Role of TLR4/NF-κB pathway for early change of synovial membrane in knee osteoarthritis rats / 中国骨伤

OBJECTIVE@#To study role of TLR4/NF-κB pathway for early change of synovial membrane in knee osteoarthritis rats.@*METHODS@#Eighteen male SD rats weighted (200±20) g were randomly divided into 2 groups, namely control and model group, and 9 in each group. Knee OA model group was established by using modified Hulth method in model group. Control group was not treated. Synovial tissue and serum was extracted at 4 and 21 d after operation. Expression of CD14, TLR4, IL-1β, TNF-α, ADAMTS-4, MMP-13 were detected by real-time PCR respectively. NF-κB p65 protein was detected by Western-blot; serum concentrations of haluronic acid (HA), N-propeptide of type III procollagen(PIIINP) was detected by Elisa.@*RESULTS@#Expression of CD14, ADAMTS-4, and NF-κB p65 in model group were higher than that of control group at 4 and 21 days after operation, while expression of TLR4, IL-1β, TNF-α and MMP-13 were higher than that of control group at 21 days after operation(<0.01). Concentration of PIIINP and HA in model group were higher than that of control group at 4 days after operation, while there was no significant difference at 21 days after operation.@*CONCLUSIONS@#NF-κB pathway could mediate occurrence of KOA by early activating and triggeringg synovial increasingly secreting inflammatory secretion CD14, TLR4, IL-1β, TNF-α, ADAMTS-4, MMP-13, PIIINP and HA.

Effect of HBP-A on meniscal injury and pathological hypertrophy and calcification of the meniscus / 南方医科大学学报

<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>

Establishment of chondrocyte degeneration model in vitro by rat serum / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a model of chondrocyte degeneration in vitro.</p><p><b>METHODS</b>Chondrocytes were isolated from articular cartilages of newly born SD rats by digestion with typeⅡ collagenase. The chondrocytes were cultured with H-DMEM medium containing 10%FBS, 50 ng/mL IL-1β+10%FBS, 2.5% rat serum and 5% rat serum, respectively; and the chondrocytes at passage one were used in the experiments. The morphology changes were investigated under phase contrast microscope after chondrocytes were treated with rat serum and IL-1β. Proliferation of chondrocytes was detected by MTT method. The protein expression levels of PCNA, typeⅡ collagen and MMP-13 were examined by Western blotting. The levels of ADAMTS5, MMP-9, Aggrecan and SOX-9 mRNA were detected by real-time PCR.</p><p><b>RESULTS</b>The cell morphology was changed from polygon to spindle in both rat serum groups and IL-1β group, and the proliferation of chondrocytes in these groups was much higher than that in control group. The results showed that the expression levels of typeⅡ collagen, Aggrecan and SOX-9 decreased while the expression levels of MMP-13, MMP-9 and ADMATS5 were up-regulated in rat serum and IL-1β-treated groups compared with control group.</p><p><b>CONCLUSION</b>The results indicate that rat serum can induce chondrocyte degeneration and may be used for osteoarthritis model in vitro.</p>

Etablishment of cartilage degeneration model by IL-1 beta in vitro / 中国骨伤

<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>

Regulating effect of anodonta glucan HBP-A on chondrocytes through Wnt pathway / 中国骨伤

