Changes in metabolic parameters and serum YKL-40 levels in Chinese rheumatoid arthritis patients during tocilizumab therapy.

OBJECTIVE: To investigate the metabolic changes during therapy of tocilizumab (TCZ) and methotrexate (MTX) in non-diabetic rheumatoid arthritis (RA) patients and for the first time explore the associations between metabolic parameters and serum YKL-40 (sYKL-40) levels. METHODS: We enrolled active non-diabetic RA patients who were refractory to MTX. Patients received intravenous TCZ (8 mg/kg) once every 4 weeks combined with MTX for 24 weeks. Metabolic parameters and sYKL-40 levels were measured before TCZ infusion at baseline, week 4, week 12, and week 24. Correlations were assessed by the Spearman's rank correlation analysis. RESULTS: A total of 91 non-diabetic RA patients were enrolled in this study. At week 24, we observed a significant elevation in body mass index (BMI), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) levels. In contrast, there was a significant decrease in TC/HDL­C ratio. No apparent changes in insulin resistance were found. Additionally, we detected a significant reduction in sYKL-40 levels during the study. At week 24, changes in sYKL-40 levels showed a significant negative correlation (r = -0.334, p = 0.002) with changes in TC levels. CONCLUSION: The combined therapy of TCZ and MTX resulted in a significant increase in BMI and lipid levels, while an evident decrease in the TC/HDL­C ratio and sYKL-40 levels in RA patients. Additionally, there was a significant correlation between the decrease in sYKL-40 levels and the increase in TC levels during treatment with TCZ and MTX. Key Points • Lipid levels elevated significantly and sYKL-40 levels decreased obviously after therapy of TCZ combined with MTX in Chinese RA patients. • There was a significant correlation between the increase in TC levels and the decrease in sYKL-40 levels during treatment with TCZ and MTX in RA patients.

Construction and evaluation of an aging-associated genes-based model for pancreatic adenocarcinoma prognosis and therapies.

Objectives: Pancreatic adenocarcinoma (PAAD) is a highly malignant tumor. Despite extensive research, the precise role of aging-related genes in the initiation, microenvironment regulation, and progression of PAAD remains unclear.Methods: Patients with PAAD were selected from the International Cancer Genome Consortium (ICGC), and The Cancer Genome Atlas (TCGA) cohorts and the cell senescence-associated genes were obtained from CellAge. ConsensusClusterPlus was utilized for cluster identification. The least absolute shrinkage and selection operator (LASSO) Cox regression analysis was performed to construct a prognosis prediction model.Results: We identified three clusters (C1, C2, and C3) based on aging-associated gene profiles. The C1 cluster had a shorter overall survival time, advanced clinical grades, lower immune ESTIMATE score, and tumor immune dysfunction and exclusion (TIDE) score than the C3 subgroup. Moreover, signaling pathways for cell cycle activation were enriched in the C1 cluster. We also identified eight hub genes and constructed a risk model. The high cellular senescence-related signature (CSRS) score subtype exhibited poor prognosis, advanced clinical grades, M2 macrophage infiltration, higher immune checkpoint gene expression, and lower immunotherapeutic benefits.Conclusion: Our risk score model shows high prediction accuracy and survival prediction ability in individual clinical prognosis and pre-immunotherapy evaluation.

Clinical features and independent predictors of Behçet's disease associated with myelodysplastic syndrome.

