Objective To estimate the influences of N-acetyl cysteine (NAC) on a chemical hypoxiamimetic agent CoCl2 induced-injury to, and expressions of inflammatory factors by, an immortal human skin keratinocyte line HaCaT. Methods HaCaT cells were treated with CoCl2 of 2000 μmol/L for 4 hours to set up a chemical hypoxia-induced cell model of skin injury. NAC of various concentrations ( 1000, 2000, 3000 μmol/L)was used to pretreat HaCaT cells for 2 hours prior to the establishment of cell model. After these treatments,cell viability was detected by cell counting kit 8 (CCK-8), the levels of interleukin 6 and 8 (IL-6 and -8) and tumor necrosis factor α (TNF-α) in culture supernatant by ELISA kits, mitochondrial membrane potential (MMP) by rhodamine 123 (Rh123) staining and photofluorography, intracellular reduced glutathione (GSH)content by glutathione detection kit. Results An obvious decline was observed in HaCaT cell viability after pretreatment with various concentrations of NAC for 2 hours. The treatment with CoCl2 of 2000 μmol/L for 4 hours induced an elevation in the supernatant levels of IL-6, IL-8 and TNF-α and a decrease in GSH content and MMP, while the pretreatment with NAC for 2 hours retarded the CoCl2-induced increase in IL-6 and IL-8 levels as well as decrease in GSH content and MMP. Conclusion The reactive oxygen species (ROS) scavenger NAC can protect against CoCl2-induced injury to and inflammatory reaction in HaCaT cells, which may be associated with a decrement in oxidative stress.

Objective:To investigate the role of sterol regulatory element binding protein-1 (SREBP1) in atorvastatin-induced reduction of nucleotide-binding oligomerization domain-like receptor protein 1 ( NLRP1 ) inflammasome expression. Methods:THP-1 cells were treated with phorbol 12-myristate 13-acetate (160 nmol/L) for 12 h to be differentiated into macrophages. The medium was then replaced with serum-free medium containing lipopolysaccharide and ( or ) atorvastatin. The mRNA expression of NLRP1 and SREBP1 were detected by Real-time PCR. The protein expression of NLRP1 and SREBP1 were determined by Western blot. Furthermore, we observed the effect of SREBP1 siRNA on atorvastatin-induced reduction of NLRP1 expression. Results:Atorvastatin inhibited the mRNA and protein expression of NLRP1 and SREBP1 in the THP-1 macrophages. SREBP1 siRNA showed no significant difference on lowering NLRP1 expression when compared with atorvastatin. Treating cells with SREBP1 siRNA and atorvastatin at the same time resulted in more obvious reduction of NLRP1 expression than single use of SREBP1 siRNA or atorvastatin. Conclusion:Atorvastatin might exert anti-inflammatory effect by repressing NLRP1 expression through the SREBP1 path-way.

Objective To explore whether the phosphorylation of NF-κB P65 subunit is involved in the cytotoxicity to and inflammation in an immortal human keratinocyte cell line HaCaT during cobalt chloride (CoCl2-induced chemical hypoxia. Methods HaCaT cells were treated with CoCl2 of 2 mmol/L to set up a chemical hypoxia-induced cell model of injury. Then, RNA interference was used to down-regulate the expression of P65 in CoCl2-induced HaCaT cells. After additional culture, cell viability was tested by cell counting kit8 (CCK-8), the levels of interleukin 6 (IL-6) and interleukin 8 (IL-8) were detected by ELISA kits, phosphorylated and total P65 protein was measured by Western blot. Results The exposure of HaCaT cells to 2 mmol/L CoCl2 for 0 to 4 hours enhanced the phosphorylation of P65, which began at 0.5 hour, peaked at 1.5 hours, and restored to the normal level at 4 hours, and the level of P65 phosphorylation was about 6.6 times that in the untreated control group. The CoCl2 of 2 mmol/L decreased the cell viability of HaCaT cells in a time dependent manner, and a significant difference was observed in the viability of HaCaT cells between CoCl2-treated and untreated HaCaT cells at 2, 4, and 6 hours (P ＜ 0.05, 0.01, 0.01 ). The release of IL-6 and IL-8 from HaCaT cells was also promoted by CoCl2 treatment. The knockdown of P65 expression with siRNA markedly suppressed the CoCl2-induced cytotoxicity to and increase in the release of IL-6 and IL-8 from HaCaT cells,despite of an increment in cell viability by about 11%. Conclusion The phosphorylated P65 subunit mediates CoCl2-induced cytotoxicity and inflammatory injury to HaCaT cells.