<p><b>OBJECTIVE</b>To investigate regulation function of anodonta glucan HBP-A on chondrocytes through Wnt pathway in vitro.</p><p><b>METHODS</b>Rat chondrocytes were cultured and differentiated induced with IL-1beta (10 ng/ml) in vitro. Chondrocytes were divided into five groups:IL-13 group,IL-1beta + IWP-2 (5 microM,Wnt pathway inhibitor) group, IL-1beta + HBP-A (0.3 mg/ml) group and IL-1beta + IWP-2 + HBP-A group. Wnt-3a, beta-catenin (24 h,48 h,72 h) and MMP-13(72 h) genes expression were detected by Rt-PCR, while beta-catenin, MMP-13, Sox-9 and coll-II (48 h) protein expression were measured by Western-blot.</p><p><b>RESULTS</b>After induction of IL-1beta, gene expression of Wnt-3a, beta-catenin and MMP-13 were increased,so were the protein expression of beta-catenin and MMP-13. In contrast,protein expression of Sox-9 and Coll-II were declined. Following addition of HBP-A, Wnt-3a, beta-catenin and MMP-13 were shown as induction of IL-1beta, but protein expression of Sox-9 and Coll-II were upgraded. Combining HBP-A with IWP-2 led to the lowest level in Wnt-3a, beta-catenin gene and beta-catenin protein expression and highest expression of Sox-9 protein.</p><p><b>CONCLUSION</b>HBP-A could not only delay the differentiation of chondrocytes through downgrading the signal expression of Wnt/beta-catenin,but also adjust the expression of Wnt-3a, beta-catenin and Sox-9 when combinated with the Wnt inhibitor.</p>

Effects of serum enatninine Gumibao (Chinese character: see text) on the aroliferation and differentiation of osteoblast induced by dexamethasone / 中国骨伤

<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>

Effect of osthole on proliferation of neonatal rat osteoblast and the relative mechanism research / 中国骨伤

<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>

Mechanism of resveratrol on the promotion of induced pluripotent stem cells / 中西医结合学报

<p><b>OBJECTIVE</b>To investigate the effects of resveratrol (RV) in reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) and the related mechanism.</p><p><b>METHODS</b>Primary MEFs were isolated from E13.5 embryos and used within three passages. Retroviruses expressing Sox2 and Oct4 were produced by transfecting GP2-293t cells with recombinant plasmids (MSCV)-Sox2 and MSCV-Oct4. Supernatants containing retroviruses were obtained after 48-hour transfection and MEFs were then infected. Different concentrations (0, 5, 10 and 20 μmol/L) of RV were added to embryonic stem cell (ESC) medium to culture MEFs 48 h post-infection. iPSC clones emerged and were further cultured. Expression of pluripotent markers of iPSCs was identified by cell immunofluorescence and reverse transcription-polymerase chain reaction. Both cytotoxicity and cell proliferation were assayed by Western blot analysis after RV was added into ESC medium. The ultrastructure change of mitochondria was observed by electron microscopy.</p><p><b>RESULTS</b>More than 2.9-fold and 1.3-fold increases in colony number were observed by treatment with RV at 5 and 10 μmol/L, respectively. The reprogramming efficiency was significantly decreased by treatment with 20 μmol/L RV. The proliferation effect on MEFs or MEFs infected by two factors Sox2/Oct4 (2 factors-MEFs, 2F-MEFs) was investigated after RV treatment. At 20 μmol/L RV, induced cell apoptosis and proliferation inhibition were more obvious than those of 5 and 10 μmol/L treatments. Clones were selected from the 10 μmol/L RV-treated group and cultured. Green fluorescent protein expression from one typical clone was silenced one month later which expressed ESC-associated marker genes Gdf3, Nanog, Ecat1, Fgf4 and Foxd3. Electron transmission microscope showed obvious cavitations in mitochondria. The expression of hypoxia-inducible factor-1α was up-regulated when 2F-MEFs were treated with RV compared to the control group.</p><p><b>CONCLUSION</b>RV improved the efficiency of reprogramming 2F-MEFs into iPSCs at low and moderate concentrations (5 and 10 μmol/L). The effect of 10 μmol/L RV on reprogramming was much greater than that of 5 μmol/L RV. However, high concentration of RV (20 μmol/L) led to more severe cavitations in mitochondria and caused cytotoxic effects. Taken together, these findings suggest that RV mimics hypoxia in cells and promotes reprogramming at a low concentration.</p>

Comparison of the activity and yield rate of osteoblast obtained by different digestion methods / 中国骨伤

<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>