This study is to develop a sensitive method by using reversed-phase high performance liquid chromatography coupled with UV detector (HPLC-UV) to simultaneously determine four bioactive compounds, iriflophenone 3-C-beta-D-glucoside, iriflophenone 3,5-C-beta-D-diglucoside, mangiferin, and iriflophenone 2-O-alpha-L-rhamnoside in the leaves of Aquilaria sinensis. An Agilent Zorbax SB-C, column (4, 6 mm x 250 mm, 5 microm) was used, and the gradient elution was performed with mobile phase of 0.1% aqueous phosphoric acid and acetonitrile at a flow rate of 1 mL x min(-1). The detection wavelength was 280 nm, and the column temperature was 25 degrees C. The four marker compounds were well separated with good linearity (R2 > 0.9990), precision, stability and repeatabili y. The-recovery rates were in the range of 98.80%-101.39%. For 15 branch of the leaves, the contents of iriflophenone 3-C-beta-D-gluoside, iriflophenone 3,5-C-beta-D-diglucoside, mangiferin, and iriflophenone 2-O-alpha-L-rhamnoside were between 0.41-14.48, 0.72-3.85, 4.30-29.07, 0.24-5.06 mg, respectivley. This method is precise, accurate and reliable, which provides an efficient way for the quality control of the leaves of A. sinensis. This will promote the comprehensive usage of this plant.
Benzophenones ; analysis ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; Plant Leaves ; chemistry ; Spectrophotometry, Ultraviolet ; methods ; Thymelaeaceae ; chemistry ; Xanthones ; analysis

The objective of this study was to screen out the effective shRNA which can inhibit the gene expression of tumour necrosis factor-alpha (TNF-alpha), to construct the recombinant plasmid and to determine its sequence so as to provide the new approach for searching gene therapy of TNF-alpha related diseases. The primary macrophages were added into 15% DMEM, then cells were adjusted as 2 x 10(7) cells/L and were inoculated in 6-well plate with 3 ml/well, and were cultured at 37 degrees C in a fully humidified atmosphere with 5% CO(2). Cells were stimulated with lipopolysaccharide (LPS) and the concentration of TNF-alpha in the supernatant at different time points was determined by enzyme-linked immunosorbent assay (ELISA). The 5 synthesized DNA sequences which can be transcripted into shRNA were transfected into cells with lipofectamine 2000, then the cells were stimulated with LPS for 24 hours. The concentration of TNF-alpha in the supernatant and the expression of TNF-alpha mRNA were determined by ELISA and reverse transcription polymerase chain reaction (RT-PCR) respectively. The most effective shRNA was inserted into plasmid, and the recombinant plasmid was identified by sequence analysis. The results showed that the concentration of TNF-alpha in the supernatant reached peak after the stimulation with LPS for 24 hours. In the RNA interference group, the shRNA 1 was the most effective one, which could inhibit the expression of TNF-alpha by 59.46% and the expression of TNF-alpha mRNA by 61.2%. The recombinant plasmid was cloned and the sequence of interest was obtained. In conclusion, the most effective shRNA targeting TNF-alpha was successfully screened out and the recombinant plasmid was constructed. The recombinant plasmid may be helpful to search new gene therapy for TNF-alpha related disease.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Macrophages ; cytology ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Recombination, Genetic ; Transfection ; Tumor Necrosis Factor-alpha ; genetics

Objective Cervical radiculopathy treatment experts' consensus to establish radiculopathy type by using the modified Delphi method. Methods Use document retrieval method to review information and articles about the treatment guidelines and articles of cervical radiculopathy including domestic and international areas, established a protocol about clini?cal consensus of the treatments for cervical radiculopathy. This protocol included 23 questions (the effective proportion of non?operating therapy, neck immobilization, physiotherapy, pharmacologic treatment, surgical indications, contraindications, anteri?or surgical decompression, anterior surgical implants). We performed a modified Delphi survey in which current professional opinions from experienced experts, representing from almost all of the Chinese provinces, were gathered. And then we modi?fied the protocol according to those professional opinions. Three rounds were performed and finally we established consensus. Consensus was achieved with ≥70% agreement. Results The panel included 30 experienced experts. The recycling question?naire's quantity of three rounds were 30(100%), 24(80%) and 16(53.3%) respectively. After three expert assessments, there were 18 questions which achieved with≥70%agreement and these questions accounted for 64.3%(18/28) of all the questions. Consen?sus of the treatments for cervical radiculopathy was reached on 7 aspects, including:the effective proportion of non?operating thera?py (1 question), neck immobilization (1 question), physiotherapy (1 question), pharmacologic treatment (5 questions), surgical indi?cations (3 questions), contraindications (4 questions), surgery (3 questions). Conclusion This modified Delphi study had reached a consensus concerning several treatment issues on cervical radiculopathy which had strong representativeness of experts and good convergence of opinions. In the absence of high?level evidence, at present, these experts' opinion findings will guide health care providers to define appropriate treatment in their regions. Areas with no consensus provide excellent insight for future research.