Aim To explore the effect of chemical hypoxia-mimetic agent,cobalt chloride(CoCl2)on inflammatory reaction in human keratinocytes(HaCat cells).Methods After HaCat cells were treated with CoCl2 at different concentrations to set up a chemical hypoxia-induced cell model of skin injury,cell viability,intracellular reactive oxygen species(ROS),mitochondrial membrane potential(MMP),the levels of both interleukin 6(IL-6)and interleukin 8(IL-8)as well as the expression of heme oxygenase-1(HO-1)were detected.Results The viability of HaCat cells was reduced by CoCl2 at the concentrations from 500 to 3 000 ?mol?L-1,and the higher CoCl2 doses,the lower cell viability was.CoCl2 induced oxidative stress reaction(increasing ROS production and decreasing MMP).CoCl2 induced inflammatory reaction,enhancing the release of IL-6 and IL-8.CoCl2 at concentrations from 1 000 to 3 000 ?mol?L-1 upregulated HO-1 expression in HaCat cells.Conclusion CoCl2 induces not only oxidative stress,but also inflammatory reaction,increasing the release of both IL-6 and IL-8,as well as HO-1 expression.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic variation. In this study, we characterized the genetic variation and evolutionary relationships among circulating PRRSV strains in southern China. We analyzed 29 NSP2 strains and 150 ORF5 strains from clinical samples collected in southern China during 2007–2014. The alignment results showed that the nucleotide identity similarities of the two genes among these strains were 80.5%–99.7% and 80.9%–100%, respectively. Phylogenetic analysis based on the NSP2 gene showed that highly pathogenic (HP)-PRRSV was still the dominant virus in southern China from 2013 to 2014. Compared with reference strains CH-1a and VR-2332, the field strain 131101-GD-SHC, which shared high homology with JXA1-P170, had a novel 12 amino acid deletion at position 499–510. Phylogenetic analysis based on the ORF5 gene showed that HP-PRRSV, VR2332-like strains, and QYYZ-like strains were simultaneously circulating in southern China from 2007 to 2014, suggesting that, in recent years, the type 2 PRRSV was more diverse in southern China. In conclusion, mutations in the decoy epitope and primary neutralizing epitope could be markers of viral evolution and used to study evolutionary relationships among PRRSV strains in China.
China ; Genetic Variation* ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus*

The spike gene of porcine epidemic diarrhea virus (PEDV) was sequenced from 55 South China field strains isolated from pigs with symptoms of diarrhea. The sequences were compared within the set of field strains as well as with reference strains available in GenBank. Within the 55 South China PEDV field strains, the deduced amino acid sequence identities ranged from 93.8% to 99.9 % and ranged from 90.7% to 99.5% when compared with the foreign reference strains in GenBank. Our phylogenetic analysis showed that 10 of the 55 South China PEDV strains belonged to G1b and 45 belonged to G2b.
Amino Acid Sequence ; China* ; Databases, Nucleic Acid ; Diarrhea ; Porcine epidemic diarrhea virus* ; Sequence Analysis* ; Swine

Outbreaks of pseudorabies (PR) have occurred in southern China since late 2011, resulting in significant economic impacts on the swine industry. To identify the cause of PR outbreaks, especially among vaccinated pigs, 11 pseudorabies virus (PRV) field strains were isolated from Guangdong province during 2013–2014. Their major viral genes (gE, TK, gI, PK, gD, 11K, and 28K) were analyzed in this study. Insertions or deletions were observed in gD, gE, gI and PK genes compared with other PRV isolates from all over the world. Furthermore, sequence alignment showed that insertions in gD and gE were unique molecular characteristics of the new prevalent PRV strains in China. Phylogenetic analysis showed that our isolates were clustered in an independent branch together with other strains isolated from China in recent years, and that they showed a closer genetic relationship with earlier isolates from Asia. Our results suggest that these isolates are novel PRV variants with unique molecular signatures.
Asia ; China* ; Disease Outbreaks ; Genes, Viral ; Herpesvirus 1, Suid* ; Pseudorabies* ; Sequence Alignment ; Swine

OBJECTIVETo study the gene polymorphism of the Auberger antigens in Lutheran blood group system in Chinese population and establish a stable, accurate molecular method detecting Auberger antigens.

