An Inhibitory Role of Osthole in Rat MSCs Osteogenic Differentiation and Proliferation via Wnt/ß-Catenin and Erk1/2-MAPK Pathways.

BACKGROUND/AIMS: Bone marrow-derived mesenchymal stem cells (MSCs) are responsible for new bone formation during adulthood. Accumulating evidences showed that Osthole promotes the osteogenic differentiation in primary osteoblasts. The aim of this study was to investigate whether Osthole exhibits a potential to stimulate the osteogenic differentiation of MSCs and the underlying mechanism. METHODS: MSCs were treated with a gradient concentration of Osthole (6.25 µM, 12.5 µM, and 25 µM). Cell proliferation was assessed by western blotting with the proliferating cell nuclear antigen (PCNA) and Cyclin D1 antibodies, fluorescence activated cell sorting (FACS), and cell counting kit 8 (CCK8). MSCs were cultured in osteogenesis-induced medium for one or two weeks. The osteogenic differentiation of MSCs was estimated by Alkaline Phosphatase (ALP) staining, Alizarin red staining, Calcium influx, and quantitative PCR (qPCR). The underlying mechanism of Osthole-induced osteogenesis was further evaluated by western blotting with antibodies in Wnt/ß-catenin, PI3K/Akt, BMPs/smad1/5/8, and MAPK signaling pathways. RESULTS: Osthole inhibited proliferation of rat MSCs in a dose-dependent manner. Osthole suppressed osteogenic differentiation of rat MSCs by down-regulating the activities of Wnt/ß-catenin and Erk1/2-MAPK signaling. CONCLUSIONS: Osthole inhibits the proliferation and osteogenic differentiation of rat MSCs, which might be mediated through blocking the Wnt/ß-catenin and Erk1/2-MAPK signaling pathways.

Osthole Inhibits Proliferation and Induces Catabolism in Rat Chondrocytes and Cartilage Tissue.

BACKGROUND/AIMS: Cartilage destruction is thought to be the major mediator of osteoarthritis. Recent studies suggest that inhibition of subchrondral bone loss by anti-osteoporosis (OP) drug can protect cartilige erosion. Osthole, as a promising agent for treating osteoporosis, may show potential in treating osteoarthritis. The purpose of this study was to investigate whether Osthole affects the proliferation and catabolism of rat chondrocytes, and the degeneration of cartilage explants. METHODS: Rat chondrocytes were treated with Osthole (0 µM, 6.25 µM, 12.5 µM, and 25 µM) with or without IL1-ß (10ng/ml) for 24 hours. The expression levels of type II collagen and MMP13 were detected by western Blot. Marker genes for chondrocytes (A-can and Sox9), matrix metalloproteinases (MMPs), aggrecanases (ADAMTS5) and genes implicated in extracellular matrix catabolism were evaluated by qPCR. Cell proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and fluorescence activated cell sorter. Wnt7b/ß-catenin signaling was also investigated. Cartilage explants from two-week old SD rats were cultured with IL-1ß, Osthole and Osthole plus IL-1ß for four days and glycosaminoglycan (GAG) synthesis was assessed with toluidine blue staining and Safranine O/Fast Green FCF staining, collagen type II expression was detected by immunofuorescence. RESULTS: Osthole reduced expression of chondrocyte markers and increased expression of MMP13, ADAMTS5 and MMP9 in a dose-dependent manner. Catabolic gene expression levels were further improved by Osthole plus IL-1ß. Osthole inhibited chondrocyte proliferation. GAG synthesis and type II collagen were decreased in both the IL-1ß groups and the Osthole groups, and significantly reduced by Osthole plus IL-1ß. CONCLUSIONS: Our data suggested that Osthole increases the catabolism of rat chondrocytes and cartilage explants, this effect might be mediated through inhibiting Wnt7b/ß-catenin pathway.

[Establishment of chondrocyte degeneration model in vitro by rat serum].