The aim of this study was to explore the effect of hyperbaric oxygen (HBO) preconditioning on the rejection of skin allograft in mice and its molecular mechanism. BALB/c donor mice and C57BL/6 recipients received hyperbaric oxygen preconditioning once a day for 7 days. After skin transplantation, the recipients were treated with cyclosporine A (CsA) intraperitoneally. Immunofluorescent staining technique and flow cytometry were used to observe the influence HBO on percentage of spleen lymphocytes CD3+, CD4+, CD8+ and cell adhesion molecule LFA-1 (CD11a/CD18). The results showed that as compared with control, the numbers of CD3+, CD4+, CD8+, CD4+CD11a+, CD4+ CD18+, CD8+CD11a+, CD8+CD18+ lymphocytes of spleen decreased in HBO preconditioning groups and CsA group, and decreased markedly in HBO preconditioning combined with CsA group (p<0.05); the general state of recipient mice in HBO preconditioning combined with CsA group was better than that of recipient mice received HBO preconditioning or CsA only. It is concluded that the method of HBO preconditioning combined with traditional immunosuppressive agent CsA has remarkable advantage in inhibiting the rejection of skin graft. Its molecular protective mechanism is correlated with the expression of adhesive molecules on T cell subsets.
Animals ; Cell Adhesion ; Cell Adhesion Molecules ; pharmacology ; Cyclosporine ; pharmacology ; Female ; Graft Rejection ; prevention & control ; Graft Survival ; Hyperbaric Oxygenation ; Lymphocyte Count ; Lymphocytes ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Skin Transplantation ; immunology ; Spleen ; cytology ; Transplantation Conditioning ; methods

The objective of this study was to investigate the function and mechanism of hyperbaric oxygen (HBO) in antagonizing acute graft-versus-host disease (aGVHD) and improving the rate of survival. The lethally irradiated C57BL/6 recipients were injected with bone marrow and lymphocyte of spleen from BALB/c donors and were treated with HBO, cyclosporine A (CsA) and methotrexate (MTX). T lymphocytes and subsets, adhesion molecules and cytokines were detected by flow cytometry, ELISA and RT-PCR respectively. The results showed that the survival rate in HBO group was much higher than that in allogenetic bone marrow transplantation (allo-BMT) group and CsA + MTX group; the numbers of CD3(+), CD4(+), CD8(+), CD4(+)CD11a(+), CD4(+)CD18(+), CD8(+)CD11a(+), CD8(+)CD18(+) lymphocytes in spleen were decreased markedly by HBO and CsA + MTX (p < 0.05); the levels of IL-2 and TNFalpha mRNA and their serum concentrations in HBO group were much lower than those in allo-BMT group but were higher than those in CsA + MTX group; the levels of IL-4 and IL-10 mRNA in HBO group were much higher than those in allo-BMT group and CsA + MTX group. It is concluded that HBO has more remarkable advantage in improving the rate of survival than CsA + MTX, its mechanism of anti-aGVHD is tightly correlated with the transform of T cell and its subsets and the expression of adhesion molecules and cytokines.
Acute Disease ; Animals ; Bone Marrow Transplantation ; adverse effects ; Cytokines ; biosynthesis ; Female ; Graft vs Host Disease ; etiology ; therapy ; Hyperbaric Oxygenation ; Lymphocyte Transfusion ; adverse effects ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes ; immunology ; Whole-Body Irradiation

