The SMAD2/miR-4256/HDAC5/p16INK4a signaling axis contributes to gastric cancer progression.

The dysregulation of exosomal microRNAs (miRNAs) plays a crucial role in the development and progression of cancer. This study investigated the role of a newly identified serum exosomal miRNA miR-4256 in gastric cancer (GC) and the underlying mechanisms. The differentially expressed miRNAs were firstly identified in serum exosomes of GC patients and healthy individuals using next-generation sequencing and bioinformatics. Next, the expression of serum exosomal miR-4256 was analyzed in GC cells and GC tissues, and the role of miR-4256 in GC was investigated by in vitro and in vivo experiments. Then, the effect of miR-4256 on its downstream target genes HDAC5/p16INK4a was studied in GC cells, and the underlying mechanisms were evaluated using dual luciferase reporter assay and Chromatin Immunoprecipitation (ChIP). Additionally, the role of the miR-4256/HDAC5/p16INK4a axis in GC was studied using in vitro and in vivo experiments. Finally, the upstream regulators SMAD2/p300 that regulate miR-4256 expression and their role in GC were explored using in vitro experiments. miR-4256 was the most significantly upregulated miRNA and was overexpressed in GC cell lines and GC tissues; in vitro and in vivo results showed that miR-4256 promoted GC growth and progression. Mechanistically, miR-4256 enhanced HDAC5 expression by targeting the promoter of the HDAC5 gene in GC cells, and then restrained the expression of p16INK4a through the epigenetic modulation of HDAC5 at the p16INK4a promoter. Furthermore, miR-4256 overexpression was positively regulated by the SMAD2/p300 complex in GC cells. Our data indicate that miR-4256 functions as an oncogene in GC via the SMAD2/miR-4256/HDAC5/p16INK4a axis, which participates in GC progression and provides novel therapeutic and prognostic biomarkers for GC.

Cyclocarya paliurus leaves extracts alleviate metabolic phenotypes in Chinese T2DM patients by modulating gut microbiota and metabolites: a clinical randomized controlled trial.

Objective: We aimed to investigate the effect of Cyclocarya paliurus leaves extracts (CP) on glucose and blood lipid metabolism and its relationship with intestinal flora in type 2 diabetes mellitus (T2DM) patients. Methods: In this open-label, 84-day randomized controlled trial, a total of 38 T2DM patients were randomly assigned to the CP group or the Glipizide group (G group) in a 2:1 ratio. T2DM-associated metabolic phenotypes, gut microbiota and metabolites including short-chain fatty acids (SCFAs) and bile acids (BAs) were detected. Results: At the end of intervention, CP, like Glipizide, significantly improved HbA1c level and other glucose metabolism parameters (fasting plasma glucose (FBG), 2-hour post-meal blood glucose (2hPBG), the area under curve of oral glucose tolerance test glucose (OGTT glucose AUC)). Moreover, CP also resulted in the significant improvement in the levels of blood lipid and blood pressure. Notably, the improvement in blood lipid(triglycerides (TG) and high-density lipoprotein cholesterol (HDL-c)) and blood pressure (diastolic blood pressure (DBP)) was significantly greater in the CP group compared with the G group. Furthermore, the liver and kidney function parameters did not significantly change in both CP group and the G group over the 84-day period. Additionally, the enrichment of potentially beneficial bacteria (Faecalibacterium and Akkermansia), SCFAs and unconjugated BAs and the depletion of potential pathogenic bacteria (Prevotella_9) and conjugated BAs were observed in the CP group, while the abundances of the gut microbial were kept stable in the G group after intervention. Conclusion: CP displays a more beneficial effect in the alleviation of T2DM-associated metabolic phenotypes than glipizide by regulating gut microbiota and metabolites in T2DM patients, with no significant effects on liver and kidney function.

