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AIM To establish a UPLC-MS/MS method for the simultaneous content determination of liquiritin apioside,alibiflorin,swertiamarin,methyl gallate,benzoylpaeoniflorin,sweroside,6′-O-β-D-glucosylgentiopicroside,isoliquiritigenin,loganic acid,liquiritigenin,gallic acid,paeoniflorin,oxypaeoniflorin,gentiopicroside,glycyrrhizic acid,isoliquiritoside and liquiritin in Yangxue Ruanjian Capsules.METHODS The analysis was performed on a 40℃thermostatic Waters BEH C18column(2.1 mm×100 mm,1.7 μm),with the mobile phase comprising of 2 mmol/L ammonium acetate(containing 0.1%formic acid)-acetonitrile flowing at 0.3 mL/min in a gradient elution manner,and electron spray ionization source was adopted in negative ion scanning with multiple reaction monitoring mode.RESULTS Seventeen constituents showed good linear relationships within their own ranges(r>0.999 6),whose average recoveries were 91.33%-104.03%with the RSDs of 1.58%-3.50%.CONCLUSION This rapid,accurate and stable method can be used for the quality control of Yangxue Ruanjian Capsules.
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This study aims to provide evidence for clinical practice by systematically reviewing the efficacy and safety of Gusongbao preparation in the treatment of primary osteoporosis(POP). The relevant papers were retrieved from four Chinese academic journal databases and four English academic journal databases(from inception to May 31, 2022). The randomized controlled trial(RCT) of Gusongbao preparation in the treatment of POP was included after screening according to the inclusion and exclusion criteria. The quality of articles was evaluated using risk assessment tools, and the extracted data were subjected to Meta-analysis in RevMan 5.3. A total of 657 articles were retrieved, in which 15 articles were included in this study, which involved 16 RCTs. A total of 3 292 patients(1 071 in the observation group and 2 221 in the control group) were included in this study. In the treatment of POP, Gusongbao preparation+conventional treatment was superior to conventional treatment alone in terms of increasing lumbar spine(L2-L4) bone mineral density(MD=0.03, 95%CI[0.02, 0.04], P<0.000 01) and femoral neck bone mineral density, reducing low back pain(MD=-1.69, 95%CI[-2.46,-0.92], P<0.000 1) and improving clinical efficacy(RR=1.36, 95%CI[1.21, 1.53], P<0.000 01). Gusongbao preparation was comparable to similar Chinese patent medicines in terms of improving clinical efficacy(RR=0.95, 95%CI[0.86, 1.04], P=0.23). Gusongbao preparation was inferior to similar Chinese patent medicines in reducing traditional Chinese medicine syndrome scores(MD=1.08, 95%CI[0.44, 1.71], P=0.000 9) and improving Chinese medicine syndrome efficacy(RR=0.89, 95%CI[0.83, 0.95], P=0.000 4). The incidence of adverse reactions of Gusongbao preparation alone or combined with conventio-nal treatment was comparable to that of similar Chinese patent medicines(RR=0.98, 95%CI[0.57, 1.69], P=0.94) or conventio-nal treatment(RR=0.73, 95%CI[0.38, 1.42], P=0.35), and the adverse reactions were mainly gastrointestinal discomforts. According to the available data, Gusongbao preparation combined with conventional treatment is more effective than conventional treatment alone in increasing lumbar spine(L2-L4) bone mineral density and femoral neck bone mineral density, reducing low back pain, and improving clinical efficacy. The adverse reactions of Gusongbao preparation were mainly gastrointestinal discomforts, which were mild.
