OBJECTIVE: Using the method of lactate dehydrogenase (LDH) assay, to observe the activities of rat cerebral microvascular endothelial cells (CMECs) intervened by Tongluo Jiunao Injection (TLJNI), a traditional Chinese compound drug removing toxin to dredge brain collaterals, and then further study the effects of different kinds of conditioned mediums (CMECs-CM) of cerebral microvascular endothelial cells on ischemia and ischemia/reperfusion cerebral cortex cells, and to probe into the drug pharmacological mechanisms of CMECs in modulating the neurons. METHODS: Three kinds of CMECs (normal, ischemic and ischemic/reperfusional) were all treated by TLJNI previously, and then the three pairs of CMECs-CM without serum were collected respectively for LDH assay. Rat cerebral cortex neurons were also primarily cultured and then divided into similar three groups (normal, ischemic and ischemic/reperfusional). The neuron responses caused by CMECs-CM at different concentrations were observed by using LDH transudation rate assay. RESULTS: The LDH release values of ischemic and ischemic/reperfusional CMECs with TLJNI treatment were obviously reduced (P<0.01) compared with the same kinds of CMECs untreated. For ischemic neurons, both conditioned medium of ischemic CMECs (Is-CM) and conditioned medium of ischemic CMECs with drug treatment (IsT-CM) in high concentration of 100% increased the LDH transudation rate (P<0.01), while in low concentration of 10%, IsT-CM reduced the transudation rate (P<0.05). For ischemia/reperfusion neurons, all kinds of CMECs-CM reduced the transudation rate respectively (P<0.05 or P<0.01). As far as each group concentration was concerned, 10% or 50% showed relatively stronger effects, and both conditioned medium of normal CMECs (N-CM) group and conditioned medium of ischemic/reperfusional CMECs (Rp-CM) group had statistical significance (P<0.05 or P<0.01). For normal neurons, all kinds of CMECs-CM increased the transudation rate respectively (P<0.05 or P<0.01). As far as each group concentration was concerned, only conditioned medium of normal CMECs (N-CM) had statistical significance (P<0.05 or P<0.01). CONCLUSION: The study shows that TLJNI is capable of preventing the damage of CMECs from both ischemia and ischemia/reperfusion states. Chinese drug can restrain the brain ischemia and ischemia/reperfusion damage by the media that CMECs modulate the neurons, demonstrating the pharmacological mechanisms of TLJNI. This work also indicates that there exist some active substances against ischemia/reperfusion injury secreted from CMECs-CM with TLJNI treatment.

OBJECTIVETo investigate the effect of nattokinase on intimal hyperplasia in rabbit abdominal artery after balloon injury and explore a novel strategy for the preventing restenosis after percutaneous transluminal angioplasty.

METHODSFifty-six New Zealand rabbits were randomly divided into 7 groups, namely the solvent control group, model group, natto extract lavage group, refined nattokinse lavage group, intravenous refined nattokinse injection group, clopidogrel group and clopidogrel-aspirin group. Balloon injury was induced by inserting the catheter through the femoral artery into the thoracic aorta of the rabbits. The platelet counts were notad and platelet aggregation was observed, and the abdominal artery was taken for pathological analysis. The expressions of MMP-2 and -9 in the abdominal artery were detected immunohistochemically.

RESULTSThere was no significant difference in the platelet counts, platelet aggregation rate or MMP-2 and -9 expression between the model group and the nattokinse-treated groups (P>0.05). The stenosis index in each nattokinse-treated group was significantly greater and the neointimal proliferation index smaller than that of the model group (P<0.01 or 0.05).

CONCLUSIONNattokinse can inhibit restenosis of rabbit abdominal artery after percutaneous transluminal angioplasty, which is independent of its actions on the platelet or MMP-2 and -9 expressions.

