Obesogenic polystyrene microplastic exposures disrupt the gut-liver-adipose axis.

Microplastics (MP) derived from the weathering of polymers, or synthesized in this size range, have become widespread environmental contaminants and have found their way into water supplies and the food chain. Despite this awareness, little is known about the health consequences of MP ingestion. We have previously shown that the consumption of polystyrene (PS) beads was associated with intestinal dysbiosis and diabetes and obesity in mice. To further evaluate the systemic metabolic effects of PS on the gut-liver-adipose tissue axis, we supplied C57BL/6J mice with normal water or that containing 2 sizes of PS beads (0.5 and 5 µm) at a concentration of 1 µg/ml. After 13 weeks, we evaluated indices of metabolism and liver function. As observed previously, mice drinking the PS-containing water had a potentiated weight gain and adipose expansion. Here we found that this was associated with an increased abundance of adipose F4/80+ macrophages. These exposures did not cause nonalcoholic fatty liver disease but were associated with decreased liver:body weight ratios and an enrichment in hepatic farnesoid X receptor and liver X receptor signaling. PS also increased hepatic cholesterol and altered both hepatic and cecal bile acids. Mice consuming PS beads and treated with the berry anthocyanin, delphinidin, demonstrated an attenuated weight gain compared with those mice receiving a control intervention and also exhibited a downregulation of cyclic adenosine monophosphate (cAMP) and peroxisome proliferator-activated receptor (PPAR) signaling pathways. This study highlights the obesogenic role of PS in perturbing the gut-liver-adipose axis and altering nuclear receptor signaling and intermediary metabolism. Dietary interventions may limit the adverse metabolic effects of PS consumption.

Sarcosine dehydrogenase as an immune infiltration-associated biomarker for the prognosis of hepatocellular carcinoma.

This study was aimed to investigate the prognostic value and clinical significance of sarcosine dehydrogenase (SARDH) in hepatocellular carcinoma (HCC) and to explore the underlying mechanisms. The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), HPA and CPTAC databases were adopted to analyze the expression of SARDH mRNA and protein between normal liver tissue and HCC, and examine their relationship with clinicopathological features. Kaplan-Meier analysis, Cox regression, as well as nomogram were adopted to explore the prognostic value of SARDH in HCC. Gene Ontology (GO), Kyoto Gene and Genome Encyclopedia (KEGG) together with Gene Set Enrichment Analysis (GSEA) were adopted to analyze the molecular mechanisms and biological functions of SARDH in HCC; while MethSurv, STRING, GeneMANIA, TIMER database data and single-sample gene set enrichment analysis (ssGSEA) algorithm were used for other bioinformatic analysis. Furthermore, immunohistochemistry was used to verify the expression of SARDH. Compared to normal liver tissue, SARDH expression was markedly lower in HCC. A lower SARDH expression was linked with Pathologic T stage (T3&T4), pathologic stage (Stage III&IV), and histologic grade (G3&4), which further indicates worse prognosis. Besides, results of bioinformatic analysis proved that SARDH expression was correlated with immune infiltration. In addition, SARDH hypermethylation was related to a poorer prognosis. SARDH expression was related to several key genes in the Ferroptosis pathway.

SART3, regulated by p53, is a biomarker for diagnosis, prognosis and immune infiltration in hepatocellular carcinoma.

