Objective To investigate the effect of multidrug resistance protein 4 (MRP4) overexpression on lipopolysaccharide (LPS)-induced vascular endothelial hyperpermeability of rat pulmonary micro-vascular endothelial cells (PMVECs) and its molecule mechanism. Methods Three to six passages of PMVECs were cultured in vitro, and they were divided into three groups: the cells in LPS group were only challenged by LPS 10 μg/mL after being cultured in serum-free medium for 24 hours; the cells in Ad-shRNA and Ad-MRP4 groups were infected with the empty virus control or recombinant adenovirus expressing MRP4 for 2 hours, and then were cultured in serum-free medium for 24 hours followed by stimulation of LPS 10 μg/mL. Endothelial permeability was assayed by the Transwell chamber models at 2, 6, 12, and 24 hours after LPS stimulation. Intracellular cyclic adenosine monophosphate (cAMP) levels were detected by enzyme-linked immunosorbent assay (ELISA). The morphological characteristics and distribution of F-actin was determined by laser confocal fluorescence microscope. The protein expressions of MRP4,β-catenin, vascular endothelium-cadherin (VE-cad) and ZO-1 were measured by Western Blot. Results ① After LPS stimulation, endothelium permeability and intracellular cAMP levels in PMVECs were significantly increased, peaked at 12 hours, and then decreased after 24 hours. Compared with LPS group and Ad-shRNA group, PMVECs of Ad-MRP4 group were exhibited a significant increase in endothelial permeability [12-hour permeability (A value):1.88±0.06 vs. 1.12±0.17, 1.10±0.18] and a significant decrease in intracellular cAMP level [12-hour cAMP (μg/L):2.39±0.02 vs. 2.97±0.01, 3.00±0.02, all P < 0.05]. There was no significant difference in endothelium permeability and intracellular cAMP levels at all time points between the LPS group and the Ad-shRNA group (all P > 0.05).② Under laser confocal fluorescence microscope, after LPS stimulation, the stress fiber formation was induced in three groups. But there were pronounced irregular aggregation of fiber in PMVECs of Ad-MRP4 group. ③ Furthermore, compared with LPS group and Ad-shRNA group, protein expression of MRP4 in Ad-MRP4 group was dramatically increased (gray value: 0.76±0.03 vs. 0.44±0.02, 0.43±0.02, both P < 0.05), and the protein expressions of β-catenin, VE-cad, and ZO-1 were significantly decreased [β-catenin (gray value): 0.14±0.03 vs. 0.23±0.04, 0.23±0.03);VE-cad (gray value): 0.21±0.01 vs. 0.34±0.02, 0.35±0.04; ZO-1 (gray value): 0.14±0.02 vs. 0.37±0.06, 0.33±0.07, all P < 0.05]. There was no significant difference in all protein expressions between the LPS group and Ad-shRNA group (all P > 0.05). Conclusion MRP4 overexpression can decrease intracellular cAMP levels, reduce intercellular junction protein expression, and then exaggerate LPS-induced vascular endothelial hyperpermeability.

Objective To investigate the protective effect of multidrug resistant associated protein 4 (MRP4) inhibitor on rats with sepsis-induced acute lung injury (ALI). Methods Sixty Sprague-Dawley (SD) rats were randomly divided into sham group, sepsis group and MRP4 inhibitor MK571 treatment group, with 20 rats in each group. Sepsis model was reproduced by cecal ligation and puncture operation (CLP), and the rats in sham group were only received celiotomy without ligation and puncture. Rats in MK571 treatment group were intraperitoneally injected with MRP4 inhibitor MK571 (20 mg/kg) 30 minutes before model reproduction, while rates in sham group and sepsis group were given the same amount of normal saline. Twenty-four hours later, the femoral artery blood of mice was collected, and arterial blood gas analysis was measured. Serum tumor necrosis-α (TNF-α) was determined by enzyme-linked immunosorbent assay (ELISA). The lung tissues were collected, and the wet/dry weight ratio (W/D) was calculated. The expression of MRP4 protein in lung tissue was determined by Western Blot. Results Compared with sham group, arterial blood pH value and arterial partial pressure of oxygen (PaO2) were significantly lowered [pH value: 7.18±0.03 vs. 7.40±0.03; PaO2 (mmHg, 1 mmHg = 0.133 kPa): 63.15±6.24 vs. 98.05±2.58], while arterial partial pressure of carbon dioxide (PaCO2) was dramatically higher in the sepsis group (mmHg: 56.60±8.30 vs. 37.85±3.18), serum TNF-α level in the sepsis group was significantly increased (ng/L: 146.24±19.99 vs. 25.77±9.83), the W/D ratio of lung tissue was significantly increased (7.75±0.47 vs. 4.09±0.58), and the expression of MRP4 protein was up-regulated in the sepsis group (gray value: 0.153±0.006 vs. 0.087±0.005, all P < 0.05). Compared with the sepsis group, arterial blood pH value (7.30±0.02 vs. 7.18±0.03) and PaO2 (mmHg: 80.30±5.34 vs. 63.15±6.24) were significantly elevated in the MK571 treatment group, while PaCO2 was dramatically decreased (mmHg: 29.25±3.24 vs. 56.60±8.30), the serum level of TNF-α was significantly decreased (ng/L: 97.96±16.72 vs. 146.24±19.99), the W/D ratio of lung tissue was significantly reduced (5.89±0.51 vs. 7.75±0.47), and MRP4 protein expression was significantly down-regulated (gray value: 0.124±0.006 vs. 0.153±0.006, all P < 0.05). Conclusion MRP4 inhibitor may improve lung function in rats with sepsis-induced ALI by down-regulating MRP4 protein expression and reducing levels of inflammatory cytokines, which exerts protective effect on ALI.