Abdomen ; blood supply ; Angioplasty, Balloon ; adverse effects ; methods ; Animals ; Arteries ; drug effects ; metabolism ; pathology ; Constriction, Pathologic ; blood ; etiology ; prevention & control ; Female ; Immunohistochemistry ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Platelet Count ; Rabbits ; Random Allocation ; Subtilisins ; pharmacology ; therapeutic use

OBJECTIVETo evaluate the protective effect of polydatin on a PC12 cell model of oxygen and glucose deprivation (OGD).

METHODSA pheochromocytoma cell injury model was induced by OGD to simulate the cerebral ischemic changes. The protective effects of polydatin were investigated in this model.

RESULTSPolydatin treatment significantly enhanced the cell viability and reduced the levels of lactate dehydrogenase, nitric oxide and the malondialdehyde of the pheochromocytoma cells as compared with the OGD group. Polydatin also increased the activity of superoxide dismutase in the cells.

CONCLUSIONPolydatin offers protective effect against OGD-induced injury in pheochromocytoma cells.

Animals ; Brain Ischemia ; metabolism ; prevention & control ; Cell Survival ; Glucose ; metabolism ; pharmacology ; Glucosides ; pharmacology ; L-Lactate Dehydrogenase ; analysis ; Malondialdehyde ; analysis ; Nitric Oxide ; analysis ; Oxygen ; metabolism ; pharmacology ; PC12 Cells ; Protective Agents ; pharmacology ; Rats ; Stilbenes ; pharmacology ; Superoxide Dismutase ; metabolism

OBJECTIVETo investigate the effect of piperphentonamine hydrochloride (PPTA) on cognitive deficits induced by ischemia-reperfusion and explore the possible mechanisms.

METHODSSD rats were randomly divided into sham-operated group, ischemia-reperfusion group (with saline injection), PPTA-treated groups (2.5, 5, 10 mg/kg) and edaravone-treated group (6 mg/kg). Cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion, and the agents were administrated 1 h after ischemia. At 24 h after ischemia, step-through passive avoidance test was carried out, and 24 h later IL-1β, TNF-α, caspase-3 and HSP-70 mRNA expressions in the ischemic brain tissues were measured with RT-PCR.

RESULTSIn the step-through passive avoidance test, the rats in the ischemia-reperfusion group showed significantly shorter latency and more error times than those in the sham group, and these behavioral changes were improved significantly by treatments with PPTA and edaravone. Cerebral ischemia-reperfusion caused significantly increased expressions of IL-1β, TNF-α, caspase-3 and HSP-70 mRNA, and these changes were obviously reversed by PPTA, but not by edaravone.

CONCLUSIONSPPTA can reverse cognitive deficits induced by cerebral ischemia-reperfusion probably by decreasing the inflammatory responses and cell apoptosis in the brain, suggesting its potential as a new therapeutic agent for improving the cognitive function following cerebral ischemia-reperfusion.

3,4-Methylenedioxyamphetamine ; analogs & derivatives ; therapeutic use ; Animals ; Brain Ischemia ; drug therapy ; Cognition Disorders ; prevention & control ; Infarction, Middle Cerebral Artery ; drug therapy ; Male ; Neuroprotective Agents ; therapeutic use ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; prevention & control

OBJECTIVETo observe the influence of either alone or combined mixed-tocopherols combined with eicosapentaenoic acid (EPA) and α-Tocopherol use on oxidized LDL (oxLDL) induced 8-hydroxy-2'-deoxyguanosine (8-OHDG) and interleukin-6 (IL-6) secretion by human umbilical vein endothelial cells (HUVECs) and to explore the potential mechanism.

METHODCultured HUVECs in vitro were incubated with oxLDL, oxLDL + α-tocopherol, oxLDL + mixed-tocopherols, oxLDL + EPA, oxLDL + α-tocopherol + EPA, oxLDL + mixed-tocopherols + EPA for 24 hours, respectively. Secretion of 8-OHDG and IL-6 were detected by cell enzyme linked immunosorbent assay (ELISA). The expressions of superoxide dismutase (SOD), protein kinase C-δ (PKC-δ), phosphorylated PKC-δ (p-PKC-δ) were analyzed by Western blot.