OBJECTIVE: This study aimed to investigate the role of squamous cell carcinoma antigen recognized by T cells 3 (SART3) in hepatocellular carcinoma (HCC). METHODS: SART3 expression and prognostic value were analyzed in TCGA and GEO datasets. The diagnostic value and prognostic significance of SART3 were determined using immunohistochemistry in the Guangxi cohort. The whole-exome mutation spectrum of SART3 was analyzed in high and low expression groups in both TCGA and Guangxi cohorts. The biological functions of the SART3 gene were validated through in vitro experiments using small interfering RNA technology to downregulate SART3 expression in HCC cell lines. RESULTS: SART3 expression was significantly higher in HCC tissues than in adjacent noncancerous liver tissues in TCGA, GEO and Guangxi cohorts. High expression of SART3 was significantly associated with poor prognosis in HCC patients. In TCGA and Guangxi cohorts, the expression of SART3 in the TP53 mutation group was significantly higher than that in the non-mutation group. Downregulation of SART3 expression significantly inhibited the migration and proliferation of HCC cells. SART3 may be involved mainly in immune infiltration of Th2 cells and macrophages in HCC. Additionally, SART3 can upregulate the expression of immune checkpoints (PD-L1 and TIM-3) and predict potential therapeutic agents for HCC. CONCLUSION: The findings of this study demonstrate the diagnostic and prognostic value of SART3 in HCC. SART3 may be associated with immune infiltration of Th2 cells and macrophages in HCC, highlighting its potential role in the development and progression of HCC.

Prognostic value and potential molecular mechanism of ITGB superfamily members in hepatocellular carcinoma.

We analyzed the prognostic value and potential molecular mechanisms of the members of integrin ß (ITGB)superfamily in hepatocellular carcinoma (HCC) using data from The Cancer Genome Atlas (TCGA), cBioPortal, Gene Expression Profiling Interactive Analysis (GEPIA), Human Protein Atlas (HPA) HPA, Search Tool for the Retrieval of Interacting Genes/Proteins, GeneMANIA, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), TIMER and Gene set enrichment analysis (GSEA) databases. ITGB4/5 mRNA was upregulated in HCC tissues in contrast to the normal liver tissues, whereas ITGB2/3/8 levels were lower in the former. ITGB4 was the most frequently mutated ITGB gene in HCC. Receiver operating characteristic curve (ROC) analysis showed that the expression levels of ITGB2/3/4/5/7/8 had significant diagnostic value in distinguishing HCC tissues from healthy liver tissues, ITGB8 had the highest diagnostic efficacy. The ITGB1/3/6/8 were also upregulated in the HCC tissues in contrast to healthy liver tissues. The expression of ITGB8 was verified by immunohistochemistry (IHC). Furthermore, ITGB6 and ITGB7 expression levels were strongly associated with the overall survival (OS) of HCC patients. The ITGB superfamily members exhibited homology and interactions in protein structure. In addition, ITGB6 together with ITGB7 were negatively related to the infiltration of multiple immune cell populations. GSEA results showed that ITGB6 was enriched in HCC migration and recurrence, whereas ITGB7 was significantly enriched in HIPPO, TOLL and JAK-STAT signaling pathways. In conclusion, ITGB6 and ITGB7 genes are possible to be prognostic biomarkers for HCC.

Nucleoporin 107 is a prognostic biomarker in hepatocellular carcinoma associated with immune infiltration.

OBJECTIVE: To assess the diagnostic value and clinical significance of nucleoporin 107 (NUP107) in hepatocellular carcinoma (HCC), and explore the possible mechanisms. METHODS: The transcriptomic and clinical data of HCC patients were retrieved from The Cancer Genome Atlas (TCGA) and GEO databases. Tissue specimens were collected from HCC patients in the Guangxi area. According to the expression levels and prognostic characteristics of NUP107, ROC curves and nomogram models were constructed using the R package. RESULTS: NUP107 was highly expressed in 26 human cancers including HCC, and was associated with advanced HCC staging and worse prognosis. NUP107 showed satisfactory ability to predict the prognosis of HCC patients (AUC >0.8). Results of gene set enrichment analysis (GSEA) further showed that NUP107 was mainly associated with cell cycle-related pathways such as the cell cycle, DNA replication, G2M checkpoint, E2F target, and mitotic spindle. In addition, NUP107 was also associated with immune infiltration in HCC and showed significant positive correlation with immune checkpoints (PD-L1 and TIM-3).