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Humanos , Densidad Ósea , Dolor de la Región Lumbar , Medicina Tradicional China , Osteoporosis/tratamiento farmacológicoRESUMEN
BACKGROUND: The 3D printed polylactic-co-glycolicacid/nano-hydroxyapatite (PLGA/nHA) scaffold carrying human recombinant bone morphogenetic protein 2 (rhBMP-2)/chitosan (CS) sustained release tissue-engineering bone has good biological activity, mechanical properties, and biological activity of its controlled release rhBMP-2. OBJECTIVE: To investigate the repair of mandibular defects with PLGA/nHA scaffold/rhBMP-2/CS sustained release tissue-engineering bone manufactured using 3D bionic printing technology. METHODS: Animal models of bilateral critical mandibular bone defects were established in 27 New Zealand white rabbits, followed by implantation of a 3D-printed PLGA/nHA scaffold/rhBMP-2/CS sustained release tissue-engineering bone on one side (experimental side), and a 3D-printed PLGA/nHA scaffold on the other side (control side). Mandibular specimens were harvested at postoperative days 30, 60 and 90 to carry out cone-beam CT, Micro CT, histological and immunohistochemical examinations. RESULTS AND CONCLUSION: The results from micro-CT analysis revealed that the volume of newly formed bone volume and the amount of bone trabeculae on the experimental side were significantly higher than those on the control side at different postoperative time points (P < 0.05). The results from cone-beam CT examination showed that at 90 postoperative days, bone density of the bone defect on the experimental side was close to that of the surrounding bone, new bone tissues were full of the original bone defect area, and the trabecular bone arranged regularly, while on the control side, worm-eaten discontinuous low-density osteoid tissues were visible in the bone defect area. Osteogenesis on the experimental side was better than that on the control side. Histological findings demonstrated that on the experimental side, a large amount of mature lamellae were detected in the bone defect area, with well-arranged trabecular bones and abundant capillaries, and moreover, the scaffold material had been completely absorbed. However, low-density, loose-meshed, irregular braided bone tissues with rare capillaries were observed on the control side, and the scaffold material had been mostly absorbed. Immunohistochemical findings indicated that the osteocalcin-dyed area on the experimental side was significantly larger than that of the control side at postoperative 90 days. To conclude, 3D-printed PLGA/nHA scaffold/rhBMP-2/CS sustained release tissue-engineering bone is favorable for the repair and reconstruction of experimental mandibular defects in rabbits.
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<p><b>OBJECTIVE</b>To investigate the effect of HBP-A on meniscal injuries and the expressions of genes associated with pathological hypertrophy and calcification of the meniscusinduced by abnormal loading.</p><p><b>METHODS</b>Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 3 h and treated with 0.6 mg/mL of HBP-A. The cell viability in the meniscus explants after 72 hin culture was determined using live/dead staining and the expression levels of genes associated with pathological hypertrophy and calcification of the meniscus (ANKH, ENPP1, ALP, MMP13, and IL-1) were measured using real-time PCR and Western blotting. The conditioned medium was collected for testing sulfated glycosaminoglycan (GAG) release.</p><p><b>RESULTS</b>The number of dead cells, loss of proteoglycan content, and the expressions of ANKH, ENPP1, ALP and MMP13, and IL-1 at both the mRNA and protein levels were all significantly lower in the meniscus explants treated with 0.6 mg/mL HBP-A than in the explants with only 25% abnormal pressure stimulation (n=3, P<0.05).</p><p><b>CONCLUSION</b>HBP-A can effectively alleviate meniscal injuries induced by abnormal loading and suppress the expressions of genes related with pathological hypertrophy and calcification of the meniscus, and can serve as a potential drug for treatment of knee osteoarthritis.</p>
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Animales , Bovinos , Calcinosis , Quimioterapia , Glucanos , Farmacología , Hipertrofia , Meniscos Tibiales , Osteoartritis de la Rodilla , Quimioterapia , Reacción en Cadena en Tiempo Real de la Polimerasa , Lesiones de Menisco Tibial , QuimioterapiaRESUMEN
<p><b>OBJECTIVE</b>To establish a reliable model for drug screening and therapy by culturing rat femoral head and inducing cartilage degeneration quickly in vitro.</p><p><b>METHODS</b>The femoral heads from the same SD rats of two-month old were divided into control group and experimental group respectively. They were cultured with DMEM medium plus 10% fetal bovine serum or DMEM medium plus 10% fetal bovine serum plus 50 ng/ml IL-1β for three days. Femoral heads were fixed in 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin and cut into slices. Specimens were stained with Toluidine blue and Safranine O-Fast Green FCF. The protein expression levels of type II collagen, MMP13, Sox9 and ADAMTS5 were analyzed by immunofluorescence.</p><p><b>RESULTS</b>Both the Toluidine blue and Safranine O staining were pale in the margin of femoral heads which were stimulated with IL-1β for three days compared to that in control group. The Fast Green FCF staining was positive at the edge of the femoral head in experimental group, which indicated that cartilage became degenerated. The expression levels of both type H collagen and Sox9 were decreased significantly while the expression levels of MMP13 and ADAMTS5 were increased in experimental group.</p><p><b>CONCLUSION</b>The model of cartilage degeneration is established by culturing and inducing the degeneration of the femoral heads quickly in vitro.</p>
