[Clinical analysis of 2 820 cases of drug-induced liver injury].

Objective: To investigate the clinical characteristics, incidence trend, underlying diseases, causative drug and prognosis of drug-induced liver injury (DILI), so as to provide basis for its prevention and treatment. Methods: A retrospective study was conducted on 2 820 DILI cases who were admitted to our hospital from January 2002 to December 2015, and their clinical characteristics, incidence trends, underlying related diseases, causative drug, treatment and outcome were analyzed. Results: Among 2 820 DILI cases, the ratio of male to female was 1:1.44, and the age was (44.00±16.32) years old. According to the clinical classification of DILI, there were 2 353 cases (83.43%) of hepatocyte injury, 353 cases (12.51%) of cholestatic type and 114 cases (4.04%) of mixed type. In the three clinical classification of DILI, there was no statistically significant difference in the ratio of male to female (χ(2) = 3.032, P > 0.05). However, the difference in the ratio of male to female between different age groups was statistically significant (χ(2) = 48.367, P < 0.001). Among the patients with liver disease and acute liver disease admitted to our hospital from January 2002 to December 2015, the proportion of DILI and acute DILI showed an overall upward trend. The main underlying related diseases of 2 820 DILI cases were fever (15.14%), skin diseases (11.84%), cardiovascular and cerebrovascular diseases (11.17%). Chinese herbal patent medicines (37.49%), antibiotics (15.85%), antipyretic-analgesics (14.37%), and so on were the main causative drugs involved, and the prognostic differences among the three clinical classifications of DILI in terms of cure, improvement, ineffectiveness, and death were statistically significant (H = 61.300, P < 0.001). Conclusion: In recent years, among the patients with liver disease in our hospital, the proportion of DILI has shown an obvious upward trend, involving a variety of underlying diseases and causative drugs, and thus it needs clinical attention.

Genomic characterization and sequence diversity of the ß2-microglobulin gene in the miiuy croaker, Miichthys miiuy.

ß2-Microglobulin (ß2m) is related to major histocompatibility complex class I alpha chains, and forms cell-surface glycoproteins that mediate a variety of functions in immune defense. In general, ß2m has no isoforms and is not polymorphic in higher vertebrates, but polymorphisms between different alleles have been found in some fish species. In this study, full-length ß2m cDNA and genomic sequences were cloned from the miiuy croaker (Miichthys miiuy). The miiuy croaker ß2m gene shares many of the same characteristics as other fish species. Three exons and two introns were identified in the miiuy croaker ß2m gene; these genomic structural features are similar to those present in other fish. The deduced ß2m amino acid sequence exhibited 34.7-90.1% identity with mammal and teleost ß2m amino acid sequences. Sequence polymorphism analysis in six individuals identified three alleles that encoded two proteins, confirming that ß2m polymorphisms exist in this species. Phylogenetic analysis elucidated the evolutionary history of the ß2m protein among warm-blooded vertebrates and bony fish.

Genetic diversity and population genetic structure of the miiuy croaker, Miichthys miiuy, in the East China Sea by microsatellite markers.

Genetic diversity and patterns of population structure of the miiuy croaker were investigated using SSR markers. A set of 10 microsatellite loci revealed 40 alleles; the number of alleles varied from 2 to 10 for each marker. A relatively high level of genetic variability was observed between miiuy croaker individuals. Genetic diversity was relatively high within populations with corresponding high average gene flow. There were genealogical branches or clusters corresponding to sampling localities according to the UPGMA tree and principal component analysis. Knowledge of the genetic diversity and population structure will be crucial for establishing appropriate fishery management stocks for this species.

Sodium selenite induces apoptosis in colon cancer cells via Bax-dependent mitochondrial pathway.

OBJECTIVES: We aimed to elucidate a possible mechanism of action by investigating the effects of selenium (Se) on cell cycle arrest and apoptosis in colorectal cancer cells (HCT116 cells and SW620 cells). MATERIALS AND METHODS: The colorectal cancer cells were treated with varying concentrations of Se (1 microM, 5 microM and 10 microM) for 24 hours. The effects of Se on cell cycle, apoptosis, mitochondrial transmembrane potential and apoptosis related proteins were examined by flow cytometry assessment and immunoblotting. RESULTS: Se induced G2/M cell cycle arrest and apoptosis in colorectal cancer cells (HCT116 cells and SW620 cells) in a dose-dependent manner. Bax (Bcl2 associated X protein) was up-regulated and Bcl-2 (B cell linphoma gene-2) was down-regulated after Se treatment in both cells in a dose-dependent manner. Se caused increased loss of MMP (matrix metalloproteinase) and induced Bax translocation from cytosol into mitochondria and caspase 3 activation in both colorectal cancer cells in a dose-dependent manner. CONCLUSIONS: Se induced G2/M cell cycle arrest and apoptosis in both colorectal cancer cells via Bax-dependent mitochondrial pathway.

