Inhibition of GAB2 expression has a protective effect on osteoarthritis:An in vitro and in vivo study.

Osteoarthritis is a chronic age-related degenerative disease associated with varying degrees of pain and joint mobility disorders. Grb2-associated-Binding protein-2 (GAB2) is an intermediate molecule that plays a role downstream in a variety of signaling pathways, such as inflammatory signaling pathways. The role of GAB2 in the pathogenesis of OA has not been fully studied. In this study, we found that GAB2 expression was elevated in chondrocytes after constructing in vivo and in vitro models of OA. Inhibition of GAB2 by siRNA decreased the expression of MMP3, MMP13, iNOS, COX2, p62, and increased the expression of COL2, SOX9, ATG7, Beclin-1 and LC3II/LC3I. Furthermore, inhibition of GAB2 expression inhibited interleukin-1ß (IL-1ß) -induced mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling. In vivo studies, we found that reduced GAB2 expression effectively delayed cartilage destruction in a mouse model of OA induced by destabilisation of the medial meniscus (DMM). In conclusion, our study demonstrates that GAB2 is a potential therapeutic target for OA.

Association between polymorphisms in vitamin D receptor gene and adolescent idiopathic scoliosis: a meta-analysis.

PURPOSE: This meta-analysis was performed to clarify whether the two single nucleotide polymorphisms (ApaI and BsmI) in vitamin D receptor (VDR) gene conferred susceptibility to adolescent idiopathic scoliosis (AIS). METHODS: A comprehensive literature search in five online databases (PubMed, EMBASE, ISI Web of Science, CNKI, and Wanfang) was performed to identify studies that analyzed the association between VDR gene polymorphisms and risk of AIS. Observational studies met the predetermined inclusion criteria were selected for meta-analysis. The most appropriate genetic model was identified using a genetic model-free approach. Meta-analysis was performed using RevMan 5.3 software. RESULTS: Five eligible studies were included in this meta-analysis, which involved a total of 717 cases and 554 controls. A statistically significant association was observed between BsmI polymorphism and AIS (OR 1.90, 95% CI 1.32, 2.62). In subgroup analysis by ethnicity, the association between BsmI polymorphism and AIS was significant in Asians (OR 2.06, 95% CI 1.56, 2.73) but not in Caucasians (OR 0.70, 95% CI 0.23, 2.19). However, the ApaI polymorphism was not associated with AIS. Moreover, no evidence of association between BMD and the two VDR gene polymorphisms was detected. CONCLUSIONS: Meta-analysis of existing data suggested that BsmI was associated with increased risk of AIS in Asian populations. Nevertheless, further studies with rigorous design and more ethnic groups are encouraged to validate our findings. These slides can be retrieved under Electronic Supplementary Material.

Single-level lumbar pyogenic spondylodiscitis treated with minimally invasive anterior debridement and fusion combined with posterior fixation via Wiltse approach.

The effect and safety of anterior debridement and fusion with a minimally invasive approach combined with posterior fixation via the Wiltse approach were assessed in the single-level lumbar pyogenic spondylodiscitis. Seventeen patients from 2007 to 2009 underwent anterior debridement and fusion with a minimally invasive approach combined with posterior fixation via the Wiltse approach. Postoperative follow-up time was 24-41 months. Data included the patients' general information, microbiology, operative time, intraoperative blood loss, postoperative complications, intervertebral fusion rate, and preoperative and final follow-up scores for American Spinal Injury Association (ASIA) impairment, visual analogue scale (VAS), and Oswestry Disability Index (ODI). Ten patients had undergone a prior spinal invasive procedure, and 7 had hematogenous infection. The infected segments included L1-2, L2-3, L3-4, and L4-5 in 1, 2, 5, and 9 cases, respectively. Thirteen bacterial cultures were positive for Staphylococcus aureus (5 cases), Staphylococcus epidermidis (4), Streptococcus (3), and Escherichia coli (1). The operative time was 213.8±45.6 min, and the intraoperative blood loss was 180.6±88.1 mL. Postoperative complications consisted of urinary retention (2 cases), constipation (3), and deep vein thrombosis (2). On the final follow-up, VAS scores and ODIs were significantly lower than those of preoperation, while the ASIA grades improved. All the cases achieved good intervertebral bony fusion. Anterior debridement and fusion with a minimally invasive approach combined with posterior fixation via the Wiltse approach can successfully treat single-level lumbar pyogenic spondylodiscitis, with less trauma and reliable immobilization. It is a viable option for clinical application.