MCM2 promotes the stemness and sorafenib resistance of hepatocellular carcinoma cells via hippo signaling.

OBJECT: A large number of studies have suggested that stemness is an essential mechanism for drug resistance, metastasis and relapse in hepatocellular carcinoma (HCC). The aim of this study was to determine the impact of MCM2 on stemness and identify potential mechanisms that complement the stemness regulatory network in HCC. METHODS: MCM2 expression features and prognostic significance were analyzed in multiple cohorts, including TCGA LIHC dataset, GSE14520 dataset, Guangxi cohort, and GSE76427 dataset. Stemness-related molecules and phenotypes were examined to evaluate the impact of MCM2 on stemness. The expression levels of key molecules of the hippo signaling pathway together with downstream target genes were examined to evaluate the effect of MCM2 on hippo signaling. This was further demonstrated by rescue experiments with hippo signaling pathway inhibitors (super-TDU). Sorafenib-resistant cells were constructed to assess the effect of MCM2 on drug resistance. A xenotransplantation model of nude mice was constructed to validate the role of MCM2 in vivo. RESULTS: MCM2, which is expressed at higher levels in HCC tissue than in normal liver tissues, is a good indicator for distinguishing tumor tissues from normal liver tissues and can help differentiate HCC patients at different BCLC stages. The annotation of the differentially expressed genes in the MCM2 high and low expression groups indicated that MCM2 may be associated with the hippo signaling pathway. In addition, the expression of MCM2 in HCC tissues was correlated with the expression of YAP1/TAZ, which are key molecules of the hippo signaling pathway. It indicated that manipulation of MCM2 expression affects hippo signaling and stemness, while the inhibition of hippo signaling significantly reversed the effect of MCM2 on stemness. Disruption of MCM2 expression significantly elevated the sensitivity of sorafenib-resistant cells to sorafenib, as evidenced by the decrease in IC50 and diminished sphere-forming capacity. The in vivo assays showed that MCM2 effectively enhanced the efficacy of sorafenib. CONCLUSION: MCM2 is a good prognostic marker. MCM2 enhances the stemness of HCC cells by affecting the Hippo signaling pathway, while the downregulation of MCM2 inhibits resistance towards sorafenib.

A novel Peng's test in reducing bile leakage after partial hepatectomy for hepatocellular carcinoma: From an animal study to a clinical cohort Propensity score matching comparative study.

BACKGROUND: Bile leakage (BL) is a common complication of partial hepatectomy for hepatocellular carcinoma (HCC). However, various intraoperative approaches to detect BL have not been widely accepted owing to uncertainty in their treatment effectiveness and complexity of use. MATERIALS AND METHODS: A novel BL-detection approach (Peng's test) was developed in a swine model to determine the pressures generated in the gallbladder and common bile duct (CBD) during the test. A comparative study was then conducted on a prospective cohort of patients using Peng's test versus a retrospective historical cohort patient group using the White Gauze test in partial hepatectomy for HCC. Propensity score matching (PSM) was performed in a 1:1 ratio to balance confounding factors. RESULTS: The maximum pressures with methylene blue injection in the gallbladder and CBD without Pringle's maneuver in the four swines were 103.8 ± 11.8 and 42.3 ± 6.1, respectively. After Pringle's maneuver, 32.0 ± 6.8 mL methylene blue injection led to a maximum pressure in the CBD of 85.3 ± 9.5 cmH2O. The pressures in CBD were 25.8 ± 3.3 and 86.0 ± 9.9 cmH2O when BL appeared at small bile ducts and around the ligation sites, respectively. Of the 206 patients enrolled in the historical control group, 31 (15.0%) developed BL, while of the 54 patients in the study group, only 1 developed grade A BL. The number of BL detected by the routine white gauze test in the control group was significantly lower than that in the study group (Z = -3.002, P = 0.003). After PSM, the incidence of BL in the control group and grade B/C BL was 20.4% and 11.1%, respectively. The corresponding incidences in the study group were 1.9% (χ2 = 7.594, P = 0.006) and 0% (P = 0.027), respectively. The length of hospital stay in the study group was significantly reduced (Z = -6.048, P < 0.001). CONCLUSION: Peng's test for intraoperative BL detection is safe and effective in reducing BL after hepatectomy.

