Dioscin induces osteosarcoma cell apoptosis by upregulating ROS-mediated P38 MAPK signaling.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Many patients with osteosarcoma readily develop resistance to chemotherapy and have an extremely dismal prognosis. Dioscin, a saponin, is known to exhibit potent anticancer activities and induce cellular death of a variety of cancer types. However, the inhibitory effect of dioscin on osteosarcoma cells and its underlying mechanisms have not been fully elucidated. We investigated the responses of human U2-OS and MG63 osteosarcoma cells to dioscin with regard to proliferation, apoptosis, migration, and invasion, and studied the effect of dioscin on MAPK-related proteins by western blot analysis assays. Dioscin inhibited osteosarcoma cell proliferation, migration, and invasion. Moreover, it induced osteosarcoma cell apoptosis via reactive oxygen species (ROS)-dependent apoptotic signaling. N-acetylcysteine, a reactive oxygen species inhibitor, suppressed dioscin-induced apoptosis, indicating that ROS play an essential role in dioscin-induced apoptosis. Western blot analysis assays showed that p38 MAPK was upregulated after dioscin treatment, and that dioscin induced apoptosis by upregulating ROS-mediated p38 MAPK signaling. Our study suggests that dioscin possesses antitumor activities against human osteosarcoma cells, inhibits osteosarcoma cell proliferation, migration and invasion, and induces osteosarcoma cell apoptosis through upregulating ROS-mediated p38 MAPK signaling. This study may provide a new therapeutic strategy and potential clinical applications for the treatment of osteosarcoma.

Galectin-3 deficiency protects lipopolysaccharide-induced chondrocytes injury via regulation of TLR4 and PPAR-γ-mediated NF-κB signaling pathway.

The aim of the present study was to identify the functional role of galectin-3 (Gal-3) in lipopolysaccharide (LPS)-induced injury in ATDC5 cells and to explore the probable molecular mechanisms. Here, we identified that LPS is sufficient to enhance the expression of Gal-3 in ATDC5 cells. In addition, repression of Gal-3 obviously impeded LPS-stimulated inflammation damage as exemplified by a reduction in the release of inflammatory mediators interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α, as well as the production of nitric oxide and prostaglandin E2 (PGE2) concomitant with the downregulation of matrix metalloproteinases (MMP)-13 and MMP-3 expression in ATDC5 cells after LPS administration. Moreover, ablation of Gal-3 dramatically augmented cell ability and attenuated cell apoptosis accompanied by an increase in the expression of antiapoptotic protein Bcl-2 and a decrease in the expression of proapoptotic protein Bax and caspase-3 in ATDC5 cells subjected with LPS. Importantly, we observed that forced expression of TLR4 or blocked PPAR-γ with the antagonist GW9662 effectively abolished Gal-3 inhibition-mediated anti-inflammatory and antiapoptosis effects triggered by LPS. Mechanistically, depletion of Gal-3 prevents the NF-κB signaling pathway. Taken together, these findings indicated that the absence of Gal-3 exerted chondroprotective properties dependent on TLR4 and PPAR-γ-mediated NF-κB signaling, indicating that Gal-3 functions as a protector in the development and progression of osteoarthritis.

Identification of co-expression modules and pathways correlated with osteosarcoma and its metastasis.

BACKGROUND: Osteosarcoma is the most common bone tumor that occurs in children. METHODS: To identify co-expression modules and pathways correlated with osteosarcoma and its clinical characteristics, we performed weighted gene co-expression network analysis (WGCNA) on RNA-seq data of osteosarcoma with 52 samples. Then we performed pathway enrichment analysis on genes from significant modules. RESULTS: A total of 5471 genes were included in WGCNA, and 16 modules were identified. Module-trait analysis identified that a module involved in microtubule bundle formation, drug metabolism-cytochrome P450, and IL-17 signaling pathway was negatively correlated with osteosarcoma and positively correlated with metastasis; a module involved in DNA replication was positively correlated with osteosarcoma; a module involved in cell junction was positively correlated with metastasis; and a module involved in heparin binding negatively correlated with osteosarcoma. Moreover, expression levels in four of the top ten differentially expressed genes were validated in another independent dataset. CONCLUSIONS: Our analysis might provide insight for molecular mechanisms of osteosarcoma.