Total spondylectomy of C2 and circumferential reconstruction via combined anterior and posterior approach to cervical spine for axis tumor surgery.

As a result of the complex anatomy in upper cervical spine, the operative treatment of axis neoplasms is always complicated. Although the procedure for the second cervical vertebra (C2) surgery had been described previously in diverse approaches and reconstruction forms, each has its own limitations and restrictions that usually result in less satisfactory conclusions. The purpose of this study was to evaluate the operation efficacy for axis tumors by using a combined anterior (retropharyngeal) cervical and posterior approach in achieving total resection of C2 and circumferential reconstruction. Eight consecutive C2 tumor patients with mean age of 47.6 years in our institute sequentially underwent vertebra resection and fixation through aforementioned approach from Jan. 2006 to Dec. 2010. No surgical mortality or severe morbidity occurred in our group. In terms of complications, 2 cases developed transient difficulty in swallowing liquids (one of them experienced dysphonia) and 1 developed cerebrospinal fluid leakage (CSFL) that was resolved later. During a mean follow-up period of 31.9 months, the visual analogue scale (VAS) and Japanese orthopedic association (JOA) score revealed that the pain level and neurological function in all patients were improved postoperatively, and there was no evidence of fixation failure and local recurrence. It is concluded that the anterior cervical retropharyngeal approach permits a visible exposure to facilitate the C2 vertebra resection and perform an effective anterior reconstruction at the same time. The custom-made mesh cage applied in our cases can be acted as a firm and convenient implant in circumferential fixation.

Inhibitory effects of high glucose/insulin environment on osteoclast formation and resorption in vitro.

Patients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 µmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 µmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.

Mulberroside A alleviates osteoarthritis via restoring impaired autophagy and suppressing MAPK/NF-κB/PI3K-AKT-mTOR signaling pathways.

Osteoarthritis (OA) is a trauma-/age-related degenerative disease characterized by chronic inflammation as one of its pathogenic mechanisms. Mulberroside A (MA), a natural bioactive withanolide, demonstrates anti-inflammatory properties in various diseases; however, little is known about the effect of MA on OA. We aim to examine the role of MA on OA and to identify the potential mechanisms through which it protects articular cartilage. In vitro, MA improved inflammatory response, anabolism, and catabolism in IL-1ß-induced OA chondrocytes. The chondroprotective effects of MA were attributed to suppressing the MAPK, NF-κB, and PI3K-AKT-mTOR signaling pathways, as well as promoting the autophagy process. In vivo, intra-articular injection of MA reduced the cartilage destruction and reversed the change of anabolic and catabolic-related proteins in destabilized medial meniscus (DMM)-induced OA models. Thus, the study indicates that MA exhibits a chondroprotective effect and might be a promising agent for OA treatment.

Physalin A alleviates intervertebral disc degeneration via anti-inflammatory and anti-fibrotic effects.