CYP2C8 Suppress Proliferation, Migration, Invasion and Sorafenib Resistance of Hepatocellular Carcinoma via PI3K/Akt/p27kip1 Axis.

BACKGROUND: Cytochrome P450 2C8 (CYP2C8) gene is one of the members of the cytochrome P450 enzymes (CYPs) gene family. The aim of this study was to reveal the function of CYP2C8 in hepatocellular carcinoma (HCC) and its effect on the sorafenib resistance. METHODS: Differential expression analysis in multiple HCC datasets all suggested that CYP2C8 expression was significantly decreased in HCC tissues, compared with para-carcinoma liver tissues. The expression level of CYP2C8 was subsequently compared between HCC tissues and para-carcinoma liver tissues of 70 patients form Guangxi, China, with the result consistent with the above. Survival analysis and ROC analysis indicated that CYP2C8 was equipped with satisfactory diagnostic and prognostic value in HCC. To examine the effect of CYP2C8 on the malignant phenotype of HCC cells, stable transcriptional cell lines with CYP2C8 over-expression were established, and then Cell Counting Kit-8 (CCK8) assay, colony formation assay, cell cycle assay, cell invasion assay and wound healing assay were performed. RESULTS: The results of aforementioned assays suggested that CYP2C8 over-expression restricted the proliferation, clonality, migration, invasion and cell cycle of HCC cells but had no significant effect on cell apoptosis. The enrichment analysis in terms of sequencing data of HCC cell lines with stable CYP2C8 over-expression suggested that CYP2C8 might be related to PI3K/Akt/p27Kip1 axis. The inhibition of CYP2C8 over-expression on PI3K/Akt/p27Kip1 axis was subsequently demonstrated with Western blot assay. In the rescue experiment, it was observed that both P27 inhibitor and PI3K agonist counteracted the repressed malignant phenotype caused by CYP2C8 over-expression, which further demonstrated that CYP2C8 played a role in HCC cells via PI3K/Akt/p27Kip1 axis. DISCUSSION: The results demonstrated that CYP2C8 enhances the anticancer activity of sorafenib in vitro assays and in tumor xenograft model, with Ki-67 down-regulation and PI3K/Akt/p27Kip1 axis inhibition. In conclusion, these findings hinted that CYP2C8 restricted malignant phenotype and sorafenib resistance in HCC via PI3K/Akt/p27kip1 axis.

Mechanisms of acrolein-induced myocardial dysfunction: implications for environmental and endogenous aldehyde exposure.

Aldehydes are ubiquitous pollutants generated during the combustion of organic materials and are present in air, water, and food. Several aldehydes are also endogenous products of lipid peroxidation and by-products of drug metabolism. Despite well-documented high reactivity of unsaturated aldehydes, little is known regarding their cardiovascular effects and their role in cardiac pathology. Accordingly, we examined the myocardial effects of the model unsaturated aldehyde acrolein. In closed-chest mice, intravenous acrolein (0.5 mg/kg) induced rapid but reversible left ventricular dilatation and dysfunction. In mouse myocytes, micromolar acrolein acutely depressed myofilament Ca(2+) responsiveness without altering catecholamine sensitivity, similar to the phenotype of stunned myocardium. Immunoblotting revealed increased acrolein-protein adducts and protein-carbonyls in both acrolein-exposed myocardium (1.8-fold increase, P < 0.002) and myocytes (6.4-fold increase, P < 0.02). Both the contractile dysfunction and adduct formation were markedly attenuated by pretreatment with the thiol donor N-acetylcysteine (5 mM). Two-dimensional gel electrophoresis and mass-assisted laser desorption/ionization time-of-flight mass spectrometry analysis revealed two groups of adducted proteins, sarcomeric/cytoskeletal proteins (cardiac alpha-actin, desmin, myosin light polypeptide 3) and energy metabolism proteins (mitochondrial creatine kinase-2, ATP synthase), indicating site-specific protein modification that was confirmed by immunohistochemical colocalization. We conclude that direct exposure to acrolein induces selective myofilament impairment, which may be, in part, related to the modification of proteins involved in myocardial contraction and energy metabolism. Myocardial dysfunction induced by acrolein and related aldehydes may be symptomatic of toxicological states associated with ambient or occupational exposures or drug toxicity. Moreover, aldehydes such as acrolein may mediate cardiac dysfunction in pathologies characterized by high-oxidative stress.