Objective To analyze the clinical effect and complication of trans-radial and femoral artery for percutaneous coronary intervention (PCI) in patients with coronary heart disease.Methods Totally 153 patients with coronary heart disease undergoing PCI were divided into radial artery and femoral artery groups.X-ray exposure time,operation time,the success rates of puncture and operation,in-bed time and complication were recorded. Results There were no significant differences in X-ray exposure time[(17±5)min vs.(16±6)min,t=0.61,P=0.57],operation time [(49± 9) min vs. (48 ± 11) min,t=0.59,P =0.61],the success rates of puncture (98.7％ vs.100.0％,x2 =0.47,P=0.53) and operation (96.0％ vs.96.2％,x2 =0.14,P=0.64) between radial artery and femoral artery groups.However,the complication rates was higher in femoral artery group than in radial artery group (17.9％ vs.2.7 ％,x2 =9.54,P=0.002),in-bed time was shorter in radial artery group than in femoral artery group [(4.5 ± 1.2)h vs.(13.2 ±4.6)h,t=2.12,P =0.003]. Conclusions The trans-radial artery PCI is safe,effective and feasible,with less complications and shorter in-bed time.

Objective To introduce a new modified percutaneous dilational tracheostomy and compare the application effect of percutaneous dilational tracheostomy with modified percutaneous dilational tracheostomy in the treatment of critically ill patients. Methods A total of 60 critically ill patients undergoing tracheotomy were selected , and they were randomly divided into two groups according to the methods of tracheotomy. Sex, age, weight, body mass index, acute physiology and chronic health evaluation, operation time, incision size, intraoperative blood loss, incision healing time, incidences of complications after operation were compared between the two groups. Results There were not statistically significant differences of in sex, age, weight, body mass index, and acute physiology and chronic health evaluation between percutaneous dilational tracheostomy group and modified percutaneous dilational tracheostomy group (P>0.05). Operation time, incision size and intraoperative blood loss of modified percutaneous dilational tracheostomy group was statistically significantly shorter than that of percutaneous dilational tracheostomy group [(5.80 ± 1.19) min vs. (7.65 ± 1.05) min, (8.33 ± 3.30) ml vs. (11.33 ± 4.34) ml, (1.08 ± 2.96) cm vs. (1.27 ± 2.54) cm] (P<0.05). The incision healing time and incidence of complications after operation of percutaneous dilational tracheostomy group had no statistical significance compared with modified percutaneous dilational tracheostomy group (P>0.05). Conclusions The modified percutaneous dilational tracheostomy can save operation time, and reduce intraoperative blood loss, so it can be widely used.

Objective To observe the changes of tear film stability,goblet cell and mucin 5AC expression in conjunctivochalasis patients,and explore the mechanism of conjunctivochalasis.Methods Conjunctivochalasis patients (30 cases) and single age-related cataract patients (15 cases) were collected as conjunctivochalasis group and normal control group.Eye symptom assessment (OSDI score),tear break-up time (BUT),Schirmer Ⅰ test,tear fern crystallization tests were performed for all the selected persons.Conjunctival rescent-shaped resections were made for all the conjunctivochalasis patients.Conjunctival tissue samples were stained by HE staining,AB staining,mucin 5AC immunohistochemical staining from the conjunctivochalasis group and norral control group respectively,and then statistical analysis was made.Results The OSDI score in the conjunctivochalasis group (37.80 ± 8.94) was significantly higher than that in the normal control group (11.40 ±4.08) (P ＜0.01).BUT in the conjunctivochalasis group (6.70 ± 2.76) s was significantly lower than that in the normal control group (13.67 ± 3.48) s (P ＜ 0.01).Schirmer Ⅰ test in the conjunctivochalasis group (6.23 ± 3.13) mum was significantly lower than the normal control group (13.40 ± 3.74)mm (P ＜ 0.01).Tear ferbing crystallization of the conjunctivochalasis group was decreased significantly compared with the normal control group (x2 =14.309,P =0.003).Light microscopic showed that conjunctival thickness was thinned,collagen fibers were less,elastic fiber was reduced,the lamina propria and interstitial were congestion and edema,the number of goblet cells was significantly reduced,and the positive staining of mucin 5AC staining was significantly lower in the conjunctivochalasis group than in normal control group (x2 =9.499,P =0.023).Conclusion For patients with conjunctivochalasis,the tear film function is affected,goblet cells are decreased,tear fern crystallization is decreased,mucin 5AC content is decreased,which finally leads the excessive conjunctival relaxation and abnormal ocular surface and tear.

