[ ABSTRACT] AIM:To explore the effects of eplerenone on the expression and activity of aortic endothelial nitric oxide synthase ( eNOS) in high salt-induced hypertensive rats .METHODS: Male Wistar rats (4 week old, weighting 50~60 g) were randomly divided into control group , high-salt diet group and eplerenone group .The rats in control group were fed with ordinary rodent animal diet , the rats in high-salt group and eplerenone group were exposed to 5%salt diet for 16 weeks and administrated with the same dosage of saline or eplerenone (40 mg? kg-1? d-1 ) by gavage for 4 weeks, re-spectively.Systolic blood pressure (SBP) was measured by tail-cuff every 2 weeks.The rats were sacrificed after 16 weeks and the thoracic aorta was collected .The aldosterone content in the aorta was measured by ELISA .The protein levels of mineralocorticoid receptor (MR) and eNOS were determined by Western blot.The activitie of constitutive NOS (cNOS) was measured by chemocolorimetry .The protein localization of eNOS , neuronal nitric oxide synthase ( nNOS) and MR was observed by immunohistochemistry .RESULTS: A process of 8-week high-salt diet increased SBP gradually .SBP in the rats exposure to high salt for 16 weeks was significantly higher than that in control group ( P<0.05 ) .After 4 weeks of eplerenone treatment, SBP in the rats was significantly lower than that before treatment (P<0.05).Compared with control group, the aldosterone content in the aorta were significantly increased in high-salt diet group and eplerenone group ( P<0.05), the expression level of MR also increased significantly (P<0.05).Compared with control group, both eNOS pro-tein expression (P<0.05) and cNOS activity in high-salt diet group were significantly decreased (P<0.05).The protein expression of eNOS as well as cNOS activity in aorta increased significantly in eplerenone group compared with high -salt diet group (P<0.05).CONCLUSION:Aldosterone content in aorta of high-salt-induced hypertensive rats increases signifi-cantly .Aldosterone attenuates the protein expression of eNOS and reduces the enzyme activity through the activation of min -eralocorticoid receptor .The selective mineralocorticoid receptor antagonist eplerenone enhances the protein expression of eNOS and its activity , thereby improves eNOS function .

Objective To investigate the effects of granulocyte macrophage colony-stimulating factor (GM-CSF) combined with interleukin-12 (IL-12) genes on apoptosis of hepatoma cells.Methods The hepatoma cell lines were cultured in vitro and were divided into four groups: GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,negative control group (empty load group),respectively.The PIB-CMV3-GM-CSF and PIB-CMV3-IL-12 eukayotic expression vector was built,and 36 h after transfection,fluorescence microscope was used to detect the transfection effect;the expression level of IL-12,GM-CSF,p53,p38 and C-JUN mRNA were detected by RT-PCR,and Western blot was used to examine the expression level of IL-12,GM-CSF,p53,p38 and C-JUN protein.In addition,the flow cytometry was applied to detect cell apoptosis.Results Through fluorescence microscope,green fluorescence was observed in cells of GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,indicating that the plasmid has successfully transferred into cells.In addition,the expression of p53mRNA in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were 1.2±0.10,4.3±0.98,4.2±0.34,9.2±0.87,and the protein expression were 1.0±0.10,3.6±0.34,3.8±0.30,5.0±0.60.Compared with the empty load group,the expression level of p53 mRNA and protein were significantly increased in the three plasmid transfection groups (P<0.01).The expression of p53 mRNA and protein were significantly increased in co-transfection group than GM-CSF group and IL-12 group (P<0.01),while in the comparison with GM-CSF transfection group and IL-12 transfection group,the expression level of p53mRNA and protein in the co-transfection group could be improved to a higher degree(P<0.01).Meanwhile,p38 C-JUN mRNA expression levels in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were as follows: 7.5± 0.9,3.5±0.45,3.7±0.25,1.0±0.11,while p38protein expression levels were 10.1±1.03,6.1± 0.67,7.1 ± 0.61,1.0 ± 0.12,respectively,C-JUN mRNA expression levels were 11.2 ± 1.20,4.1 ± 0.19,3.3 ± 0.30,1.0 ± 0.01,separately,C-JUN protein expression levels were 2.25 ± 0.2,1.8 ± 0.13,1.4 ± 0.12,1.0 ± 0.09.P38, C-JUN mRNA and protein levels were significantly reduced in the three plasmid transfection groups compared with the empty load group (P<0.01).The expression of p38,C-JUN mRNA and protein were reduced to a lower degree in co-transfection group than in GM-CSF transfection group and IL-12 transfection group (P<0.01).Flow cytometer showed that the hepatoma cell apoptosis rate of the empty load group,GM-CSF transfection group,IL-12 transfection group,co-transfection group were (3.43±0.9)%,(5.87±1.02)%,(7.32±1.1)%,(17.47±2.11)%,the rates of the three plasmid transfection groups were significantly higher than that of the empty load group (P<0.01).And the apoptosis rate was significantly increased in the co-transfected group compared with other plasmid groups (P<0.01). Conclusion The combination of GM-CSF and IL-12 could significantly accelerate the apoptosis of hepatoma cells by up-regulating the expression of p53,and down-regulating the expression of p38 and C-JUN.