Background: The incidence of intervertebral disc degeneration (IVDD) is a common degenerative disease with inflammation, decreased autophagy, and progression of fibrosis as its possible pathogenesis. Physalin A (PA) is a widely studied anti-inflammatory drug. However, its therapeutic effects on IVDD remain unexplored. Therefore, we aimed to explore the therapeutic potential of PA in IVDD progression. Materials and methods: In vivo, we investigated PA bioactivity using a puncture-induced IVDD rat model. IVDD signals and height changes were detected using X-ray, micro-CT, and MRI, and structural and molecular lesions using histological staining and immunohistochemistry of intervertebral disc sections. In vivo, interleukin-1 beta (IL-1ß) and TGF-ß1 were employed to establish inflammation fibrotic nucleus pulposus (NP) cells. The PA effect duration, concentration, influence pathways, and pathological changes in IVDD treatment were elucidated using western blotting, real-time PCR, and immunofluorescence. Results: PA exerted significant effects on IVDD remission due to anti-inflammation, fibrosis reduction, and autophagy enhancement. In vitro, PA improved inflammation by blocking the NF-κB and MAPK pathways, whereas it promoted autophagy via the PI3K/AKT/mTOR pathway and affected fibrotic progression by regulating the SMAD2/3 pathway. Moreover, PA improved the disc degeneration process in IVDD model. Conclusions: PA exhibited significant anti-inflammatory and anti-fibrotic effects and improved autophagy in vivo and in vitro IVDD models, thus effectively relieving IVDD progression, indicating it is a promising agent for IVDD treatment. The translational potential of this article: This study successfully reveals that PA, a natural bioactive withanolide, effectively relieved IVDD progression via inflammation inhibition, fibrosis reduction, and autophagy enhancement, indicating it is a promising agent for IVDD treatment.

A Novel Low Air Pressure-Assisted Approach for the Construction of Cells-Decellularized Tendon Scaffold Complex.

OBJECTIVE: The goal of this study was to develop a decellularized tendon scaffold (DTS) and repopulate it with adipose-derived stem cells (ADSCs) assisted by low air pressure (LP). METHODS: The porcine superficial flexor tendons were processed into the DTSs using a combination of physical, chemical, and enzymatic treatments. The effectiveness of decellularization was verified by histological analysis and DNA quantification. The properties of the DTSs were evaluated by quantitative analysis of biochemical characterization, porosimetry, in vitro biocompatibility assessment, and biomechanical testing. Subsequently, the ADSCs-DTS complexes were constructed via cell injection assisted by LP or under atmospheric pressure. The differences in cell distribution, biomechanical properties, and the total DNA content were compared by histological analysis, biomechanical testing, and DNA quantification, respectively. RESULTS: Histological analysis confirmed that no cells or condensed nuclear materials were retained within the DTSs with widened interfibrillar space. The decellularization treatment resulted in a significant decrease in the content of DNA and glycosaminoglycans, and a significant increase in the porosity. The DTSs were cytocompatible in vitro and did not show reduced collagen content and inferior biomechanical properties compared with the fresh-frozen tendons. The assistance of LP promoted the broader distribution of cells into the adjacent interfibrillar space and cell proliferation in DTSs. The biomechanical properties of the scaffolds were not significantly affected by the recellularization treatments. CONCLUSION: A novel LP-assisted approach for the construction of cells-DTS complex was established, which could be a methodological foundation for further bioreactor and in vitro studies.

Oroxin B alleviates osteoarthritis through anti-inflammation and inhibition of PI3K/AKT/mTOR signaling pathway and enhancement of autophagy.