OBJECTIVE: To establish a model of chondrocyte degeneration in vitro. METHODS: 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-1ß+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-1ß. 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. RESULTS: The cell morphology was changed from polygon to spindle in both rat serum groups and IL-1ß 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-1ß-treated groups compared with control group. CONCLUSION: The results indicate that rat serum can induce chondrocyte degeneration and may be used for osteoarthritis model in vitro.

Osthole exhibits anti-cancer property in rat glioma cells through inhibiting PI3K/Akt and MAPK signaling pathways.

AIMS: The purpose of this study was to investigate how Osthole affects glioma cell proliferation, apoptosis, invasion and migration. METHODS: Rat glioma cells were treated with different concentrations of Osthole (0 µM, 25 µM, 50 µM, and 100 µM). Cell proliferation was assessed by measuring PCNA expression and CCK8 assay at different time points. Apoptosis was evaluated by measuring the expression of pro-apoptotic protein including Bax, Bcl2, PARP, and cleaved Caspase3, and of anti-apoptotic protein Survivin. Cell migration and invasion were assessed using different methods. Signaling pathways such as PI3K/Akt and MAPK, which are involved in the development of glioma cells, were also investigated in this study. RESULTS: Treatment with Osthole markedly inhibits glioma cell proliferation, as assessed by western blot with the PCNA antibody. Osthole also induces cell apoptosis by upregulating the expression of pro-apoptotic proteins, and by reducing the expression of anti-apoptotic factors. Moreover, C6 cell migration and invasion were efficiently inhibited in groups treated with Osthole, compared to the control group. Additionally, inhibition of PI3K/Akt and MAPK signaling pathway was also observed in C6 cells treated with Osthole. CONCLUSIONS: Our findings showed an anti-cancer effect of Osthole on glioma cells, including the proliferation inhibition, apoptosis induction, and migration/invasion inhibition. Further investigation in C6 glioma cells implicated the role of Osthole in essential pathways controlling glioma cell progression. Taken together, our data suggested that Osthole may have a potential application in glioma therapy.

Over-expression of Sox2 in C3H10T1/2 cells inhibits osteoblast differentiation through Wnt and MAPK signalling pathways.

PURPOSE: Many Sox proteins play important roles both in mesoderm and ectoderm development. It is reported that Sox2, a member of this family, is essential for the maintenance of the self-renewal of embryonic stem cells (ES) and neural stem cells (NSCs). To investigate whether Sox2 participates in mesoderm development besides ectoderm, Sox2 was introduced into C3H10T1/2 cells. METHODS: We produced recombinant retrovirus expressing Sox2 in GP2-293t cells and infected the virus into C3H10T1/2 cells. Growth property, alkaline phosphatase (ALP) staining, mineralized nodules, osteogenic gene expression and related signal pathways were analysed and compared between Sox2-expressing cells and control cells. RESULTS: Sox2 over-expression led to increased proliferation of C3H10T1/2 cells, activation of Wnt/ß-catenin and p38MAPK pathways. When cultured in osteogenic differentiation medium, ALP and mineralized nodules formation were inhibited in Sox2 over-expressing cells with down-regulation of osteogenic gene expression as well as inhibition of Wnt/ß-catenin and p38MAPK pathways. CONCLUSIONS: All these data suggested that over-expression of Sox2 promoted proliferation and inhibited osteoblast differentiation of C3H10T1/2 cells.

[Inhibition effect of osthole on proliferation of rat chondrocytes].