Objective To investigate the effect and mechanism of soluble epoxide hydrolase inhibitor (sEHI) for NF-κB pathway and cell circle arrest of tubular epithelial cell in unilateral ureteral obstruction (UUO) mice model.Methods Thirty-two healthy C57BL/6 male mice performed UUO surgery to induce renal interstitial fibrosis.Animals were randomly divided into 4 groups:sham group (n=8),sEHI (1 mg· kg-1·d-1) group (n=8),UUO group (n=8) and UUO+sEHI (1 mg· kg-1· d-1) group (n=8).Daily sEHI [1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea,TUPS] or 2％ DMSO was applied to mice by oral gavage from day 1 to day 14 after surgery.All mice were sacrificed at day 14 and kidneys were harvested for further analysis.The changes of renal tissue morphology and pathology were observed by Hematoxylin and eosin (HE) and sirius red staining.The expressions of sEH,nuclear factor κB p65 (NF-κB p65) and IκB were measured by Western blotting.The expressions of TNF-α,IL-1β,MCP-1,IL-6,TGF-β,CTGF,collagen-Ⅳ and α-SMA were analyzed by real-time PCR.Immunofluorescence staining of phospho-histone H3 (p-HH3) and Ki67 was performed to determine the stage of cell cycle G2/M arrest.Results The expression and activity of sEH increased in UUO group (P ＜ 0.05).Administration of sEHI inhibited activity of sEH and infiltration of inflammatory cell in tubular interstitial,as well as attenuated tubular damage and tubular interstitial fibrosis.Western blotting analysis revealed administration of sEHI inhibited up-regulated NF-κB p65 and down-regulated IκB in UUO group (P ＜ 0.05).Real-time PCR demonstrated that administration of sEHI obviously decreased the mRNA expression of cytokines and fibrosis markers,including of TNF-α,IL-1 β,MCP-1,IL-6,TGF-β,CTGF,Collagen-Ⅳ,α-SMA (P ＜ 0.05).Immunofluorescence staining showed that there were much more p-HH3 and Ki67 double positive nuclear tubular epithelial cells and interstitial cells in UUO group,compared with Sham group (P ＜ 0.05).Administration of sEHI reduced the number of double positive nuclear cell only in tubular epithelial cells (P ＜ 0.05),but not in interstitial cells.Conclusions In UUO tubular interstitial fibrosis model,sEHI inhibits the activation of NF-κB pathway by down-regulating p65 and up-regulating IκB and ameliorates the infiltration of inflammatory cells.In addition,sEHI plays anti-fibrosis effect by moderating cell cycle G2/M arrest and reducing the excrete of pro-fibrosis factors of tubular epithelial cells.