Background: Osteoarthritis (OA) is a common aging-related degenerative joint disease with chronic inflammation as its possible pathogenesis. Oroxin B (OB), a flavonoid isolated from traditional Chinese herbal medicine, possesses anti-inflammation properties which may be involved in regulating the pathogenesis of OA, but its mechanism has not been elucidated. Our study was the first to explore the potential chondroprotective effect and elucidate the underlying mechanism of OB in OA. Methods: In vitro, primary mice chondrocytes were stimulated with IL-1ß along with or without the administration of OB or autophagy inhibitor 3-methyladenine (3-MA). Cell viability assay was measured with a cell counting kit-8 (CCK-8). The phenotypes of anabolic-related (Aggrecan and Collagen II), catabolic-related (MMP3, MMP13, and ADAMTS5), inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1ß), and markers of related signaling pathways in chondrocytes with different treatment were detected through western blot, RT-qPCR, and immunofluorescent staining. In vivo, the destabilized medial meniscus (DMM) operation was performed to establish the OA mice model. After knee intra-articular injection with OB for 8 weeks, the mice's knee joints were obtained for subsequent histological staining and analysis. Results: OB reversed the expression level of anabolic-related proteins (Aggrecan and Collagen II) and catabolic-related (MMP3, MMP13, and ADAMTS5) in IL-1ß-induced chondrocytes. Mechanistically, OB suppressed the inflammatory response stimulated by IL-1ß, as the inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1ß) markers were downregulated after the administration of OB in IL-1ß-induced chondrocytes. Besides, the activation of PI3K/AKT/mTOR signaling pathway induced by IL-1ß could be inhibited by OB. Additionally, the autophagy process impaired by IL-1ß could be rescued by OB. What's more, the introduction of 3-MA to specifically inhibit the autophagic process impairs the protective effect of OB on cartilage. In vivo, histological staining revealed that intra-articular injection of OB attenuated the cartilage degradation, as well as reversed the expression level of anabolic and catabolic-related proteins such as Aggrecan, Collagen II, and MMP13 induced in DMM-induced OA models. Conclusions: The study verified that OB exhibited the chondroprotective effect by anti-inflammatory, inhibiting the PI3K/AKT/mTOR signaling pathway, and enhancing the autophagy process, indicating that OB might be a promising agent for the treatment of OA.

Endogenous conversion of n-6 to n-3 polyunsaturated fatty acids facilitates the repair of cardiotoxin-induced skeletal muscle injury in fat-1 mice.

In this study, we investigated the beneficial effects of high endogenous levels of n-3 polyunsaturated fatty acids (PUFAs) on skeletal muscle repair and regeneration using a mouse cardiotoxin (CTX, 20 µM/200 µL) -induced gastrocnemius muscle injury model. Transgenic fat-1 mice expressing the Caenorhabditis elegans fat-1 gene, encoding n-3 fatty acid desaturase, showed higher n-3 PUFA levels and lower n-6/n-3 PUFA ratios in gastrocnemius muscle tissues. Hematoxylin and eosin and Masson's trichrome staining of gastrocnemius sections revealed increased muscle fiber size and reduced fibrosis in fat-1 mice on days 7 and 14 after CTX injections. Gastrocnemius muscle tissues from fat-1 mice showed reduced inflammatory responses and increased muscle fiber regeneration reflecting enhanced activation of satellite cells on day 3 after cardiotoxin injections. Gastrocnemius muscle tissues from cardiotoxin-treated fat-1 mice showed reduced levels of pro-apoptotic proteins (Caspase 3 and Bax) and increased levels of anti-apoptotic proteins (Bcl-2 and Survivin). Moreover, eicosapentaenoic acid (EPA) reduced the incidence of apoptosis among cardiotoxin-treated C2C12 mouse myoblasts. These findings demonstrate that higher endogenous n-3 PUFA levels in fat-1 mice enhances skeletal muscle repair and regeneration following cardiotoxin-induced injury.

Physalin A Inhibits MAPK and NF-κB Signal Transduction Through Integrin αVß3 and Exerts Chondroprotective Effect.

Osteoarthritis (OA) is a common articular ailment presented with cartilage loss and destruction that is common observed in the elderly population. Physalin A (PA), a natural bioactive withanolide, exerts anti-inflammatory residences in more than a few diseases; however, little is known about its efficacy for OA treatment. Here, we explored the therapeutic effects and potential mechanism of PA in mouse OA. After the in vitro administration of PA, the expression of inflammation indicators including inducible nitric oxide synthase and cyclooxygenase-2 was low, indicating that PA could alleviate the IL-1ß-induced chondrocyte inflammation response. Moreover, PA reduced IL-1ß-induced destruction of the extracellular matrix by upregulating the gene expression of anabolism factors, including collagen II, aggrecan, and sry-box transcription factor 9, and downregulating the gene expression of catabolic factors, including thrombospondin motif 5 and matrix metalloproteinases. In addition, the chondroprotective effect of PA was credited to the inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Furthermore, in vivo experiments showed that intra-articular injection of PA could alleviate cartilage destruction in a mouse OA model. However, the anti-inflammatory, anabolism enhancing, catabolism inhibiting, and MAPK and NF-κB signaling pathway inhibiting properties of PA on IL-1ß-induced chondrocytes could be reversed when integrin αVß3 is knocked down by siRNA. In conclusion, our work demonstrates that PA exhibits a chondroprotective effect that may be mediated by integrin αVß3. Thus, PA or integrin αVß3 might be a promising agent or molecular target for the treatment of OA.