Downregulation of CuZn-superoxide dismutase contributes to beta-adrenergic receptor-mediated oxidative stress in the heart.

OBJECTIVE: Sustained beta-adrenergic receptor (beta-AR) activation augments oxidative stress in the heart; whether alterations in antioxidant enzymes contribute to this effect is unknown. METHODS AND RESULTS: Adult male Wistar rats were implanted with osmotic minipumps to infuse either l-isoproterenol (ISO, 25 microg/kg/h) or saline (SAL). After 7-days, ISO-treated hearts exhibited significant (p<0.005): 1) concentric hypertrophy and augmentation of systolic function, 2) reductions of end-systolic wall stress, and 3) augmentation of oxidative stress, with a approximately 3-fold increase in 4-hydroxy-2-nonenal-and malondialdehyde-protein adducts. ISO-treated hearts also exhibited significant (p<0.01) reductions of CuZn-superoxide dismutase (SOD) enzyme activity (30%), protein (40%), and mRNA (60%), without changes in Mn-SOD, catalase, or glutathione peroxidase. Elk-1 and YinYang1 (YY1) are transcription factors that positively and negatively regulate CuZn-SOD expression, respectively. ISO-treated hearts exhibited a 3-fold increase in YY1 and a 2-fold reduction in Elk-1 DNA binding activity, strongly favoring CuZn-SOD gene repression. In isolated cardiomyocytes, sustained (24 h) ISO stimulation significantly (p<0.01) increased reactive oxygen species (ROS), an effect blocked by CGP20712A, a beta1-AR antagonist, but not by ICI118,551, a beta2-AR antagonist. CuZn-SOD downregulation paralleled the increase in ROS, and were similarly blocked by beta1- but not beta2-AR blockade. There were no changes in CuZn-SOD mRNA stability or myocyte size with ISO treatment. However, nuclear run-on revealed a 40% reduction in CuZn-SOD mRNA expression (p<0.01), consistent with transcriptional repression. ISO also depressed total cellular antioxidant capacity, reduced glutathione (GSH) levels, and the GSH:GSSG ratio. Moreover, CuZn-SOD siRNA transfection of H9c2 cardiomyocytes to suppress CuZn-SOD protein by approximately 40-50% (analogous to the in vivo changes) induced cellular apoptosis. CONCLUSIONS: Sustained beta-AR stimulation transcriptionally downregulates CuZn-SOD in myocardium via the beta1-AR, thereby contributing to beta-AR-mediated oxidative stress.

Prolonged oxidative stress inverts the cardiac force-frequency relation: role of altered calcium handling and myofilament calcium responsiveness.