OBJECTIVE: To investigate the effects of osthole on chondrocyte proliferation in vitro. METHODS: Primary rat chondrocytes were isolated from the femoral head of newborn rats using collagenase digestion and cultured in Dulbecco's modified Eagle's medium. The proliferation of primary chondrocytes was assessed in second-passage cultures using cell counting kit-8 and the growth curve was drawn. Type II procollagen gene (Col2a1) expression in chondrocytes was also identified using cell immunofluorescence assay. The second-passage chondrocytes were divided into five groups, including control group and osthole groups at 6.25, 12.5, 25 and 50 µmol/L. The growth property of rat chondrocytes was observed after 24, 48 and 72 h of culture with osthole at corresponding dose. Both protein and mRNA expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 was measured by Western blot and polymerase chain reaction methods. RESULTS: The second-passage chondrocytes were viable and showed Col2a1 expression in the cytoplasm. The proliferation of rat chondrocytes was inhibited by osthole in a dose-dependent manner. Meanwhile, there were significant decreases in both protein and mRNA expression of PCNA and cyclin D1 in the osthole groups compared with the control group. CONCLUSION: Osthole exhibits inhibitory effect on proliferation of rat chondrocytes by down-regulating PCNA and cyclin D1 expression.

A comparative analysis of the osteogenic capacity of osteoblasts from newborn and two-week-old rats.

AIM: To compare the proliferation and osteogenic differentiation of osteoblasts between newborn rats (1d group) and two-week-old rats (14d group) and to clarify the mechanism underlying these effects. METHOD: The endogenous expression of osteogenic marker genes was detected by qPCR, including ALP, OCN, Col1a1, and Runx2. The osteoblasts proliferation was evaluated by EdU assay and Western Blotting [PCNA and Cyclin D1]. ALP activities in osteoblasts were detected using a PNPP kit, ALP staining and qPCR. Mineralized nodule formation and intracellular calcium levels were assessed by Alizarin Red staining and calcium colorimetric assay respectively while OCN, Col1a1 and Runx2 levels in osteoblasts were analyzed by immunostaining. Osteogenesis-associated pathways including Wnt/ß-Catenin, Akt/PPAR and Smad were analyzed via Western Blotting. RESULT: Endogenous ALP, OCN, Col1a1, and Runx2 expression levels were significantly higher in osteoblasts from 14d group than those from 1d group. After treatment with osteogenic induction medium, osteoblast proliferation, ALP activity, mineralized nodule formation, and intracellular calcium levels were markedly increased in osteoblasts from 1d group, with similar results also being observed for the expression of OCN, Col1a1, and Runx2. Wnt3a, ß-catenin, p-Akt, p-Smad1/5/8, and p-Smad5 protein levels were also higher in osteoblasts from 1d group relative to those from 14d group, while the expression of PPARγ was lower. CONCLUSION: The superior osteogenic differentiation capacity in osteoblasts was associated with the higher activation levels of Wnt/ß-Catenin, Akt/PPAR and Smad signaling pathways, and the enhanced proliferative activity in osteoblasts from 1d group.

Similarities and differences between rat and mouse chondrocyte gene expression induced by IL-1ß.

BACKGROUND: Osteoarthritis (OA) is the most prevalent degenerative joint disease. In vitro experiments are an intuitive method used to investigate its early pathogenesis. Chondrocyte inflammation models in rats and mice are often used as in vitro models of OA. However, similarities and differences between them in the early stages of inflammation have not been reported. OBJECTIVE: This paper seeks to compare the chondrocyte phenotype of rats and mice in the early inflammatory state and identify chondrocytes suitable for the study of early OA. METHODS: Under similar conditions, chondrocytes from rats and mice were stimulated using the same IL-1ß concentration for a short period of time. The phenotypic changes of chondrocytes were observed under a microscope. The treated chondrocytes were subjected to RNA-seq to identify similarities and differences in gene expression. Chondrocytes were labelled with EdU for proliferation analysis. Cell proliferation-associated proteins, including minichromosome maintenance 2 (MCM2), minichromosome maintenance 5 (MCM5), Lamin B1, proliferating cell nuclear antigen (PCNA), and Cyclin D1, were analysed by immunocytochemical staining, cell immunofluorescence, and Western blots to verify the RNA-seq results. RESULTS: RNA-seq revealed that the expression patterns of cytokines, chemokines, matrix metalloproteinases, and collagen were similar between the rat and mouse chondrocyte inflammation models. Nonetheless, the expression of proliferation-related genes showed the opposite pattern. The RNA-seq results were further verified by subsequent experiments. The expression levels of MCM2, MCM5, Lamin B1, PCNA, and Cyclin D1 were significantly upregulated in rat chondrocytes (P < 0.05) and mouse chondrocytes (P < 0.05). CONCLUSIONS: Based on the findings, the rat chondrocyte inflammation model may help in the study of the early pathological mechanism of OA.