Characterization of nine novel microsatellite loci for the Venus clam (Cyclina sinensis).

The Venus clam, Cyclina sinensis, is one of the most important bivalves in China marine aquaculture. Using (CA)(15)-enriched genomic libraries of this species, nine novel polymorphic microsatellite loci were isolated and characterized. The mean number of observed alleles per locus was 16 (range 8-24). The observed and expected heterozygosity ranged from 0.119 to 0.872 and from 0.626 to 0.931, respectively. Three loci had significant departure from Hardy-Weinberg equilibrium and non-significant linkage disequilibrium was found among all nine loci. These highly informative microsatellite markers should be useful for population genetic analyses of C. sinensis.

Development of microsatellite markers for the small yellow croaker Larimichthys polyactis (Sciaenidae) by cross-species amplification.

The small yellow croaker (Larimichthys polyactis) is a highly valued fish for human consumption found in the Western Pacific that was considered endangered until recently because of overfishing. We selected microsatellite markers for this species from markers developed for Miichthys miiuy, also of the family Sciaenidae. Among 43 markers polymorphic for M. miiuy, 11 were found to be polymorphic for L. polyactis. Characterization of these 11 loci was made based on 30 L. polyactis individuals collected by trawling in the Zhoushan Fishing Ground, Zhejiang Province, China. Total genomic DNA was isolated from fin clips. The number of alleles per locus ranged from 4 to 10, with a mean of 5.82, while the effective number of alleles ranged from 1.64 to 10.00, with a mean of 3.22. Observed and expected heterozygosities ranged from 0.17 to 0.72 and from 0.39 to 0.81, respectively. Significant deviation from Hardy-Weinberg equilibrium was found at four loci, after applying Bonferroni's correction. There was no significant association between any of the pairs of microsatellite loci, hence allelic variation at these loci was considered independent. These 11 polymorphic microsatellite loci will be useful for genetic diversity analysis and molecular-assisted breeding for L. polyactis.

Genetic diversity of Setipinna taty (Engraulidae) populations from the China Sea based on mitochondrial DNA control region sequences.

The genetic diversity of Setipinna taty, which is commercially fished in the China Sea, was studied based on mitochondrial DNA control region sequences. PCR was used to amplify the control region fragment in 100 individuals of S. taty collected from Weihai (WH), Yantai (YT), Zhoushan (ZS), Xiangshan (XS), and Ninghai (NH) in China. A control region fragment of 656 bp was successfully sequenced in these 100 individuals. The A+T content of this S. taty control region was 71.7%; 172 variable sites and 62 haplotypes were found. Nucleotide diversity in the WH, YT, ZS, XS, and NH groups was 0.0228, 0.0247, 0.0441, 0.0126, and 0.0238, respectively. The haplotype diversity was 0.984, 0.911, 0.989, 0.926, and 0.979, respectively. Analysis of molecular variance showed that 97.95% of genetic variation was within populations, and only 2.05% among populations. The neighbor-joining phylogenetic tree obtained based on genetic distance showed that no significant genealogical structure exists throughout this range of S. taty. These results indicate no apparent geographical differentiation in the comparison of Yellow Sea and East China Sea populations of S. taty. Within the control region, we identified an extended termination-associated sequence domain, a central conserved sequence block domain and a conserved sequence block domain; insertions of short tandem repeat sequence segments were found at the 5' end of the control region.

Universal primers for amplification of the complete mitochondrial control region in marine fish species.

Through multiple alignment analysis of mitochondrial tRNA-Thr and tRNA-Phe sequences from 161 fishes, new universal primers specially targeting the entire mitochondrial control region were designed. This new primer set successfully amplified the expected PCR products from various kinds of marine fish species, belonging to various families, and the amplified segments were confirmed to be the control region by sequencing. These primers provide a useful tool to study the control region diversity in economically important fish species, the possible mechanism of control region evolution, and the functions of the conserved motifs in the control region.

