Nanwei Xu

Follistatin-like protein 1 is elevated in systemic autoimmune diseases and correlated with disease activity in patients with rheumatoid arthritis

IntroductionFollistatin-like protein 1 (FSTL1) is a proinflammation mediator implicated in arthritis in rodent animal models. The present study is aimed at assessing FSTL1 levels in systemic autoimmune diseases and correlating them with disease activity in patients with rheumatoid arthritis (RA).MethodsSerum FSTL1 levels from 487 patients with systemic autoimmune diseases and 69 healthy individuals were measured by enzyme-linked immunosorbent assay (ELISA). FSTL1 expression in synovial fluid (SF) and synovial tissues (STs) was determined by ELISA, immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and western blot analysis in RA patients and trauma controls. FSTL1 levels in fibroblast-like synoviocytes (FLSs) from RA patients were determined by real-time PCR and western blot analysis.ResultsSerum FSTL1 levels were significantly elevated in patients with RA, ulcerative colitis, systemic lupus erythematosus, Sjögrens syndrome (SS), systemic sclerosis and polymyositis/dermatomyositis. Serum FSTL1 levels in the RA and secondary SS patients were substantially higher than those in other patients. Serum FSTL1 levels were increased in early RA, rheumatoid factor (RF)- and anti-cyclic citrullinated peptide antibody (ACPA)-negative patients compared to healthy controls. Moreover, serum FSTL1 concentrations were significantly higher in long-standing RA patients than in early RA patients and in the RF- and ACPA-positive RA patients than in RF- and ACPA-negative RA patients. Elevated FSTL1 levels in the STs and SF of RA patients were also observed. FSTL1 levels in serum were markedly higher than those in SF in RA patients. The strongest FSTL1 staining was detected in the cytoplasm of synovial and capillary endothelial cells from RA synovium. Furthermore, FSTL1 was induced in FLSs by inflammatory mediators. Importantly, serum FSTL1 levels were correlated with several important biologic and clinical markers of disease activity, including erythrocyte sedimentation rate, C-reactive protein, RF, ACPA, swollen joint count, patient global visual analogue scale score and Disease Activity Score 28 in the adult RA patient population. Notably, serum FSTL1 levels were significantly diminished following successful treatment and clinical improvement.ConclusionsElevated FSTL1 levels reflect not only joint diseases but also inflammation and tissue degradation in systemic autoimmune diseases. Serum FSTL1 levels may thus serve as a serological inflammatory marker of disease activity in RA patients.

Follistatin-like protein 1: a serum biochemical marker reflecting the severity of joint damage in patients with osteoarthritis

IntroductionFollistatin-like protein 1 (FSTL1) is a secreted glycoprotein that has been implicated in arthritis pathogenesis in a mouse model. The aim of this study is to detect FSTL1 expression and to further assess its potential utility as a biomarker of joint damage in osteoarthritis (OA) patients.MethodsFSTL1 expression was detected by real-time PCR, western blot and immunohistochemistry (IHC) in the synovial tissues (STs) and by IHC in the articular cartilage from OA patients and control trauma patients. The serum and synovial fluid (SF) FSTL1 concentrations were measured by ELISA in OA patients and control individuals. Linear regression analyses were used to assess correlations between the serum FSTL1 levels and the clinical characteristics in OA patients.ResultsThe FSTL1 mRNA and protein levels were substantially elevated in the STs from OA patients compared with those from control trauma patients. The FSTL1 expression was strong in the cytoplasm of the synovial and capillary endothelial cells of the STs, but weak in the chondrocytes of the articular cartilage from OA patients. Furthermore, the serum and SF FSTL1 concentrations were significantly higher in OA patients than in respective control subjects. Interestingly, the serum and SF FSTL1 levels were markedly higher in female OA patients than in males. Importantly, bivariate regression analysis revealed that the serum FSTL1 levels in female OA patients had significant correlations with Kellgren and Lawrence (KL) grade, joint space narrowing (JSN) and the Western Ontario McMaster and Universities Osteoarthritis (WOMAC) stiffness subscale, an inverse correlation with height, and marginal correlations with the total WOMAC score and the WOMAC function subscale. Multivariate regression analysis revealed that the serum FSTL1 levels correlated independently with KL grade in female OA patients. Bivariate analysis also revealed that the serum FSTL1 levels correlated significantly with age and disease duration, and they correlated marginally with high sensitivity C-reactive protein (hs-CRP) and KL grade in male OA patients.ConclusionsIncreased FSTL1 expression may be a characteristic of OA patients. FSTL1 is a potential serum biomarker that may reflect the severity of joint damage, and further studies are required to evaluate its potential application for monitoring the course of the disease and the efficacy of therapies in OA patients.

