Objective To study the characteristics of liver function damage in brucellosis and provide theoretical basis for clinical treatment.Methods One hundred and thirty-six hospitalized patients with brucellosis from Endemic Disease Prevention and Control Center of Wulanchabu City from January 2011 to October 2012 were selected randomly as the research subjects.Parameters of Alanine transaminase (ALT),Aspartate transaminase (AST),Alkaline phosphatase (ALP),Gamma glutamyltransferase (γ-GT),Total protein (TP),Albumin (ALB),Albumin/globulin (A/G),total bilirubin (TBIL) and Direct bilirubin (DBIL) were determined.The means of liver function parameters with mid-value of all the reference ranges were compared by one sample t test;Parameters of liver function were analyzed in 70 acute and 66 chronic brucellosis by two independent-samples t test.Results The means of ALT,AST,ALP,γ-GT,TP,ALB,TBIL and DBIL ((x):36.18,28.20,95.87,29.29 U/L,65.08,40.84 g/ L and 19.76,5.86 μmol/L) in the 136 samples of brucellosis serum were significantly different from the medians of their reference ranges ((x):20.50,18.50,85.00,24.50 U/L,69.00,43.50 g/L and 11.80,3.40 μ mol/L,t =9.46,9.19,4.55,2.22,-2.71,-4.99,5.58,11.32,all P ＜ 0.05);A/G was not significantly different ((x):2.14,2.00;t =0.94,P ＞ 0.05);Parameters of liver function were not significantly different between acute and chronic brucellosis (all P ＞ 0.05).Conclusions This study confirmed that the liver function of the 136 patients with brucellosis is impaired.The level of the liver function impairment is usually mild and moderate and no difference in acute and chronic brucellosis is found.

Objective To explore the change and value of interleukin (IL)-2,IL-6 and tumor necrosis factor (TNF)-α in the serum samples of patients with brucellosis.Methods The levels of serum IL-2,IL-6 and TNF-α were measured using the enzyme linked immunosorbent assay (ELISA) method in 155 patients with brucellosis in different clinical stages (including 69 cases in acute,34 cases in subacute and 52 cases in chronic periods) and 50 healthy controls.IL-2 was used to represent the helper T cell (Th)-type 1 cytokines,IL-6 represent Th2 type cytokines,and the Th1/Th2 ratio in different clinical stages was compared.Results The expression level of serum IL-2 in patients with brucellosis was different (ng/L:acute:10.15 ± 2.01;subacute:9.53 ± 1.68;chronic:6.76± 1.31;control:47.25 ± 5.68),the differences were statistically significant (F =74.921,P ＜ 0.05).The expression levels of serum IL-2 in acute,subacute and chronic periods of brucellosis patients were significantly lower than that of healthy controls (all P ＜ 0.05).The expression levels of serum IL-2 in acute and subacute periods of brucellosis patients were higher than that of chronic period patients (all P ＜ 0.05).The expression of IL-6 and TNF-α in serum of patients in different clinical stages were different (ng/L:acute:615.22± 341.07,802.55 ± 479.53;subacute:478.45 ± 105.33,680.21 ± 366.95;chronic:306.37 ± 96.12,455.36 ± 176.27;control:121.45 ± 30.16,87.51 ± 24.03),the differences were statistically significant (F =57.692,63.210,all P ＜ 0.05).The levels of serum IL-6 and TNF-o were significantly higher in patients with brucellosis in acute,subacute and chronic clinical stages than that of healthy controls (all P ＜ 0.05).Serum IL-6 and TNF-α levels in acute period of brucellosis patients were higher than those of subacute and chronic periods patients (all P ＜ 0.05);the levels of serum IL-6 and TNF-α in subacute period of brucellosis patients were also significantly higher than that of chronic period patients (all P ＜ 0.05).The Th1/Th2 ratio was different (acute:0.02 ± 0.00;subacute:0.02 ± 0.00;chronic:0.23 ± 0.02;control:0.41 ± 0.06) in clinical patients with brucellosis,the differences were statistically significant (F =17.843,P ＜ 0.05),the value of Th1/Th2 in acute and subacute period patients were lower than that of chronic period (all P ＜ 0.05).Conclusion IL-2,IL-6 and TNF-α may play an important role in the pathogenesis of brucellosis,and their dynamic changes can reflect the progression and outcome of brucellosis.