RESULTS8-OHDG and IL-6 secretion of HUVECs was significantly increased significantly after incubated with oxLDL for 24 hours which could be significantly attenuated in the presence of tocopherols and EPA (alone or in combination, all P < 0.05) while the strongest inhibition effects were seen with combined use of mixed-tocopherols and EPA. Moreover, combination of mixed-tocopherols and EPA could also significantly increase SOD activity and decrease PKC activity (all P < 0.05). However, the protein expression of SOD and PKC-was similar among groups.

CONCLUSIONCombined mixed-tocopherols + EPA use enhanced the inhibiting effects on the secretion of 8-OHDG and IL-6 in oxLDL stimulated HUVECs which might be linked with increased SOD activity and reduced p-PKC activity.

Antioxidants ; Cells, Cultured ; Deoxyguanosine ; analogs & derivatives ; secretion ; Eicosapentaenoic Acid ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Interleukin-6 ; secretion ; Lipoproteins, LDL ; adverse effects ; Protein Kinase C ; metabolism ; Superoxide Dismutase ; metabolism ; alpha-Tocopherol ; pharmacology

Acquired hemophilia A is a deficiency disease caused by blood coagulation factor Ⅷ antibodies due to a variety of causes, The disease incidence rate is extremely low, but the mortality rate is high. This disease is often misdiagnosed,resulting in delays in treatment, so early diagnosis and timely treatment is crucial for reducing the mortality. The principle of treatment for the disease is mainly to control acute bleeding, eradicate inhibitor, and treat the primary disease,therefore, rational and personalized choices of treatment are also critical.

OBJECTIVETo establish a rapid method for detecting MTHFR gene 677C>T polymorphisms with high-resolution melting curve method (HRM) and pyrosequencing.

METHODSPeripheral blood samples were collected from 155 Down syndrome patients and 182 normal controls from Children's Hospital of Shanghai. The accuracy of three methods including regular HRM, internal control HRM and artificial heterozygosity HRM was compared. Meanwhile, allele frequencies in 10, 30 and 50 mixed samples were measured with pyrosequencing, and the results were compared with that of HRM.

RESULTSHeterozygosity of 677C>T polymorphism could be distinguished by various HRM methods. However, homozygotes CC and TT were only identifiable by internal control HRM and artificial heterozygosity HRM. The accuracy of pyrosequencing for allele frequency has improved with increased sample number. When the number of mixed samples has exceeded 30, the difference between pyrosequencing results and actual values became less than 4%. TT genotype was more frequent in Down syndrome patients than controls (25.2% vs. 14.3%). No significant difference was found in T allele frequency between the two groups (44.9% vs. 40.1%).

CONCLUSIONRespectively, internal control HRM and pyrosequencing may be ideal methods for determination of genotypic and allelic frequencies.

Child ; Child, Preschool ; Down Syndrome ; diagnosis ; enzymology ; genetics ; Female ; Humans ; Male ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Point Mutation ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA ; methods ; Transition Temperature

OBJECTIVETo assess the accuracy and reliability of the nest-PCR-sequence specific primer(SSP) method in HLA-A site genotyping of single blastomeres retrieved from human pre-implantation embryos.

METHODSBy nest PCR on HLA-A exon 2, the success rate of first-round amplification was estimated for single blastomeres. Based on the first-round amplification, the HLA-A genotype of every single blastomeres was analyzed by commercially available PCR-SSP kits.

