Objective To develop the UHPLC-MS/MS method for the determination of amygdalin, paeoniflorin, ferulic acid, calycosin glucosidase, quercetin and formononetin in Buyang-Huanwu decoction. Methods Isocratic elution was carried out with mobile phase consisting of methanol- 2 mM ammonium formate. The separation was performed on Agilent ZORBAX SB-C18 maintained at 35 ℃. The flow rate was 200 μl/min, and the injection volume was 2 μl. The mass spectrometer was operated in the positive and negative ionization electrospray (ESI) mode using multiple monitoring (MRM) for analysis of six components. The mass spectrometric conditions were that ion source temperature 400 ℃, dry gas flow 500 L/h, atomization gas flow rate 75.8 Kpa, spray voltage 4000 V, dry gas temperature 400 ℃. Results The amygdalin, paeoniflorin, ferulic acid, calycosin glucosidase, quercetin and formononetin were all analyzed exactly, and the linear ranges were 0.5-32, 0.2-12.8, 0.1-6.4, 0.8-51.2, 0.4-25.6, 0.08-5.12 ng, respectively. The r were 0.9921, 0.9945, 0.9928, 0.9958, 0.9947, 0.9966, respectively. The recoveries of six analytes ranged from 99.21％ to 101.44％ and the relative standard deviations were all below 2.05％. Conclusions A sensitive, accuracy and suitable UHPLC-MS/MS method has been developed, and the method could be applied for the determination of amygdalin, paeoniflorin, ferulic acid, calycosin glucosidase, quercetin and formononetin in Buyang-Huanwu decoction.

Objective To assess the efficacy and safety of carotid ligation in the treatment of the carotid artery rupture(CAR). Methods A series of 30 patients who had CAR treated with carotid ligation were reviewed. There were 24 males and 6 females ranging in age from 32 to 76 years, with a mean of 53.9 years. The original sites of tumours were hypopharynx(n=11), larynx(n=5), thyroid (n= 6) and others(n =8). Of the 30 patients, 24 patients had received radiotherapy from 40 - 126 Gy and 10 patients underwent one or more surgical treatments. CAR in all cases occurred after surgical operation. CAR occurred in 5 -21 days after operation. Results By pressing the carotid and keeping breathe of the patients immediately after CAR, 25 patients were conscious, 2 patients in coma, and 3 patients died before carotid ligation. Seven cases were applied with carotid ligation, 3 cases with the combined ligation of carotid and brachiophatic arteries, and 17 cases with carotid ligation plus the repair by pectoralis major myocutaneous flap. The causes of CAR concluded fistula, wound dehiscence, wound infection and transferred flap necrosis. The omende hemorrhage occurred in 14 patients. Of 25 cases with the treatments of carotid ligation, 22 patients survived with no complication, 1 with brief muscle weakness and 2 with unconscious.Clinical follow-up period lasted more than 5 years at least in 6 patients. Conclusions CAR is the most dangerous complication in advanced carcinoma of the head and neck. The prompt hemostasis and carotid ligation are effective methods to rescue patients of CAR. It is important to keep patients conscious before carotid ligation surgery, with low rates of death and hemiplegia postoperatively.

BACKGROUNDClinical observations have shown that the complex fractionated atrial electrogram (CFAE) associates with ganglionated plexus activity in the cardiac autonomic nervous system. This study aimed to investigate the impact of CFAE ablation on vagal modulation to atria and vulnerability to develop atrial fibrillation (AF).

METHODSTen adult mongrel dogs were involved. Cervical sympathovagal trunks were decentralized and sympathetic effects were blocked. CFAE was color tagged on the atrial 3-dimensional image and ablated during AF induced by S1S2 programmed stimulation plus sympathovagal trunk stimulation. Atrial effective refractory period (ERP) and vulnerability window (VW) of AF were measured on baseline and at vagal stimulation at 4 atrium sites. Serial tissue sections from ablative and control specimens received hematoxylin and eosin staining for microscopic examination.

RESULTSMost CFAE areas were localized at the right superior pulmonary quadrant, distal coronary sinus (CS(d)) quadrant, and proximal coronary sinus (CS(p)) quadrant (21.74%, separately). Sinus rhythm cycle length (SCL) shortening did not decrease significantly after ablation at the sites, including right atrial appendage, left atrial appendage, CS(d), and CS(p) (P > 0.05). ERP shortening during vagal stimulation significantly decreased after ablation (P < 0.01); the VW to vagal stimulation significantly decreased after ablation (P < 0.05). The architecture of individual ganglia altered after ablation.

CONCLUSIONSCFAE has an autonomic basis in dogs. The decreased SCL and ERP shortening to vagal stimulation after CFAE ablation demonstrate that CFAE ablation attenuates vagal modulation to the atria, thereby suppressing AF mediated by enhanced vagal activity. CFAE ablation could suppress AF mediated by enhanced vagal activity.

