Insights into the effect of benzotriazoles in liver using integrated metabolomic and transcriptomic analysis.

Benzotriazoles (BTRs) are a class of benzoheterocyclic chemicals that are frequently used as metal-corrosive inhibitors, both in industry and daily use. However, the exposure effect information on BTRs remains relatively limited. In this study, an integrated metabolomic and transcriptomic approach was utilized to evaluate the effect of three BTRs, benzotriazole, 6-chloro-1-hydroxi-benzotriazole, and 1-hydroxy-benzotriazole, in the mouse liver with results showing disrupted basal metabolic processes and vitamin and cofactor metabolism after 28 days. The expression of several genes that are related to the inflammatory response and aryl hydrocarbon receptor pathways, such as Gstt2 and Arntl, was altered by the exposure to BTRs. Exposure to BTRs also affected metabolites and genes that are involved in the immune system and xenobiotic responses. The altered expression of several cytochrome P450 family genes reveal a potential detoxification mechanism in the mouse liver. Taken together, our findings provide new insights into the multilayer response of the mouse liver to BTRs exposure as well as a resource for further exploration of the molecular mechanisms by which the response occurs.

Normobaric hyperoxia alleviates complement C3-mediated synaptic pruning and brain injury after intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a common cerebrovascular disease, and the complement cascade exacerbates brain injury after ICH. As the most abundant component of the complement system, complement component 3 (C3) plays essential roles in all three complement pathways. However, the effects of C3 on neurological impairment and brain injury in ICH patients and the related mechanism have not been fully elucidated. Normobaric hyperoxia (NBO) is regarded as a treatment for ICH patients, and recent clinical studies also have confirmed the neuroprotective role of NBO against acute ICH-mediated brain damage, but the underlying mechanism still remains elusive. AIMS: In the present study, we investigated the effects of complement C3 on NBO-treated ICH patients and model mice, and the underlying mechanism of NBO therapy in ICH-mediated brain injury. RESULTS: Hemorrhagic injury resulted in the high plasma C3 levels in ICH patients, and the plasma C3 levels were closely related to hemorrhagic severity and clinical outcomes after ICH. BO treatment alleviated neurologic impairments and rescued the hemorrhagic-induced increase in plasma C3 levels in ICH patients and model mice. Moreover, the results indicated that NBO exerted its protective effects of on brain injury after ICH by downregulating the expression of C3 in microglia and alleviating microglia-mediated synaptic pruning. CONCLUSIONS: Our results revealed that NBO exerts its neuroprotective effects by reducing C3-mediated synaptic pruning, which suggested that NBO therapy could be used for the clinical treatment of ICH.

Association of plasma MMP-2 levels and prognosis of patients with intracerebral hemorrhage: a prospective cohort study.

Objective: The role of MMP-2 in patients with ICH is controversial and the impact of plasma MMP-2 level on clinical outcome is still unclear. Materials and methods: In this study, the peripheral venous blood was acquired from 93 patients with ICH and 88 healthy controls within 24 h of hospitalization and at enrollment. We retrospectively investigated plasma MMP-2 levels of patients and healthy controls. The edema volume, the NIHSS score, the GCS score, and mRS were used to assess and quantify neurological deficit following ICH. Logistic regression analysis was configured to determine the independent relation of plasma MMP-2 levels with clinical outcomes. In addition, the plasma MMP-14 levels and complement C4 level were tested to explore the relationship with plasma MMP-2 level. Results: There was a significant reduction of plasma MMP-2 levels in ICH patients than that in healthy controls (38.02 ± 1.71 vs. 54.03 ± 2.15, p < 0.0001), and MMP-2 is negatively correlated with the edema volume (r = -0.2187, p < 0.05), NIHSS score (r = -0.2194, p < 0.05), blood leucocyte count (r = -0.2549, p = 0.012), and complement C4 level (r = -0.2723, p = 0.005). There is positive correlation between MMP-2 level and GCS score (r = 0.2451, p = 0.01) and MMP-14 level (r = 0.7013, p = 0.005). The multivariate analysis revealed that reduced plasma MMP-2 level is associated with elevated edema volume (OR = 0.2604, 95% CI [0.07 to 0.84], p = 0.02). Conclusion: The plasma MMP-2 level in patients with ICH is significantly lower than that of healthy controls, and plasma MMP-2 level may be a prognostic factor. Plasma MMP-2 levels are correlated with the clinical outcomes of patients and negatively correlated with blood leucocyte count and complement C4 level in patients with ICH.