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Animales , Humanos , Masculino , Ratas , Enfermedades de los Cartílagos , Genética , Metabolismo , Colágeno Tipo II , Genética , Metabolismo , Modelos Animales de Enfermedad , Cabeza Femoral , Metabolismo , Técnicas In Vitro , Interleucina-1beta , Genética , Metabolismo , Metaloproteinasa 13 de la Matriz , Genética , Metabolismo , Ratas Sprague-Dawley , Factor de Transcripción SOX9 , Genética , MetabolismoRESUMEN
<p><b>OBJECTIVE</b>To investigate the effects of serum containing Gumibao (Chinese character: see text) on the proliferation and differentiation of osteoblast induced by dexamethasone.</p><p><b>METHODS</b>Osteoblasts were extracted from skulls in newly born (within 24 hours) SD rats, and digested with collagenase. The first passage of cells were used for experiments. Cells were cultured in the medium containing different concentrations of dexamethasone (0, 10(-8), 10(-7), 10(-6), 10(-5) ,10(-4) mol/L). Alkaline phosphatase staining were carried out after 1 week and numbers of mineralized nodes with alizarin red staining were observed after 3 weeks. Accordingly, following the treatment of 10(-5) mol/L dexamethasone for 1 week, cells were cultured in the medium with serum containing Gumibao (Chinese character: see text). One week after Cumibao (Chinese character: see text) treatment, cells were stained with Alkaline phosphatase and collagen I and PCNA were examined by Western-blot. However, the observation of numbers of mineralized nodes with alizarin red stain required one more week.</p><p><b>RESULTS</b>High concentration of dexamethasone could inhibit the expression of PCNA, collagen I, alkaline phosphatase and reduce the number of mineralized nodes of osteoblast, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.</p><p><b>CONCLUSION</b>High concentration of dexamethasone could inhibit the proliferation and differentiation of osteoblastic cells, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.</p>
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Animales , Femenino , Ratas , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Colágeno Tipo I , Dexametasona , Farmacología , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos , Farmacología , Osteoblastos , Biología Celular , Fisiología , Antígeno Nuclear de Célula en Proliferación , Ratas Sprague-DawleyRESUMEN
Objective To further understand the biomechanical relationship between activities of cervical spine and blood flow of vertebral artery (VA) by developing the VA finite element model and calculating the fluid-structure interaction. Methods Based on the normal model of cervical spine and the developed C0-T1 finite element model with bilateral VA, the flexion and extension, right and left lateral bending, right and left axial rotation movement of cervical spine at physiological velocity were simulated. The effects of cervical activities on stress of vertebral arterial wall were observed, and the biomechanical interaction between the vessel wall and fluid was calculated by fluid-structure interaction equation to obtain the hemodynamic parameters. Results The maximum stress was usually concentrated on the both sides of C2 transverse foramen, where the second arc of vertebral arterial wall protruded into the cranial direction during cervical activities. The maximum strain of the vessel wall was most obvious during the extension and lateral bending movement, with strain ratio of 23.04% and 35.5%, respectively. The maximum stress on the vessel was located in the position of contralateral transverse foramen during lateral bending movement, while the maximum strain on the vessel was located in the position of ipsilateral transverse foramen during rotation movement. In aspect of cervical spine range of motion (ROM), the minimum volume flow rate occurred within 30%-40% of the physiological ROM. The volume flow rate-time curve of bilateral VA was similar during flexion and extension movement, when the circulation of flow rate was completed for two times within 0.5 s. The peak and valley of ipsilateral blood flow in volume flow rate-time curve occurred earlier than that of contralateral blood flow during lateral bending movement, while the results of rotation movement were opposite. Conclusions The obtained stress features of bilateral VA vessel and the law of the volume flow rate-time curve validated the experimental results with those in the literature, which could reasonably explain the clinical phenomenon. The established model would provide an ideal platform for researches on vertebral artery-related diseases.