RESULTSThe amplification of HLA-A exon 2 were performed to 120 blasotmeres retrieved from in vitro fertilization(IVF) surplus embryos donated by 10 couples. The average success rate of family 1-5 and 6-10 was 78.2%(43/55) and 93.8%(61/65), respectively. And 86.7%(104/120) in total. Eighty blastomeres were further tested by nest-PCR-SSP, among which 11 blastomeres failed to HLA-A exon 2 amplification and then failed to genotyping while the other 69 blastomeres succeed in HLA-A exon 2 amplification and succeed in genotyping. Except for 6 blastomeres that were uncertain for allele lost because of parents' homozygosity, the left 63 blastomeres had accurate HLA genotyping. Among these 63 blastomeres, 59 blastomeres had genotypes confirmed from their parents(93.6%), 3 blastomeres lost one of parents' alleles(4.8%), and only one blastomere had two more than parents' alleles(1.6%).

CONCLUSIONThe above research results indicated that based on the successful first round amplification of single blastomeres, nest-PCR-SSP strategy offers a convenient and reliable option for HLA genotyping on single blastomeres, which is a key process in pre-selecting HLA-identical sibling for allogeneic cord blood cell transplantation.

Base Sequence ; Blastomeres ; metabolism ; DNA ; analysis ; DNA Fingerprinting ; methods ; DNA Mutational Analysis ; Female ; HLA Antigens ; analysis ; HLA-A Antigens ; analysis ; genetics ; Histocompatibility Antigens Class I ; analysis ; genetics ; Humans ; Male ; Polymerase Chain Reaction ; methods ; Single Person

BACKGROUNDTendon adhesion is one of the most common causes of disability following tendon surgery. Therefore, prevention of peritendinous adhesion after surgical repair of tendon is a major challenge. The aim of this study was to explore the possible application of a collagen membrane for the prevention or attenuation of peritendinous adhesions.

METHODSSprague-Dawley (SD) rat Achilles tendon was cut and sutured by a modified Kessler's technique with or without the collagen membrane wrapped. Macroscopic, morphological and biomechanical evaluations were applied to examine the recovery of the injured tendon at 4 and 8 weeks after surgery.

RESULTSThe surgery group wrapped by collagen membranes had a better outcome than the group with surgery repair only. In the collagen membrane-treated group, less adhesion appeared, stronger tensile strength was detected, and more tendon fibers and collagen I expression were observed morphologically.

CONCLUSIONWrapping the tendon with a collagen membrane may be an efficient approach for tendon repair and preventing tendon adhesion after its ruptures.

Achilles Tendon ; injuries ; Animals ; Collagen ; Male ; Rats ; Rats, Sprague-Dawley ; Tendon Injuries ; surgery ; Tissue Adhesions ; prevention & control ; Wound Healing

OBJECTIVETo explore significance of high-risk human papillomavirus (HR-HPV) testing in atypical squamous cells, cannot exclude high grade squamous intraepithelial lesion (ASC-H).

METHODSPresence of HR-HPV DNA was examined in 45 patients with ASC-H using hybrid capture II (HC-II) test. Colposcopic examination and biopsy were taken all results were evaluated.

RESULTSOverall, 33 of 45 (73.3%) ASC-H cases were biopsy proven cervical intraepithelial lesion (CIN). 36 of 45 ASC-H cases were HPV-DNA positive, including 19 cases of HSIL and over lesion; whereas no HSIL or over was found in 9 HR-HPV negative cases. Sensitivity and negativity predictive value of HR-HPV in ASC-H with HSIL and over lesion were both 100%.

CONCLUSIONSASC-H strongly predicts the presence of HSIL, HR-HPV may serve as a predict select whether a patient with ASC-H should take colposcopic examination immediately, patients with positive HR-HPV should undergo immediate colposcopic examination, while negative HR-HPV is an excellent predictor of the absence of HSIL.

Adult ; Aged ; Biopsy ; Carcinoma, Squamous Cell ; pathology ; virology ; Cervical Intraepithelial Neoplasia ; pathology ; virology ; Colposcopy ; DNA, Viral ; analysis ; Female ; Humans ; Middle Aged ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; Precancerous Conditions ; pathology ; virology ; Uterine Cervical Neoplasms ; pathology ; virology ; Uterine Cervicitis ; pathology ; virology ; Vaginal Smears ; Young Adult