Objective To sequence an atypical Brucella strain 16S rDNA,and to evaluate the feasibility of 16S rDNA sequencing method for identification of Brucella.Methods Preliminary identification of atypical strains was carried out with conventional method.Strain DNA was extracted,and 16S rDNA complete sequence was bidirectional sequenced,and Blast in NCBI and DNAMAN software were used for comparison of the sequence identities of the 16S rDNA.Moreover,16S rDNA complete sequence of the stains those were known to cross-react serologically with Brucella was downloaded from GenBank,MEGA 6.0 was used to construct the phylogenetic tree.Results The conventional identification results revealed that it was an atypical Brucella,the gene similarity between the sequences of the test strain 16S rDNA and Brucella was 99％,between 16S rDNA sequence of Brucella abortus 544A and Brucella melitensis 16M was 96.99％.Phylogenetic tree revealed that the test stain was a Brucella,and closely related to Brucella abortus 544A and Brucella melitensis 16M.Conclusion The test strain is an atypical Brucella,and 16S rDNA sequencing analysis is a simple,rapid,and accurate identification method for atypical Brucella.

Objective This study was designed to compare the mRNA expression of Toll-like receptors 2 (TLR2) and Toll-like receptors 4 (TLR4) in monocytes between brucellosis cases and healthy controls,to explore the role and value of TLR2 and TLR4 in the pathogenesis of brucellosis.Methods From March to June in 2015,a total of 155 brucellosis cases at different clinical stages were diagnosed in Ulanqab Center for Endemic Disease Prevention and Control and 50 healthy controls were selected as research subjects.Subjective clinical symptoms of all patients were observed.Serological detection of all cases was done using serum tube agglutination test (SAT).The mRNA expression of TLR2 and TLR4 in peripheral blood cell was detected by real-time PCR technique.Results Fever,hyperhidrosis,weak,bone ache and hepatosplenomegaly were found in patients of acute (69 people),subacute (34 people) and chronic (52 people) periods.The incidences of fever and hyperhidrosis in acute and subacute periods were higher than that in chronic period,and the differences were statistically significant (x2 =58.427,26.190,all P ＜ 0.01).The incidence of bone ache in chronic period was higher than that in acute period,and the difference was statistically significant (x2 =9.264,P ＜ 0.01).In the agglutination titer of 1:800 and higher,the positive rate of chronic period was lower than those in acute and subacute periods,and the differences were statistically significant (x2 =14.302,8.682,all P ＜ 0.01).The mRNA expression levels of TLR2 and TLR4 were different among different clinical periods of brucellosis patients,and the differences were statistically significant (F =17.502,24.931,all P ＜ 0.01).The expression levels of TLR2 mRNA (8.67 ± 2.39) and TLR4 mRNA (12.38 ± 3.87) in acute period of brucellosis patients were higher than those of subacute period (5.21 ± 1.76,7.62 ± 2.21),chronic period (1.25 ± 0.47,1.72 ± 0.55) and healthy control (1.17 ± 0.23,1.43 ± 0.62),and the differences were statistically significant (all P ＜ 0.01).The expression levels of TLR2 and TLR4 mRNA in subacute period of brucellosis patients were higher than those of chronic period and healthy controls,and the differences were statistically significant (all P ＜ 0.01).The expression levels of TLR2 and TLR4 mRNA were not significantly different (all P ＞ 0.05) between chronic period and healthy controls.Conclusion TLR2 and TLR4 may be the specific recognizing receptors participated in the immune response in brucellosis and associated with the occurrence and development of brucellosis.

Objective To investigate the effects of ginkgo flavone on the expression of NF-κB and TLR4 in the liver of mice with nonalcoholic fatty liver disease (NAFLD). Methods 120 KM mice were randomly divided into the control group, model group, as well as high, medium and low dosage of ginkgo flavone groups. The animal model of NAFLD in mice was constructed with high fat diet. The pathological changes of liver, liver index , the serum TNF-α, IL-6 , TG , NF-κB and TLR4 in hepatic tissue was observed after 8 weeks of administration. Results Compared with the model group, the level of liver index, serum TG, TNF-α, IL-6 and the expression of NF-κBp65 in the ginkgo flavone groups dramatically decreased 8 weeks after the administration. And the hepatic steatosis was milder. There was no statistical differences in the expression of TLR4 between the ginkgo flavone groups and the control group (P > 0.05). Conclusions These results suggested the closely relationship between TLR4/NF-κB inflammatory pathway and NAFLD. Ginkgo flavone had the therapeutical effects on NAFLD by anti-inflammatory and lipid-lowering action, but no effect was observed on the expression of TLR4 in hepatic tissue.