[Effect of short-hairpin RNA expression vector-mediated osteopontin RNA interference on proliferation and invasion of prostate cancer PC-3 cells].

BACKGROUND AND OBJECTIVE: Studies showed that osteopontin (OPN) regulates cell migration and invasion in a variety of cancers, which associates with the activities of matrix metalloproteinase (MMP)-2 and MMP-9. This study was to investigate the role of OPN in the proliferation and invasion of human prostate cancer PC-3 cells and the possible functions of IgammaB kinase (IKK) in nuclear factor kappa B (NF-kappaB)-mediated signaling pathways. METHODS: OPN short-hairpin RNA (shRNA) recombinant plasmids were transfected into PC-3 cells and different concentrations of IKK inhibitors were used to inhibit the activities of IKKalpha and IKKbeta. The mRNA and protein expression levers of OPN, MMP-2, and MMP-9 were detected by real-time polymerase chain reaction (PCR) and Western blot. Cell cycle was detected by flow cytometry, cell proliferation by MTT assay, and cell invasion by Transwell chamber assay. RESULTS: Compared with untreated cells, the protein levers of OPN, MMP-2, and MMP-9 in OPN shRNA-transfected PC-3 cells were reduced by 55.22%, 51.71%, and 28.35%, respectively, and the ability of cell migration and invasion were decreased by 45.48% and 51.96%, respectively (P<0.05). Moreover, the inhibition of IKKbeta inhibited the expressions of MMP-2 and MMP-9. CONCLUSION: A shRNA expression vector-mediated OPN gene silencing can inhibit the malignant biological behaviors of PC-3 cells. IKKbeta may play a crucial role in the OPN-induced activation of MMP-2 and MMP-9 via NF-kappaB-mediated IkappaB/IKKbeta pathways.

[One-stage total en bloc spondylectomy by anterior and posterior approaches for lumbar vertebral tumors].

OBJECTIVE: To investigate the surgical results of one stage total en bloc spondylectomy (TES) by anterior and posterior approaches for lumbar vertebral tumors and evaluate its benefit for these tumors. METHODS: A total of 21 patients with the lumbar vertebral tumor treated with on stage TES by posterior and anterior approaches from April 2003 to August 2007 were reviewed, which included 14 males and 7 females with an average age of 47.6 years. Thirteen patients were suffered with the primary lumbar vertebral tumors and 8 patients were diagnosed for the lumbar vertebral metastasis tumors. There were 8 of S3, 3 of I A and 2 of II according to Ennekinng stage system. And there were 1 of Grade B, 4 of Grade C, 8 of Grade D, and 6 of Grade E according to Frankel grade system. The spinal reconstruction was obtained by titanium mesh filled with autograft for benign and low-grade malignant tumors and methylmethacrylate-filled titanium mesh for malignant tumors. The spinal stability was enhanced by posterior internal fixation with rod-screw system. RESULTS: The operation time was 250 min and bleed loss was 2100 ml on average. The follow-up period lasted from 1.0 to 5.5 years. All cases had pain before operation, among which 14 cases obtained complete relief and 7 cases obtained partly relief after operation. In all cases with neurological deficit, they improved neurologically by more than one grade using the Frankel grading system. Up to now, 1 patient had be local recurrence after operation and 4 patients were dead on the following time. The others still are alive and no local recurrence. CONCLUSION: One-stage TES by anterior and posterior approaches for lumbar vertebral tumor is feasible, safe and effective to lumbar vertebral tumor resection and stability reconstruction, which has many advantages such as controlling local recurrence, spinal cord decompression thoroughly, relieving the pain, improving the life quality and prolonging the lifetime.