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<p><b>OBJECTIVE</b>To compared the activity and yield rate of osteoblast obtained by different collagenase digestion methods, to find a better way to extract osteoblast for the experimental researches of osteoporosis.</p><p><b>METHODS</b>Ten 24-hour-old SD rats were were euthanized. The cranium of rats were removed and cuted into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, all the cranium were divided into two equal parts, and randomly divided into two groups which would be digested by type I collagenase and type II collagenase separately for two times. The rat cells of the two groups were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. The primary culture osteoblasts were counted by using a haemacytometer after digestion and 72 hours later. The second generation osteoblasts cultured 48 h were dyed by NBT/BCIP staining solution, and were detected by quantitative measurement with PNPP.</p><p><b>RESULTS</b>The cells had irregular shapes. The results of cell counting showed that the cell number of type I group was larger than type 11 group. Alkaline phosphatase dyeing were positive. Detecting of alkaline phosphatase using the method of PNPP showed that the absorbance value in type I group were higher than type II group (P<0.05).</p><p><b>CONCLUSION</b>Two types of collagenase are both suitable for the in vitro culture of rat osteoblasts. The activity and yield rate of osteoblasts in type I group are higher which could provide more stable seed cells for the treatment of osteoporosis.</p>
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Animales , Femenino , Masculino , Ratas , Recuento de Células , Técnicas de Cultivo de Célula , Métodos , Colagenasas , Metabolismo , Osteoblastos , Biología Celular , Ratas Sprague-DawleyRESUMEN
<p><b>OBJECTIVE</b>To investigate the effects of osthole on proliferation of neonatal rat osteoblast and the mechanism.</p><p><b>METHODS</b>Ten 24 hours old SD rats were executed by dislocating. The cranium of rats were removed and cut into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, the cranium were digested by type I collagenase for one hour two times. The mixed cells were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. To identify the cells, ALP staining and alizarin red staining were performed after cultured 48 h and 28 d. The osteoblasts were randomly divided into five groups. Cells were treated with osthole at concentrations of 100, 50, 25, 12.5, 0 micromol/L. CCK-8 method was used to evaluate the proliferation after 24 h,48 h and 72 h. The expression of PCNA and beta-catenin protein were detected through the method of Western Blot after one week.</p><p><b>RESULTS</b>The cells had irregular shapes and showed typical features of osteoblast. The results of ALP staining and alizarin red staining were both positive. CCK-8 detection showed that the osthole with final concentration of 100 micromol/L inhibited the proliferation of osteoblast after 24 h, while the osthole with final concentrations of 50 micromol/L and 25 micromol/L displayed the inhibition effect after 48 h. The osthole of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast. The result of Western Blot showed that osthole reduced the expression of PCNA and beta-catenin protein in a dose-dependent manner.</p><p><b>CONCLUSION</b>The osthole with final concentrations of 100, 50, 25 micromol/L inhibited the proliferation of osteoblast (P < 0.05). The osthole with final concentrations of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast (P > 0.05). These findings demonstrate that osthole may inhibit the proliferation of osteoblast by regulating the Wnt/beta-catenin signaling in osteoblast.</p>
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Animales , Femenino , Masculino , Ratas , Animales Recién Nacidos , Diferenciación Celular , División Celular , Proliferación Celular , Células Cultivadas , Cumarinas , Farmacología , Osteoblastos , Biología Celular , Metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Sincalida , Metabolismo , beta Catenina , MetabolismoRESUMEN