Stenosing tenosynovitis : Evaluation of percutaneous release with a specially designed needle vs. open surgery.

PURPOSE: The purpose of this study was to evaluate the effectiveness of conventional open surgery and percutaneous release with a specially designed needle for treating stenosing tenosynovitis in terms of cure, relapse and complication rates. METHODS: In this study 89 fingers from 76 patients were randomly assigned and allocated to one of the treatment groups. A total of 37 patients were treated with open surgery in group 1 and 39 patients with percutaneous release using a specially designed needle in group 2. A patient-based 4-inch visual analogue scale (VAS), Quinnell grading (QG), disability of arm shoulder and hand (DASH) score and finger total joint range of motion (FTROM) score were evaluated before treatment and after 7, 30 and 180 days. When finger QG scores were equal or greater than 2 points at follow-up at 180 days this was defined as recurrence.. RESULTS: There were no significant differences between the two groups (P > 0.05) in terms of VAS, DASH and QG scores and the degree of FTROM. At 7 days all the data were significantly different (p < 0.05) compared with preoperative data, 30 days was significantly different (p < 0.05) compared with 7 days while at 180 days no significant differences could be found (p > 0.05) compared with 30 days. The recurrence rate in group 1 was 4.65% and 6.55% in group 2. CONCLUSION: The percutaneous release and open surgery methods displayed similar effectiveness regarding the cure and recurrence of trigger finger disorder. The use of a specially designed needle for release is a safe and reliable method.

Connexin 43 Modulates Osteogenic Differentiation of Bone Marrow Stromal Cells Through GSK-3beta/Beta-Catenin Signaling Pathways.

BACKGROUND/AIMS: Bone marrow stromal cells (BMSCs) are multipotent precursors that give rise to osteoblasts, and contribute directly to bone formation. Connexin 43 (Cx43) is the most ubiquitous gap junction protein expressed in bone cell types, and plays crucial roles in regulating intercellular signal transmission for bone development, differentiation and pathology. However, the precise role and mechanism of Cx43 in BMSCs are less known. Here, we investigate the function of Cx43 in osteogenic differentiation of BMSCs in vitro. METHODS: BMSCs were isolated by whole bone marrow adherent culture. Knock down of Cx43 was performed by using lentiviral transduction of Cx43 shRNA. BMSCs were induced to differentiate by culturing in a-MEM, 10% FBS, 50 µM ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone. Alkaline phosphatase (ALP) activity and alizarin red S staining were used to evaluate osteogenic differentiation in calcium nodules. Target mRNAs and proteins were analyzed by using real-time quantitative PCR (qPCR) and western blotting. RESULTS: Cx43 expression markedly increased during osteogenic differentiation. Osteogenic differentiation was suppressed following lentiviral-mediated knockdown of Cx43 expression, as judged by decreased levels of Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteocalcin (Bglap), Osterix (Osx), alkaline phosphatase (ALP) activity and the number of calcium nodules in response to osteogenic differentiation stimuli. Knock down of Cx43 reduced the level of phosphorylation of GSK-3beta at Ser9 (p-GSK-3beta), resulting in decreased beta-catenin expression and activation. Furthermore, treatment of Cx43-knockdown cells with lithium chloride (LiCl), a GSK-3beta inhibitor, reduced osteogenic differentiation and decreased GSK-3beta levels, as well as partially rescued levels of both total and activated beta-catenin. CONCLUSION: These findings indicate that Cx43 positively modulates osteogenic differentiation of BMSCs by up-regulating GSK-3beta/beta-catenin signaling pathways, suggesting a potential role for Cx43 in determining bone mass and bone mineral density by modulating osteogenesis.

Quercetin triggers apoptosis of lipopolysaccharide (LPS)-induced osteoclasts and inhibits bone resorption in RAW264.7 cells.