The normally positive force- and Ca2+ -frequency responses (FFR and CaFR) are inverted in heart failure (HF); whether oxidative stress contributes to these abnormalities is unknown. We evaluated the impact of acute and prolonged oxidative stress on contraction and Ca2+ handling in adult rat cardiomyocytes. Acute (30 min) exposure to H2O2 (100 microM) induced a twofold increase (P<0.025) in intracellular oxyradicals together with contractile depression despite preservation of the Ca2+ transient and the FFR and CaFR to 3 Hz, indicating reduced myofilament Ca2+ responsiveness. In contrast, prolonged (24 h) exposure to the copper-zinc superoxide dismutase inhibitor diethyldithiocarbamic acid (DDC, 1 microM) similarly augmented oxyradicals but also increased cell size, and contraction and Ca2+ transient duration (P<0.025). DDC-treated myocytes displayed inverted FFRs and attenuated (though still positive) CaFRs as compared to control, indicating reduced myofilament Ca2+ responsiveness coupled with altered Ca2+ handling. Protein levels of the Na+ -Ca2+ exchanger (NCX), sarcoplasmic reticular (SR) Ca2+ ATPase (SERCA2), and serine-16 phosphorylated phospholamban (pSer16-PLB) were increased (P<0.025), whereas dihydropyridine receptor abundance was decreased. Total PLB and ryanodine receptor protein expression were unchanged. Caffeine-induced Ca2+ release showed increased NCX activity (P<0.025) without changes in total releasable SR Ca2+, suggesting compensatory changes in SERCA2 and pSer16-PLB to maintain SR Ca2+ load. The superoxide scavenger Tiron attenuated these effects. Thus, acute oxyradical exposure rapidly depresses myofibrillar Ca2+ responsiveness. Prolonged oxidative stress further induces alterations in Ca2+ handling that combined with extant reductions in myofibrillar responsiveness invert the FFR. With regard to Ca2+ handling, reduced transsarcolemmal Ca2+ flux rather than reduced SR Ca2+ uptake was the primary determinant of a negative FFR. Analogous changes may be operative in HF, a state characterized by both oxidative stress and Ca2+ dysregulation.

Beta-adrenergic receptor blockade modulates Bcl-X(S) expression and reduces apoptosis in failing myocardium.

The mechanisms by which beta-adrenergic receptor (beta-AR) blockade modulates apoptosis in heart failure (HF) are unclear. We examined the impact of beta-AR blockade with metoprolol on myocardial remodeling, apoptosis, pro-apoptotic (Fas, Fas ligand, Bax, and Bcl-X(S)) and anti-apoptotic (Bcl-X(L)and Bcl-2) gene expression, and Bcl-X(L) and Bcl-X(S) protein in post-infarction HF in rats. In untreated rats, there was significant (P < 0.001) LV dilatation and systolic dysfunction compared to sham. Myocardial apoptosis was significantly increased (P < 0.005). Fas, Bax, and Bcl-2 mRNA expression was unchanged. However, Fas ligand mRNA and Bcl-X(S) mRNA and protein, all undetectable in sham, were markedly elevated (P < 0.001), whereas Bcl-X(L) mRNA and protein was unchanged. Immunohistochemistry confirmed increased Bcl-X(S) staining in failing myocardium, with unchanged Bcl-X(L). Metoprolol treatment resulted in: (1) improved LV remodeling (P < 0.025), (2) reduced myocardial apoptosis (P < 0.005), and (3) selective reduction in myocardial Bcl-X(S) expression (P < 0.001) without change in Fas, Fas ligand, Bax, Bcl-2, or Bcl-X(L). Studies in isolated rat myocytes revealed that prolonged isoproterenol (ISO) stimulation significantly increased Bcl-X(S) protein, reducing the Bcl-X(L)/X(S) ratio and myocyte survival (P < 0.005). ISO-induced Bcl-X(S) expression was significantly attenuated (P < 0.001) by both metoprolol and CGP20712A, a beta1-AR selective antagonist, but not by ICI118,551, a beta2-AR selective antagonist. We conclude that adrenergic activation, such as occurs in HF, increases pro-apoptotic Bcl-X(S) expression via the beta1-AR. beta-AR blockade in HF reduces myocardial apoptosis; attenuation of Bcl-X(S) expression may be one mechanism underlying this effect.