Recent advances in enzyme-related biomaterials for arthritis treatment.

Arthritis is a group of highly prevalent joint disorders, and osteoarthritis (OA) and rheumatoid arthritis are the two most common types. The high prevalence of arthritis causes severe burdens on individuals, society and the economy. Currently, the primary treatment of arthritis is to relieve symptoms, but the development of arthritis cannot be effectively prevented. Studies have revealed that the disrupted balance of enzymes determines the pathological changes in arthritis. In particular, the increased levels of matrix metalloproteinases and the decreased expression of endogenous antioxidant enzymes promote the progression of arthritis. New therapeutic strategies have been developed based on the expression characteristics of these enzymes. Biomaterials have been designed that are responsive when the destructive enzymes MMPs are increased or have the activities of the antioxidant enzymes that play a protective role in arthritis. Here, we summarize recent studies on biomaterials associated with MMPs and antioxidant enzymes involved in the pathological process of arthritis. These enzyme-related biomaterials have been shown to be beneficial for arthritis treatment, but there are still some problems that need to be solved to improve efficacy, especially penetrating the deeper layer of articular cartilage and targeting osteoclasts in subchondral bone. In conclusion, enzyme-related nano-therapy is challenging and promising for arthritis treatment.

Recent Advances in Reactive Oxygen Species (ROS)-Responsive Polyfunctional Nanosystems 3.0 for the Treatment of Osteoarthritis.

Osteoarthritis (OA) is an inflammatory and degenerative joint disease with severe effects on individuals, society, and the economy that affects millions of elderly people around the world. To date, there are no effective treatments for OA; however, there are some treatments that slow or prevent its progression. Polyfunctional nanosystems have many advantages, such as controlled release, targeted therapy and high loading rate, and have been widely used in OA treatment. Previous mechanistic studies have revealed that inflammation and ROS are interrelated, and a large number of studies have demonstrated that ROS play an important role in different types of OA development. In this review article, we summarize third-generation ROS-sensitive nanomaterials that scavenge excessive ROS from chondrocytes and osteoclasts in vivo. We only focus on polymer-based nanoparticles (NPs) and do not review the effects of drug-loaded or heavy metal NPs. Mounting evidence suggests that polyfunctional nanosystems will be a promising therapeutic strategy in OA therapy due to their unique characteristics of being sensitive to changes in the internal environment.

Identification of thrombin as a key regulator of chondrocyte catabolic activity through RNA-Seq and experimental verification.

The present study was designed to explore the relationship between thrombin and catabolic activity in chondrocytes. Primary rat chondrocytes were cultured for 24 h with rat serum (RS), rat plasma (RP), or rat plasma supplemented with thrombin (RPT). RNA-sequencing was then performed. Cell proliferation was analyzed by EdU uptake, CCK-8 assays and protein-protein interaction (PPI) network of proliferation-related genes. Heatmaps were used to visualize differences in gene expression. Gene Ontology (GO) enrichment analyses of up- and down-regulated differentially expressed genes were conducted. Molecular probes were used to label the endoplasmic reticulum in chondrocytes from three treatment groups. Immunofluorescence and Safranin O staining were used to assess type II collagen (Col2a1) expression and proteoglycan synthesis, whereas Lox expression was assessed by immunocytochemistry. The expression of enzymes involved in the synthesis and maturation of extracellular matrix (ECM) components and chemokines were measured by qPCR while matrix metalloproteinases (MMPs) levels were evaluated by Western blotting. Relevant nodules were selected through further PPI network analyses. A total of 727 and 1162 genes were up- and down-regulated based on the Venn diagrams comparison among groups. Thrombin was thus able to promote chondrocyte proliferation and a shift towards fibrotic morphology, while upregulating MMPs and chemokines linked to ECM degradation. In addition, thrombin decreased the enzyme expression involved in the synthesis and maturation of ECM.