[Treatment of adolescent idiopathic scoliosis with posterior simultaneous correction by bilateral corrective rod on the convex and concave sides].

OBJECTIVE: To evaluate the efficacy and clinical value of the strategy of posterior simultaneous correction by bilateral corrective rod on the convex and concave sides in the treatment of adolescent idiopathic scoliosis (AIS). METHODS: From February 2006 to August 2008, posterior fusion was performed to 48 AIS patients. There were 16 males and 32 females, with an average age at the time of surgery of 17.1 years. Lenke Type I was found in 17 cases, Type II in 9, Type III in 14 and Type IV in 8. There were 27 patients used selective posterior fusion in thoracic, 21 cases without selective fusion. Observation index: the Cobb angle on coronal plane, translation and rotation of apical vertebrae, the coronal balance, the Cobb angle on sagittal plane, obliquity between lowest instrumented vertebrae (LIV) and the pelvis, intervertebral angle and rotation of the LIV. The patients were followed up at an average time of 15.1 months (12-27 months). RESULTS: In the 27 cases with selective fusion, thoracic coronal Cobb angle was (17 + or - 8) degrees after the operation, with an average correction rate of (76 + or - 11)% at final follow up. The lumbar Cobb angle was (13 + or - 7) degrees after the operation, with an average correction rate of (72 + or - 9)% at final follow up. In the 21 cases without selective fusion, the thoracic Cobb angle was (20 + or - 7) degrees after the operation, with an average correction rate of (74 + or - 15)% at final follow up. The lumbar Cobb angle was (16 + or - 8) degrees after the operation, with an average correction rate of (69 + or - 9)% at final follow up. The average number of vertebrae retained below LIV was 4.4. There was 1 case developing thoracolumbar kyphosis. During the follow up, there were no major complication of neurological injury, no pseudarthrosis and no spine decompensation. CONCLUSION: Posterior bilateral segmental pedicle screw simultaneous correction technique as a technique for correcting thoracic and lumbar curves scoliosis can improve the treatment of idiopathic scoliosis with fewer vertebral fusion and complications.

Comprehensive proteomic analysis of exosomes derived from human bone marrow, adipose tissue, and umbilical cord mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cell (MSC)-derived exosomes have shown comprehensive application prospects over the years. Despite performing similar functions, exosomes from different origins present heterogeneous characteristics and components; however, the relative study remains scarce. Lacking of a valuable reference, researchers select source cells for exosome studies mainly based on accessibility and personal preference. METHODS: In this study, exosomes secreted by MSCs derived from different tissues were isolated, by ultracentrifugation, and proteomics analysis was performed. A total of 1014 proteins were detected using a label-free method. RESULTS: Bioinformatics analysis revealed their shared function in the extracellular matrix receptor. Bone marrow MSC-derived exosomes showed superior regeneration ability, and adipose tissue MSC-derived exosomes played a significant role in immune regulation, whereas umbilical cord MSC-derived exosomes were more prominent in tissue damage repair. CONCLUSIONS: This study systematically and comprehensively analyzes the human MSC-derived exosomes via proteomics, which reveals their potential applications in different fields, so as to provide a reference for researchers to select optimal source cells in future exosome-related studies.

Differentiation of rat adipose tissue-derived mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro.

