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OBJECTIVE@#To explore the mechanism of ursolic acid in treating sepsis using myeloid differentiation protein-2 (MD-2) as the research carrier.@*METHODS@#The affinity of ursolic acid and MD-2 was determined by biofilm interferometry technique, and the bonding mode between ursolic acid and MD-2 was tested with the aid of molecular docking technique. Raw 264.7 cells were cultured in RPMI 1640 medium and subcultured was conducted when the cell density reached 80%-90%. The second-generation cells were used for in the experiment. The effects of 8, 40 and 100 mg/L ursolic acid on cell viability were assessed by methyl thiazolyl tetrazolium (MTT) method. Cells were divided into blank group, lipopolysaccharide (LPS) group (LPS 100 μg/L) and ursolic acid group (100 μg/L LPS treatment after addition of 8, 40 or 100 mg/L ursolic acid). The effect of ursolic acid on the release of cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) were evaluated by enzyme-linked immunosorbent assay (ELISA). The influence of ursolic acid on the mRNA expressions of TNF-α, IL-6, IL-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). The implication of ursolic acid on the protein expressions of LPS-Toll-like receptor 4 (TLR4)/MD-2-nuclear factor-κB (NF-κB) pathway were tested by Western blotting.@*RESULTS@#Ursolic acid could bind to the hydrophobic cavity of MD-2 through hydrophobic bond with the amino acid residues of the protein. Therefore, ursolic acid showed high affinity with MD-2 [dissociation constant (KD) = 1.43×10-4]. The cell viability were decreased slightly, with the concentration of ursolic acid increasing, and the cell viability of 8, 40 and 100 mg/L ursolic acid were 96.01%, 94.32% and 92.12%, respectively, and there was no significant difference compared with the blank group (100%). Compared with the blank group, the cytokine level of the LPS group was significantly increased. The level of cytokines were significantly reduced by the treatment of 8, 40 and 100 mg/L ursolic acid, and the higher the concentration, the more obvious effect [compared between 100 mg/L ursolic acid group and LPS group: IL-1β (μmol/L): 38.018±0.675 vs. 111.324±1.262, IL-6 (μmol/L): 35.052±1.664 vs. 115.255±5.392, TNF-α (μmol/L): 39.078±2.741 vs. 119.035±4.269, NO (μmol/L): 40.885±2.372 vs. 123.405±1.291, all P < 0.01]. Compared with the blank group, the mRNA expressions of TNF-α, IL-6, IL-1β, iNOS and COX-2 in the LPS group were significantly increased, and the protein expressions of MD-2, myeloid differentiation factor 88 (MyD88), phosphorylation NF-κB p65 (p-NF-κB p65) and iNOS in the LPS-TLR4/MD-2-NF-κB pathway were significantly up-regulated. Compared with the LPS group, the mRNA expressions of TNF-α, IL-6, IL-1β, iNOS and COX-2 were significantly reduced by the treatment of 100 mg/L ursolic acid bound with MD-2 protein [TNF-α (2-ΔΔCt): 4.659±0.821 vs. 8.652±0.787, IL-6 (2-ΔΔCt): 4.296±0.802 vs. 11.132±1.615, IL-1β (2-ΔΔCt): 4.482±1.224 vs. 11.758±1.324, iNOS (2-ΔΔCt): 1.785±0.529 vs. 4.249±0.811, COX-2 (2-ΔΔCt): 5.591±1.586 vs. 16.953±1.651, all P < 0.01], and the proteins expressions of MD-2, MyD88, p-NF-κB p65 and iNOS in the LPS-TLR4/MD-2-NF-κB pathway were significantly down-regulated (MD-2/β-actin: 0.191±0.038 vs. 0.704±0.049, MyD88/β-actin: 0.470±0.042 vs. 0.875±0.058, p-NF-κB p65/β-actin: 0.178±0.012 vs. 0.571±0.012, iNOS/β-actin: 0.247±0.035 vs. 0.549±0.033, all P < 0.01). However, there was no difference in protein expression of NF-κB p65 among the three groups.@*CONCLUSIONS@#Ursolic acid inhibits the release and expression of cytokines and mediators and regulates LPS-TLR4/MD-2-NF-κB signaling pathway by blocking MD-2 protein, and thus plays an anti-sepsis role.