Animals ; Atrial Fibrillation ; therapy ; Autonomic Nervous System ; Catheter Ablation ; methods ; Dogs ; Electrophysiologic Techniques, Cardiac ; methods ; Electrophysiology ; Female ; Male

BACKGROUNDValve replacement or repair is recommended in patients with tricuspid regurgitation when deterioration of their clinical status occurs as a consequence of right ventricular dysfunction. Percutaneous valve replacement was developed in recent years. To investigate the feasibility, effectiveness and long-term results of percutaneous tricuspid valve replacement, an experimental model with tricuspid regurgitation is needed. We developed a simple and reproducible percutaneous approach for the creation of tricuspid regurgitation in sheep.

METHODSA specially designed grasping forceps were used to grasp chordae tendineae or the tricuspid valve leaflets through a catheter. Transcatheter creation of tricuspid regurgitation was performed on 7 healthy sheep. These sheep were followed up shortly after the procedure and at 6th month post-procedure with echocardiography. Additionally, all the sheep were sacrificed for anatomic evaluation at 6th month after the procedure.

RESULTSCreation of tricuspid regurgitation was successfully accomplished in all sheep. Necropsy confirmed that damage was done to the tricuspid valve apparatus in all animals (tearing of the anterior leaflet of the tricuspid valve in five animals and posterior leaflet of the tricuspid valve in two animals). At the six-month follow-up, there was no significant increase in the right ventricle dimension and ejection fraction measured by echocardiography. Autopsy examinations demonstrated the tearing of tricuspid valve leaflets.

CONCLUSIONSThe creation of an animal model of tricuspid regurgitation via a percutaneous approach using forceps to sever one or more tricuspid leaflets is feasible and will allow investigation of devices designed replace the tricuspid valve via a percutaneous approach. Despite significant tricuspid regurgitation, the hemodynamics did not change during the follow-up period in this model.

Animals ; Catheterization ; Disease Models, Animal ; Echocardiography ; Female ; Heart Valve Prosthesis Implantation ; methods ; Male ; Sheep ; Tricuspid Valve Insufficiency ; therapy

Adolescent ; Adult ; Aged ; Child ; Female ; Humans ; Influenza A Virus, H1N1 Subtype ; Influenza, Human ; complications ; pathology ; physiopathology ; Liver ; pathology ; physiopathology ; Male ; Middle Aged ; Retrospective Studies ; Young Adult

BACKGROUNDPercutaneous aortic valve replacement is a promising strategy in the treatment of patients with aortic valve stenosis. And many kinds of valved stents have been implanted in selected patients worldwide. However, the clinical experience is still limited. We developed a W-model valved stent and evaluated the feasibility and safety of percutaneous implantation of the device in the native aortic valve position.

METHODSA self expanding nitinol stent with W-model, containing porcine pericardium valves in its proximal part, was implanted in six sheep by means of a 14 French catheter through the right common iliac artery under guidance of fluoroscopy. During stent deployment the original aortic valve was pushed against the aortic wall by the self expanding force of the stent while the new valve was expanded. These sheep were followed up shortly after procedure with supra-aortic angiogram and left ventriculography. Additionally, one sheep was sacrificed after the procedure for anatomic evaluation.

RESULTSIt was possible to replace the aortic valve in the beating heart in four sheep. The procedure failed in two sheep due to coronary orifice occlusion in one case and severe aortic valve regurgitation in the other case. One sheep was killed one hour after percutaneous aortic valve replacement for anatomic evaluation. There were no signs of damage of the aortic intima, or of obstruction of the coronary orifice.

CONCLUSIONSPercutaneous aortic valve replacement with a W-model valved stent in the beating heart is possible. Further studies are mandatory to assess safety and efficacy of this kind of valved stent in larger sample size and by longer follow-up period.

Animals ; Aortic Valve ; pathology ; surgery ; Aortic Valve Stenosis ; surgery ; Feasibility Studies ; Female ; Fluoroscopy ; Heart Valve Prosthesis Implantation ; instrumentation ; methods ; Male ; Sheep ; Stents ; adverse effects

OBJECTIVETo investigate the changing regular of specific cytokeratin (CK) markers expressing in human pseudoepitheliomatous hyperplasia (PEH), keloids (Ke) and hypertrophic scar (HS) lesion, and to explore the correlation between such changes and the different outcomes of wound repair.

METHODSHistopathology and immunohistochemistry (IHC) double staining methods were used in samples of human PEH, Ke, HS and NS to determine the distribution characteristics and changing regularity of CKs in epidermal tissues.

RESULTSNo CK8&18 and CK17 expressed in epidermis of NS group, while CK8&18(+) cells and CK17(+) cells were detected in epidermis of active-stage Ke, HS and PEH. The quantities of CK8&18(+) cells and CK17(+) cells ranked as follows: PEH > Ke > HS and HS > Ke > PEH (P < 0.05). CK19(+) cells and CK5&6(+) cells expressed similar changing trend, while reverse trend of CK10(+) cells was detected in epidermal cells, with local epidermal hyperplasia, cells morphological changes and sub-epidermal inflammatory reaction.

CONCLUSIONDifferent degree of de-differentiation and terminal differentiation imbalance are found in epidermal cells of active-stage PEH, Ke and HS, which hint the correlation between the abnormal proliferation and differentiation of epidermal cells and the different outcomes of wound repair.

