UBE2L3 Promotes Oxidative Stress-regulated Necroptosis to Accelerate Osteosarcoma Progression.

BACKGROUND: Osteosarcoma is a highly invasive bone marrow stromal tumor with limited treatment options. Oxidative stress plays a crucial role in the development and progression of tumors, but the underlying regulatory mechanisms are not fully understood. Recent studies have revealed the significant involvement of UBE2L3 in oxidative stress, but its specific role in osteosarcoma remains poorly investigated. OBJECTIVE: This study aimed to explore the molecular mechanisms by which UBE2L3 promotes oxidative stress-regulated necroptosis to accelerate the progression of osteosarcoma using in vitro cell experiments. METHODS: Human osteoblast hFOB1.19 cells and various human osteosarcoma cell lines (MG-63, U2OS, SJSA-1, HOS, and 143B) were cultured in vitro. Plasmids silencing UBE2L3 and negative control plasmids were transfected into U2OS and HOS cells. The cells were divided into the following groups: U2OS cell group, HOS cell group, si-NC-U2OS cell group, si-UBE2L3-U2OS cell group, si-NC-HOS cell group, and si-UBE2L3-HOS cell group. Cell viability and proliferation capacity were measured using the Tunnel method and clonogenic assay. Cell migration and invasion abilities were assessed by Transwell and scratch assays. Cell apoptosis was analyzed by flow cytometry, and ROS levels were detected using immunofluorescence. The oxidative stress levels in various cell groups and the expression changes of necroptosis-related proteins were assessed by PCR and WB. Through these experiments, we aim to evaluate the impact of oxidative stress on necroptosis and uncover the specific mechanisms by which targeted regulation of oxidative stress promotes tumor cell necroptosis as a potential therapeutic strategy for osteosarcoma. RESULTS: The mRNA expression levels of UBE2L3 in human osteosarcoma cell lines were significantly higher than those in human osteoblast hFOB1.19 cells (p <0.01). UBE2L3 expression was significantly decreased in U2OS and HOS cells transfected with si-UBE2L3, indicating the successful construction of stable cell lines with depleted UBE2L3. Tunnel assay results showed a significant increase in the number of red fluorescent-labeled cells in si-UBE2L3 groups compared to si-NC groups in both cell lines, suggesting a pronounced inhibition of cell viability. Transwell assay demonstrated a significant reduction in invasion and migration capabilities of si-UBE2L3 groups in osteosarcoma cells. The clonogenic assay revealed significant suppression of proliferation and clonogenic ability in both U2OS and HOS cells upon UBE2L3 knockdown. Flow cytometry confirmed that UBE2L3 knockdown significantly enhanced apoptosis in U2OS and HOS cells. Immunofluorescence results showed that UBE2L3 silencing promoted oxidative stress levels in osteosarcoma cells and facilitated tumor cell death. WB analysis indicated a significant increase in phosphorylation levels of necroptosis-related proteins, RIP1, RIP3, and MLKL, in both osteosarcoma cell lines after UBE2L3 knockdown. In addition, the expression of necrosis-associated proteins was inhibited by the addition of the antioxidant N-acetylcysteine (NAC). CONCLUSION: UBE2L3 is upregulated in osteosarcoma cells, and silencing of UBE2L3 promotes oxidative stress in these cells, leading to enhanced necroptosis and delayed progression of osteosarcoma.

ADAM19 and TUBB1 Correlate with Tumor Infiltrating Immune Cells and Predicts Prognosis in Osteosarcoma.

BACKGROUND: Osteosarcoma is the most common type of primary malignant bone tumor. INTRODUCTION: This study aimed to explore potential key prognostic genes and their roles in osteosarcoma. METHODS: Three microarray datasets for osteosarcoma were downloaded from the GEO database. Differentially expressed genes (DEGs) were screened by the Limma package. Functional enrichment analysis was performed based on DAVID, GeneMANIA, and Metascape databases. Prognostic value of DEGs was elevated by survival analysis. CIBERSORT was used to assess the infiltrating abundance of 22 immune cells, followed by the Pearson correlation analysis between immune cells and prognosis-related genes. Gene set enrichment analysis and drug-gene interactions prediction were performed for prognosis-related genes. RESULTS: A total of 8 common up-regulated DEGs and 13 common down-regulated DEGs were screened in the GSE36001 and GSE56001 datasets. Enrichment analysis showed these DEGs were implicated in platelet activation, SMAD protein phosphorylation, lymphocyte/leukocyte/T cells activation, and cell migration. Survival analysis indicated that elevated expression of ADAM19 and TUBB1 were associated with a favorable prognosis. CIBERSORT algorithm revealed the higher infiltrating level of CD8 T cells, macrophages M0, and M2 in osteosarcoma. ADAM19 expression positively correlated with naïve B cells and negatively correlated with activated dendritic cells infiltrating abundance. TUBB1 expression positively correlated with gamma delta T cells while negatively correlated with helper follicular T cells infiltrating abundance. A total of 56 drugs were found to target TUBB1. CONCLUSION: ADAM19 and TUBB1 could be prognostic biomarkers in osteosarcoma. Both their expression correlates with tumor infiltrating immune cells. TUBB1 was a multi-drug target that might be a therapeutic target in osteosarcoma.