AIMS: Quercetin, a flavonoid present in vegetables, has anti-inflammatory properties and potential inhibitory effects on bone resorption. Up to date, the effect of quercetin on lipopolysaccharide (LPS)-induced osteoclastogenesis has not yet been reported. In the current study, we evaluated the effect of quercetin on LPS-induced osteoclast apoptosis and bone resorption. METHODS: RAW264.7 cells were non-treated, treated with LPS alone, or treated with both LPS and quercetin. After treatment, the number of osteoclasts, cell viability, bone resorption and osteoclast apoptosis were measured. The expressions of osteoclast-related genes including tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP9) and cathepsin K (CK) were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of receptor activator of nuclear factor-ĸB (RANK), tumor necrosis factor receptor-associated factor 6 (TRAF6), cyclooxygenase-2 (COX-2), Bax, Bcl-2 and mitogenactivated protein kinases (MAPKs) were measured using Western blotting assays. The MAPK signaling pathway was blocked by pretreatment with MAPK inhibitors. RESULTS: LPS directly promoted osteoclast differentiation of RAW264.7 cells and upregulated the protein expression of RANK, TRAF6 and COX-2; while quercetin significantly decreased the number of LPS-induced osteoclasts in a dose-dependent manner. None of the treatments increased cytotoxicity in RAW264.7 cells. Quercetin inhibited mRNA expressions of osteoclast-related genes and protein levels of RANK, TRAF6 and COX-2 in LPS-induced mature osteoclasts. Quercetin also induced apoptosis and inhibited bone resorptive activity in LPS-induced mature osteoclasts. Furthermore, quercetin promoted the apoptotic signaling pathway including increasing the phosphorylation of p38-MAPK, c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK), and Bax, while inhibited Bcl-2 expression. CONCLUSIONS: Quercetin could supress LPS-induced osteoclast bone resorption through blocking RANK signaling and inhibiting the expression of osteoclast-related genes. Quercetin also promoted LPS-induced osteoclast apoptosis via activation of the MAPK apoptotic signaling pathway. These findings suggest that quercetin could be of potential use as a therapeutic agent to treat bacteria-induced bone resorption.

Retraction: MicroRNA-182 downregulates Wnt/ß-catenin signaling, inhibits proliferation, and promotes apoptosis in human osteosarcoma cells by targeting HOXA9.

[This retracts the article DOI: 10.18632/oncotarget.21167.].

Panax notoginseng saponins promote osteogenic differentiation of bone marrow stromal cells through the ERK and P38 MAPK signaling pathways.

The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. Here, we investigated whether PNS could promote osteogenesis of bone marrow stromal cells (BMSCs) through modulating the MAPK signaling pathways, which are implicated in BMSC osteogenesis. We found that PNS markedly increased the mineralization of BMSCs by alizarin red S assays and stimulate alkaline phosphatase activity of these cells. Additionally, PNS significantly increased the mRNA levels of alkaline phosphatase, core-binding factor a1, and bone sialoprotein while decreasing PPARγ2 mRNA levels. Furthermore, inhibitors of ERK, PD98059, and p38, SB203580 inhibited the osteogenesis-potentiating effects by PNS. PNS stimulated the activation of ERK and p38 as evidenced by increased phosphorylation of these proteins, which was inhibited by PD98059 and SB203580. Our findings indicate that PNS could promote BMSC osteogenesis by activating the ERK and p38 signaling pathways.

Clinical application of the flap based on the distal cutaneous branch of the ulnar artery.

OBJECTIVE: To introduce our experiences of using the flap based on the distal cutaneous branch of the ulnar artery. METHODS: Twenty-four patients sought surgical treatment for soft tissue defects of the hand at our medical institution between January 2003 and December 2008. Fifteen cases had soft tissue defect on the palmar aspect of the hand, and nine cases had soft tissue defects on the dorsal aspect of the hand. The flap based on the distal cutaneous branch of the ulnar artery was performed in all these patients. The size of the flaps ranged from 5 cm to 12 cm in length and from 4 cm to 8 cm in width. RESULTS: Two flaps developed partial necrosis (25-35% of their area). In the other cases, both the donor and recipient sites healed successfully. No patient complained of cold intolerance of the hand or any altered sensation in the forearm. The range of motion of the wrist and hand joints was within normal limits in most cases, with 14 cases with excellent, 8 cases with good, 2 cases with fair, and 0 case with poor results according to the total active motion (TAM) criteria. None of the patients had limitations in activities of daily living. CONCLUSION: Because the flap does not compromise the dominant hand arteries and provides a reliable blood supply, it is a good choice for soft tissue reconstruction of defects in the dorsal and palmar aspects of the hand.

