methods The experimental study was conducted.The CSCs expressing Lgr5+ were sorted by fluorescence activated cell sorting.Lgr5+ cells that were transfected with Lgr5-shRNA lentiviral vector and nontarget shRNA lentiviral vector were respectively allocated into the experimental group and control group.The percentage of Lgr5+ cells was analyzed by flow cytometery.The relative expression of Lgr5 mRNA was detected by fluorescence quantitative real-time polymerase chain reaction (qRT-PCR).The capacity of self-renewal was detected by sphere forming assay.The tumorigenesis in vitro and in vivo were respectively measured by colony formation assay and xenografting experiment.The mRNA expressions of stem cells related genes (Oct4,Sox2,Nanog,KLF4),CSCs genes (CD133,CD44,ALDH) and Wnt/β-catenin pathway key genes (Axin2,Wnt5a,Wnt3a,Fzd3,c-myc,VEGF,Ascl2,claudin-1) were detected by qRT-PCR.Measurement data with normal distribution were represented as-x±s.Comparison between groups was analyzed using the t test.Results (1)Transfection efficiency of shRNA lentiviral vector induced Lgr5 by flow cytometery was respectively 6.8％± 1.0％ in the experimental group and 92.7％±3.3％ in the control group,with a statistically significant difference (t =43.148,P＜0.05).The relative expression of Lgr5 mRNA measured by qPT-PCR was respectively 0.168±0.057 in the experimental group and 1.148±0.004 in the control group,with a statistically significant difference (t=28.778,P＜0.05).(2) The capacity of self-renewal was detected by sphere forming assay.The results of sphere forming assay:the number of spheres was 29±6 in the experimental group and 410± 10 in the control group,with a statistically significant difference (t =41.070,P＜0.05).The results of colony formation assay:the numbers of colonies in the experimental group and control group were respectively 72±4 and 412± 19,showing a statistically significant difference (t =31.433,P＜ 0.05).The results of tumorigenesis:the volumes of tumors in the experimental group and control group were respectively (81± 15)mm3 and (328±24)mm3,with a statistically significant difference (t=11.304,P＜0.05).(3) The effects of Lgr5 down-regulation on related genes,results of qRT-PCR detection:① The mRNA relative expressions of Oct4,Sox2,Nanog and KLF4 (stem cells related genes) were 0.377±0.093,0.662±0.104,3.591±0.300,0.425±0.091 in the experimental group and 1.957± 0.026,2.137±0.015,5.831±0.165,1.536±0.014 in the control group,with statistically significant differences (t=23.079,22.261,8.446,19.186,P＜0.05).② The mRNA relative expressions of CD133,CD44 and ALDH (CSCs genes) were 1.490±0.155,5.535±0.487,1.640±0.039 in the experimental group and 2.488± 0.061,9.908±0.332,5.718±0.292 in the control group,with statistically significant differences (t =8.170,9.667,27.849,P＜0.05).③The mRNA relative expressions of Axin2,Wnt5a,Wnt3a,Fzd3,c-myc,VEGF,Ascl2 and claudin-1 genes in the Wnt/β-catenin pathway were respectively 1.592±0.267,0.528±0.138,2.153±0.078,1.480±0.064,0.248±0.128,1.492±0.025,0.658±0.095,1.647±0.087 in the experimental group and 3.651±0.224,2.570±0.093,2.301±0.157,1.636±0.058,1.415±0.080,2.610±0.159,2.480±0.123,3.432±0.273 in the control group.There were statistically significant differences in the mRNA relative expressions of Axin2,Wnt5a,c-myc,VEGF,Ascl2 and claudin-1 genes between the 2 groups (t =7.316,15.332,12.649,12.320,14.831,9.063,P＜0.05),and no statistically significant difference in the mRNA relative expressions of Wnt3a and Fzd3 between the 2 groups (t =2.887,2.242,P＞0.05).Conclusion The malignant behaviors of colorectal CSCs are suppressed after shRNA lentivirus interfered with expression of Lrg5,and its mechanism is related to inhibiting activity of Wnt/β-catenin pathway.

Objective To investigate the polymorphisms of human cytomegalovirus ( HCMV ) UL146 gene in asymptomatic children. Methods Urine samples were collected from 47 asymptomatic chil-dren who were positive for HCMV DNA. PCR was performed to amplify the open reading frame ( ORF) of UL146 gene. Positive bands were sequenced and variations in UL146 gene were analyzed by using bioinfor-matics software. Results Seventeen samples were successfully amplified and sequenced. Variations spread all over the sequence of UL146 gene and the variability in nucleotide and amino acid sequences ranged from 0％ to 42. 5％ and 0％ to 67. 7％ respectively. Compared with the Towne strain, there was diversity in sig-nal sequence and C-terminal region. Phylogenetic analysis indicated that UL146 in the 17 asymptomatic chil-dren belonged to four genotypes, which were G1, G8, G9 and G11. Forms of post-translational modification varied greatly among the four genotypes, while the important functional region of ELRCXC chemokine was highly conservative. Secondary structure prediction showed that random-coli conformation was the predomi-nant structure of active proteins. Isoelectric point ( PI) and molecular weight ( MW) were dissimilar among the four genotypes. Conclusion HCMV UL146 gene in asymptomatic children was hypervariable in both nucleotide sequence and amino acid structure. However, the important functional region was highly con-served. The predominant genotypes of UL146 in these children were G1, G8, G9 and G11, and the geno-type distribution in them showed no significant difference with previous findings in children with symptomatic HCMV infection.