Single-Cell Transcriptome Analysis Reveals the Importance of IRF1/FSTL1 in Synovial Fibroblast Subsets for the Development of Rheumatoid Arthritis.

Objectives: This study aimed to investigate the potential role of synovial fibroblasts (SFs) in the development of rheumatoid arthritis (RA) to identify potential molecular targets and provide a theoretical basis for the treatment of RA. Methods: GSE109449, a fibroblast transcriptome dataset of synovial tissue from RA and osteoarthritis (OA), were obtained from the GEO database. After standard cell quality control, this single-cell transcriptome data was used to perform routine single-cell analysis processes. After completing dimensionality reduction, clustering, and cell subset identification of fibroblasts, the SCENIC analysis helped calculate the significant gene regulatory networks in fibroblasts and their subsets. From these computed gene regulatory networks, the regulon in which follistatin-like protein 1 (FSTL1) resides was extracted and used to analyze the transcriptional regulatory status of fibroblasts. Finally, the gene set enrichment analysis (GSEA) was used to calculate the respective enriched gene sets of IRF1 and FSTL1. Results: Three SF subgroups were identified from the single-cell transcriptome analysis; SF subset 3 was more abundant in RA than in OA (p < 0.001). From the SCENIC analysis, we obtained 269 regulons and the corresponding gene regulatory networks in SF from the RA datasets. Next, we screened and obtained a regulon-containing FSTL1, where IRF1 was the major transcription factor. The top five regulons in SF subset 3 were TWIST1, MECOM, KLF6, MAFB, and RUNX1. Among the 3 SF subsets, IRF1 regulon was ranked the highest in SF subset 3. Differential analysis of pseudobulk RNA-seq showed that IRF1 was up-regulated in RA compared to OA. Between the three SF subgroups, IRF1 and FSTL1 expression was more up-regulated in SF subset 3 compared to the other two subgroups. Conclusions: IRF1 was found to regulate the invasiveness of SFs by regulating FSTL1, which may influence the disease progression of RA.

Analysis of the function and mechanism of DIRAS1 in osteosarcoma.

BACKGROUND: Osteosarcoma is a prevalent malignant bone tumor with a tendency to metastasize to the lungs. In this study, we intend to detect the function and mechanism of DIRAS family GTPase 1 (DIRAS1) in osteosarcoma cells. METHODS: Expression level of DIRAS1 in osteosarcoma cells was analyzed by western blot. Cell location of DIRAS1 in osteosarcoma cells was detected by immunofluorescence. Small interfering RNAs (siRNA)-DIRAS1 and pcDNA3.1-DIRAS1 were employed to regulate DIRAS1 expression. The malignant behaviors of osteosarcoma cells were examined by cell counting kit-8, colony formation, transwell, and wound healing assays. The expression of related proteins was measured by western blot. ELISA and dot blot assays were used to detect the methylation level of m6A. Rescue assays were performed to detect the function of METTL3/METTL14 and DIRASI on osteosarcoma cells. RESULTS: DIRAS1 was located in the nucleus of osteosarcoma cells. Silencing of DIRAS1 in MG63 cells strengthened the proliferation, invasion and migration abilities, as well as blocked the apoptosis ability. Also, p-ERK expression was regulated by DIRAS1 expression, while p-AKT was not affected. Furthermore, DIRAS1 expression was suppressed by METTL3 or/and METTL14 treatment. Moreover, the inhibitory effect of DIRAS1 overexpression on HOS cells malignant behaviors can be reversed by METTL3 and METTL14 joint treatment. The reduced expression of p-ERK induced by DIRAS1 overexpression can be inversed by METTL3 and METTL14 co-treatment. CONCLUSIONS: Taken together, our findings illustrated that DIRAS1 regulated by METTL3 and METTL14 can obviously modulate the malignant behaviors of osteosarcoma cells by inactivating ERK pathway.

LINC01119 negatively regulates osteogenic differentiation of mesenchymal stem cells via the Wnt pathway by targeting FZD4.