Three kinds of forearm flaps for hand skin defects: experience of 65 cases.

INTRODUCTION: Reverse radial forearm flap has been proven reliable and effective for hand reconstruction. Here we report our experience with the use of reverse forearm flap that does not contain the radial or ulnar artery for reconstruction of hand defects in 65 cases with soft tissue defects of the hand. METHOD: Sixty-five patients who sought surgical treatment for soft tissue defects of the hand at our hospital between January 2003 and December 2008 were included in the study. 39 cases had soft tissue defect on the dorsal aspect of the hand and 26 cases on the palmar aspect of the hand. 65 flaps were performed with the posterior interosseous artery flap in 26 cases, island flap supplied by the distal cutaneous branch of the ulnar artery in 23 cases, and the flap based on distally perforator of the radial artery in sixteen cases with the size of the flaps ranging from 5 to 12 cm in length and from 4 to 8 cm in width. RESULTS: The distal cutaneous branch of the ulnar artery flap showed partial necrosis (25-35% of their area) in two cases. Both the donor and the recipient sites healed successfully in other cases. At 8.4 months of follow up, all patients had insensitivity in recipient sites. No patient complained of cold intolerance, pain, numbness and so on in the forearm and hand. According to the TAM criteria (the total active motion of the finger joint) and DASH (Disability of the Arm, Shoulder, and Hand) score showed that postoperative functions were excellent and symptoms were minor, with no significant differences among the groups (P > 0.05). CONCLUSION: Our results indicated that the reverse forearm flap preserving the radial and ulnar artery is a reliable and effective method to cover skin defects of the hand.

All-trans-retinoic acid suppresses rat embryo hindlimb bud mesenchymal chondrogenesis by modulating HoxD9 expression.

In vertebrates, 5'-Hoxd genes (Hoxd9), which are expressed in the hindlimb bud mesenchyme, participate in limb growth and patterning in early embryonic development. In the present study, We investigated the mechanisms by which ATRA regulates cultured E12.5 rat embryo hindlimb bud mesenchymal cells (rEHBMCs). Following exposure to ATRA over 24 h, mRNA and protein expression levels of HoxD9 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), and western blotting. Flow cytometry was used to detect apoptosis. ATRA inhibited the condensation and proliferation, and promoted the apoptosis rate of the rEHBMCs in a dose-dependent manner. Sox9 and Col2a1 in rEHBMCs were downregulated by ATRA in a dose-dependent manner at both mRNA and protein levels. Similarly, HoxD9 was downregulated by ATRA in a dose-dependent manner, in parallel with the cartilage-specific molecules Sox9 and Col2a1. Both qPCR and western blotting showed that both Shh and Gli3 were downregulated. Overexpression of HoxD9 reversed the effects of ATRA. These results demonstrate that ATRA suppresses chondrogenesis in rEHBMCs by inhibiting the expression of HoxD9 and its downstream protein targets, including Sox9 and Col2a1. This effect may also be correlated with inhibition of the Shh-Gli3 signaling pathway.

Panax notoginseng saponins potentiate osteogenesis of bone marrow stromal cells by modulating gap junction intercellular communication activities.

AIMS: The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. We investigated the effects of PNS on gap junction intercellular communication (GJIC) and osteogenesis-associated genes in rat bone marrow stromal cells (BMSCs). METHODS AND RESULTS: Our MTT assays demonstrated that PNS enhanced BMSC proliferation under basal medium culture in vitro. Alkaline phosphatase (ALP) assays and alizarin Red staining showed that PNS stimulated ALP activity and calcium deposition by BMSCs. Measurement of the traversing of Lucifer yellow through intercellular junctions revealed that PNS significantly stimulated GJIC activities. RT-PCR assays further showed that PNS augmented the increase in the mRNA levels of ALP, core-binding factor a1, and bone sialoprotein while decreasing the mRNA level of PPARγ2. PNS also reduced RANKL levels and increased osteoprotegerin levels. Gap junction inhibitor, 18a-glycyrrhetinic acid, could partially reverse the actions of PNS on BMSCs. CONCLUSIONS: Our findings indicate that PNS could promote osteogenesis of BMSCs by targeting osteogenesis-associated genes, which could be mediated by their actions on GJIC.