Identification of Cuproptosis-Related Genes in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent hepatic pathology worldwide. However, the precise molecular mechanisms for NAFLD are still not sufficiently explained. Recently, a new mode of cell death (cuproptosis) is found. However, the relationship between NAFLD and cuproptosis remains unclear. We analyzed three public datasets (GSE89632, GSE130970, and GSE135251) to identify cuproptosis-related genes stably expressed in NAFLD. Then, we performed a series of bioinformatics analyses to explore the relationship between NAFLD and cuproptosis-related genes. Finally, 6 high-fat diet- (HFD-) induced NAFLD C57BL/6J mouse models were established to carry out transcriptome analysis. The results of gene set variation analysis (GSVA) revealed that the cuproptosis pathway was abnormally activated to a certain degree (p = 0.035 in GSE89632, p = 0.016 in GSE130970, p = 0.22 in GSE135251), and the principal component analysis (PCA) of the cuproptosis-related genes showed that the NAFLD group separated from the control group, with the first two principal components accounting for 58.63%-74.88% of the variation. Among three datasets, two cuproptosis-related genes (DLD and PDHB, p < 0.01 or 0.001) were stably upregulated in NAFLD. Additionally, both DLD (AUC = 0.786-0.856) and PDHB (AUC = 0.771-0.836) had favorable diagnostic properties, and the multivariate logistics regression model further improved the diagnostic properties (AUC = 0.839-0.889). NADH, flavin adenine dinucleotide, and glycine targeted DLD, and pyruvic acid and NADH targeted PDHB in the DrugBank database. The DLD and PDHB were also associated with clinical pathology, especially with steatosis (DLD, p = 0.0013-0.025; PDHB, p = 0.002-0.0026) and NAFLD activity score (DLD, p = 0.004-0.02; PDHB, p = 0.003-0.031). What is more, DLD and PDHB were correlated with stromal score (DLD, R = 0.38, p < 0.001; PDHB, R = 0.31, p < 0.001) and immune score (DLD, R = 0.26, p < 0.001; PDHB, R = 0.27, p < 0.001) in NAFLD. Furthermore, Dld and Pdhb were also significantly upregulated in the NAFLD mouse model. In conclusion, cuproptosis pathways, especially DLD and PDHB, could be potential candidate genes for NAFLD diagnostic and therapeutic options.

Difference in hepatotoxicity of furan-containing components in cortex dictamni correlates the efficiency of their metabolic activation.

BACKGROUND: Cortex Dictamni (CD) has been associated with an increased risk of liver injury, which may be attributable to the metabolic activation of its furan-containing components (FCC). However, the hepatotoxic potencies of these FCCs and the mechanisms behind the differences in their toxicity intensity remain unknown. METHODS: The constituents of CD extract were determined by LC-MS/MS. Potentially toxic FCCs were screened by a previously published method. Hepatotoxicity of potentially toxic FCCs was evaluated in cultured mouse primary hepatocytes and mice. The ability to deplete hepatic glutathione (GSH), along with the formation of the corresponding GSH conjugates, resulting from the metabolic activation was determined ex vivo in mice. Intrinsic clearance rates (CLint,Vmax/Km) were assessed by a microsome-bases assay. RESULTS: A total of 18 FCCs were detected in CD extract. Among them, four FCCs, including rutaevin (RUT), limonin (LIM), obacunone (OBA) and fraxinellone (FRA) were found to be bioactivated in microsomal incubations. Only FRA displayed significant hepatotoxicity in vitro and in vivo. Similarly, FRA caused GSH depletion and GSH conjugation the most in vivo. The order of CLint for the four FCCs was FRA>>OBA>LIM>RUT. CONCLUSION: FRA is the major toxic FCC component of hepatotoxic CD extract. The hepatotoxicity of FCCs is closely related to the efficiency of their metabolic activation.

Immunochemical and LC-MS/MS Detection of Protein Adduction Resulting from Metabolic Activation of Columbin In Vitro and In Vivo.