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into diverse cell types under specific conditions. Dysfunction in the osteogenic differentiation of MSCs can result in bone metabolism-related diseases, including osteoporosis. Accumulating evidence has revealed that long non-coding RNA (lncRNAs) play critical regulatory roles during MSC differentiation. METHODS: In the present study, we identified an evolutionarily conserved lncRNA expressed during the osteogenic differentiation of MSCs, which we termed LINC01119. We first identified LINC01119 as a negative regulator of the osteogenic differentiation of MSCs. RESULTS: LINC01119 knockdown markedly induced calcium deposition in bone marrow MSCs and promoted the osteogenic differentiation of MSCs. More importantly, we demonstrated the underlying molecular basis through which LINC01119 regulates osteogenesis via the Wnt pathway by targeting FZD4. Furthermore, we observed that transcription factor EBF3 could directly bind the promoter site of LINC01119. CONCLUSIONS: We first explored the molecular regulatory mechanism of LINC01119 during the osteogenic differentiation of MSCs and revealed that LINC01119 negatively regulates osteogenesis through the Wnt pathway by targeting FZD4.

Bioinformatics integrated analysis to investigate candidate biomarkers and associated metabolites in osteosarcoma.

BACKGROUND: This study hoped to explore the potential biomarkers and associated metabolites during osteosarcoma (OS) progression based on bioinformatics integrated analysis. METHODS: Gene expression profiles of GSE28424, including 19 human OS cell lines (OS group) and 4 human normal long bone tissue samples (control group), were downloaded. The differentially expressed genes (DEGs) in OS vs. control were investigated. The enrichment investigation was performed based on DEGs, followed by protein-protein interaction network analysis. Then, the feature genes associated with OS were explored, followed by survival analysis to reveal prognostic genes. The qRT-PCR assay was performed to test the expression of these genes. Finally, the OS-associated metabolites and disease-metabolic network were further investigated. RESULTS: Totally, 357 DEGs were revealed between the OS vs. control groups. These DEGs, such as CXCL12, were mainly involved in functions like leukocyte migration. Then, totally, 38 feature genes were explored, of which 8 genes showed significant associations with the survival of patients. High expression of CXCL12, CEBPA, SPARCL1, CAT, TUBA1A, and ALDH1A1 was associated with longer survival time, while high expression of CFLAR and STC2 was associated with poor survival. Finally, a disease-metabolic network was constructed with 25 nodes including two disease-associated metabolites cyclophosphamide and bisphenol A (BPA). BPA showed interactions with multiple prognosis-related genes, such as CXCL12 and STC2. CONCLUSION: We identified 8 prognosis-related genes in OS. CXCL12 might participate in OS progression via leukocyte migration function. BPA might be an important metabolite interacting with multiple prognosis-related genes.

ZFAS1 knockdown inhibits fibroblast-like synoviocyte proliferation, migration, invasion and inflammation, and promotes apoptosis via miR-3926/FSTL1 in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by joint disorders. Long non-coding RNA zinc finger antisense 1 (ZFAS1) is aberrantly expressed in numerous human diseases, including RA. The present study aimed to investigate the functions and underlying mechanisms of ZFAS1 in RA. Reverse transcription-quantitative PCR was performed to determine the expression levels of ZFAS1, microRNA (miR)-3926 and follistatin-like protein 1 (FSTL1). MTT assay, flow cytometric analysis and Transwell assay were performed to examine the proliferation, apoptosis, migration and invasion of fibroblast-like synoviocytes (FLSs), respectively. Western blotting was employed to measure the protein expression levels of cleaved caspase-3, interleukin (IL)-6, IL-1ß, tumor necrosis factor-α and FSTL1. Dual-luciferase reporter assay was performed to verify the interaction between miR-3926 and ZFAS1 or FSTL1. The results demonstrated that ZFAS1 and FSTL1 were upregulated, and miR-3926 was downregulated in RA synovial tissues and RA-FLSs. ZFAS1 knockdown suppressed cell proliferation, migration, invasion and inflammatory cytokine production, and induced apoptosis in RA-FLSs. ZFAS1 acted as a sponge for miR-3926, and ZFAS1 overexpression abolished the impact of miR-3926 on the development of RA-FLSs. FSTL1 was a direct target of miR-3926, and the effect of FSTL1 knockdown on the progression of RA-FLSs was rescued by miR-3926 inhibition. Furthermore, ZFAS1 regulated FSTL1 expression levels via sponging miR-3926 in RA-FLSs. In conclusion, ZFAS1 knockdown inhibited RA-FLS proliferation, migration, invasion and inflammatory cytokine production, and induced apoptosis in RA via the miR-3926/FSTL1 axis.