Objective We compared peripheral blood nucleotide-binding oligomerization domain-leucinerich repeats containing pyrin domain 3 (NLRP3),cysteinyl aspartate specific proteinase-1 (Caspase-1),interleukin-1β (IL-1β) and IL-18 between acute and chronic brucellosis patients before treatment and revealed their immune characteristics,to find targets for immune intervention of brucellosis.Methods From March to April 2016,42 acute and 42 chronic brucellosis patients were selected from Ulanqab Center for Endemic Disease Prevention and Control as the research subjects,and 20 local healthy persons were selected as healthy control.Brucellosis were diagnosed according to the "Diagnostic Criteria of Brucellosis".Enzyme-linked immunesorbent assay (ELISA) method was used to determine serum levels of NLRP3,Caspase-1,IL-1β and IL-18.Results The expression levels of NLRP3,Caspase-1,IL-1β and IL-18 [(1 264.40 ± 424.74),(1 350.67 ± 468.93),(192.96 ± 61.52),(162.74 ±54.23),(172.44 ± 60.56),(120.10 ± 61.52),(47.23 ± 13.79),(46.68 ± 14.72),(27.71-± 8.71),(202.23 ± 65.24),(169.19 ± 54.33),(108.62 ± 41.39) ng/L] were compared in acute,chronic and control groups,the differences were statistically significant (F =61.96,6.26,16.68,18.31,P ＜ 0.01).Compared to control group,the levels of NLRP3,Caspase-1,IL-1β and IL-18 in acute and chronic groups were significantly increased (P ＜ 0.05);compared to chronic group,the levels of IL-18 in acute group were significantly increased (P ＜ 0.05),and NLRP3,Caspase-1 and IL-1 β levels were not statistically different (P ＞ 0.05).Conclusions This study has showed that the expression of NLRP3 inflammasome in innate immunity is not significantly different between acute brucellosis and chronic brucellosis.The difference of IL-18 levels between acute and chronic brucellosis may affect their immune response.

Objective To study the expression of high mobility group box protein 4 (SOX4) and β-catenin in lung tissues of preterm rats with hyperoxia exposure,and to study its significance.Method One-day-old preterm Sprague Dawley rats were randomly assigned into 4 groups:95％ hyperoxia group,70％ hyperoxia group,45％ hyperoxia group and air group.After three days,pathological changes of the lung tissues were observed by hematoxylin eosin staining,the mRNA expressions of SOX4 and β-catenin in lung tissues were detected by semi-quantitative reverse transcription polymerase chain reaction,and the protein expressions of SOX4 and β-catenin in the lung tissues were measured by western blot.Result The structure of lung tissues in air group was normal.45％ hyperoxia group represented mainly effusion and inflammatory cell infiltration,70％ and 95％ hyperoxia group showed acute lung injury characterized by inflammatory cell infiltration,hyperaemia,hemorrhagic change,disorganization and collapse of alveolar.The expressions of SOX4 mRNA and protein were highly elevated in all hyperoxia groups than that in air group (P ＜ 0.01);Compared with 45％ hyperoxia group,the expressions of SOX4 mRNA and protein were higher in 95％ hyperoxia group (P ＜ 0.01).β-catenin mRNA,total β-catenin protein and nuclear β-catenin protein were also highly elevated in hyperoxia groups compared with air group (P ＜ 0.05).Compared with 45％ hyperoxia group,the expressions of β-catenin mRNA and total β-catenin protein were also highly raised in 70％ hyperoxia group and 95％ hyperoxia group (P ＜ 0.05).The expression of nuclear β-catenin protein was higher in 95％ hyperoxia group than that in 45％ hyperoxia group (P ＜ 0.01).Conclusion The mRNA and protein expressions of SOX4 and β-catenin in lung tissues of preterm rats were increased by hyperoxia exposure.This mechanism may take part in hyperoxia-induced preterm rats lung injury.