OBJECTIVES: To investigate the correlation of Behçet's disease (BD) with myelodysplastic syndrome (MDS) and identify the predictive risk factors in Chinese patients. METHODS: A retrospective study of BD associated with MDS (BD-MDS) patients from the First Affiliated Hospital of Zhengzhou University was conducted. RESULTS: Among 15 BD-MDS patients, 10 were females and 5 males. While 13 (86.7%) patients had abnormal karyotype, 11 patients with trisomy 8. 10 (66.7%) had gastrointestinal (GI) involvement. Compared with 60 general BD patients without MDS, the BD-MDS patients were significantly older. In addition, fever and GI involvement were more common in BD-MDS patients, whereas these patients had lower levels of leukocyte count, haemoglobin, and platelet count (p<0.05). Logistic regression analysis showed that GI involvement, low haemoglobin, and high ESR level were independently associated with the development of MDS in BD patients. BD-MDS patients with GI involvement (IBD-MDS) were usually much older and have more fever than IBD patients without MDS, as well as lower leukocyte count, haemoglobin level, platelet count, and higher erythrocyte sedimentation rate (ESR) and C-reactive protein levels (p<0.05). By comparison with 60 primary MDS patients without BD, the BD-MDS patients had more abnormal karyotypes and more trisomy 8 (p<0.05), while the distribution of 2016 WHO subtypes of MDS and IPSS-R categories were similar. CONCLUSIONS: Our findings suggest that cytogenetic abnormalities, especially trisomy 8, may play a role in the association of GI involvement, BD, and MDS. GI involvement, low haemoglobin, and high ESR level were independent predictors for MDS development in BD patients.

Disturbance of hepatocyte growth and metabolism in a hyperammonemia microenvironment.

BACKGROUND: We found through previous research that hyperammonemia can cause secondary liver damage. However, whether hepatocytes are target cells of ammonia toxicity and whether hyperammonemia affects hepatocyte metabolism remain unknown. AIMS: The purpose of the current study is to examine whether the hepatocyte is a specific target cell of ammonia toxicity and whether hyperammonemia can interfere with hepatocyte metabolism. METHODS: Cell viability and apoptosis were analyzed in primary hepatocytes and other cells that had been exposed to ammonium chloride. Western blotting was adopted to examine the expression of proteins related to ammonia transport. We also established a metabolomics method based on gas chromatography-mass spectrometry to understand the characteristics of the hepatocyte metabolic spectrum in a hyperammonemia microenvironment, to screen and identify differential metabolites, and to determine the differential metabolic pathway. Different technologies were used to verify the differential metabolic pathways. RESULTS: Hepatocytes are target cells of ammonia toxicity. The mechanism is related to the ammonia transporter. Hyperammonemia interferes with hepatocyte metabolism, which leads to TCA cycle, urea cycle, and RNA synthesis disorder. CONCLUSIONS: This study demonstrates that hepatocyte growth and metabolism are disturbed in a hyperammonemia microenvironment, which further deteriorates hepatocyte function.

Evaluation of the reporting quality of clinical practice guidelines on melanoma using the RIGHT checklist.

BACKGROUND: The International Reporting Items for Practice Guidelines in Healthcare (RIGHT) instrument was launched in 2016 to improve the reporting of clinical practice guidelines (CPGs). We aimed to systematically evaluate the reporting quality of CPGs on melanoma using RIGHT. METHODS: We systematically searched electronic databases, guideline databases and medical society websites until November 2020 to identify guidelines for melanoma published since 2018. The reporting quality of included guidelines was assessed by calculating the percentages of the 35 items of the RIGHT checklist that were appropriately reported. We stratified the results by selected characteristics to describe the correlation of these factors with reporting quality. RESULTS: A total of 20 guidelines were identified and analyzed. The mean reporting rate was greater than 50% in five of the seven domains of the RIGHT checklist; the remaining two domains (Other information, Review and quality assurance) both had a mean reporting rate of 35.0%. The mean overall reporting rate was 63.7%. No CPG considered equity, feasibility or acceptability of the recommendations (item 14c), and only one CPG described the role of funders (item 18b). Guidelines that reported funding or were published in higher-impact journals tended to have a higher reporting quality, whereas the reporting rate in the one included Chinese-language CPG was low. CONCLUSIONS: Reporting quality of melanoma CPGs tends to be relatively good. The CPGs developed in China were however an exception. The use of the 2016 RIGHT tool in guideline development should be encouraged to support rigorous and transparent reporting.

m6A Regulator-Associated Modification Patterns and Immune Infiltration of the Tumor Microenvironment in Hepatocarcinoma.