Subject(s)
Humans , Tumor Necrosis Factor-alpha , Actins , Cyclooxygenase 2 , Interleukin-6 , Lipopolysaccharides , Lymphocyte Antigen 96 , Molecular Docking Simulation , Myeloid Differentiation Factor 88 , NF-kappa B , Toll-Like Receptor 4 , Sepsis , Cytokines , Cell Differentiation , RNA, MessengerABSTRACT
Objective:To screen and identify the potential targets of carthamin against sepsis by studying the characteristics of carthamin.Methods:The pharmacological parameters and molecular characteristics of carthamin were analyzed with the aid of Traditional Chinese Medicine Systems Pharmacology (TCMSP). The targets of carthamin were screened by SwissTargetprediction (a website providing compound target prediction) and Drug Repositioning and Adverse drug Reaction via Chemical-Protein Interactome (DRAR-CPI). The anti-sepsis targets were selected from the three databases of Online Mendelian Inheritance in Man (OMIM), Comparative Toxicogenomics Database (CTD) and Therapeutic Targets Database (TTD). The targets of carthamin screened by the two websites and disease targets selected from the three databases were matched to screen the targets of carthamin against sepsis. The anti-sepsis potential targets of carthamin were identified by molecular docking software.Results:The oral bioavailability of carthamin was 41.15%, the drug-likeness was 0.24, and the rotational bond number was 1, which indicated that carthamin was well absorbed by oral administration and showed good drug formation. A total of 115 potential targets of carthamin were screened by SwissTargetprediction and DRAR-CPI; 149 disease targets were found from OMIM, CTD and TTD databases; 115 target proteins of carthamin screened by the two websites were matched with the disease targets , and 10 target proteins were found to be both molecular targets and disease targets. The 10 target proteins were coagulation factor Ⅸ (F9), adenosine A1 receptor (ADORA1), nitric oxide synthase 2 (NOS2), mitogen activity protein kinase 1 (MAPK1), cathepsin G (CTSG), neutrophil elastase (ELANE), protein C (PROC), lipocalin 2 (LCN2), glucose-6-phosphate dehydrogenase (G6PD) and prostaglandin endoperoxidase 2 (PTGS2). Molecular docking software analysis showed that carthamin had the ability to bind to the above 10 target proteins, which were potential targets of carthamin against sepsis. Carthamin could interact with the key amino acid residues of the targeted proteins, so as to play the corresponding efficacy.Conclusion:Carthamin combines with the targets could reduce the tissues and organs damage of sepsis by regulating CTSG, ELANE and LCN2, reduce inflammatory response of sepsis by regulating ADORA1, PTGS2, NOS2, MAPK1 and mediating PROC and F9 to inhibit clotting, and improve oxidative stress, reduce the incidence of sepsis by regulating G6PD, finally, prevented and treated sepsis.
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Objective To determine the spectrum of mutations responsible for Phenylalanine hydroxylase (PAH) deficiency on phenylketonuria (PKU) patients of Han Chinese people in the Huaihai region of central China.Methods One hundred and one patients diagnosed with PKU were referred to Xuzhou Maternity and Child Health Care Hospital for genetic counseling/analysis from January 2003 to December 2013.Thirteen exons of PAH gene mutations,as well as their flanking introns,were identified in 202 of chromosomes using polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) and sequencing.Results (1) The spectrum was composed of 24 different mutation types,which had been submitted to the National Center for Biotechnology Information(NCBI) dbSNP databases under accession number SS#2137543837_SS#C2137543860.(2)The most commonly affected region was exon 7 and its flanking introns.The most prevalent mutations were c.728G ＞ A (p.R243Q),followed by c.721C ＞ T (p.R241C),c.1155G ＞ C(p.L385L),c.1068C ＞ A(p.Y356X),c.-71A ＞ C(-71A ＞ C) and c.60 + 62C ＞ T (IVS1 +62C ＞T),accounting for 18.317％,8.416％,4.950％,3.960％,3.465％ and 2.970％ of the mutant chromosomes,respectively.(3)Two novel mutations were identified in PAH gene in PKU patients of Han Chinese people:c.60+62C＞T(IVS1 +62C ＞T) and c.782G ＞T(p.R261L).Conclusions The vast majority of PAH mutations identified corresponded to those observed for the PKU populations in the other regions in China,whereas a few are considerably different from others.The mutational spectrum of PAH gene found in patients with PKU in the Huaihai region exhibit regional association.