Downregulation of RAB7 and Caveolin-1 increases MMP-2 activity in renal tubular epithelial cells under hypoxic conditions.

Tubulointerstitial fibrosis leads to tubular basement membrane thickening and accumulation of interstitial extracellular matrix (ECM). Matrix metallopeptidase-2 (MMP-2) is involved in the breakdown of ECM. Chronic hypoxia often occurs in the kidney tissues of patients with chronic kidney disease. Our previous study reported the effect of autophagy and endocytosis on MMP-2 activity in hypoxia-treated HK-2 cells. In this study, the relationship between the Ras-related protein Rab-7a (RAB7) and MMP-2 activity was further investigated. RAB7 overexpression decreased MMP-2 activity. In contrast, the results for RAB7 knockdown displayed the opposite pattern. Short hairpin RNA technology was used to knockdown Caveolin-1 (Cav-1) or Beclin-1 (Bec-1) in HK-2 cells. The two genes displayed differential effects on MMP-2 activity. Cav-1 and RAB7 interference increased MMP-2 activity. This study suggested that autophagy and endocytosis, RAB7, Cav-1, and Bec-1 may serve as potential mediators for altered MMP-2 activity.

Different effects of total flavonoids from Arachniodes exilis on human umbilical cord mesenchymal stem cells in vitro.

Traditional Chinese medicines are used in promotion of fractured bone healing and bone diseases. Some studies reported total flavonoids from plant can be used as an auxiliary source of exogenous.Use different methods to identify and verify effects of total flavonoids from Arachniodes exilis (TFAE) on human umbilical cord mesenchymal stem cells (HUCMSCs) in vitro.Concentrations of 1 and 5 µg/mL TFAE significantly increased ALPase activity in HUCMSCs compared to the other concentrations at days 3 and 7 (P < .05). RT-PCR showed that expression levels of osteogenic genes (Col1a1, OPN, Runx2 and Osx) were remarkably enhanced in HUCMSCs following treatment with different concentrations of TFAE for 9 days compared with 0 µg/mL TFAE group (control). The results showed that concentration < 5 µg/mL of TFAE induced osteogenic differentiation in HUCMSCs Alizarin red staining assays revealed that both TFAE and S1191 was significantly decreased (7.80 ±â€Š0.66) compared with the TFAE group (16.00 ±â€Š0.97) (P < .01). ALPase activity on days 3 and 7 was relatively lower in HUCMSCs grown in media supplemented with both S1191 and TFAE than that of in TFAE group only. The results indicated that osteogenic markers (Col1a1, OPN, Runx2 and Osx) were significantly downregulated in the TFAE + S1191 group in comparison to the control group. The expressions of Col1a and OPN in the TFAE + S1191 group decreased significantly (P < .01) by Western blotting.TFAE promotes the odonto/osteogenic differentiation of human UCMSCs via activation of ER.

Molecular characteristic analysis for the VP1 region of coxsackievirus A6 strains isolated in Jiujiang area, China, from 2012 to 2013.

BACKGROUND: Enterovirus 71 (EV-A71) and Coxsackievirus A16 (CV-A16) are the most common causative agents causing hand, foot, and mouth disease (HFMD). However, coxsackievirus A6 (CV-A6), previously largely ignored, became the predominant pathogen in China in 2012. The objective of this study is to investigate the genetic characteristics and molecular epidemiology of HFMD caused by CV-A6 to guide the diagnosis and treatment of the disease, as well as disease prevention. MATERIAL AND METHODS: A total of 138 suspected HFMD cases were enrolled in this study and analyses based on complete VP1 nucleotide sequences were performed to determine the evolutionary trajectory of emerging CV-A6. RESULTS: Among 138 samples in Jiujiang, 125 (90.58%) were positive for enterovirus, the most frequently presented serotypes were CV-A6 (77, 61.60%), CV-A16 (28, 22.40%), EV-A71 (6, 4.80%) and untyped enteroviruses (14, 11.20%). Seventy-seven CV-A6 positive specimens were analyzed for the complete VP1 sequences by sequencing and 36 representative isolates were selected to perform nucleotide sequence similarity analysis. The results showed that 36 strains isolated from HFMD patients were clustered closely to the mainland China and were far from prototype strain CV-A6/Gdula (AY421764) and other international subtypes. Moreover, phylogenetic analysis of the VP1 gene revealed that 36 circulating strains were not significantly concentrated in one branch, but were widely distributed in each branch. CONCLUSIONS: Continuous surveillance of HFMD etiological agents other than EV-A71 and CV-A16 is necessary. CV-A6 is emerging as the most common pathogen causing HFMD. Closely monitoring the magnitude and trend of CV-A6 epidemic and the trend of pathogenic spectrum changes can provide scientific basis for this disease prevention and control to the department of disease control.