Objective Previous studies indicated that activation of Toll-like receptor4 (TLR4) was involved in the progression and instability of atherosclerotic plaque.Anti-inflammatory effects were shown in statins. However,the mechanisms underlying these effects have not been well explored.We test the hypothesis that a por- tion of these anti-inflammatory effects are mediated by regulation of TLR4 expression.Methods One hundred twenty-one subjects (22 normal persons,17 patients with stable angina and 82 patients with ACS) were recruited. 41 patients with ACS were randomized to atorvastatin 10 mg/d or atorvastatin 40 mg/d on top of routine anti-anginal treatment.Serum level of hsCRP,blood lipids,TLR4 expression on CD_(14)~+ monocytes were measuered before and after one month treatment.TLR4 expression on CD_(14)~+ monocytes were quantified via flow-cytometry.Results hsCRP and TLR4 expression on CD_(14)~+ monocytes in patients with ACS were higher than patients with stable angina and normal persons(hsCRP,ACS:11.1?14.3 vs stable angina:2.5?2.7 mg/L vs normal:2.3?4.2 mg/L,P

OBJECTIVETo investigate the protective effect of suppressive oligodeoxynucleotides (Sup ODN) on interferon-γ (IFN-γ) and signal transducers and activators of transcription (pSTAT4) expression of Silica-induced pulmonary inflammation in Mice.

METHODSSixty Balb/c mice were randomly divided into 4 groups, normal control group, silicious group, suppressive oligodeoxynucleotides (Sup ODN) group, control oligodeoxynucleotides (Con ODN) group. Except the normal control group injected normal saline, the rest groups were induced by the intratracheal instillation of 0.1 ml (5 g/L) of sterilized silica suspension. Sup ODN group and Con ODN group were treated by i.p. injection of 0.3 ml (1mg/mL) of suppressive or control ODN 3 h before silica administration. After 7 days, the animals were killed and levels of IFN-γ were detected by ELISA. The pathologic changes in lung tissues of mice were observed with HE staining. Expressions of IFN-γ and pSTAT4 in lung tissue were detected with immunohistochemistry and quantified by Image-Pro Plus 7.0.

RESULTSHE staining showed that the lung tissue of silicious group were damaged seriously than Sup ODN group. Compared with the normal control group (serum: (280.1±41.3) pg/ml, lung tissue: (0.249±0.373), IFN-γ increased in silicious group (serum: (886.3±81.7) pg/ml, lung tissue: (0.270±0.300) (P < 0.05). Compared with the normal control group and Con ODN group [(894.5±91.6) pg/ml], IFN-γ in the serum of Sup ODN group decreased significantly (P < 0.01). Compared with the silicious group , IFN-γ in lung tissue decreased in Sup ODN group (0.241±0.250) (P < 0.05). Compared with the normal control group (0.279±0.353), pSTAT4 in lung tissue increased significantly in silicious group (0.313±0.231) (P < 0.01). Compared with the silicious group, pSTAT4 in lung tissue decreased significantly in Sup ODN group (0.269±0.523) (P < 0.01).

CONCLUSIONSup ODN attained protective effect on Silica treated mice by suppressing expression of IFN-γ and pSTAT4.

Animals ; Female ; Inflammation ; metabolism ; Interferon-gamma ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Oligodeoxyribonucleotides ; pharmacology ; Phosphorylation ; STAT4 Transcription Factor ; metabolism ; Silicon Dioxide ; toxicity

OBJECTIVETo observe different effects of moxibustion on extracellular potassium ion in acupoint under physiological and pathological status and provide experimental evidence for exploring action mechanism of moxibustion on acupoint local.

METHODSForty female SD rats were randomly divided into a blank group, a blank-moxibustion group, a model group and a model-moxibustion group, 10 cases in each one. The complete Freund's adjuvant(CFA) was adopted to establish model of adjuvant arthritis (AA) in the model group and model-moxibustion group. No treatment was given in the blank group and model group while moxibustion was applied at "Zusan-li" (ST 36) for 30 min in the blank-moxibustion group and model-moxibustion group. The tissue fluid in "Zusanli" (ST 36) was collected with microdialysis and real-time analyzed by electrolytic analyzer. The change of concentration of potassium ion in "Zusanli" (ST 36) was observed.