The Effect of Manipulation Under Anesthesia for Secondary Frozen Shoulder: A Randomized Controlled Trial.

INTRODUCTION: Manipulation under anesthesia (MUA) is often used for frozen shoulder treatment, but controversy still exists regarding MUA compared with conservative treatment. This research was conducted to compare the outcome between MUA and celecoxib (CLX) in secondary frozen shoulder. METHODS: Patients with secondary frozen shoulder were randomized into two groups, an MUA plus exercise (EX) group and a CLX plus EX group. Clinical outcomes were documented at baseline and at 1 day, 2, 4, and 12 weeks after intervention, including Constant-Murley Score (CMS) for function, Pain Rating Index (PRI) and Present Pain Intensity (PPI) for pain, passive range of motion (ROM) measurements including external rotation, internal rotation, forward flexion, and abduction. Primary outcome was CMS. Secondary outcomes were PRI, PPI, and passive ROM. RESULTS: Sixty-seven patients out of 68 in the MUA group and 66 out of 68 in the CLX group finished the entire study period. There were no significant differences in basic properties of the two groups before intervention. As the primary outcome, CMS changes in the MUA group improved faster than the CLX group. Secondary outcomes, passive ROM, and pain PPI were faster and significant in the MUA group from 1 day after intervention compared with CLX (P < 0.05). At 12 weeks, a statistically significant difference was not observed in the PPI (P > 0.05). A statistically significant difference was not observed in the PRI between groups in 1 day (P > 0.05). For the primary outcome, from 0 to 12 weeks the mean changes in CMS were 44.00 for MUA plus EX (95% CI 43.07-44.93, P < 0.001) and 27.09 for CLX plus EX (26.20-27.98, P < 0.001). The significant difference in improvement appeared from 2 weeks. CONCLUSION: To treat secondary frozen shoulder with MUA, this treatment could achieve better therapeutic effects on improvement of function, pain, and passive ROM than CLX did. CLINICAL TRIAL REGISTRATION: The trial was registered at www.chictr.org.cn , identifier ChiCTR2200060269.

Nanodevices for deep cartilage penetration.

Osteoarthritis (OA) is a degenerative joint disease and is the main cause of chronic pain and functional disability in adults. Articular cartilage is a hydrated soft tissue that is composed of normally quiescent chondrocytes at a low density, a dense network of collagen fibrils with a pore size of 60-200 nm, and aggrecan proteoglycans with high-density negative charge. Although certain drugs, nucleic acids, and proteins have the potential to slow the progression of OA and restore the joints, these treatments have not been clinically applied owing to the lack of an effective delivery system capable of breaking through the cartilage barrier. Recently, the development of nanotechnology for delivery systems renders new ideas and treatment methods viable in overcoming the limited penetration. In this review, we focus on current research on such applications of nanotechnology, including exosomes, protein-based cationic nanocarriers, cationic liposomes/solid lipid nanoparticles, amino acid-based nanocarriers, polyamide derivatives-based nanocarriers, manganese dioxide, and carbon nanotubes. Exosomes are the smallest known nanoscale extracellular vesicles, and they can quickly deliver nucleic acids or proteins to the required depth. Through electrostatic interactions, nanocarriers with appropriate balance in cationic property and particle size have a strong ability to penetrate cartilage. Although substantial preclinical evidence has been obtained, further optimization is necessary for clinical transformation. STATEMENT OF SIGNIFICANCE: The dense cartilage matrix with high-negative charge was associated with reduced therapeutic effect in osteoarthritis patients with deep pathological changes. However, a systematic review in nanodevices for deep cartilage penetration is still lacking. Current approaches to assure penetration of nanosystems into the depth of cartilage were reviewed, including nanoscale extracellular vesicles from different cell lines and nanocarriers with appropriate balance in cationic property and size particle. Moreover, nanodevices entering clinical trials and further optimization were also discussed, providing important guiding significance to future research.