Columbin (CLB) is a diterpenoid furanolactone compound occurring in some herbal medicines. Administration of CLB has been reported to induce liver injury. The reported CLB hepatotoxicity is suggested to require metabolism to a cis-enedial intermediate. We successfully detected hepatic protein adduction resulting from the metabolic activation of CLB and found that the intermediate reacted with lysine residues or lysine/cysteine residues to produce the corresponding pyrroline derivative or pyrrole derivative. The detection was achieved by proteolysis- and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods. Furthermore, we prepared a polyclonal antibody approach which allowed us to detect the protein adduction in the forms of protein immunoblot as well as tissue- and cell-based immunostaining. The antibody technique verified the protein adduction detected by LC-MS/MS.

Mechanistic Study of Xanthotoxin-Mediated Inactivation of CYP1A2 and Related Drug-Drug Interaction with Tacrine.

Xanthotoxin (XTT) is a biologically active furanocoumarin widely present in foods and plants. The present study is designed to systematically investigate the enzymatic interaction of XTT with CYP1A2, along with pharmacokinetic alteration of tacrine resulting from the co-administration of XTT. The results showed that XTT induced a time-, concentration-, and NADPH-dependent inhibition of CYP1A2, and the inhibition was irreversible. Co-incubation of glutathione (GSH) and catalase/superoxide dismutase was unable to prevent enzyme inactivation. Nevertheless, competitive inhibitor fluvoxamine exhibited a concentration-dependent protective effect against the XTT-induced CYP1A2 inactivation. A GSH trapping experiment provided strong evidence for the production of epoxide or/and γ-ketoenal intermediates resulting from the metabolic activation of XTT. Furthermore, pretreatment of rats with XTT was found to significantly increase the Cmax and area under the curve of plasma tacrine relative to those of tacrine administration alone.

DNA damage and up-regulation of PARP-1 induced by columbin in vitro and in vivo.

Columbin (CLB) is the most abundant (>1.0%) furan-containing diterpenoid lactone in herbal medicine Tinospora sagittate (Oliv.) Gagnep. The furano-terpenoid was found to be hepatotoxic, but the exact mechanisms remain unknown. The present study demonstrated that administration of CLB at 50 mg/kg induced hepatotoxicity, DNA damage and up-regulation of PARP-1 in vivo. Exposure to CLB (10 µM) induced GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1 and cell death in cultured mouse primary hepatocytes in vitro. Co-treatment of mouse primary hepatocytes with ketoconazole (10 µM) or glutathione ethyl ester (200 µM) attenuated the GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1, and cell death induced by CLB, while co-exposure to L-buthionine sulfoximine (BSO, 1000 µM) intensified such adverse effects resulting from CLB exposure. These results suggest that the metabolic activation of CLB by CYP3A resulted in the depletion of GSH and increase of ROS formation. The resultant over-production of ROS subsequently disrupted the DNA integrity and up-regulated the expression of PARP-1 in response to DNA damage, and ROS-induced DNA damage was involved in the hepatotoxicity of CLB.

Formation of RNA adducts resulting from metabolic activation of spice ingredient safrole mediated by P450 enzymes and sulfotransferases.

Safrole (SFL) is an IARC class 2B carcinogen. To better understand the mechanism involved in SFL toxicity, we explored the potential interactions between SFL metabolites and RNA. Three guanosine adducts (G1-G3), two adenosine adducts (A1-A2), and two cytosine adducts (C1-C2) were detected by LC-MS/MS in mouse liver S9 incubations, cultured mouse primary hepatocytes, and liver tissues of mice after exposure to SFL. These adducts were chemically synthesized, and one of the guanosine adducts was structurally characterized by 1H-NMR. Studies in vitro and in vivo showed that SFL was oxidized by cytochrome P450 enzymes to the corresponding 1'-hydroxyl metabolite which was further metabolized by sulfotransferases to form allylic sulfate esters. The formed reactive intermediate(s) subsequently reacted with bases of RNA, leading to RNA adduction, which could play a partial role in the toxicities of SFL through the alteration of RNA biochemical properties and interruption of RNA functions.