Effects of autophagy and endocytosis on the activity of matrix metalloproteinase­2 in human renal proximal tubular cells under hypoxia.

Tubulointerstitial fibrosis is characterized by tubular atrophy with basement membrane thickening and accumulation of interstitial extracellular matrix (ECM). A decrease in the activity of matrix metalloproteinase­2 (MMP­2) may promote this process. Although proximal tubular cells are sensitive to oxygen deprivation, whether cellular autophagy or endocytosis induced by hypoxia can alter the activity of MMP­2 remains to be elucidated. The aim of the present study was to investigate whether autophagy and endocytosis induced by hypoxia can have an effect on the activity of MMP­2 in HK­2 cells. The investigations involved exposing the HK­2 cell line to an autophagy inhibitor, 3­MA, or an endocytotic inhibitor, filipin. The mRNA expression of MMP­2 was elevated in the hypoxic milieu. Furthermore, it was found that filipin increased the activity of MMP­2 under hypoxia. These results suggested that autophagy and endocytosis were potential mediators for the altered expression of MMP­2, and endocytosis was a potential target for regulating the activity of MMP­2. These data suggested that hypoxia may be an important pro­fibrogenic stimulus, which acts in part via endocytosis.

Transcriptomic changes in human renal proximal tubular cells revealed under hypoxic conditions by RNA sequencing.

Chronic hypoxia often occurs among patients with chronic kidney disease (CKD). Renal proximal tubular cells may be the primary target of a hypoxic insult. However, the underlying transcriptional mechanisms remain undefined. In this study, we revealed the global changes in gene expression in HK­2 human renal proximal tubular cells under hypoxic and normoxic conditions. We analyzed the transcriptome of HK­2 cells exposed to hypoxia for 24 h using RNA sequencing. A total of 279 differentially expressed genes was examined, as these genes could potentially explain the differences in HK­2 cells between hypoxic and normoxic conditions. Moreover, 17 genes were validated by qPCR, and the results were highly concordant with the RNA seqencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these differentially expressed genes. The upregulated genes appeared to be significantly enriched in the pathyway of extracellular matrix (ECM)-receptor interaction, and in paticular, the pathway of renal cell carcinoma was upregulated under hypoxic conditions. The downregulated genes were enriched in the signaling pathway related to antigen processing and presentation; however, the pathway of glutathione metabolism was downregulated. Our analysis revealed numerous novel transcripts and alternative splicing events. Simultaneously, we also identified a large number of single nucleotide polymorphisms, which will be a rich resource for future marker development. On the whole, our data indicate that transcriptome analysis provides valuable information for a more in depth understanding of the molecular mechanisms in CKD and renal cell carcinoma.

Molecular epidemiology of human coxsackievirus A16 strains.

The hand, foot and mouth disease (HFMD) epidemics have mainly been caused by human enterovirus 71 and human coxsackievirus A16 (CA16), which circulated alternatively or together in the epidemic area. The aim of the present study was to provide guidance in the prevention and control of HFMD from CA16 infection. The molecular epidemiology of the human CA16 strains was investigated. Overall, 1,151 specimens (throat swabs) were collected from 1,151 patients with HFMD symptoms. The results of the homology comparison in the VP1 of CA16 strains showed that the CA16 strains belonged to the B1b subgenotype. The difference of the 6 CA16 strains analyzed showed that the most prominent strain was the A genotype, and the most close strains were the B1 gene subtype, particularly the B1b gene subtype. With regards to the amino acids, in addition to the A genotype, the differences of amino acids with other gene subtype was not significant. The present data suggest that more effective and highly targeted intervention mechanisms could be developed for the prevention and control of HFMD.

Overendocytosis of gold nanoparticles increases autophagy and apoptosis in hypoxic human renal proximal tubular cells.