METHODSPeripheral blood samples from 162 randomly collected and unrelated volunteer blood donors were directly sequenced for the exon 12 at the gene locus of Auberger antigens. PCR products with novel nucleotide were further investigated by restriction fragment length polymorphism (RFLP) analysis.

RESULTSAuberger genotypes in the 162 Chinese individuals were obtained: Au(a+ b- )(nt1615A) was found in 119 individuals, Au(a+ b+ ) (nt1615A/G) in 40 individuals and Au(a- b+ ) (nt1615G) in 3 individuals. The allele frequencies of the Au(a) and Au(b) were 0.8580 and 0.1420, respectively. An individual with homozygous Au(a) genotype had a nucleotide mutation (1595 G to T). The mutation was confirmed by digesting the DNA with Hha I.

CONCLUSIONThe distribution of gene polymorphism of Auberger antigens in a Chinese population was investigated and obtained. And a molecular method determining the Auberger antigen was established. A novel Lutheran allele was deposited in GenBank (accession number EU260043).

Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; DNA Mutational Analysis ; Female ; Gene Frequency ; Haplotypes ; Humans ; Lutheran Blood-Group System ; chemistry ; genetics ; Male ; Molecular Sequence Data ; Polymorphism, Genetic

We designed the specific primers and TaqMan probes targeting cytochrome b genes of mitochondrial DNA from bovine, goat and sheep. We used different fluorescents to label the probes. After optimization of reaction conditions, we set up a multiplex fluorescent real-time PCR method to detect bovine, goat and sheep derived materials, simultaneously. We finished the detection tests of 17 kinds of animal DNA and 200 DNA samples from different sources with the developed method and the National Standard GB/T 20190Y-2006 routine PCR method. The coincidence rate of these two methods was 100%. Without electrophoresis or restriction digestion, the developed method could reduce the test time to one third as routine PCR and identify three kinds of animal derived materials including bovine, goat and sheep in one reaction. The developed method was approximately 10 times more sensitive than routine PCR, and was applicable to identifications of bovine, goat and sheep derived materials in feed stuff, meat, milk, pelt and grease, etc. The study showed that the developed real-time PCR method is a rapid, sensitive and efficacious detection assay for bovine, goat and sheep derived materials in animal products.
Animal Feed ; analysis ; Animals ; Cattle ; Cytochromes b ; genetics ; DNA Primers ; DNA, Mitochondrial ; analysis ; genetics ; Food Contamination ; analysis ; Genes, Mitochondrial ; Goats ; Meat ; analysis ; Mitochondria, Muscle ; enzymology ; genetics ; Polymerase Chain Reaction ; methods ; Sheep

Objecfive:To investigate the clinical characteristics and surgical management of tumor invasion on recurrent laryngeal nerve at the entrance of larynx in thyroid cancer.Methods:The clinical data of 30 patients with recurrent laryngeal nerve invasion by thyroid cancer from Aug 2012 to Aug 2018 in Cangzhou Central Hospital were analyzed retrospectively. Patients were divided into groups A (14 cases ,nerve was invaded at the larynx) and group B (16 cases,nerve was involved in other parts).Results:Between the two groups, operation time, intraoperative blood loss, number of tumor focus, adhesion and infiltration were not statistically different ( P>0.05). The tumor size in group A was smaller ( t=-3.614, P=0.001), the lymph node metastasis rate was lower ( χ2=5.593, P=0.018), and the microcancer rate was higher ( χ2=4.051, P=0.044).Follow up data up to 24 months showed there were no significant difference in postoperative hoarseness , laryngoscope abnormality and recurrence rate between the two groups (all P>0.05). Conclusion:Patients of thyroid cancer with recurrent laryngeal nerve invasion at the larynx had relatively small tumor size, higher proportion of microcancer, lower rate of lymph node metastasiss.