Self-Reported Weather Sensitivity is Associated with Clinical Symptoms and Structural Abnormalities in Patients with Knee Osteoarthritis: A Cross-Sectional Study.

INTRODUCTION: Patients with knee osteoarthritis (KOA) often complain about clinical symptoms affected by weather-related factors. The purpose of the present study was to use cross-sectional analysis to determine whether weather sensitivity was associated with clinical symptoms, as well as structure abnormalities, in KOA patients. METHODS: Data from 80 participants were obtained from the Feng Hans Shi Effects on OA (FHS) study, an OA cohort study initiated in China in 2015. The weather sensitivity of each participant was determined by a self-reported questionnaire. The following measurements were used to assess clinical outcomes: Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) for symptoms, and semi-quantitative Whole-Organ Magnetic Resonance Imaging Score (WORMS) for cartilage defects and marrow abnormalities of magnetic resonance imaging (MRI). Chi-square with Cochran-Armitage test for trend and regression analysis were used to evaluate the associations between weather sensitivity and WOMAC and WORMS of KOA patients. RESULTS: Most of the KOA participants (57.5%) perceived the weather as affecting their knee-joint clinical symptoms. After adjusting for age, gender, and body mass index (BMI), weather sensitivity was not only associated with knee pain [OR = 3.3 (95% CI 1.1, 9.9), P = 0.032], dysfunction [OR = 5.5 (95% CI 1.8, 16.8), P = 0.003], and overall clinical symptoms [OR = 3.3 (95% CI 1.1, 10.2), P = 0.034], but also associated with cartilage defect [OR = 3.1 (95% CI 1.1, 8.5), P = 0.027] and marrow abnormality [OR = 3.0 (95% CI 1.1, 8.1), P = 0.029]. CONCLUSIONS: In KOA patients, weather sensitivity was associated with clinical symptoms and structural abnormalities. Future longitudinal study is warranted for the causal relationship. INFOGRAPHIC.

Intermittent pressure imitating rolling manipulation ameliorates injury in skeletal muscle cells through oxidative stress and lipid metabolism signalling pathways.

BACKGROUND: Traditional Chinese medicine manipulation (TCMM) is often used to treat human skeletal muscle injury, but its mechanism remains unclear due to difficulty standardizing and quantifying manipulation parameters. METHODS: Here, dexamethasone sodium phosphate (DSP) was utilized to induce human skeletal muscle cell (HSkMC) impairments. Cells in a three-dimensional environment were divided into the control normal group (CNG), control injured group (CIG) and rolling manipulation group (RMG). The RMG was exposed to intermittent pressure imitating rolling manipulation (IPIRM) of TCMM via the FX­5000™ compression system. Skeletal muscle damage was assessed via the cell proliferation rate, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and creatine kinase (CK) activity. Isobaric tagging for relative and absolute protein quantification (iTRAQ) and bioinformatic analysis were used to evaluate differentially expressed proteins (DEPs). RESULTS: Higher-pressure IPIRM ameliorated the skeletal muscle cell injury induced by 1.2 mM DSP. Thirteen common DEPs after IPIRM were selected. Key biological processes, molecular functions, cellular components, and pathways were identified as mechanisms underlying the protective effect of TCMM against skeletal muscle damage. Some processes (response to oxidative stress, response to wounding, response to stress and lipid metabolism signalling pathways) were related to skeletal muscle cell injury. Western blotting for 4 DEPs confirmed the reliability of iTRAQ. CONCLUSIONS: Higher-pressure IPIRM downregulated the CD36, Hsp27 and FABP4 proteins in oxidative stress and lipid metabolism pathways, alleviating excessive oxidative stress and lipid metabolism disorder in injured HSkMCs. The techniques used in this study might provide novel insights into the mechanism of TCMM.