Objective To investigate the effects of multidrug resistance-associated protein 4 (MRP4) on the cytoskeleton and cellular permeability of rat pulmonary microvascular endothelial cells (PMVECs) induced by lipopolysaccharide (LPS).Methods PMVECs were cultured for 3 to 6 generations were randomly divided into 4 groups:control group,LPS group,Ad-shMRP4 group (adenoviral expression of a short-hairpin RNA directed against MRP4),Ad-shRNA group.The infection rate of cells was detected by fluorescence microscope observation.The level of MRP4 was assayed by Western botting.Monolayer permeability was determined by the Transwell assay.The morphological characteristic and distribution of Factin was measured by laser confocal fluorescence microscope.Results Compared with control group,the expression of MRP4 protein was up-regulated (P ＜ 0.05) and the significant increase in the permeability of endothelial cells (2 h,6 h,12 h and 24 h respectively:0.28 ±0.02 vs.0.41 ±0.04,0.32 ±0.02,0.30 ±0.01 vs.0.53±0.04,0.39±0.03,0.33 ±0.04 vs.1.12±0.17,0.70 ±0.07,0.32±0.03 vs.0.79 ± 0.02,0.57 ± 0.05,P ＜ 0.05),the F-actin was remodeled,and the stress fibers were formed in LPS group and Ad-shMRP4 group.However,compared with LPS group,the expression of MRP4 protein was down-regulated (P ＜ 0.05) and the markedly decrease in the permeability of endothelial ceils (2 h,6 h,12 h and 24 h respectively:0.41 ± 0.04 vs.0.32 ± 0.02,0.53 ± 0.04 vs.0.39 ± 0.03,1.12 ± 0.17 vs.0.70 ± 0.07,0.79 ± 0.02 vs.0.57 ± 0.05,P ＜ 0.05) was found,and the remodeling of F-actin,and the formation of stress fibers were observed in Ad-shMRP4 group.Conclusions Silencing of MRP4 gene can effectively attenuates LPS-induced increase in the endothelial cell permeability and the destruction of cytoskeleton,thus playing an important role in the protection of endothelial cell barrier.

Objective To investigate the effect of estrogen-related receptor alpha(ERRα)on lipopolysaccharide-induced inflammatory response in rat pulmonary microvascular endothelial cells (PMVECs) and its mechanism.Methods PMVECs were cultured in vitro.When the cells were in the logarithmic growth phase,the cell were ransfected with lentivirus,and a stable low-expression ERRα cell line was constructed.The cells were divided into four groups:Ctr group (normal control group),Ctr+LPS group (normal celI+LPS treatment group),shERRα1 (shERRα1 gene knockdown group),and shERRα1+LPS group (shERRα1 gene knockdown +LPS treatment group).After 20 μg/mL LPS stimulated cells in the control group and shERRal group for 6,12 and 24 h,cell counting kit-8 (cck-8) was used to detect the cell proliferation ability of each group,and enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of tumor necrosis factor alpha (TNF-α) and Interleukin-1β (IL-1β) in cell culture fluid.After 12 h LPS stimulation,the expression levels of ERRα and NF-κB related proteins (p-p65,p65,P-IKBα,IKBα) were measured by Western blot.Pairwise comparisons were performed with SNK-q test (two-tailed),and multiple-group comparisons were performed with one-way ANOVA.The non-parametric test of rank transformation was used when homogeneity of variance were not met.P value＜0.05 was considered significantly different.Results Compared with the control group,ERRα expression in the shERRα group was significantly decreased (0.09±0.01 vs 0.15±0.01).At 6,12 and 24 h after LPS stimulation,compared with the control group,the cell proliferation ability (％) of the shERRαl+LPS group was significantly reduced (99.68±4.53 vs 48.62±1.60) and the concentration of TNF-α (ng/mL) (15.76±3.38 vs 5 498.91±367.95) and IL-1β (ng/mL) (14.41±3.86 vs 6 014.92±277.33) in the cell culture supematant were significantly increased.The change was most obvious after 12 h stimulation.Meanwhile the expression of p-p65 (0.30±0.50 vs 1.05±0.07) and p-IKBα (0.27±0.04 vs 0.77±0.06) were increased significantly,while the expression of IKBα (0.96±0.07 vs 0.14±0.04) was decreased significantly in the shERRαl+LPS group (all P＜0.05).Conclusion ERRα gene attenuates LPS-induced inflammatory response in rat pulmonary microvascular endothelial cells by inhibiting NF-κB signaling pathway activation.