Effect of insulin in combination with selenium on Irs/PI3K-mediated GLUT4 expression in cardiac muscle of diabetic rats.

BACKGROUND: Oral administration of selenium is an effective treatment for diabetes in animal models. However, selenium exerts these effects at high doses and several toxic effects are produced. Low doses of selenium are relatively safe but are unable to elicit any antidiabetic effect. OBJECTIVES: The present study explored the prospect of using low doses of insulin in combination with selenium to evaluate their antidiabetic effect, and to evaluate their effect on IRS-1, PI3K and GLUT4 levels in cardiac muscle of diabetic rats. MATERIALS AND METHODS: Diabetic rats were treated with insulin, selenium, and insulin and selenium in combination for four weeks. The effect of these antidiabetic compounds was examined on general physiological parameters and distribution of IRS-1, PI3K and GLUT4 in cardiac muscle by immunoblotting and immunohistochemistry. RESULTS: Insulin in combination with selenium could significantly revive normoglycemia, and restore the disturbances in IRS-1, PI3K and GLUT4 levels in cardiac muscle. Treatment with insulin was only partially effective in the restoration of diabetic alterations. CONCLUSION: The treatment of diabetic rats with combined doses of insulin and selenium was most effective in controlling glycaemia, and remarkably restored GLUT4 distribution by IRS-1/PI3K-dependent pathway in cardiac muscle of diabetic rat.

Effect of insulin in combination with selenium on blood glucose and PI3K-mediated GLUT4 expression in skeletal muscle of streptozotocin-induced diabetic rats.

OBJECTIVES: We evaluated the effect of low doses of insulin (1 U/kg/day) and selenium (180 microg/kg/day) in combination on general physiological parameters, and on PI3K and GLUT4 levels in skeletal muscle of streptozotocin (STZ)-induced diabetic rats. MATERIALS AND METHODS: Diabetic rats were treated with insulin, selenium, and insulin and selenium in combination for four weeks. The levels of blood glucose and hemoglobin A1c were estimated, and the levels of P13K and GLUT4 in skeletal muscle were examined by immunoblotting and immunohistochemistry. RESULTS: Insulin in combination with selenium could significantly lower blood glucose and HbAlc levels, and restore the disturbances in PI3K and GLUT4 levels in skeletal muscle. Treatment with insulin was only partially effective in the restoration of diabetic alterations. CONCLUSION: We conclude that there was cooperation between insulin and selenium, and that treatment of diabetic rats with combined doses of insulin and selenium was effective in the normalization of blood glucose and correction of altered PI3K and GLUT4 distribution in skeletal muscle of diabetic rats.

Allelic polymorphism, gene duplication and balancing selection of the MHC class II DAB gene of Cynoglossus semilaevis (Cynoglossidae).

Major histocompatibility complex (MHC) genes play an important role in the immune response of vertebrates. Allelic polymorphism and evolutionary mechanism of MHC genes have been investigated in many mammals, but much less is known in teleosts. We examined the polymorphism, gene duplication and balancing selection of the MHC class II DAB gene of the half-smooth tongue sole (Cynoglossus semilaevis); 23 alleles were found in this species. Gene duplication manifested as three to six distinct sequences at each domain in the same individuals. Non-synonymous substitutions occurred at a significantly higher frequency than synonymous substitutions in the PBR domain, suggesting balancing selection for maintaining polymorphisms at the MHC II DAB locus. Many positive selection sites were found to act very intensely on antigen-binding sites of MHC class II DAB gene.

Development and characterization of microsatellite markers for the lizardfish known as the Bombay duck, Harpadon nehereus (Synodontidae).

The Bombay duck, or bummalo (Harpadon nehereus), is a lizardfish native to the Arabian sea, but also common in the China sea. It is normally dried and salted before consumption and export. To provide molecular information on this economically important fish species, we developed and characterized microsatellite markers. Ninety positive clones from the (CA)(15)-enriched genomic library were sequenced; 62 sequences contained sufficient repeat motifs (di-, tri- and tetra-nucleotide). Twenty-eight primer pairs were designed and 21 were successfully amplified; five loci were polymorphic, but with a low number of alleles (three or four). The observed and expected heterozygosities ranged from 0.3500 to 0.8421 and from 0.5244 to 0.6244, respectively. All of the five polymorphic loci were at Hardy-Weinberg equilibrium (adjusted), while linkage disequilibrium between Hane-97 and Hane-175 (P< 0.05, adjusted P value = 0.01) was significant. The low degree of polymorphism of microsatellite markers may be due to the large size of the repeat motifs. These markers will be useful for genetic diversity analysis of H. nehereus.