Comparison of copper concentration between non-alcoholic fatty liver disease patients and normal individuals: A meta-analysis.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide. Copper metabolism plays an important role in the pathogenesis of NAFLD. However, the relationship between serum/hepatic copper concentration and NAFLD is still debated. A literature search was performed using electronic databases to find publications up to September 2022, where the relationship between serum/hepatic copper or ceruloplasmin concentration and NAFLD was evaluated. Finally, 6 articles with 9 unique outcomes involving 2,607 NAFLD patients and 1,441 non-NAFLD normal individuals were included. The pooled results showed that hepatic copper concentration was significantly decreased in NAFLD patients (SMD = -0.98, 95% CI = [-1.21; -0.74], p < 0.0001), and the sensitivity analysis also confirmed this. Nevertheless, serum copper (SMD = -0.02, 95% CI = [-0.32; 0.28], p = 0.88) and ceruloplasmin (SMD = -0.03, 95% CI = [-0.69; 0.63], p = 0.93) were not associated with NAFLD. This meta-analysis revealed that low hepatic copper concentration was found in NAFLD patients and serum copper and ceruloplasmin were not associated with NAFLD. Larger cohort studies and related trials are needed to further validate the result of this meta-analysis in the future.

Bioactivity profiling of the extremolyte ectoine as a promising protectant and its heterologous production.

Ectoine is a compatible solutes that is diffusely dispersed in bacteria and archaea. It plays a significant role as protectant against various external pressures, such as high temperature, high osmolarity, dryness and radiation, in cells. Ectoine can be utilized in cosmetics due to its properties of moisturizing and antiultraviolet. It can also be used in the pharmaceutical industry for treating various diseases. Therefore, strong protection of ectoine creates a high commercial value. Its current market value is approximately US$1000 kg-1. However, traditional ectoine production in high-salinity media causes high costs of equipment loss and wastewater treatment. There is a growing attention to reduce the salinity of the fermentation broth without sacrificing the production of ectoine. Thus, heterologous production of ectoine in nonhalophilic microorganisms may represent the new generation of the industrial production of ectoine. In this review, we summarized and discussed the biological activities of ectoine on cell and human health protection and its heterologous production.

Differential Effects of Dietary White Meat and Red Meat on NAFLD Progression by Modulating Gut Microbiota and Metabolites in Rats.

Objective: To assess the effects of dietary white meat (grass carp and chicken) and red meat (pork and beef) on metabolic parameters, including the intestinal microbiota and its metabolites (SCFAs and bile acids) in NAFLD rats induced by high-fat diet. Methods: NAFLD rats were randomly assigned to five groups: NAFLD group, grass carp group, chicken group, pork group, and beef group (10 rats in each group), and these rats were fed for 8 weeks using the high-fat diet, grass carp-based diet, chicken-based diet, pork-based diet, and beef-based diet, respectively. At the end of the intervention, NAFLD-related metabolic indexes, intestinal flora, and its metabolites were measured. Results: The grass carp-based diet significantly improved hepatic pathological changes and glycolipid metabolism, and the chicken-based diet only partially improved the metabolic parameters. However, NAFLD progression was observed in the pork group and the beef group. What is more, the white meat-based diet-mediated changes in the enrichment of beneficial bacteria (such as Lactobacillus or Akkermansia), SCFAs, and unconjugated BAs (such as UDCA) and the depletion of pathogenic bacteria (such as Bilophila and Prevotella_9) and conjugated BAs were observed, while the red meat-based diet-induced changes in the enrichment of pathogenic bacteria (Prevotella_9 or Lachnospiraceae_UCG-010) and conjugated BAs and the depletion of SCFAs and unconjugated BAs were found. Conclusion: The dietary white meat and red meat modulating gut microbiota and its metabolites may favor and aggravate NAFLD in rats, respectively.

Nonalcoholic fatty liver disease and health outcomes: An umbrella review of systematic reviews and meta-analyses.