The involvement of follistatin-like protein 1 in osteoarthritis by elevating NF-κB-mediated inflammatory cytokines and enhancing fibroblast like synoviocyte proliferation

IntroductionOur previous work has revealed that expression of follistatin-like protein 1 (FSTL1) is elevated in the synovial tissues from osteoarthritis (OA) patients. The aim of this study was to elucidate the underlying molecular mechanisms by which FSTL1 plays a role in the pathogenesis of OA.MethodsCultured fibroblast-like synoviocytes (FLSs) from synovial tissues of OA patients were stimulated with human recombinant FSTL1, and then the expression of inflammatory cytokines in FLS and their concentrations in the cell supernatants were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Nuclear factor kappa B (NF-κB) activation was examined by western blot and chromatin immunoprecipitation (ChIP) assay at the p65 binding site. Finally, the proliferation of FLSs and the expression level of the proliferation-related tumor suppressors (p53 and p21) were determined by MTS assay kit and western blot in the presence or absence of FSTL1, respectively.ResultsFSTL1 remarkably promoted expression levels of several inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6)) in vitro. Western blot analysis showed that FSTL1 activated the inflammatory-related NF-κB signaling pathway, as validated by ChIP assay detecting p65-binding level on the cytokine promoter region. Moreover, FSTL1 promoted the proliferation of OA FLS by downregulating the expression of p53 and p21. Interestingly, the concentration of synovial fluid IL-6 was remarkably elevated in OA patients, and was correlated with synovial fluid and serum FSTL1 levels.ConclusionsThese findings show that FSTL1 functions as an important proinflammatory factor in the pathogenesis of OA by activating the canonical NF-κB pathway and enhancing synoviocytes proliferation, suggesting that FSTL1 may be a promising target for the treatment of OA.

Polysaccharide from Angelica sinensis protects chondrocytes from H2O2-induced apoptosis through its antioxidant effects in vitro

This study aimed to explore the protective effects of Angelica sinensis polysaccharide (ASP) on rat chondrocyte injury induced by hydrogen peroxide (H2O2). Rat chondrocytes were cultured and treated with different concentrations of ASP alone or in combination with H2O2, and they were measured with cell viability, apoptosis, release of inflammatory cytokines, such as interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), activity of superoxide dismutase (SOD), and catalase (CAT), and levels of malondialdehyde (MDA) production, respectively. In addition, quantitative real-time reverse transcription polymerase chain reaction was used to estimate the relative expression levels of osteoarthritis (OA)-associated genes, such as collagen type II (Col2a1), aggrecan, SOX9, matrix metalloproteinase (MMP)-1, -3, and -9, as well as tissue inhibitor of matrix metalloproteinase (TIMP)-1, respectively. Results indicated that ASP protected chondrocytes from H2O2-induced oxidative stress and subsequent cell injury through its antioxidant, antiapoptotic and anti-inflammatory effects in vitro. Our study suggests that ASP could become a therapeutic supplementation for the treatment of OA.

Periodic Mechanical Stress Activates PKCδ-Dependent EGFR Mitogenic Signals in Rat Chondrocytes via PI3K-Akt and ERK1/2.