Background: Immunotherapy elicits durable responses in many tumors. Nevertheless, the positive response to immunotherapy always depends on the dynamic interactions between the tumor cells and infiltrating lymphocytes in the tumor microenvironment (TME). Currently, the application of immunotherapy in hepatocellular carcinoma (HCC) has achieved limited success. The ectopic modification of N6-methyladenosine (m6A) is a common feature in multiple tumors. However, the relationship between m6A modification with HCC clinical features, prognosis, immune cell infiltration, and immunotherapy efficacy remains unclear. Materials and Methods: Here, we comprehensively evaluated m6A modification clusters based on 22 m6A regulators and systematically explored the relationship between m6A modification with tumor progression, prognosis, and immune cell infiltration characteristics. The m6Ascore was calculated by principal component analysis to quantify the m6A modifications of individual patients. Key regulators involved in immunoregulation in HCC were identified using immunohistochemistry and immunofluorescence. Results: Three distinct m6A modification clusters were identified. The m6A clusters were significantly associated with clinical features, prognosis, and immune cell infiltration. The three clusters were highly consistent with the three tumor immune phenotypes, i.e., immune-excluded, immune-inflamed, and immune-desert. Comprehensive bioinformatics analysis revealed that high m6Ascore was closely associated with tumor progression, poor prognosis, and immunotherapy non-response. m6A regulators were dysregulated in HCC tissues. Hence, they play a role as predictors of poor prognosis. Tissue microarray demonstrated that overexpressed YTHDF1 was associated with low CD3+ and CD8+ T cell infiltration in HCC. Conclusion: Our findings demonstrate that m6A modification patterns play a crucial role in the tumor immune microenvironment and the prognosis of HCC. High YTHDF1 expression is closely associated with low CD3+ and CD8+ T cell infiltration in HCC.

ARNTL2 promotes pancreatic ductal adenocarcinoma progression through TGF/BETA pathway and is regulated by miR-26a-5p.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and the therapeutic outcomes remain undesirable. Increasing evidence shows that aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2) plays crucial roles in tumorigenesis of multiple tumors. However, the expression status and functions of ARNTL2 in PDAC remain elusive. Here we showed that ARNTL2 expression was markedly upregulated in PDAC tissues and cell lines. elevated expression of ARNTL2 was positively related to unfavorable prognosis. Knockdown of ARNTL2 could suppress motility and invasive ability of PDAC cells in vitro, as well as tumor development in vivo. In addition, microRNA-26a-5p (miR-26a-5p) was identified as the crucial specific arbitrator for ARNTL2 expression and the expression of miR-26a-5p was inversely correlated with ARNTL2 expression in PDAC tissues. Functionally, elevated expression of miR-26a-5p was found to inhibit the proliferation, migration, and invasion of PDAC cells in vitro, while ARNTL2 increased expression could partially abolish the suppressive effect of miR-26a-5p. Mechanism study indicated that elevated expression of miR-26a-5p suppressed TGF/BETA signaling pathway by targeting ARNTL2 in PDAC cells. In conclusion, our data suggested that ARNTL2 acted as an oncogene to regulate PDAC growth. MiR-26a-5p/ARNTL2 axis may be a novel therapeutic candidate target in PDAC treatment.

Expression Status And Prognostic Value Of M6A-associated Genes in Gastric Cancer.

Purpose: Gastric cancer (GC) is a primary cause of cancer-associated mortality worldwide. N6-methyladenosine (m6A) is one of the most common RNA modifications that involves in the progression of numerous cancers. However, the expression status and function of m6A-related genes in gastric cancer is still not well understood. The current study is aimed to investigate the expression status and determinate prognostic value of m6A-related genes in gastric cancer. Methods: m6A-asssociated gene expression was evaluated via analyzing the expression data of GC patients from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. The protein expression levels of m6A-associated molecules were further validated by immunohistochemical (IHC) staining data from GC tissue microarray (TMA) cohort and Human Protein Atlas (HPA) database. Kaplan-Meier analysis was performed to assess the prognostic value of m6A-associated genes in gastric cancer. Risk score model was established by lasso COX regression analysis and its prognostic predicted efficiency was assessed by the receiver-operator characteristic (ROC) curve. Cox regression analyses were used for exploring risk factors related to GC patient prognosis. Results: Most of m6A-related genes were upregulated at both mRNA and protein levels in gastric cancer tissues compared with that in normal gastric tissues. The expression levels of m6A-related genes were associated with clinicopathological features including race, age and TNM stage. High expression of WTAP and FTO predicted poor prognosis of GC patients. Survival analysis demonstrated that patients with high-risk scores had worse overall survival (OS) and ROC curves suggested the prediction performance for gastric patients. Moreover, Cox regression analyses indicated that m6A risk model score was a prognostic factor for OS and FTO upregulation might be a potential independent prognostic factor for recurrence-free survival (RFS) in gastric cancer patients. Conclusion: m6A-related genes were dysregulated in GC and were closely associated with prognosis of GC patients. FTO might serve as a novel prognostic biomarker for gastric cancer, while the m6A-related risk score might be informative for risk assessment and prognostic stratification.