Quantitative evaluation in vivo of the degree of differentiation of hindlimb cartilage in a rat clubfoot model.

The Limb Morphogenetic Differentiation Scoring system introduced by Neubert and Barrach in 1977 has been used in drug testing as a measure of the degree of cartilage growth inhibition especially for forelimb in vitro. There is no scoring system to quantify the degree of hindlimb bud cartilage differentiation in vivo. A total of 60 female Sprague-Dawley rats weighing 220-250 g were assigned at random to six control groups and six experimental groups on day 0 of pregnancy. The experimental groups were treated with all-trans-retinoic acid (ATRA). A new limb morphogenetic differentiation scoring system was developed and used to quantify the degree of development of the hindlimb buds from the fetuses at embryonic days E13 to E18. The differentiation of cartilages assessed by the new scoring system showed a statistically significant difference between the experimental group and the control group from E13 to E18 (T-test, p < 0.05). Cartilage growth (the proximodistal length) in the control group increased gradually from E14, reaching its peak at E17, but in the experimental group the growth at E13, E16, E17, and E18 was significantly shorter (p < 0.05). In conclusion, the new limb morphogenetic differentiation scoring system described here can be used to quantify the degree of inhibition of the hindlimb bud development by teratogenic drugs or materials, and morphogenetic differentiation in vivo.

Reliability, Validity, and Responsiveness of the Chinese Version of the Knee Injury and Osteoarthritis Outcome Score (KOOS) in Patients with Anterior Cruciate Ligament Reconstruction in Mainland China.

OBJECTIVE: The aim of this study was to perform a cross-cultural adaption of the KOOS into Chinese and to evaluate its psychometric properties in patients with anterior cruciate ligament reconstruction (ACL reconstruction) in mainland China. DESIGN: A cross-sectional study. SETTING: Patients completed the Chinese version of the KOOS and the SF-36 questionnaire three times. We evaluated the reliability, checked the validity, and assessed the responsiveness. PARTICIPANTS: A total of 42 patients who had undergone ACL reconstruction. MAIN OUTCOME MEASURES: The results of the questionnaire survey. RESULTS: The Chinese version of the KOOS was well accepted, with ideal test-retest reliability and internal consistency. The test-retest reliability was significant, with high ICC values ranging from 0.888 to 0.941. Additionally, we found that the internal consistency was adequate, with Cronbach's alpha coefficient ranging from 0.740 to 0.975. All a priori hypotheses were supported by a high correlation between the KOOS and SF-36. Furthermore, responsiveness was demonstrated since the ES and SRM between subscales following ACL reconstruction was found in the expected pattern. CONCLUSIONS: The Chinese version of the KOOS showed psychometric properties demonstrating acceptable reliability and validity similar to the original version. We conclude that the Chinese version is a reliable and valid instrument for research and clinical assessments of ACL reconstruction patients in mainland China.

Investigating age­induced differentially expressed genes and potential molecular mechanisms in osteosarcoma based on integrated bioinformatics analysis.

Osteosarcoma (OS) is the most common primary bone malignancy. It predominantly occurs in adolescents, but can develop at any age. The age at diagnosis is a prognostic factor of OS, but the molecular basis of this remains unknown. The current study aimed to identify age­induced differentially expressed genes (DEGs) and potential molecular mechanisms that contribute to the different outcomes of patients with OS. Microarray data (GSE39058 and GSE39040) obtained from the Gene Expression Omnibus database and used to analyze age­induced DEGs to reveal molecular mechanism of OS among different age groups (<20 and >20 years old). Differentially expressed mRNAs (DEMs) were divided into up and downregulated DEMs (according to the expression fold change), then Gene Ontology function enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed. Furthermore, the interactions among proteins encoded by DEMs were integrated with prediction for microRNA­mRNA interactions to construct a regulatory network. The key subnetwork was extracted and Kaplan­Meier survival analysis for a key microRNA was performed. DEMs within the subnetwork were predominantly involved in 'ubiquitin protein ligase binding', 'response to growth factor', 'regulation of type I interferon production', 'response to decreased oxygen levels', 'voltage­gated potassium channel complex', 'synapse part', 'regulation of stem cell proliferation'. In summary, integrated bioinformatics was applied to analyze the potential molecular mechanisms leading to different outcomes of patients with OS among different age groups. The hub genes within the key subnetwork may have crucial roles in the different outcomes associated with age and require further analysis.