Objective To observe the levels of peripheral blood cytokines interferon-γ (IFN-γ)and interleukin-4 (IL-4) in patients with acute and chronic brucellosis before and during treatment,and to understand the differences of two immunocytokines in acute and chronic stage of brucellosis,and the effect of antibacterial therapy on these two cytokines to provide immunological basis for clinical evaluation of the therapeutic effect of brucellosis.Methods Research subjects were 36 pre-treatment acute brucellosis and 36 pre-treatment chronic brucellosis and 36 dur-treatment acute brucellosis and 36 dur-treatment chronic brucellosis,which were selected from Ulanqab Center for Endemic Disease Prevention and Control of Jining City with 25 local healthy persons as healthy controls.The levels of IFN-γ and IL-4 in serum were measured by enzyme-linked immunosorbent assay (ELISA) in patients with acute and chronic brucellosis and control group before and during treatment.Parameters of IFN-γ and IL-4 were analyzed with One-Way ANOVA analysis in pre-treatment and dur-treatment acute brucellosis,chronic brucellosis and control groups.Results The means of IFN-γ [(462.79 ± 47.94),(431.92 ± 40.39),(280.50 ± 40.48) ng/L] and IL-4 [(606.11 ± 51.86),(550.66 ± 51.56),(383.24 ± 53.98) ng/L] were significantly different in the three groups before treatment (F =141.84,139.28,P ＜ 0.05);Compared to control group,the levels of IFN-γ and IL-4 in acute brucellosis and chronic brucellosis were significantly increased before treatment (P ＜ 0.05).The levels of IFN-γand IL-4 in the acute brucellosis were significantly increased compared to those of chronic brucellosis before treatment (P ＜ 0.05).After about ten days antibiotic therapy,the means of IFN-γ [(356.05 ± 43.75),(368.61 ± 35.69),(280.50 ± 40.48) ng/L] and IL-4 [(487.31 ± 51.59),(496.73 ± 48.70),(383.24 ± 53.98) ng/L] were significantly different in the three groups (F =39.57,41.99,P ＜ 0.05).Compared to control group,the levels of IFN-γ and IL-4 in acute brucellosis and chronic brucellosis were significantly increased during treatment (P ＜ 0.05).The levels of IFN-γand IL-4 in the acute brucellosis were not significantly different compared to those of chronic brucellosis during treatment (P ＞ 0.05).Conclusion Different immunological characteristics of cytokines in peripheral blood of patients with acute and chronic brucellosis before treatment have affected the therapeutic effect and clinical outcomes.

OBJECTIVETo investigate the effect of immunization with prokaryotically expressed recombinant fusion protein of extracellular near-transmembrane domain of Tibet minipig leptin receptor (OBR) on fat deposition in SD rats.

METHODSA pair of specific primers containing BamHI and HindIII restriction enzyme sites was designed to amplify the extracellular near-transmembrane domain (1705-2364 bp) of Tibet minipig OBR gene. After digestion, the amplified fragment was inserted into the plasmid pRSETA between BamHI and HindIII sites. The recombinant plasmid was transformed and expressed in E.coli BL21(DE3) and the product was analyzed by SDS-PAGE and Western blotting. SD rats were immunized with the fusion protein, and the changes in body weight, feed intake, body length, Lee's index, percentage of abdominal fat, liver fat deposition and subcutaneous fat deposition were assessed.

RESULTSThe recombinant fusion protein obtained (about 27.6 kD) was expressed in E.coli induced by IPTG and identified by SDS-PAGE and Western blotting. The rats immunized with the fusion protein showed no significant changes in body weight, body length, Lee's index, percentage of abdominal fat or liver fat deposition as compared with the control rats. Nevertheless, the immunization caused significantly increased feed intake and significantly decreased volume of subcutaneous fat cells.

CONCLUSIONImmunization with the fusion protein of extracellular near-transmembrane domain of Tibet minipig OBR can promote feed intake and suppress subcutaneous fat deposition in SD rats.

Adiposity ; drug effects ; Animals ; Base Sequence ; Female ; Gene Expression ; Genetic Vectors ; Obesity ; Plasmids ; Rats ; Rats, Sprague-Dawley ; Receptors, Leptin ; administration & dosage ; genetics ; Recombinant Fusion Proteins ; administration & dosage ; genetics ; Subcutaneous Fat ; physiology ; Swine ; Swine, Miniature

Objective To investigate the effects of recombinant human erythropoietin (rhEPO) on the expression of vascular endothelial growth factor (VEGF) protein and mRNA in lung tissue of premature rat model of the new type bronchopulmonary dysplasia (BPD).Methods Lipopolysaccharide (LPS) or PBS was injected into 60 pregnant rats on the 15th day of gestation.The premature rats by cesarean delivery on the 21th day of gestation were divided into 5 groups:PBS+ air group,PBS+ hyperoxia group,LPS+hyperoxia group,PBS+hyperoxia+rhEPO group,LPS+hyperoxia+rhEPO group.In rhEPO intervention groups,after 6 h exposure to hyperoxia,rhEPO (1 200 IU/kg) was administrated subcutaneously,once every other day for 7 times.The survival rate,body weight,lung weight and lung weight/body weight ratio and pathological changes of lung tissue were observed,the expression levels of VEGF protein and mRNA in the lung tissue were determined by Western blot and RT-PCR at the 1st,7th and 14th day after hyperoxia exposure.Results Compared with the PBS+air group,the survival rate and body weight were decreased,the lung weight was increased,the lung pathological damages were more serious,the expression levels of VEGF protein and mRNA in lung tissue were decreased after hyperoxia exposure.These changes were more notable in the LPS+hyperoxia group (P＜0.05).The lung weight/body weight ratio showed an increasing tendency (P＞0.05).The above indexes were improved after rhEPO intervention (P＜0.05).Conclusion rhEPO could increase the survival rate and produce certain intervention and treatment effects by regulating the VEGF relative pathway in BPD model rats.