MEK1/2 Inhibitor (GDC0623) Promotes Osteogenic Differentiation of Primary Osteoblasts Inhibited by IL-1ß through the MEK-Erk1/2 and Jak/Stat3 Pathways.

OBJECTIVE: We evaluated the effects and mechanisms of GDC0623 on osteogenic differentiation of osteoblasts induced by IL-1ß. Methodology. Osteoblasts were treated with 20 ng/ml IL-1ß and 0.1 µM GDC0623. Cell proliferation levels were evaluated by the cell counting kit 8 (CCK8), EdU assay, and western blotting [proliferating cell nuclear antigen (PCNA) and Cyclin D1]. Osteoblasts were cultured in an osteogenic induction medium for 1-3 weeks after which their differentiations were assessed by alkaline phosphatase (ALP) staining, Alizarin Red staining, calcium concentration, immunocytochemistry staining, real-time quantitative PCR (RT-qPCR), and immunofluorescence staining. The osteogenesis-associated mechanisms were further evaluated by western blotting using appropriate antibodies. RESULTS: Relative to the control group, IL-1ß induced the rapid proliferation of osteoblasts and suppressed their osteogenic differentiations by upregulating the activities of MEK-Erk1/2 as well as Jak-Stat3 pathways and by elevating MMP13 and MMP9 levels. However, blocking of the MEK-Erk1/2 signaling pathway by GDC0623 treatment reversed these effects. CONCLUSION: Inhibition of Jak-Stat3 pathway by C188-9 downregulated the expression levels of MMP9 and MMP13, activated MEK-Erk1/2 pathway, and inhibited osteogenic differentiation.

Fibroblast growth factor 2 contributes to the effect of salidroside on dendritic and synaptic plasticity after cerebral ischemia/reperfusion injury.

Ischemic stroke, a serious neurological disease, is associated with cell death, axonal and dendritic plasticity, and other activities. Anti-inflammatory, anti-apoptotic, promote dendritic and synaptic plasticity are critical therapeutic targets after ischemic stroke. Fibroblast growth factor-2 (FGF2), which is involved in the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/CAMP response element (CRE)-binding protein (CREB) pathway, has been shown to facilitate dendritic and synaptic plasticity. Salidroside (Sal) has been reported to have anti-inflammatory, anti-oxidative, and anti-apoptotic effects; however, the underlying mechanisms of Sal in promoting dendritic and synaptic plasticity remain unclear. Here, the anti-inflammatory, anti-apoptotic, dendritic and synaptic plasticity effects of Sal were investigated in vitro in PC12 cells under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions and in vivo in rats with middle cerebral artery occlusion/reperfusion (MCAO/R). We investigated the role of Sal in promoting dendritic and synaptic plasticity in the ischemic penumbra and whether the FGF2-mediated cAMP/PKA/CREB pathway was involved in this process. The present study demonstrated that Sal could significantly inhibit inflammation and apoptosis, and promote dendritic and synaptic plasticity. Overall, our study suggests that Sal is an effective treatment for ischemic stroke that functions via the FGF2-mediated cAMP/PKA/CREB pathway to promote dendritic and synaptic plasticity.

[Role of TLR4/NF-κB pathway for early change of synovial membrane in knee osteoarthritis rats].

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-1ß, 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-1ß, TNF-α and MMP-13 were higher than that of control group at 21 days after operation(P<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-1ß, TNF-α, ADAMTS-4, MMP-13, PIIINP and HA.