Purpose: A large number of systemic reviews and meta-analyses have explored the relationship between nonalcoholic fatty liver disease (NAFLD) and multiple health outcomes. The aim of this study is to conduct an umbrella review to assess the strength and evidence for the association between NAFLD and health outcomes. Methods: We systematically identified the present meta-analyses of observational studies reporting an association between NAFLD and health outcomes. For each meta-analysis, we assessed the quality with AMSTAR2 and graded the epidemiologic evidence. Results: Fifty-four articles comprising 111 unique meta-analyses were included in this study. Eighty-five unique outcomes showed significant associations (P ← 0.05), whereas 26 unique outcomes showed insignificant associations, and we cannot assess the epidemiologic evidence. For 85 significant health outcomes, four outcomes (carotid intima-media thickness (C-IMT), peak A velocity, left ventricle end-diastolic diameter, incident chronic kidney disease (CKD) in adult patients) was graded as high quality of evidence, 23 outcomes were graded as the moderate quality of evidence, and the remaining 58 outcomes were graded as weak quality of evidence. Fourty-seven (87.03%) studies showed critically low methodological quality. Conclusion: In this umbrella review, only four statistically significant health outcomes showed high epidemiologic evidence. NAFLD seems to relate to an increased risk of C-IMT, peak A velocity, left ventricle end-diastolic diameter, and incident CKD in adult patients.

Metabolic Activation of Militarine In Vitro and In Vivo.

Bletilla striata is consumed as food and herbal medicine. Militarine (MLT) is a major ingredient in B. striata. Previous studies demonstrated that MLT showed teratogenic toxicity to zebrafish embryos. The present study aimed to identify reactive metabolites possibly involved in the cytotoxicity of MLT and determine the metabolic pathways involved. MLT was found to be hydrolyzed to p-hydroxybenzyl alcohol (HBA) by ß-glucosidase and esterases. The resulting HBA further underwent spontaneous dehydration to form quinone methide. HBA was also metabolized to the corresponding sulfate, followed by departure of the sulfate to generate a quinone methide. The resultant quinone methide reacted with hepatic glutathione (GSH) and protein to form the corresponding GSH conjugate and protein adduction. Additionally, inhibition of sulfotransferases (SULTs) attenuated the susceptibility of hepatocytes to the toxicity of MLT. This study provides that the hydrolytic enzymes ß-glucosidase, esterases, and SULTs participate in the metabolic activation of MLT.

Metabolic activation of 3-aminodibenzofuran mediated by P450 enzymes and sulfotransferases.

3-Aminodibenzofuran (3-ADBF) is a potent bladder carcinogen. This study aimed to identify reactive metabolites and the metabolic pathways of 3-ADBF. The in vitro and in vivo studies demonstrated that 3-ADBF was oxidized to the corresponding hydroxylamine by cytochrome P450 enzymes, followed by sulfation of the hydroxyl group mediated by sulfotransferases. The resulting sulfate conjugate was chemically reactive to GSH and cysteine residues of hepatic protein to form the corresponding GSH conjugate and protein adduction. Exposure of 3-ADBF to primary hepatocytes caused protein covalent binding and decreased cell viability. The resultant protein adduction was found to correlate the observed cytotoxicity of 3-ADBF.

Bioinformatics Analysis Explores Potential Hub Genes in Nonalcoholic Fatty Liver Disease.

Background: Nonalcoholic fatty liver disease (NAFLD) is now recognized as the most prevalent chronic liver disease worldwide. However, the dysregulated gene expression for NAFLD is still poorly understood. Material and methods: We analyzed two public datasets (GSE48452 and GSE89632) to identify differentially expressed genes (DEGs) in NAFLD. Then, we performed a series of bioinformatics analyses to explore potential hub genes in NAFLD. Results: This study included 26 simple steatosis (SS), 34 nonalcoholic steatohepatitis (NASH), and 13 healthy controls (HC). We observed 6 up- and 19 down-regulated genes in SS, and 13 up- and 19 down-regulated genes in NASH compared with HC. Meanwhile, the overlapping pathways between SS and NASH were PI3K-Akt signaling pathway and pathways in cancer. Then, we screened out 10 hub genes by weighted Gene Co-Expression Network Analysis (WGCNA) and protein-protein interaction (PPI) networks. Eventually, we found that CYP7A1/GINS2/PDLIM3 were associated with the prognosis of hepatocellular carcinoma (HCC) in the TCGA database. Conclusion: Although further validation is still needed, we provide useful and novel information to explore the potential candidate genes for NAFLD prognosis and therapeutic options.