OBJECTIVE: To differentiate rat adipose tissue-derived mesenchymal stem cells (ADSCs) into cells with a nucleus pulposus-like phenotype in vitro, so as to lay a foundation for the cell-based transplantation therapy of degenerated intervertebral discs. METHODS: Rat ADSCs were isolated only from the subcutaneous inguinal region and purified by limited dilution. ADSCs of the third passages were analyzed by fluorescence activated cell sorter (FACS) to detect the cell surface markers (Sca-1, CD44, CD45, CD11b). To induce ADSCs towards a nucleus pulposus-like phenotype, ADSCs were immobilized in 3-dimensional alginate hydrogels and cultured in an inducing medium containing transforming growth factor-beta1 (TGF-beta1) under hypoxia (2% O(2)), while control groups under normoxia (21% O(2)) in alginate beads in medium with or without the presence of TGF-beta1. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate phenotypic and biosynthetic activities in the process of differentiation. Meanwhile, Alcian blue staining were used to detect the formation of sulfated glycosaminoglycans (GAGs) in the differentiated cells. RESULTS: The purified ADSCs were fibroblast-like and proliferated rapidly in vitro. The flow cytometry showed that ADSCs were positive for Sca-1 and CD44, negative for CD45 and CD11b. The results of RT-PCR manifested that the gene expressions of Sox-9, aggrecan and collagen II, which were chondrocyte specific, were upregulated in medium containing TGF-beta1 under hypoxia (2% O(2)). Likewise, gene expression of HIF-1a, which was characteristics of intervertebral discs, was also upregulated. Simultaneously, Alcian blue staining exhibited the formation of many GAGs. CONCLUSIONS: The approach in our experiment is a simple and effective way to acquire a large quantity of homogenous ADSCs. Rat ADSCs can be differentiated into nucleus pulposus-like cells. ADSCs may replace bone marrow mesenchymal stem cells as a new kind of seed cells in regeneration of degenerated intervertebral discs using cell transplantation therapy.

Decreased RIPK1 expression in chondrocytes alleviates osteoarthritis via the TRIF/MyD88-RIPK1-TRAF2 negative feedback loop.

Osteoarthritis (OA) is the most common degenerative joint disease and involves the loss of articular cartilage integrity, formation of articular osteophytes, remodeling of subchondral bone, and synovitis. Knockdown of receptor interacting serine/threonine kinase (RIPK) 1 leads to anti-inflammatory and anti-apoptotic effects. However, the involvement of RIPK1 in the pathogenesis of OA is unclear. Here, we evaluated the effect of RIPK1 on chondrocytes and elaborated the underlying molecular mechanism. Knockdown of RIPK1 protected chondrocytes against inflammation and apoptosis induced by interleukin (IL)-1ß in vitro and in vivo. RIPK1 was required for myeloid differentiation primary response 88 (MyD88)- and TIR-domain-containing adapter-inducing interferon b (TRIF)-mediated production of matrix metalloproteinases (MMPs) in OA. Moreover, overexpression of RIPK1 promoted the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2), which blocked the expression and phosphorylation of RIPK1. Upregulation of TRAF2 decreased the expression of TRIF, MyD88, and MMPs in chondrocytes. Furthermore, knockdown of RIPK1 blocked activation of the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) signaling pathways. In summary, knockdown of RIPK1 alleviated OA in a manner mediated by the TRIF/MyD88-RIPK1-TRAF2 negative feedback loop and activation of the NF-κB and JNK signaling pathways.

Peimine suppresses interleukin­1ß­induced inflammation via MAPK downregulation in chondrocytes.

Osteoarthritis (OA) is the most common type of degenerative joint disease and secreted inflammatory molecules serve a pivotal role in it. Peimine has been reported to have anti­inflammatory activity. In order to investigate the potential therapeutic role of Peimine in OA, mouse articular chondrocytes were treated with IL­1ß and different doses of Peimine in vitro. The data revealed that Peimine not only suppressed IL­1ß­induced production of nitric oxide (NO) and prostaglandin E2, but also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase­2 (COX­2). In addition, Peimine inhibited the IL­1ß­induced mRNA expression of matrix metalloproteinase (MMP)­1, MMP­3, MMP­9, MMP­13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)­4 and ADAMTS­5. Furthermore, Peimine inhibited IL­1ß­induced activation of the mitogen­activated protein kinase (MAPK) pathway. The protective effect of Peimine on IL­1ß­treated chondrocytes was attenuated following activation of the MAPK pathway, as demonstrated by the increased expression levels of MMP­3, MMP­13, ADAMTS­5, iNOS and COX­2 compared with the Peimine group. The in vivo data suggested that Peimine limited the development of OA in the mouse model. In general, the data indicate that Peimine suppresses IL­1ß­induced inflammation in mouse chondrocytes by inhibiting the MAPK pathway, suggesting a promising therapeutic role for Peimine in the treatment of OA.