Objective To evaluate the feasibility of quantitative analysis software for myocardial contrast echocardiography (MCE) in assessment of myocardial perfusion.Methods According to coronary occlusion and reperfusion at different times,rabbits were divided into two groups:15 min occlusion / 30 min reperfusion (group Ⅰ) and 120 min occlusion / 60 min reperfusion (group Ⅱ).MCE was performed on all rabbits at baseline,occlusion and after reperfusion,and its images were analyzed by a new quantitative analysis software based on eliminating particle swarm optimization (EPSO) clustering algorithm,by which obtain myocardial perfusion parameters.Results (1) The values of calibrated contrast intensity (CI) in risk segments of Groups Ⅰ and Ⅱ were significantly lower than those at baseline during occlusion (t =5.104 and t =4.327,P<0.01).After reperfusion,calibrated CI in risk segments significantly improved in Group Ⅰ (t =2.933,P<0.01) while those remained unchanged in Group Ⅱ (P>0.05).(2) The areas of red-coded region in color-coded map and myocardial infarction in triphenyl-tetrazolium chloride (TTC) were (21.4±12.3)% and (18.0±9.5)%,respectively.The correlation between color-coded image and TTC was 0.89 (P<0.01).(3) The histogram in all risk segments was skew distribution during occlusion.After reperfusion,the histogram in Group Ⅰ was normal distribution while that was still skewed distribution in Group Ⅱ.Conclusion The MCE image analysis software based on EPSO clustering algorithm in the quantitative assessment of myocardial microperfusion and identification of myocardial perfusion abnormalities was feasible and of high value.
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<p><b>OBJECTIVE</b>To evaluate the feasibility and value of determining myocardial perfusion and regional systolic function by myocardial contrast stress echocardiography (MCSE) with computer-assisted technique in a rabbit model of ischemia/reperfusion injury.</p><p><b>METHODS</b>Rabbits underwent 30-(Group I, n = 15) and 120-(Group II, n = 15) minute left ventricular branch of the left circumflex coronary artery occlusion foll owed by 60-minute reperfusion, dobutamine at increasing doses (5, 10, 15 and 20 microg.kg(-1).min(-1)) was then infused after reperfusion for 15 min. Bolus myocardial contrast agent was injected and MCSE performed at baseline, at the end of coronary occlusion and reperfusion, at the end of each dobutamine infusion. Images were analyzed by computer-assisted technique and myocardial calibrated contrast intensity (CI) of each segment was measured and a color-coded map was then obtained automatically (yellow: from 0 to -20 pix, blue:from -21 to -40 pix, green: from -41 to -70 pix, red: < -70 pix). The area at risk and infarct area obtained by red-coded map were compared with ex vivo results determined by fluorescent microsphere and triphenyl-tetrazolium chloride (TTC) staining. Percentage wall thickening (WT) of each risk segment at each stage were also measured.</p><p><b>RESULTS</b>(1) During occlusion, WT in the areas at risk decreased to zero or negative and the calibrated CI values were significantly lower than those at baseline. Area at risk obtained by red-coded map correlated well with that obtained by fluorescent staining (r = 0.91, P < 0.01). (2) After reperfusion and 5 microg.kg(-1).min(-1) dobutamine administration, WT and calibrated CI in all rabbits remained depressed. Calibrated CI at -70 pix was an optimal cutoff point to identify infarcted segments (sensitivity 95%, specificity 87%). The correlation between the infarct size by red-coded image and TTC was 0.89 (P < 0.01). (3) Calibrated CI and WT significantly improved in Group I rabbits while these parameters remained unchanged in Group II rabbits after increasing doses of dobutamine post ischemia.</p><p><b>CONCLUSIONS</b>Myocardial contrast stress echocardiography in combination with computer-assisted analysis technique are valuable techniques to quantitatively assess myocardial perfusion and regional systolic function and exactly identify stunned myocardium and infarcted myocardium.</p>