Objective To investigate influences of estrogen-related receptor α(ERRα) on pulmonary vascular endothelium of rats undergoing sepsis. Methods Male Sprague-Dawley (SD) rats were divided into four groups according to the random number table method (12 in each group): normal control group (NC group), sham operation group (Sham group), sepsis model caused by cecal ligation and puncture (CLP) group (CLP group), XCT790 intervention group (XCT790 group, given the XCT790 2.5 mg/kg via intraperitoneal injection 30 minutes before CLP). After 24 hours, rats were sacrificed and the organs were harvested. The pathological changes of lung tissue were observed using hematoxylin and eosin (HE) staining, and the ultrastructural changes of lung tissue were observed by double staining of uranium citrate with lead acetate, the degree of apoptosis of pulmonary capillary endothelial cells were observed by TdT-mediated dUTP nike end labeling stain (TUNEL), the permeability of lung vascular endothelial was detected by Evans blue (EB) staining, the levels of serum cytokines were detected by enzyme linked immunosorbent assay (ELISA), and white blood cell count in bronchial alveolar lavage fluid (BALF) was detected. Results Compared with NC group and Sham group, the CLP group and XCT790 group had severe pathological damage and increased lung tissue permeability, the levels of serum cytokines and white blood cell count in BALF were increased. Compared with CLP group, the pathological changes of lung tissue, the degree of ultrastructural damage of lung tissue, the degree of apoptosis of lung capillary endothelial cells in XCT790 group further intensified, the permeability of lung endothelial barrier further increased [the content of EB (μg/g): 116.00±15.46 vs. 60.19±19.79, P < 0.05], and the level of serum cytokines further increased [interleukin-1β(IL-1β, ng/L): 71.38±4.01 vs. 56.58±2.45, interleukin-6 (IL-6, ng/L): 741.62±88.94 vs. 534.22±72.70, tumor necrosis factor-α(TNF-α, ng/L):188.55±7.41 vs. 143.33±11.27, all P < 0.05], the white blood cell count in the BALF increased further (×104/L:193.79±27.46 vs. 99.34±36.41, P < 0.05). Conclusion ERRα can aggravate inflammation in sepsis rats, destroy lung tissue and increase pulmonary permeability.

Objective To investigate the effect of Berberine on insulin resistance and its mechanism in Otsuka Long-Evans Tokushima Fatty(OLETF) rats with metabolic syndrome(MS).Methods LETO(Long-evans Tokushima Otsuka)rats(the control group receiving standard normal diet,n=10)and diabetic OLETF rats(the MS group receiving high-fat diet for 24 weeks,n=30).Rats in the MS group were randomly divided into 3 subgroups(n=10,each subgroup).Each subgroup was gavaged with normal saline,high-dose Berberine(100 mg · kg-1 · d-1)and low-dose Berberine (50 mg · kg-1 · d-1) respectively,and the high-fat diet remained unchanged.After 6 weeks of berberine treatment,body weight,blood glucose and lipid metabolism parameters were determined.The oral glucose tolerance test(OGTT) and insulin tolerance test(ITT) were used to detect insulin resistance.Expression levels of the protein and mRNA of 78 kDa glucose-regulated protein (GRP7 8),Caspase-12 and CCAAT/enhancer-binding protein(C/EBP) homologous protein(CHOP) in skeletal muscles were detected by Western blot and RT-PCR.Results After Berberine treatment,the body weight,fasting plasma glucose,fasting insulin[(28.9 ± 2.0) mU/L,(31.5± 2.4) mU/L vs.(36.9 ± 4.7) mU/L],total cholesterol,triglycerides,and low-density lipoprotein cholesterol were decreased,while the high-density lipoprotein cholesterol(HDL-C) levels were increased in MS rats with high-dose berberine and low-dose berberine as compared with the control group (P ＜ 0.05) respectively.Berberine treatment could reduce the protein and mRNA expression levels of GRP78,Capase-12 and CHOP in the skeletal muscle of MS rats(P＜0.05).Conclusions Berberine may alleviate insulin resistance in rats with metabolic syndrome by reducing endoplasmic reticulum stress in skeletal muscle.

Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARS-CoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us.
Amino Acid Motifs ; Amino Acid Substitution ; Base Composition ; Codon ; genetics ; Computational Biology ; DNA Mutational Analysis ; Evolution, Molecular ; Gene Transfer, Horizontal ; Genetic Variation ; Genome, Viral ; Phylogeny ; SARS Virus ; genetics