Background/Aims: The present study aimed to analyze the mechanisms by which periodic mechanical stress is translated into biochemical signals, and to verify the important role of signaling molecules including phosphatidylinositol-3-kinase (PI3K)-Akt, protein kinase C (PKC), and epidermal growth factor receptor (EGFR) in chondrocyte proliferation. The effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively in recent years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to periodic mechanical stress need further investigation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, GF109203X, Gö6976, rottlerin, and AG1478. They were maintained under static conditions or periodic mechanical stress for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. In the second step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, AG1478, and rottlerin. They were maintained under static conditions or periodic mechanical stress for 1 h prior to Western blot analysis. Results: Proliferation was inhibited by pretreatment with PKC or PKCδ inhibitor GF109203X or rottlerin and by short hairpin RNA (shRNA) targeted to PKCδ, but not by PKCα inhibitor Gö6976 in chondrocytes in response to periodic mechanical stress. Meantime, rottlerin and shRNA targeted to PKCδ also attenuated EGFR, Akt, and ERK1/2 activation. Furthermore, inhibiting EGFR activity by AG1478 and shRNA targeted to EGFR abrogated chondrocyte proliferation and phosphorylation levels of Akt and extracellular signal-regulated kinase (ERK)1/2 subjected to periodic mechanical stress, while the phosphorylation site of PKCδ was not affected. In addition, pretreatment with the PI3K-Akt-selective inhibitor LY294002 and shRNA targeted to Akt reduced periodic mechanical stress-induced chondrocyte proliferation and phosphorylation of ERK1/2, while the phosphorylation levels of EGFR and PKCδ were not inhibited. Conclusion: These findings suggested that periodic mechanical stress promoted chondrocyte proliferation through PKCδ-EGFR-PI3K-Akt-ERK1/2. They provide a stronger viewpoint for further investigations into chondrocyte mechanobiology under periodic mechanical stress and the ways to improve the quality of tissue-engineered cartilage.

SLIT2/ROBO1 axis contributes to the Warburg effect in osteosarcoma through activation of SRC/ERK/c-MYC/PFKFB2 pathway

Cellular metabolic reprogramming is the main characteristic of cancer cells and identification of targets using this metabolic pattern is extremely important to treat cancers, such as osteosarcoma (OS). In this study, SLIT2 and ROBO1 were upregulated in OS, and higher expression of ROBO1 was associated with worse overall survival rate. Furthermore, in vitro and in vivo experiments demonstrated that the SLIT2/ROBO1 axis promotes proliferation, inhibits apoptosis, and contributes to the Warburg effect in OS cells. Mechanistically, the SLIT2/ROBO1 axis exerted cancer-promoting effects on OS via activation of the SRC/ERK/c-MYC/PFKFB2 pathway. Taken together, the findings reveal a previously unappreciated function of SLIT2/ROBO1 signaling in OS, which is intertwined with metabolic alterations that promote cancer progression. Targeting the SLIT2/ROBO1 axis may be a potential therapeutic approach for patients with OS.

MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease with features of inflammatory cell infiltration, synovial cell invasive proliferation, and ultimately, irreversible joint destruction. It has been reported that the p53 pathway is involved in RA pathogenesis. MDM4/MDMX is a major negative regulator of p53. To determine whether MDM4 contributes to RA pathogenesis, MDM4 mRNA and protein expression were assessed in fibroblast-like synoviocytes (FLS) by real-time PCR, western blotting, and in synovial tissues by immunohistochemistry. Furthermore, MDM4 was knocked down and overexpressed by lentivirus-mediated expression, and the proliferative capacity of FLS was determined by MTS assay. We found that cultured FLS from RA and osteoarthritis (OA) patients exhibited higher levels of MDM4 mRNA and protein expression than those from trauma controls. MDM4 protein was highly expressed in the synovial lining and sublining cells from both types of arthritis. Finally, MDM4 knockdown inhibited the proliferation of RA FLS by enhancing functional p53 levels while MDM4 overexpression promoted the growth of RA FLS by inhibiting p53 effects. Taken together, our results suggest that the abundant expression of MDM4 in FLS may contribute to the hyperplasia phenotype of RA synovial tissues.