BACKGROUND: Gold nanoparticles (GNPs) can potentially be used in biomedical fields ranging from therapeutics to diagnostics, and their use will result in increased human exposure. Many studies have demonstrated that GNPs can be deposited in the kidneys, particularly in renal tubular epithelial cells. Chronic hypoxic is inevitable in chronic kidney diseases, and it results in renal tubular epithelial cells that are susceptible to different types of injuries. However, the understanding of the interactions between GNPs and hypoxic renal tubular epithelial cells is still rudimentary. In the present study, we characterized the cytotoxic effects of GNPs in hypoxic renal tubular epithelial cells. RESULTS: Both 5 nm and 13 nm GNPs were synthesized and characterized using various biophysical methods, including transmission electron microscopy, dynamic light scattering, and ultraviolet-visible spectrophotometry. We detected the cytotoxicity of 5 and 13 nm GNPs (0, 1, 25, and 50 nM) to human renal proximal tubular cells (HK-2) by Cell Counting Kit-8 assay and lactate dehydrogenase release assay, but we just found the toxic effect in the 5 nm GNP-treated cells at 50 nM dose under hypoxic condition. Furthermore, the transmission electron microscopy images revealed that GNPs were either localized in vesicles or free in the lysosomes in 5 nm GNPs-treated HK-2 cells, and the cellular uptake of the GNPs in the hypoxic cells was significantly higher than that in normoxic cells. In normoxic HK-2 cells, 5 nm GNPs (50 nM) treatment could cause autophagy and cell survival. However, in hypoxic conditions, the GNP exposure at the same condition led to the production of reactive oxygen species, the loss of mitochondrial membrane potential (ΔΨM), and an increase in apoptosis and autophagic cell death. CONCLUSION/SIGNIFICANCE: Our results demonstrate that renal tubular epithelial cells presented different responses under normoxic and hypoxic environments, which provide an important basis for understanding the risks associated with GNP use-especially for the potential GNP-related therapies in chronic kidney disease patients.

Molecular characterization, expression patterns, and polymorphism of a differentially expressed porcine gene (PYGM) isolated by suppression subtractive hybridization and two-dimensional gel electrophoresis analysis.

Suppression subtractive hybridization was performed to detect the differences in gene expression of porcine longissimus dorsi muscles between Large White and Chinese Meishan pigs. An upregulated gene in Large White that shared high homology with human muscle glycogen phosphorylase (PYGM) was identified. The porcine PYGM gene contains an open reading frame encoding 842 amino acid residues with 26 and 283 nucleotides in the 5' and 3' untranslated regions, respectively. Tissue distribution analysis indicated that porcine PYGM mRNAs are highly expressed in all tissues. Expression pattern of PYGM was similar in the two breeds. Both breeds had the highest expression levels when 120 days old (p<0.01), and PYGM was upregulated during skeletal muscle development. A similar expression pattern of PYGM in protein level was also observed by differential proteome analysis of skeletal muscle development using two-dimensional gel electrophoresis and mass spectroscopy. The mRNA abundance of PYGM in Large White was higher than Meishan at all four stages (p<0.05). Moreover, a G/T mutation in exon 8 was identified and association analysis with meat quality traits showed that it was significantly associated with lean meat percentage (p<0.05). Our data may provide further insight into the molecular mechanisms responsible for breed-specific differences in porcine growth and meat quality.

Molecular characterization and expression patterns of serine/arginine-rich specific kinase 3 (SPRK3) in porcine skeletal muscle.

SRPK3 is a protein kinase belonging to serine/arginine protein kinases (SRPK) family, which phosphorylates serine/arginine repeat-containing proteins, and is controlled by a muscle-specific enhancer directly regulated by MEF2. In this study, a full-length cDNA of the porcine SRPK3 gene encoding a 566 amino acid protein was isolated. It contains 14 exons over approximately 4.3 kb. The deduced amino acid sequence of porcine SRPK3 contains a bipartite kinase domain, and shows high similarities to their corresponding human and cattle homologues. Tissue distribution analysis indicated that porcine SRPK3 mRNAs are highly expressed in heart and skeletal muscle especially in uterus and parorchis, but at low level in brain, stomach, small intestine, and ovary. Expression pattern of SRPK3 was similar in Large White and Chinese Meishan breeds. Both the two breeds had the highest expression levels at fetal 65 days (P < 0.01), and decreased while the age increased until 60 days old, then increased at 120 days (P < 0.01) and decreased at 180 days (P < 0.05). However, at fetal 65 days, the mRNA abundance of SRPK3 in Large White was 12.5-fold higher than in Meishan pigs (P < 0.01), whereas at 180 days, the abundance in Meishan was 3.4-fold higher than in Large White pigs (P < 0.01). These results suggest that the SRPK3 gene might be an important gene of skeletal muscle development and also provides basic molecular information useful for further studies on its roles in porcine skeletal muscle.