OBJECTIVE:To investigate the effects of Glycyrrhiza uralensis extract (GE) on the expression of uridine diphosphate glucuronyltransferase 1A (UGT1A) and multidrug resistance associated protein 2 (MRP2) in human liver L-02 cells damaged by triptolide (TP),and to study attenuated mechanism of G.uralensison for TP.METHODS:The survival rates of L-02 cells were determined by MTT assay after cultured with 0 (blank control),40,80,160 nmol/L TP for 12,18,24 h.L-02 cells were divided into blank control group (blank culture medium),model control group (80 nmol/L TP) and GE pretreatment group (adding 80 nmol/ L TP after pretreated with 30,60,90 mg/L GE for 24 h);after cultured for 18 h,survival rates of L-02 cells were determined by MTT assay.Rifampin (RIF) group (positive control,adding 80 nmoi/L TP after pretreated with 10 μmol/L RIF for 24 h) was added on the basis of the above grouping (GE concentration of 60 mg/L in GE pretreatment group).After cultured for 24 h,the protein expressions of UGT1A and MRP2 were detected.RESULTS:The inhibition effect of TP on cell proliferation was positively correlated with the concentration and the time.Compared with blank control group,cell survival rate of model control group was decreased significantly (P＜0.05),and the protein expression of MRP2 was decreased significantly (P＜0.01).Compared with model control group,cell survival rates of 30,60,90 mg/L GE pretreatment groups were all increased significantly (P＜0.01).The protein expressions of UGT1A and MRP2 were increased significantly in 60 mg/L GE pretreatment group (P＜0.01).CONCLUSIONS:GE pretreatment can relieve TP-induced human liver L-02 cell damage,and its attenuated mechanism may be associated with the increase the expression of UGT1A and MRP2.

Liver disease during pregnancy is substantially different from common liver diseases, and it is very important to raise the awareness of such disease and improve the diagnostic level. The literature on liver disease during pregnancy published in recent years is reviewed in this article, and the research advances in the pathogenesis, diagnosis, and treatment of liver disease during pregnancy are summarized.

Objectives To investigate whether osteoarthritis (OA) progression can be delayed by casticin in rodent models of anterior cruciate ligament transection (ACLT).Methods Eighteen 2-month-old male C57BL/6J mice were randomised into 3 even groups (n =6) subjected respectively to sham-operation,ACLT-vehicle-treatment and ACLT-casticin-treatment.The knee capsule was dissected in the sham-operation group and ACLT on the right knee was conducted in the ACLT-vehicle-treatment and ACLT-casticin-treatment groups.Intragastric administration of the same amount of Tween-80 solution was conducted for the sham-operation and ACLT-vehicle-treatment groups;Intragastric administration of casticin of 20mg/kg was conducted once per day for the ACLT-casticin-treatment group.Bone micro CT (μCT) was quantitated to detect alterations in microarchitecture of femoral condyle subchondral bone.Tartrate resistant acid phosphatase(TRAP) stain and NOX4 immunostaining were conducted to detect relative proteins and the osteoclast changes on the subchondral bone.Articular cartilage degeneration was graded using HE and safranin O-green staining and the Mankin score criteria.Results Compared with the the sham-operation group,the subchondral bone density,trabecular bone volume fraction and trabecular thickness were decreased,and the trabecular space,positive rates of TRAP stain and NOX4 immunostaining and Mankin scores were increased in the ACLT-vehicle-treatment group.All the above comparisons were statistically significant (P ＜ 0.05).Compared with ACLT-vehicle-treatment group,the subchondral bone density and trabecular bone volume fraction were increased,and the trabecular space,positive rates of TRAP stain and NOX4 immunostaining and Mankin scores were decreased in the ACLT-casticin-treatment group.All the above comparisons were statistically significant (P ＜0.05).Conclusion As casticin may attenuate early OA progression by inhibiting NOX4 activity in subchondral bone and formation of osteoclasts,it may be a new clue to preventive therapy for OA.

Mild cognitive impairment (MCI) is considered to be the early stage of dementia. About 10%-15% of MCI patients eventually develop dementia each year. Alzheimer's disease (AD) is the leading cause of dementia, and also the final outcome of most MCI. Early identification and intervention of MCI with high risk of conversion to AD is of great significance for controlling the progress of cognitive impairment. The author summarizes the latest research progress of core and other new biomarkers that predict the conversion of MCI to AD from levels of body fluid, imaging and genetics, to provide guidance for predicting the outcomes of MCI or early diagnoses of AD.