Development and characterization of microsatellite markers for the walking goby (Scartelaos viridis; Gobiidae).

Scartelaos viridis (walking goby) is a small edible fish that inhabits warm inshore environments. To provide molecular information of S. viridis, we developed and characterized microsatellite markers for this species. Using (CA)15-enriched genomic libraries of Scartelaos viridis, 44 positive clones were sequenced; 34 sequences contained multiple repeat motifs (di-, tri- and tetra-nucleotide). In all, 23 primer pairs were designed and 15 were successfully amplified. Forty-two S. viridis individuals collected from the East China Sea were used to characterize the polymorphism at each locus. Three loci (13%) were polymorphic, with three to six alleles. The observed and expected heterozygosity ranged from 0.1000 to 0.4500 and from 0.4487 to 0.7580, respectively. The polymorphism information content per locus ranged from 0.4214 to 0.7510. Three loci significantly deviated from the Hardy-Weinberg equilibrium (adjusted P value=0.017); the pairwise tests for linkage disequilibrium between Scvi-1-13 and Scvi-2-11 were significant (P<0.05, adjusted P value=0.017). The low number of polymorphic microsatellite loci may be due to the close genetic relationship of the individuals that we collected and the large size of the motifs.

Development and characterization of polymorphic microsatellite markers in the gray mullet (Mugil cephalus).

Using an (AG)13 enriched genomic library of Mugil cephalus, 12 polymorphic microsatellite loci were isolated and characterized in a test population; the number of alleles ranged from 2 to 11. The observed and expected heterozygosities ranged from 0.2593 to 0.8966 and from 0.3047 to 0.8454, respectively. Two loci deviated from Hardy-Weinberg equilibrium; linkage disequilibrium among the 12 loci was non-significant. These polymorphic microsatellite loci will be useful for genetic diversity analysis and molecule-assisted breeding of the gray mullet.

Isolation and characterization of polymorphic microsatellite loci in the hard-shelled mussel, Mytilus coruscus (Mytilidae).

Mytilus coruscus is one of the most important cultured species of marine shellfish in China. Using an expressed sequence tag-library and two microsatellite-enriched genomic libraries of M. coruscus, we isolated and characterized 12 polymorphic microsatellites in a test population; the number of alleles ranged from three to seven, and the observed and expected heterozygosities ranged from 0.0333 to 0.8571 and from 0.3452 to 0.8267, respectively. Four loci deviated from Hardy-Weinberg equilibrium. These polymorphic microsatellite loci can be employed in genetic diversity analysis and molecular marker-assisted breeding of M. coruscus.

Polymorphic microsatellite loci from two enriched genomic libraries for the genetic analysis of the miiuy croaker, Miichthys miiuy (Sciaenidae).

Twelve polymorphic microsatellites from the (AG)(13) and (CA)(13) enriched genomic libraries of Miichthys miiuy were isolated and characterized in a test population; the number of alleles ranged from two to nine. The observed and expected heterozygosities ranged from 0.1923 to 1.0000 and from 0.2633 to 0.8337, respectively. Three loci deviated from Hardy-Weinberg equilibrium, and linkage disequilibrium between five pairs of loci was significant. These polymorphic microsatellite loci can be used for genetic diversity analysis and molecular-assisted breeding of M. miiuy.

Microsatellite-centromere mapping in Cynoglossus semilaevis using gynogenetic diploid families produced by the use of homologous and non-homologous sperm.

Twenty-one microsatellite markers were studied in three meiogynogenetic families of Cynoglossus semilaevis gunther for centromere mapping using half-tetrad analysis. Among the 13 mapped loci, 10 were estimated to be located in the telomeric region, one in the centromeric region, and two in the intermediate region of the chromosome. This study provides a basis for constructing a linkage map of C. semilaevis.

miR-132 weakens proliferation and invasion of glioma cells via the inhibition of Gli1.