Adolescent ; Adult ; Aged ; Cell Differentiation ; Cell Proliferation ; Child ; Child, Preschool ; Cicatrix ; metabolism ; pathology ; Epidermis ; metabolism ; pathology ; Epithelial Cells ; metabolism ; pathology ; Female ; Humans ; Hyperplasia ; metabolism ; pathology ; Infant ; Keratins ; metabolism ; Male ; Middle Aged ; Wound Healing ; Young Adult

OBJECTIVETo construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells.

METHODSThe designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method.

RESULTSThe proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation.

CONCLUSIONSThese new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.

Antigens, Surface ; analysis ; Base Sequence ; Biological Assay ; Cells, Cultured ; DNA ; analysis ; genetics ; Gene Expression Profiling ; methods ; Humans ; Lipopolysaccharide Receptors ; analysis ; Lymphocyte Antigen 96 ; Membrane Glycoproteins ; analysis ; Molecular Probe Techniques ; Molecular Sequence Data ; Monocytes ; metabolism ; RNA Probes ; analysis ; genetics ; Receptors, Cell Surface ; analysis ; Receptors, Immunologic ; analysis ; Ribonucleases ; Toll-Like Receptor 4 ; Toll-Like Receptors

OBJECTIVETo explore the efficacy on knee osteoarthritis (KOA) treated with different acupuncture methods at different stages.

METHODSOne hundred and eighty cases of KOA were divided into stagnation stage, fascia cramp stage and tendon lesion stage according to the condition of disease, 60 cases in each stage. Each stage was randomized into a staging treatment group and an electroacupuncture (EA) group, 30 cases in each one. In the staging treatment group, acupuncture at the tendon points of meridians and electric thermal needling method were adopted for the cases at the stagnation stage; the small needle-knife therapy and bleeding method were used for the cases at the fascia cramp stage; and the electric thermal therapy with thick silver needles was applied for the cases at the tendon lesion stage. In the EA group, EA was applied for the cases of all the three stages at Liangqiu (ST 34), Xuehai (SP 10), Yanglingquan (GB 34), etc.

RESULTSThe total effective rate was 96.1% (87/90) in the staging treatment group and was 91.1% (82/90) in the EA group, without significant difference in comparison (P > 0.05). The controlled and remarkably effective rate (88.9%, 80/90) in the staging treatment group was higher apparently than (62.2%, 56/90) in the EA group (P < 0.001). And in the EA group, with the disease progression and the further disease stages, the controlled and remarkably effective rate was reduced. After treatment, the scores of symptoms and signs were decreased in both groups (both P < 0.001), the improvement in the staging treatment group was superior to that in the EA group (P < 0.001).

CONCLUSIONThe different acupuncture methods at the three stages improve obviously the clinical effect and are highly targeted. The mechanism of the three stages on "meridian muscle region pathology" and the treatment based on the disease stages can be the effective approach to KOA.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Aged ; Disease Progression ; Female ; Humans ; Male ; Middle Aged ; Osteoarthritis, Knee ; pathology ; therapy

OBJECTIVETo investigate the role of TGF-beta(1)/MAPK signaling pathways in the expression of type I collagen and activity of MMP-2, 9 in human lung fibroblasts.

METHODSHuman lung fibroblasts cell line (HLF-02) was cultured and and then stimulated with 10 ng/ml TGF-beta(1) for different time; SB203580 or PD98059 was added into culture medium to block p38 or ERK kinase pathway before incubated with TGF-beta(1); the expression of type I collagen was detected by Western blotting and RT-PCR; zymogram analysis was used to analyze the activity of MMP-2 and MMP-9.

RESULTS(1) In the process of stimulation by TGF-beta(1), the type I collagen mRNA level of 24 h, 48 h and 72 h group was: 1.33 +/- 0.07, 2.46 +/- 0.09 and 2.39 +/- 0.08 respectively; and the type I collagen protein level of 24 h, 48 h and 72 h group was: 114.89 +/- 8.95, 208.16 +/- 6.75 and 211.46 +/- 8.05 respectively; and the activity of MMP-2 of 24 h, 48 h and 72 h group was: 190.33 +/- 5.86, 214.33 +/- 8.39 and 212.67 +/- 11.59 respectively. (2) SB203580 significantly inhibited the TGF-beta(1)-induced expression of type I collagen mRNA, protein and MMP-2 activity (inhibition ratio: 51%, 24% and 20%); (3) PD98059 also significantly attenuated the TGF-beta(1)-induced expression of type I collagen mRNA, protein and MMP-2 activity (inhibition ratio: 42%, 13% and 16%).

CONCLUSIONTGF-beta(1) is capable of inducing the expression of type I collagen mRNA and protein and up-regulating MMP-2 activity in HLF-02 cells. p38 and ERK kinase signaling pathways play important role in regulation and control for this process.

Blotting, Western ; Cell Line ; Collagen Type I ; biosynthesis ; genetics ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; physiology ; Fibroblasts ; drug effects ; metabolism ; Flavonoids ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Lung ; cytology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Pyridines ; pharmacology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; physiology ; Transforming Growth Factor beta1 ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; physiology