Objective: To determine the extract from Radix Astragali. Methods : Astragaloside IV in extract from Radix Astragali was determined by TLC-Scanning. Results : The linear relationship was at the rang of 0.78 ?g～ 4.68 ?g. The average recovery was 99.06% ( RSD=1.17% and n =5). Conclusion : The method is available with a good reproducibility. The pretreatment is simple and easy to operate.

Background Chondrocytes' phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure.Microfilaments,microtubules,and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes.The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro.Methods Chondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods (n=24).The isolated cells were stabilized for three days and then exposed to low,medium,and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest:colchicine for microtubules,acrylamide for intermediate filaments,and cytochalasin D for actin microfilaments.A group of control cells were treated with carrier.Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents.The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining on day 3.The mRNA levels of aggrecan (AGG) and type Ⅱ collagen (Col-2) and their levels in culture medium were analyzed using real-time PCR and enzymelinked immunosorbent serologic assay (ELISA) on days 3,6,and 9.Results In the initial drug-dose-response study,there was no significant difference in the vitality of cells treated with 0.1 μmol/L colchicine,2.5 mmol/L acrylamide,and 10 μg/L cytochalasin D for two days when compared with the control group of cells.The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei.Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were significantly decreased for cultures treated with colchicine and acrylamide when compared with untreated cells at three,six,and nine days,and this inhibition was correlated with higher matrix metalloprotease-13 expression in these cells.Cellular proliferation,monolayer morphology,and matrix metabolism were unaffected in cytochalasin D-treated cells when compared with control cells over the nine-day culture period.Conclusions The disruption of the microtubulin and intermediate filaments induced chondrocyte apoptosis,increased matrix metalloprotease expression,and decreased AGG and Col-2 expression in rabbit knee chondrocyte cultures.Our findings suggest that microtubulin and intermediate filaments play a critical role in the synthesis of cartilage matrix by rabbit knee chondrocytes.

Objective:To determine the median effective dose (ED 50) of 0.5% ropivacaine based on femoral nerve cross-sectional area for ultrasound-guided femoral nerve block. Methods:Patients of both sexes, aged 18-64 yr, of American Society of Anesthesiologists physical status I or Ⅱ, with body mass index of 20-30 kg/m 2, scheduled for elective open reduction and internal fixation for patella fracture or removal of patella fracture by internal fixation, were enrolled in this study.Ultrasonic localization of femoral nerve was performed for measurement of the femoral nerve cross-sectional area, and 0.5% ropivacaine was injected based on the area.ED 50 was determined by Dixon′s up-and-down sequential method.The initial dose was 0.22 ml/mm 2, and the difference between the two successive doses was 0.02 ml/mm 2.The effective block was defined as complete loss of pain sensation in the areas of anterior skin of knee joint, skin on the inner side of the calf and dorsal medial skin of the foot and the degree of motor block was in stages 1-3 assessed using Brunnstrom motor function within 30 min after nerve block.Nerve block was considered ineffective if pain occurred in any nerve distribution area mentioned above.The study was terminated if 7 effective and ineffective alternating waves occurred.ED 50 and 95% confidence interval (CI) were calculated using Probit analysis. Results:Twenty-seven patients were enrolled in the study with the femoral nerve cross-sectional area (75±5) mm 2.ED 50 (95%CI) of 0.5% ropivacaine for ultrasound-guided femoral nerve block was 0.106 (0.069-0.125) ml/mm 2. Conclusion:ED 50 of 0.5% ropivacaine based on femoral nerve cross-sectional area for ultrasound-guided femoral nerve block is 0.106 ml/mm 2.

BACKGROUNDChondrocytes' phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure. Microfilaments, microtubules, and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes. The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro.

METHODSChondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods (n = 24). The isolated cells were stabilized for three days and then exposed to low, medium, and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest: colchicine for microtubules, acrylamide for intermediate filaments, and cytochalasin D for actin microfilaments. A group of control cells were treated with carrier. Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents. The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining on day 3. The mRNA levels of aggrecan (AGG) and type II collagen (Col-2) and their levels in culture medium were analyzed using real-time PCR and enzyme-linked immunosorbent serologic assay (ELISA) on days 3, 6, and 9.

RESULTSIn the initial drug-dose-response study, there was no significant difference in the vitality of cells treated with 0.1 µmol/L colchicine, 2.5 mmol/L acrylamide, and 10 µg/L cytochalasin D for two days when compared with the control group of cells. The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei. Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were significantly decreased for cultures treated with colchicine and acrylamide when compared with untreated cells at three, six, and nine days, and this inhibition was correlated with higher matrix metalloprotease-13 expression in these cells. Cellular proliferation, monolayer morphology, and matrix metabolism were unaffected in cytochalasin D-treated cells when compared with control cells over the nine-day culture period.

CONCLUSIONSThe disruption of the microtubulin and intermediate filaments induced chondrocyte apoptosis, increased matrix metalloprotease expression, and decreased AGG and Col-2 expression in rabbit knee chondrocyte cultures. Our findings suggest that microtubulin and intermediate filaments play a critical role in the synthesis of cartilage matrix by rabbit knee chondrocytes.

Animals ; Cartilage, Articular ; cytology ; metabolism ; Chondrocytes ; cytology ; Collagen ; metabolism ; Cytoskeleton ; metabolism ; Knee Joint ; cytology ; metabolism ; Microscopy, Confocal ; Rabbits