Curcumin induces apoptosis and inhibits proliferation in infantile hemangioma endothelial cells via downregulation of MCL-1 and HIF-1α.

BACKGROUND: Curcumin has been used as an alternative medicine for the treatment of infantile hemangiomas (IHs); however, the mechanism underlying the effectiveness of curcumin in IHs remains largely unclear. METHODS: In this study, we isolated primary human hemangioma endothelial cells (HemECs) from fresh surgical specimens of 3 patients. We treated HemECs by curcumin and investigated the alterations in proliferative and apoptotic signaling pathways with cell counting kit-8, flow cytometry, western blotting, immunofluorescence, and real-time polymerase chain reaction. RESULTS AND CONCLUSION: We found that curcumin potently inhibited proliferation in HemECs, achieving low-micromolar IC50 (the half maximal inhibitory concentration) value. We also observed that treatment with curcumin induced apoptosis in HemECs, as evidenced by positively Annexin-V-FITC staining, caspase-3 activation, and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP) in the treated cells. Moreover, we showed that curcumin suppressed the expression of antiapoptotic protein myeloid cell leukemia-1 (MCL-1), hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF).Altogether, our study suggests that the effectiveness of curcumin in IHs may be associated with its potent antiproliferative and apoptotic activities in HemECs.

Lowering blood ammonia prevents hepatocyte injury and apoptosis.

UNLABELLED: To study hepatocyte injure through establishing the rat model of acute hepatic failure (ALF). ALF rat model was established by administration with D-galactosamine and LPS, and then giving lowering blood ammonia (LBA) treatment. Besides, the intervention groups were injected with ornithine and aspartate. The control groups were injected saline. Blood ammonia, ALT, AST, TNF-α and IL-6 in blood samples were test at 12 hrs and 24 hrs after treatment with LBA. Hepatocyte apoptosis were tested by TUNEL and DNA Ladder. Expression of P53 and SPP1 were detected by RT-PCR. RESULTS: showed that blood ammonia in hepatic failure group and intervention group compared with blank control group was significantly increased at 12 h, 24 h; intervention group compared with hepatic failure group was significantly reduced (P<0.05). Serum ALT, AST in 24 h group were higher than 12 h. 12 h intervention group was decreased compared with hepatic failure group, but there was no significant statistically difference (P>0.05). 24 h intervention group compared with hepatic failure group was significantly reduced (P<0.05). Except the control group, DNA ladder and the TUNEL results showed hepatocyte apoptosis rate increased in 24 h compared with 12 h. Intervention group compared with hepatic failure group was significantly reducing (P<0.05). IL-6, TNF-α, p53 expression levels were increased with time (24 h>12 h). The hepatic failure and intervention group compared with blank control group was significantly increased; Intervention group compared with hepatic failure group was significantly reducing (P<0.05). SPP1 gene was high expression in ALF rat model. SPP1 level in hepatic failure and intervention group compared with control group was significantly increased, and intervention group compared with hepatic failure group was significantly reducing (P<0.05). In conclusion, hepatocyte apoptosis is an important pathological change in ALF rat mode, and lowing ammonia can reduce liver injury and apoptosis. Blood TNF-α, IL-6 and SPP1 may be more sensitive injure indicators.

Poly (ADP­ribose) polymerase­ and cytochrome c­mediated apoptosis induces hepatocyte injury in a rat model of hyperammonia­induced hepatic failure.