MicroRNA-182 downregulates Wnt/ß-catenin signaling, inhibits proliferation, and promotes apoptosis in human osteosarcoma cells by targeting HOXA9.

We investigated the mechanisms by which microRNA (miR)-182 promotes apoptosis and inhibits proliferation in human osteosarcoma (OS) cells. Levels of miR-182 and Homeobox A9 (HOXA9) expression were compared between human OS and normal cells. Subjects were divided into OS and normal groups. We analyzed the target relationship of miR-182 and Homeobox A9 (HOXA9). Cells were then assigned into blank, negative control, miR-182 mimics, miR-182 inhibitors, siRNA-HOXA9, or and miR-182 inhibitors + siRNA-HOXA9 groups. Cell function was assayed by CCK-8, flow cytometry and wound healing assay. Additionally, we analyzed OS tumor growth in a xenograft mouse model. Dual-luciferase reporter assays indicated miR-182 directly targets HOXA9. Reverse transcription quantitative PCR and western blotting revealed elevated expression of miR-182, WIF-1, BIM, and Bax, and reduced expression of HOXA9, Wnt, ß-catenin, Survivin, Cyclin D1, c-Myc, Mcl-1, Bcl-xL, and Snail in osteosarcoma cells treated with miR-182 mimic or siRNA-HOXA9 as compared to controls. Osteosarcoma cells also exhibited decreased cell proliferation, migration, and tumor growth, and increased apoptosis when treated with miR-182 mimic or siRNA-HOXA9. Correspondingly, in a xenograft mouse model, osteosarcoma tumor volume and growth were increased when cells were treated with miR-182 inhibitor and decreased by miR-182 mimic or siRNA-HOXA9. These results indicate that miR-182 downregulates Wnt/ß-catenin signaling, inhibits cell proliferation, and promotes apoptosis in osteosarcoma cells by suppressing HOXA9 expression.

Antitumor activity of resveratrol against human osteosarcoma cells: a key role of Cx43 and Wnt/ß-catenin signaling pathway.

Osteosarcoma is a high-grade bone sarcoma with strong invasive ability. However, treatment with traditional chemotherapeutic drugs is limited by low tolerability and side effects. Resveratrol has been reported previously to have selective antitumor effect on various tumor cells while little is known about its effects and underlying mechanism in osteosarcoma biology. In this study, we found that resveratrol inhibits proliferation and glycolysis, induces apoptosis and reduces the invasiveness of U2-OS cells in vitro. After treatment with resveratrol, the expression of related Wnt/ß-catenin signaling pathway target genes, such as ß-catenin, c-myc, cyclin D1, MMP-2 and MMP-9, was downregulated and an increased E-cadherin level was observed as well. Additionally, the dual luciferase assay results also indicated that resveratrol suppressed the activity of Wnt/ß-catenin signaling pathway. Interestingly, we noticed that the expression of connexin 43 (Cx43) increased with the prolongation of resveratrol treatment time. To further investigate the relationship between Cx43 and the Wnt/ß-catenin signaling pathway in osteosarcoma, we used lentiviral-mediated shRNA to knockdown the expression of Cx43. Knockdown of Cx43 activated the Wnt/ß-catenin signaling pathway, promoted proliferation and invasion, and inhibited apoptosis of U2-OS cells. Taken together, our results demonstrate that the antitumor activity of resveratrol against U2-OS cells in vitro occurs through up-regulating Cx43 and E-cadherin, and suppressing the Wnt/ß-catenin signaling pathway. Moreover, Cx43 expression is negatively related to the activity of the Wnt/ß-catenin pathway in U2-OS cells.

All-trans-retinoic acid activates SDF-1/CXCR4/ROCK2 signaling pathway to inhibit chondrogenesis.