OBJECTIVE: Transcriptional factor Gli1 in Hedgehog signal pathway facilitates epithelial mesenchymal transition (EMT) and is associated with invasion or proliferation of multiple tumor cells. The previous study showed the correlation between miR-132 down-regulation and glioma pathogenesis. We investigated the role of miR-132 in mediating Gli1 expression and in affecting proliferation or invasion of glioma cells. PATIENTS AND METHODS: Dual luciferase reporter gene assay was used to confirm the targeted regulation between miR-132 and Gli1. Tumor tissues at different pathological grades (grade II, III and IV) were collected from glioma patients, in parallel with brain tissues from contusion surgery. The expression of miR-132 and Gli1 was measured by RT-PCR. Glioma cell line U251 was treated with miR-132 or si-Gli1 followed by measuring the expression of Gli1, E-cadherin, Vimentin and Cyclin D1. In addition, flow cytometry and transwell assay were performed to evaluate cell invasion potency. RESULTS: Bioinformatics analysis showed the complementary binding sites between miR-132 and 3'-UTR of Gli1 mRNA. Transfection of miR-132 mimic significantly reduced luciferase activity, indicating the targeted regulatory relationship between miR-132 and Gli1 mRNA. Compared with control group, miR-132 expression was decreased and Gli1 level was elevated in glioma tissues, both of which were correlated with the pathological grade. Transfection of miR-132 mimic or si-Gli1 remarkably suppressed the expression of Gli1, Vimentin or Cyclin D1 in U251 cells, up-regulated E-cadherin expression, suppressed cell proliferation and invasion. CONCLUSIONS: Our data indicated that over-expression of miR-132 could inhibit proliferation or invasion of glioma cells via targeted inhibition of Gli1 expression.

Insulin in combination with selenium inhibits HG/Pal-induced cardiomyocyte apoptosis by Cbl-b regulating p38MAPK/CBP/Ku70 pathway.

OBJECTIVE: In this study, we investigated whether insulin and selenium in combination (In/Se) suppresses cardiomyocyte apoptosis and whether this protection is mediated by Cbl-b regulating p38MAPK/CBP/Ku70 pathway. MATERIALS AND METHODS: Firstly, H9c2 cardiomyocytes were treatment with high glucose (25 mmol/L) and palmitate (600 µmol/L) (HG/Pal). Next, H9c2 cardiomyocytes were treatment with HG/Pal+In/Se (10 nmol/L Insulin in combination with 10 nmol/L selenium). Finally, cells were treated with siRNA against Cbl-b, followed by HG/Pal and HG/Pal+In/Se treatment. Then, Cell apoptosis was observed by flow cytometry (FCM). The levels of Cbl-b, p-p38MAPK, CBP and Bax were examined by Western blotting. The acetylated Ku70 was detected by immunoprecipitation. RESULTS: Insulin and selenium in combination reduced cell apoptosis, up-regulated Cbl-b expression, down-regulated p38MAPK, CBP and acetylated Ku70 expressions and prevented Bax translocation, whereas Cbl-b knockdown strongly suppressed In/Se-induced these effects in HG/Pal-treated cardiomyocytes. CONCLUSIONS: Insulin and selenium synergistically suppressed cardiomyocyte apoptosis by Cbl-b regulating p38MAPK/CBP/Ku70 pathway.

Acetylsalicylic acid enhances the anti-inflammatory effect of fluoxetine through inhibition of NF-κB, p38-MAPK and ERK1/2 activation in lipopolysaccharide-induced BV-2 microglia cells.

The latest advancements in neurobiological research provide increasing evidence that inflammatory and neurodegenerative pathways play an important role in depression. According to the cytokine hypothesis, depression could be due to the increased production of pro-inflammatory cytokines by microglia activation. Thus, using the BV-2 microglial cell line, the aim of the present study was to investigate whether fluoxetine (FLX) or acetylsalicylic acid (ASA) could inhibit this microglia activation and could achieve better results in combination. Our results showed that FLX could attenuate lipopolysaccharide (LPS)-induced production of interleukin-1ß (IL-1ß), the expression of the indoleamine 2,3 dioxygenase (IDO) enzyme and the depletion of 5-HT. Moreover, FLX could inhibit phosphorylation of nuclear factor-κB (NF-κB) and phosphorylation of p38 mitogen-activated protein kinase (MAPK), and the combined use with ASA could enhance these effects. Notably, the adjunctive agent ASA could also inhibit phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2). Taken together, our results suggest that FLX may have some anti-inflammatory effects by modulating microglia activation and that ASA served as an effective adjunctive agent by enhancing these therapeutic effects.