[Differential proteomic analysis and function study of human prostate carcinoma cells with different osseous metastatic tendency].

OBJECTIVE: To investigate the differential protein expression profiles of human prostatic carcinoma cells with different metastatic tendency and to screen the osseous metastasis associated proteins and investigate their function. METHODS: Proteomics and Western blotting were applied to screen and identify the differentially expressed proteins in the prostatic carcinoma cells of different lines: line T3B with high osseous metastasis potential, line P2-4 with high lymphatic metastasis potential, and their common parent cell line PC-3. The eukaryotic expression vector carrying human Pgenesil-1/HMGB1 siRNA was constructed and transfected into T3B cells by Lipofectamine 2000 and the positive clones was screened by G418. Pgenesil-1/HMGB1 siRNA/T3B, Pgenesil-1/T3B, and T3B cells were inoculated into the left ventricles of nude mice. Twelve weeks later the mice were killed. The number of osseous metastatic nodules and osseous metastasis inhibition rate were calculated. The mice metastatic tumor cells were identified by immunohistochemistry. RESULT: Six differential expressed proteins, correlated with cytoskeleton, transcriptional control, cellular metabolism, and phosphorylation were identified by proteomics and Western blotting. The recombinant plasmid Pgenesil-1/HMGB1 siRNA was successfully constructed. The HMGB1 expression of the T3B cells transfected with Pgenesil-1/HMGB1 siRNA was significantly lower than those of the other 2 groups (both P <0.05). The number of osseous metastatic nodules of the mice inoculated with Pgenesil-1/HMGB1 siRNA/T3B was significantly less than those of the other 2 groups (both P < 0.05). The metastatic osseous tumor cells were identified as the human prostatic carcinoma cells. CONCLUSION: Osseous metastasis associated proteins exist in prostatic carcinoma cells of the line with high osseous metastasis potential. HMGB1 is closely related to the osseous metastasis of human prostatic carcinoma cells. siRNA targeting HMGB1 specifically suppresses the expression of HMGB1 gene in the human prostatic carcinoma cells with high osseous metastasis potential and effectively inhibits the osseous metastasis.

Association between polymorphism rs12722 in COL5A1 and musculoskeletal soft tissue injuries: a systematic review and meta-analysis.

The rs12722 polymorphism in COL5A1 gene has been implicated in the etiology of musculoskeletal soft tissue injuries in several association studies with limited sample size and conflicting results. The purpose of the present systematic review and meta-analysis was to evaluate and synthesize the currently available data on the association between rs12722 and musculoskeletal soft tissue injuries. Five electronic databases including Pubmed, EMBASE, ISI Web of Science, CNKI and Wanfang were searched to identify relevant studies published before 15 May, 2017. Summary odds ratios (ORs) and corresponding 95% confidence intervals (95% CIs) were estimated using the RevMan 5.3 software. Nine studies comprising 1140 cases and 1410 healthy controls met the eligibility criteria. Recessive model was confirmed to be the optimum model (TT vs TC + CC). The results indicated that rs12722 SNP was significantly associated with musculoskeletal soft tissue injuries (OR 1.58, 95% CI 1.33, 1.89; P < 0.00001). When stratified by injury sites, modest but statistically significant association was found in Achilles tendon pathology (ATP), anterior cruciate ligament injuries (ACLI) and tennis elbow (TE). Subgroup-analysis by ethnicity suggested that TT genotype of rs12722 was associated with tendon and ligament injuries in Caucasians (OR 1.59, 95% CI 1.33, 1.90; P < 0.00001) but not in Asians (OR 1.46, 95% CI 0.46, 4.60; P = 0.52). Our findings indicated that rs12722 of COL5A1 was positively associated with tendon and ligament injuries, especially in Caucasian subjects. Individuals with TT genotype were predisposed to higher risk of ATP, ACLI and TE.