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Objective To analyze the correlation between the tubular gastric width and the anti gastroesophageal reflux after esophageal cancer operation,and to provide reference for the choice of surgical methods in treatment of esophageal cancer.Methods Selected 60 patients who received radical surgery for esophageal carcinoma combined with gastric tube reconstruction surgery in our hospital from January 2015 to October 2015,and divided them into two groups according to the way of stomach tube anastomosis (cervical anastomosis,thoracic anastomosis) and different width of gastric tube (greater than or equal to or less than 3 cm).Namely:cervical anastomosis + greater than or equal to 3 cm group(14 cases),cervical anastomosis + less than 3 cm group(15 cases),thoracic anastomosis + greater than or equal to 3 cm group(15 cases) and thoracic anastomosis + less than 3 cm group(16 cases).All patients recieved esophageal pH monitoring for 3 days continuously from the 11 th day after operation.The monitoring indicators include:number of reflux,accumulation time of pH ＜ 4,whether there were clinical symptoms (heartburn,chest pain,pharyngeal foreign body sensation,cough,asthma,etc.) after surgery,and the frequency and time of these clinical symptoms appeared.All the patients were given endoscopic examination at the 14th days postoperatively.Observed the esophageal mucosa of patients and conducted histopathological grading of gastric mucosal inflammation.And then made a correlation analysis of gastric tube width and esophageal mucosal inflammation grade among all the patients with reflux symptoms.Results The cumulative time and number of reflux,incidence rate of clinical symptoms,and pH values less than 4 were significantly different(P ＜ 0.05).The cumulative time and number of reflux,incidence rate of clinical symptoms,and pH values less than 4 in the cervical anastomosis + less than 3 cm group were significantly lower than that in the other 3 groups(P ＜ 0.05),with statistical significance between different groups of endoscopic esophageal mucosa inflammation grade difference (P ＜ 0.05).Esophageal mucosal inflammation grading in patients of the cervical anastomosis + less than 3 cm group was the lightest.It showed a linear correlation between the gastric tube width and esophageal mucosal inflammation grading in patients with reflux symptoms.Conclusion Postoperative gastroesophageal reflux is closely related to stomach esophagus width after resection of esophageal carcinoma with tubular stomach reconstruction of stomach esophagus,because it is unable to control gastric tube width to the appropriate range.And it should be strengthened in patients with reflux related indicators for monitoring,so as to take measures to prevent gastroesophageal reflux as soon as possible to improve the prognosis of patients with quality.
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Objective To discuss the relationships between regular pattern changes of plasma fibrinogen (Fib),D-dimer and fibrinogen degradation products (FDP) levels and the recent dissolution of thrombus in patients with pulmonary embolism (PE) in 14 days after treatment.Methods A prospective study was conducted.PE patients admitted to Departments of Respiratory Disease in 4 hospitals from January 2015 to March 2016 were enrolled and all of them were treated with thrombolysis and/or anticoagulation after admission.The computed tomographic pulmonary angiography (CTPA) was examined pre-treatment and 14 days post-treatment in PE patients.The pulmonary artery obstruction index (PAOI) was assessed according to the Mastora scoring method to estimate the thrombus load.The plasma Fib,D-dimer and FDP levels were measured before and on 1,2,3,5,7 and 14 days after treatment,and the relationships between the change regularities of these parameters and PAOI were also analyzed.Results A total of 42 PE patients were enrolled.The curve change of coagulation-fibrinolytic system parameters in 14 days after treatment showed that the Fib level was raised to its peak on the 3rd day after treatment and then decreased (g/L:4.24 ± 1.45 vs.3.83 ± 1.56),representing that its curve change was in accordance with the quadratic model (P =0.095).After treatment,the D-dimer and FDP levels were kept declining,they were reached the valley on 14th day [D-dimer (mg/L):1.58 ± 1.38vs.8.84 ± 6.35,FDP (mg/L):4.23 ± 3.63 vs.23.41 ± 16.54],and their curve changes were in accordance with the cubic model (F was 32.190 and 34.326,respectively both P =0.000).The PAOI variation before and 14 days after treatment [(18.77 ± 14.22)％] was not correlated with Fib variation [(1.20 ± 0.93) g/L,r =-0.194,P =0.219],but was positively correlated with D-dimer variation [(7.29 ± 7.10) mg/L] and FDP variation [(19.29 ± 18.67) mg/L,r was 0.556 and 0.460,respectively;P was 0.020 and 0.002,respectively].Conclusions The D-dimer and FDP levels are kept falling in PE patients after treatment,suggesting that the pulmonary artery embolus is being dissolved.
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ObjectiveTo investigate the clinical effect of nape acupuncture on post-stroke dysphagia by a meta-analysis.Method Domestic literature on clinical randomized controlled trials of nape acupuncture treatment of post-stroke dysphagia published from Jun. 2004 to Jun. 2014 were obtained by a computer search ofChina National Knowledge Internet(CNKI), Wanfang Data medical information system (WF), VIP information resource system (VIP) and Chinese Biomedical Literature Database (CBM) combined with a manual search of Jinan University library journal database. Literature inclusion and exclusion criteria were established to extract data and the qualities of the included studies were assessed using a Jadad rating scale. A meta-analysis was made using the software ReviewManager 5.2.ResultA total of 17 articles were included with 1158 patients. The results of meta-analysis showed that there were statistically significant differences in the total efficacy rate [OR=3.99, 95%CI(2.83, 5.63),P<0.00001] and the cure rate[OR=2.67, 95%CI(2.03, 3.53),P<0.00001] between the nape acupuncture and control groups.ConclusionNape acupuncture has clinically a good therapeutic effect on post-stroke dysphagia. Multi-center, large-sample and high-quality studies are still needed for validation because the qualities of the included articles are lower, which is one of the factors influencing the assessment.