RESULTS(1) Under physiological status, the concentration of extracellular potassium ion in the blank group was not changed within 150 min (P > 0.05); before the moxibustion, the concentration of extracellular potassium ion in the blank-moxibustion group was (1.21 +/- 0.31) mmol/L, and after treatment it was gradually increased and reached its peak at (2.38 +/- 0.42) mmol/L after 60 min (P < 0.05), then it was reduced. 150 min after the treatment, concentration of potassium ion was slightly higher than that before moxibustion as well as that in the blank group. The concentration in the blank-moxibustion group at 60 min was statistically significant compared with that in the blank group (P < 0.05). (2) Under pathological status, the concentration of extracellular potassium ion in the model group was not changed within 150 min, differences of which at each time point was not statistically significant (all P > 0.05). Before the moxibustion, the concentration of extracellular potassium ion was (1.09 +/- 0.12) mmol/L in the model-moxibustion group, and it was immediately increased to (1.96 +/- 0.18) mmol/L after moxibustion. 60 min and 90 min after the moxibustion, it still maintained a higher level, which was (1.87 +/- 0.29) mmol/L and (1.59 +/- 0.16) mmol/L respectively (both P < 0.05). The differences of each time point after moxibustion in the model-moxibustion group were statistically significant compared with those in the model group (all P < 0.05).

CONCLUSIONThe moxibustion could increase the concentration of potassium ion in rat's acupoint local under physiological status but time of effect is short; with moxibustion at "Zusanli" (ST 36) under pathological status, the concentration of local potassium ion is obviously increased and maintains for a long time.

Acupuncture Points ; Animals ; Arthritis, Experimental ; metabolism ; therapy ; Disease Models, Animal ; Female ; Humans ; Moxibustion ; Potassium ; metabolism ; Rats ; Rats, Sprague-Dawley

With the introduction of assisted reproductive technology (ART), sperm assessment has developed progressively, from conventional semen routine tests to novel cellular and molecular measures. Sperm DNA damage is a new marker of male fertility, whose genetic mechanism involves abnormal package and segregation of chromatin, oxidative stress, abnormal cell apoptosis, etc. Sperm chromatin structure assay (SCSA) is one of the common techniques to measure sperm DNA damage. Sperm DNA damage might be associated with the pregnancy outcome of ART, recurrent spontaneous abortion and potential genetic risk of ICSI offspring. Some treatment strategies might reduce the percentage of sperm DNA damage and increase the success rate of ART, including oral administration of antioxygen drugs, ICSI with testis sperm, sperm freezing and preservation, removing of etiological factors, traditional Chinese medicine, and so on. This review focuses on the mechanism and detection of sperm DNA damage, its association with reproductive outcomes, and relevant treatment strategies in assisted reproductive technology.
DNA Damage ; Humans ; Infertility, Male ; therapy ; Male ; Sperm Injections, Intracytoplasmic ; adverse effects ; methods ; Spermatozoa ; metabolism

OBJECTIVETo explore the prediction of the site for microsurgical vasoepididymostomy (VE) in the treatment of epididymal obstructive azoospermia (OA).

METHODSThis study involved 56 infertile men with confirmed OA whose obstruction was suspected to be in the epididymis. Based on their medical history and results of preoperative physical examination and ultrasonography, we predicted the sites for VE. We performed surgical scrotal exploration for the status of epididymal obstruction, conducted palpation and microscopic observation for the epididymal tubules to be anastomosed, and finally decided on the sites for VE by making sure of the presence of motile sperm in the epididymal fluid of the patients. After surgery, we followed up the patients for the rate of pregnancy.

RESULTSAll the patients received bilateral scrotal ultrasonography and surgical scrotal exploration, totaling 112 procedures, including 98 VE procedures. The accuracy rate of the predicted sites for VE was 80.5% (153/190) by medical history and physical examination, 80.3% (90/112) based on the results of ultrasonography, and 87.4% (90/103) according to the first selected epididymal tubules. Of the 28 patients followed up for more than 12 months, motile sperm were found in 19 (67.9% ) at 2 to 12 months and spontaneous pregnancies were achieved in 10 (35.7%), all with the anastomotic sites in the corpus or cauda.

CONCLUSIONMedical history and physical examination contribute to the selection of anastomotic sites and non-invasive scrotal ultrasonography is effective and practical for positioning epididymal obstruction. The epididymal tubules with motile sperm for anastomosis could be easily obtained from the most dilated ones in indurated epididymides.

Azoospermia ; surgery ; Body Fluids ; Epididymis ; diagnostic imaging ; surgery ; Female ; Humans ; Male ; Microsurgery ; methods ; Pregnancy ; Pregnancy Rate ; Scrotum ; diagnostic imaging ; Ultrasonography ; Vas Deferens ; diagnostic imaging ; surgery