Hepatic failure (HF) is caused by several factors, which induce liver cell damage and dysfunction. However, the specific mechanism of HF remains to be fully elucidated. The present study aimed to investigate the underlying cause of hepatocyte injury and liver dysfunction. Liver cells were isolated from healthy female Sprague­Dawley rats, aged between 6 and 8 weeks, weighing ~230 g. The liver cells were cultured in RPMI­1640 medium containing 10% fetal bovine serum. An MTT assay was used to examine the inhibitory rate of liver growth in each group. Flow cytometric analysis was performed to detect liver cells undergoing apoptosis. The protein expression levels of poly (ADP­ribose) polymerase (PARP) and cytochrome c (Cyt C) were detected by western blotting. The level of calmodulin­dependent kinase (CaMK) was assessed using an ELISA. The results indicated that the growth inhibitory rate of rat liver cells was significantly increased following treatment with increasing concentrations of NH4Cl. The results of flow cytometric analysis demonstrated that the apoptotic rate in the BAPTA­acetoxymethyl ester group was significantly lower compared with the NH4Cl group (P<0.05). Treatment with NH4Cl increased the protein expression levels of PARP and Cyt C in the liver cells. The mRNA expression of CaMK decreased gradually following treatment with increasing concentrations of NH4Cl for 6, 12 and 24 h. The results suggested that hepatocyte injury and liver dysfunction may be caused by inducing apoptosis via the PARP and Cyt C pathways. Additionally, downregulation of CaMK may be associated with the apoptosis observed in hepatocyte injury.

Ammonium chloride inhibits autophagy of hepatocellular carcinoma cells through SMAD2 signaling.

Autophagy is a cellular degradation process for the clearance of damaged or superfluous proteins and organelles, the recycling of which serves as an alternative energy source during periods of metabolic stress to maintain cell homeostasis and viability. The anti-necrotic function of autophagy is critical for tumorigenesis of many tumor cells, including hepatocellular carcinoma (HCC). However, the underlying mechanism is not clarified yet. Ammonium chloride (NH4Cl) is a well-known autophagy inhibitor, whereas its interaction with SMAD2 signaling pathway has not been reported previously. Here, we show that NH4Cl significantly inhibited rapamycin-induced autophagy in HCC cells through decreasing the levels of Beclin-1, autophagy-related protein 7 (ATG7), p62, and autophagosome marker LC3 and significantly decreased the level of phosphorylated SMAD2 in rapamycin-treated HCC cells. In order to find out whether NH4Cl may inhibit the autophagy in rapamycin-treated HCC cells through inhibition of SMAD2 signaling, we used transforming growth factor ß1 (TGFß1) to induce phosphorylation of SMAD2 in HCC cells. We found that induction of SMAD2 in HCC cells completely abolished the inhibitory effect of NH4Cl on rapamycin-induced autophagy in HCC cells, suggesting that NH4Cl inhibits autophagy of HCC cells through inhibiting SMAD2 signaling.

Ammonia-induced energy disorders interfere with bilirubin metabolism in hepatocytes.

Hyperammonemia and jaundice are the most common clinical symptoms of hepatic failure. Decreasing the level of ammonia in the blood is often accompanied by a reduction in bilirubin in patients with hepatic failure. Previous studies have shown that hyperammonemia can cause bilirubin metabolism disorders, however it is unclear exactly how hyperammonemia interferes with bilirubin metabolism in hepatocytes. The purpose of the current study was to determine the mechanism or mechanisms by which hyperammonemia interferes with bilirubin metabolism in hepatocytes. Cell viability and apoptosis were analyzed in primary hepatocytes that had been exposed to ammonium chloride. Mitochondrial morphology and permeability were observed and analyzed, intermediates of the tricarboxylic acid (TCA) cycle were determined and changes in the expression of enzymes related to bilirubin metabolism were analyzed after ammonia exposure. Hyperammonemia inhibited cell growth, induced apoptosis, damaged the mitochondria and hindered the TCA cycle in hepatocytes. This led to a reduction in energy synthesis, eventually affecting the expression of enzymes related to bilirubin metabolism, which then caused further problems with bilirubin metabolism. These effects were significant, but could be reversed with the addition of adenosine triphosphate (ATP). This study demonstrates that ammonia can cause problems with bilirubin metabolism by interfering with energy synthesis.