The diagnostic and clinicopathological value of trefoil factor 3 in patients with gastric cancer: a systematic review and meta-analysis.

OBJECTIVE: To assess the diagnostic value of Trefoil factor 3 (TFF3) and the correlation between TFF3 expression and clinicopathological features in patients with gastric cancer (GC). METHODS: PubMed, The Cochrane, EMbase, and Web of Science were retrieved comprehensively to collect relevant literature. The search ended on 31 May 2020. All data were analyzed using PubMed, The Cochrane, EMbase, and Web of Science were retrieved to collect relevant articles. All data from the included studies were subjected to meta-analysis using Stata 12.0 software. RESULTS: Seventeen studies involved 4654 subjects were included. For the diagnostic value of TFF3 for GC, the sensitivity, specificity, and AUC were 0.71, 0.80, and 0.80, respectively. For the clinicopathological value of TFF3, tissue TFF3 expression showed a higher risk of lymph node metastasis (OR 2.20, 95% CI 1.75-2.78, p < 0.001) and muscularis propria invasion (≥T2) (1.51, 1.13-2.03, p = 0.006), as well as worse TNM stage (2.26, 1.63-3.12, p < 0.001) and histological type (1.72, 1.34-2.20, p < 0.001), while no apparent relationship was found for serous membrane invasion (T4), venous invasion, and peritoneal metastasis. CONCLUSION: TFF3 may be a promising biomarker for GC, and the TFF3 expression is likely to be involved in the invasion, metastasis, and differentiation of GC.

Antifungal peptides produced by actinomycetes and their biological activities against plant diseases.

Antibacterial peptides are a class of naturally occurring peptides produced by eukaryotes and prokaryotes. Some of them exhibit broad-spectrum antifungal activity. Antifungal peptides (AFPs) can be developed as antibiotic to control fungal infections in agriculture due to their different antifungal mechanisms. As actinomycetes are still one of the most important sources of novel antibiotics, in this review, the mechanisms of action of AFPs are explained. Characterization of several AFPs produced by actinomycetes and their biological activities against plant diseases are summarized. Furthermore, the pathway for total synthesis of naturally occurring cyclodepsipeptide, valinomycin, is proposed. Finally, the pathway for biosynthesis of kutzneride 2 is proposed and the structure-activity relationship of kutznerides is discussed.

Comparative Proteome Identifies Complement Component 3-Mediated Immune Response as Key Difference of Colon Adenocarcinoma and Rectal Adenocarcinoma.

Colorectal cancer (CRC) is one of the most lethal diseases with high morbidity and mortality worldwide. Clinically, tumors located in colon and rectum have diverse prognosis and therapeutic outcome. Here, we performed data mining derived from 20 CRC patient samples to compare proteomic difference between colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ). We found that differential expressed proteins (DEPs) upregulated in COAD were mainly enriched in immune response, moreover, higher immune scores were found in COAD than READ, as calculated by The Cancer Genome Atlas (TCGA) data. To identify the core protein of DEPs with high prognostic value for COAD, we performed topological overlap matrix (TOM) to investigate the hub proteins using 77 immune-relevant DEPs, and identified complement component 3 (C3) as the core protein in the immune-relevant DEPs matrix between the COAD and READ. Moreover, we found that C3 was up-regulated in COAD, and its expression was negatively associated with overall survival of COAD patients but not READ. In conclusion, we identified C3-mediated immune response as key feature to distinguish COAD and READ, and highlighted C3 as potential biomarker with high prognostic value for clinical application, which provided new clue for precise treatment of COAD.