Exploration about changes of IL-10, NF-κB and MMP-3 in a rat model of cervical spondylosis

Objectives To investigate the relationship and mechanism between IL‐10, NF‐&kgr;B and MMP‐3 in cervical degenerative disease induced by unbalanced dynamic and static forces in rats. Methods Sixty Sprague Dawley rats were randomized into test (n = 45) and control (n = 15) groups, which were randomly subdivided into three groups corresponding to one‐month, three‐month and six‐month post‐operation. Test group included 10, 15, 20 rats at corresponding postoperative stage and control group had five rats at each time point. By excising cervicodorsal muscles and ligaments of rats to establish unbalanced dynamic and static rat model in test group. The expression of IL‐10, NF‐&kgr;B and MMP‐3 in the intervertebral disc tissue samples of both test and control group rats were detected by immunohistochemistry at one‐month, three‐month and six‐month post‐operation. The results were analyzed and compared among groups. Results Compared with the control group, the positive expression of IL‐10 in test group was significantly higher at three‐month (P < 0.05). In the same model group, IL‐10 was highest at one‐month. Compared with the control group, NF‐kB in test group was higher at one‐month, three‐month and six‐month. In the same model group, NF‐kB was the highest at one‐month, followed by the time at three‐month and six‐month. And, compared with the control group, MMP‐3 was significantly higher in test group at one‐month (P < 0.05). Conclusion Cervical degeneration may accompanied with the changes of IL‐10, NF‐&kgr;B and MMP‐3. HighlightsWe successfully established the rat cervical model with non‐equilibrium of dynamic and static forces, histologic evidence of different degrees of cervical disc degeneration was seen in all test group at each time point postoperation.Cervical degeneration may accompanied with the changes of IL‐10, NF‐&kgr;B and MMP‐3.On the basis of our research, we speculate that some IL‐10 were produced in the process of IVD degeneration, which may activate NF‐&kgr;B signaling pathway.Activated NF‐&kgr;B drives expression of target genes that release of IL‐10 to activate the immune response in turn.IL‐10 suggests a dysregulation of aggrecan aseactivity in human degenerative IVD by damaging the balance between MMPs and TIMPs.

A new technique for arthroscopic reduction and fixation of displaced tibial intercondylar eminence fractures, using suture anchor and EndoButton system

Aim: The study aims to describe a less invasive technique for displaced tibial intercondylar eminence fractures, using only one bone tunnel with suture anchor and EndoButton system. Methods: Seventeen patients were followed up after arthroscopic fixation for tibial eminence fractures using suture anchor and EndoButton. The patients were followed with clinical examinations such as Lysholm, Tegner, and International Knee Documentation Committee (IKDC) rating scales. Radiographic assessments were also performed during the follow-up for evaluating the healing of the fracture. Results: There were 14 male patients and 3 female patients with 10 right knees, and 7 left knees included. All the fractures healed anatomically at final follow-up. Also, no clinical signs of anterior cruciate ligament deficiency were detected. The mean Lysholm score improved significantly from 74.72 ± 3.24 (range from 70 to 79) to 96.2 ± 2.54 (range from 89 to 98) (p < 0.001). The IKDC category was abnormal or severely abnormal preoperatively and all the patients improved to normal or nearly normal at final follow-up. The Tegner score also improved significantly postoperatively from 3.45 ± 1.02 to 6.34 ± 1.22 (p < 0.001). Conclusion: The study demonstrated that the procedure is safe and effective, which can be another option for tibial intercondylar eminence fractures.

Microarray Profiling analysis of long non-coding RNAs expression in tendinopathy: identification for potential biomarkers and mechanisms

The role of lncRNAs in pathologies of tendinopathy has not been researched so far, this study aims to identify the role and potent mechanism of lncRNAs in tendinopathy with a bioinformatic analysis. The gene profile of GSE26051 based on the platform of Affymetrix Human Genome U133B Array condensed was downloaded from Gene Expression Omnibus. A total of 46 specimens (including 23 normal samples and 23 tendinopathy specimens) were available. Compared with the control samples, differentially expressed genes (DEGs) of tendinopathy was identified the by packages in R. The selected DEGs were further analysed using bioinformatics methods including co‐expression and enrichment analysis to detect the potential role of lncRNAs. A total of 40 different expressed lncRNAs were identified. However, most of the identified lncRNAs have not been researched before. And this study only annotate one of the identified lncRNAs successfully, the LOC100507027 (myoregulin), with the potential role in regulating skeletal muscle tissue development and skeletal muscle organ development.