Objective:To investigate the potential correlation between miR-223 level in leukocytes and platelet responses to clopidogrel in patients with coronary artery disease.Methods:A cohort of 188 outpatients,who conducted percutaneous coronary intervention (PCI) and received dual antiplatelet therapy,were recruited.The patient's electronic health data were collected,and their blood samples were obtained for measurement of adenosine diphosphate (ADP)-induced whole-blood platelet aggregation.Extreme cases ofplatelet responses to clopidogrel (ultra-vs.non-responder) were measured with miR-223-3p levels in leukocytes.Results:Both groups had similar miR-223-3p levels in leukocytes.There were no significant differences in other demographic and clinical data except for metrics of ADP-induced whole-blood platelet aggregation between the 2 group.Conclusion:MiR-223-3p in peripheral leukocytes is not associated with the altered platelet responses to clopidogrel in PCI outpatients.

Objective To investigate the correlation of fibulin 5(FBLN5)and Rho GTPase activating protein 4(ARHGAP4)in gastric cancer tissues and their relationship with the prognosis of patients.Methods One hundred gastric cancer patients admitted to the Department of General Surgery of Shijiazhuang First Hospital were selected for the study,and gastric cancer tissues＞1 cm from the tumor margin and paracancerous tissues 5 cm from the tumor margin were collected.Retrospective analysis of the relationship between FBLN5 and ARHGAP4 expression profiles and clinicopathological indices of gastric cancer,as well as their effects on survival,was performed with univariate and Cox regression analyses of the patients'clinical data.Immunohistochemical staining was used to detect the expression of FBLN5 and ARHGAP4.The relationship between the expression levels of different FBLN5 and ARHGAP4 and the survival time of patients.Results The positive expression rate of ARHGAP4 was lower than that of paracancerent tissues,and the positive expression rate of FBLN5 was higher than that of paracancerent tissues(P＜0.05).ARHGAP4 expression was associated with tumor site,Lauren's staging,lymph node metastasis,TNM stage,differentiation degree,CE,CA19-9,CA125,and immune score(P＜0.05);FBLN5 expression was associated with Lauren's staging,lymph node metastasis,TNM stage,differentiation degree,CE,CA19-9,CA125,and immune score(P＜0.05);Kaplan-Meier analysis showed that the 3-year cumulative survival rate of FBLN5 positive group was,which was lower than that of negative group(P=0.044).The 3-year cumulative survival rate of ARHGAP4 positive patients was higher than that of negative patients(P=0.021).Cox results showed that positive ARHGAP4 was protective factor for survival(P＜0.05).Conclusion The prognosis of patients in the FBLN5-positive group is worse than that in the negative group,high expression of FBLN5 is a risk factor for survival,and the prognosis of patients with ARHGAP4 negative group is better than that in the positive group,which are closely related to the development and prognosis of gastric cancer patients.

Background: There is an urgent need to find reliable and rapid bovine tuberculosis (bTB) diagnostics in response to the rising prevalence of bTB worldwide. Toll-like receptor 2 (TLR2) recognizes components of bTB and initiates antigen-presenting cells to mediate humoral immunity. Evaluating the affinity of antigens with TLR2 can form the basis of a new method for the diagnosis of bTB based on humoral immunity. Objectives: To develop a reliable and rapid strategy to improve diagnostic tools for bTB. Methods: In this study, we expressed and purified the sixteen bTB-specific recombinant proteins in Escherichia coli. The two antigenic proteins, MPT70 and MPT83, which were most valuable for serological diagnosis of bTB were screened. Molecular docking technology was used to analyze the affinity of MPT70, MPT83, dominant epitope peptide of MPT70 (M1), and dominant epitope peptide MPT83 (M2) with TLR2, combined with the detection results of enzyme-linked immunosorbent assay to evaluate the molecular docking effect. Results: The results showed that interaction surface Cα-atom root mean square deviation of proteins (M1, M2, MPT70, MPT83)-TLR2 protein are less than 2.5 A, showing a high affinity.It is verified by clinical serum samples that MPT70, MPT83, MPT70-MPT83 showed good diagnostic potential for the detection of anti-bTB IgG and M1, M2 can replace the whole protein as the detection antigen. Conclusions Molecular docking to evaluate the affinity of bTB protein and TLR2 combined with ELISA provides new insights for the diagnosis of bTB.