Recent studies have indicated that ATRA inhibits chondrogenesis and can lead to congenital clubfoot (CCF). The molecular mechanism of ATRA-induced chondrogenesis is not clear. As RhoA/ROCK and SDF-1/CXCR4 signaling play important molecular roles for a variety of cellular processes, we hypothesized that RhoA/ROCK2 and SDF-1/CXCR4 signaling are involved in ATRA-induced chondrogenesis in rat embryo hind limb bud mesenchymal cells (rEHBMCs). We found that ATRA dose-dependently inhibits proliferation and expression of chondrogenic transcription factors (SOX9 and COL2A1) in rEHBMCs. In contrast, ATRA increases the expression of ROCK2, SDF-1 and CXCR4. Pharmacological inhibition of ROCK signaling and SDF-1/CXCR4 signaling by Y27632 and AMD3100, respectively, resulted in elevated expression of SOX9 and COL2A1. In addition, we found that disturbing SDF-1/CXCR4 signaling by AMD3100 decreases ATRA-induced ROCK2 expression. In vivo studies we also confirm that SOX9 expression of early-stage cartilage progenitors in the proliferative zone and COL2A1 expression in prehypertrophic chondrocytes are decreased in ATRA-treated rat embryo hind limb. Together, these results show that ATRA activates SDF-1/CXCR4/ROCK2 signaling to inhibit chondrogenesis to lead to CCF by suppressing differentiation through down-regulation of SOX9 and COL2A1 expression in rat embryo hind limb bud mesenchymal cells.

Expert consensus for PVCR in severe, rigid and angular spinal deformity treatment: The Kunming consensus.

In the past decades, an increasing number of surgeons started using posterior vertebral column resection (PVCR) to treat severe, rigid and angular spinal deformities. Little high-level evidence is available to guide surgical treatment. The aims of our study were to identify important surgical strategies and key technical points of Chinese experts who have extensive experience in the management of severe, rigid and angular spinal deformities using PVCR, and to standardize and unify the current core concepts. Workgroups of consensus were formed by selecting nationwide representing experts and comprehensive consultations. Eight task forces for major issues were established, then retrieval of literature, collection of expert opinions and writing of review articles were carried out. A modified Delphi process was chosen in round-table forum with three face-to-face meetings. Consensus was reached with items graded more than seven points including: indications and contraindications of PVCR; review PVCR in the evolution of spinal osteotomies; The corrective mechanism and safety of spinal cord; monitoring and responses of spinal cord crisis; characteristics and therapeutic outcome of pulmonary function; management of bleeding during PVCR; relationship of pedicle screw insertion and spinal cord safety; and analysis of non-neurologic complications and prevention strategies. In conclusion, The essential properties regarding PVCR procedure are tightly linked with various factors such as medical and surgical indication, range and level of vertebral column resection, strategies of correction, corrective efficiency and control of neurological risk. PVCR is used mainly for severe, rigid spinal deformity that is not manageable by other osteotomy techniques.

Naringin Stimulates Osteogenic Differentiation of Rat Bone Marrow Stromal Cells via Activation of the Notch Signaling Pathway.

Naringin is a major flavonoid found in grapefruit and is an active compound extracted from the Chinese herbal medicine Rhizoma Drynariae. Naringin is a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of naringin involves the Notch signaling pathway. Rat bone marrow stromal cells (BMSCs) were cultured in osteogenic medium containing-naringin, with or without DAPT (an inhibitor of Notch signaling), the effects on ALP activity, calcium deposits, osteogenic genes (ALP, BSP, and cbfa1), adipogenic maker gene PPARγ2 levels, and Notch expression were examined. We found that naringin dose-dependently increased ALP activity and Alizarin red S staining, and treatment at the optimal concentration (50 µg/mL) increased mRNA levels of osteogenic genes and Notch1 expression, while decreasing PPARγ2 mRNA levels. Furthermore, treatment with DAPT partly reversed effects of naringin on BMSCs, as judged by decreases in naringin-induced ALP activity, calcium deposits, and osteogenic genes expression, as well as upregulation of PPARγ2 mRNA levels. These results suggest that the osteogenic effect of naringin partly involves the Notch signaling pathway.