The MEK-ERK1/2 signaling pathway regulates hyaline cartilage formation and the redifferentiation of dedifferentiated chondrocytes in vitro.

The aim of this study was to investigate the role of the mitogen-activated protein kinase kinase-extracellular signal-regulated kinases 1/2 (MEK-ERK1/2) signaling pathway in chondrocyte differentiation and cartilage tissue construction in vitro. Chondrocytes were stimulated with rat serum (RS) and fetal bovine serum (FBS), and chondrocyte phenotypes were investigated microscopically. Chondrocyte proliferation was analyzed using fluorescence activated cell sorting (FACS) and the CCK8 method. Protein and mRNA expressions were assessed by western blot and RT-qPCR. Constructed cartilage tissues were examined by Safranin O-Fast Green FCF staining and immunofluorescence. In contrast to FBS, RS induced rapid dedifferentiation of chondrocytes and decreased type II collagen expression and proteoglycan synthesis. ERK1/2 and type I collagen expression increased during dedifferentiation and decreased during redifferentiation. Increased MEK-ERK1/2 pathway activity resulted in chondrocyte dedifferentiation, and inhibition of ERK1/2 by the inhibitor PD0325901 reversed dedifferentiation and led to redifferentiation. These data suggest strongly that inhibition of MEK-ERK1/2 activation prevents chondrocyte dedifferentiation and fibrocartilage formation.

The Effectiveness of Manual Therapy for Relieving Pain, Stiffness, and Dysfunction in Knee Osteoarthritis: A Systematic Review and Meta-Analysis.

BACKGROUND: Knee osteoarthritis (KOA) is the most common form of arthritis, leading to pain disability in seniors and increased health care utilization. Manual therapy is one widely used physical treatment for KOA. OBJECTIVE: To evaluate the effectiveness and adverse events (AEs) of manual therapy compared to other treatments for relieving pain, stiffness, and physical dysfunction in patients with KOA. STUDY DESIGN: A systematic review and meta-analysis of manual therapy for KOA. METHODS: We searched PubMed, EMBASE, the Cochrane Library, and Chinese databases for relevant randomized controlled trials (RCTs) of manual therapy for patients with KOA from the inception to October 2015 without language restrictions. RCTs compared manual therapy to the placebo or other interventional control with an appropriate description of randomization. Two reviewers independently conducted the search results identification, data extraction, and methodological quality assessment. The methodological quality was assessed by PEDro scale. Pooled data was expressed as standard mean difference (SMD), with 95% confident intervals (CIs) in a random effects model. The meta-analysis of manual therapy for KOA on pain, stiffness, and physical function were conducted. RESULTS: Fourteen studies involving 841 KOA participants compared to other treatments were included. The methodological quality of most included RCTs was poor. The mean PEDro scale score was 6.6. The meta-analyses results showed that manual therapy had statistically significant effects on relieving pain (standardized mean difference, SMD = -0.61, 95% CI -0.95 to -0.28, P = 76%), stiffness (SMD = -0.58, 95% CI -0.95 to -0.21, P = 81%), improving physical function (SMD = -0.49, 95% CI -0.76 to -0.22, P = 65%), and total score (SMD = -0.56, 95% CI -0.78 to -0.35, P = 50%). But in the subgroups, manual therapy did not show significant improvements on stiffness and physical function when treatment duration was less than 4 weeks. And the long-term information for manual therapy was insufficient. LIMITATIONS: The limitations of this systematic review include the paucity of literature and inevitable heterogeneity between included studies. CONCLUSION: The preliminary evidence from our study suggests that manual therapy might be effective and safe for improving pain, stiffness, and physical function in KOA patients and could be treated as complementary and alternative options. However, the evidence may be limited by potential bias and poor methodological quality of included studies. High-quality RCTs with long-term follow-up are warranted to confirm our findings.Key words: Knee osteoarthritis, manual therapy, systematic review.