Downregulation of TAF9B by miR-7-5p Inhibits the Progression of Osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is a malignant bone tumor with high metastatic potential. As a regulatory factor of apoptosis, TATA-box binding protein (TBP) associated factor 9B (TAF9B) is rarely studied in tumors. METHODS: We investigated the role and mechanism of TAF9B in OS cells by overexpression and knockdown. CCK8, colony formation, transwell, and flow cytometry analysis were performed to detect proliferation, migration, invasion, and apoptosis. RESULTS: TAF9B overexpression promotes the proliferation, migration, and invasion of OS cells, while TAF9B knockdown gives the opposite result. TAF9B inhibits apoptosis by upregulating Bcl-2 and downregulating Bax and Cleaved-caspase-3. Through starBase analysis, it was found that miR-7-5p can bind to the 3'UTR region of TAF9B, which is further confirmed by the dual luciferase reporter system assay. MiR-7-5p downregulates the expression of TAF9B in MG63 and U2OS cells. The proliferation and invasion of OS cells are inhibited after miR-7-5p mimics transfection and are promoted after miR-7-5p inhibitor transfection. TAF9B rescues the inhibitory effect of miR-7-5p on OS cells. TAF9B also activates the AKT/mTOR signaling pathway. CONCLUSION: According to our results, miR-7-5p inhibits the translation of TAF9B and then suppresses growth and metastasis through the AKT/mTOR signaling pathway in OS cells, thereby indicating the potential value of miR-7-5p and TAF9B as therapeutic targets for human OS.

Immune-related prognostic genes signatures in the tumor microenvironment of sarcoma.

Sarcomas are a heterogeneous group of malignant mesenchymal neoplasms. This study aimed to investigate the immune-related prognostic gene signatures in the tumor microenvironment of sarcoma. The RNA-sequencing data and clinical phenotype data of 260 sarcoma samples and two normal samples were downloaded from The Cancer Genome Atla (TCGA) database. Tumor purity and immune cells infiltration were evaluated by Estimation of Stromal and Immune cells in Malignant Tumors using Expression data (ESTIMATE) deconvolution algorithm. Differentially expressed genes (DEGs) were screened in high vs. low immune score groups. Survival analysis was performed using Kaplan-Meier curve with log-rank test. Tumor infiltrating of immune cells was analyzed by Tumor Immune Estimation Resource (TIMER). High immune score and ESTIMATE score were associated with favorable prognosis. A total of 623 immune DEGs were screened. The majority of these genes (532 genes accounting for 85% of the DEGs) were up-regulated, and these genes were significantly enriched in various immune related biological processed and pathways, such as neutrophil activation, T cell activation, antigen processing and presentation. A total of 146 prognosis-related immune DEGs, and seven hub genes were identified, including B2M, HLA-DRB1, HLA-DRA, HLA-E, LCK, HLA-DPA1, and VAV1. Survival analysis showed that high expression of these genes was associated with a favorable prognosis. There were negative correlations between the expression of these hub genes and tumor purity, while positive correlations between expression of these hub genes and f infiltration levels of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages and dendritic cells. These results help to stratify patients with different immune subtypes and help to design immunotherapy strategies for these patients in sarcoma.

Circ_0001103 alleviates IL-1ß-induced chondrocyte cell injuries by upregulating SIRT1 via targeting miR-375.

BACKGROUND: Osteoarthritis (OA) is a common inflammatory disease characterized by articular cartilage degeneration and injury. Circular RNAs (circRNAs) are widely involved in the development of human diseases, including OA. The objective of this study was to investigate the function and functional mechanism of circ_0001103 in OA. METHODS: Cell model of OA was established by treating chondrocytes with interleukin-1ß (IL-1ß). The expression of circ_0001103, miR-375 and sirtuin 1 (SIRT1) mRNA was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was assessed using cell counting kit-8 (CCK-8) assay. Cell apoptosis was determined using flow cytometry assay. The expression levels of inflammatory factors were quantified by qRT-PCR. The expression of extracellular matrix (ECM) metabolism-related markers, including Collagen Type II Alpha 1 Chain (COL2A1) and A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), was detected by western blot. Predicted target relationship between miR-375 and circ_0001103 or SIRT1 by the bioinformatics tools was validated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: Circ_0001103 was downregulated in OA tissues and IL-1ß-induced chondrocytes. Overexpression of circ_0001103 attenuated IL-1ß-induced chondrocyte apoptosis, inflammatory responses and ECM degradation. MiR-375 was targeted by circ_0001103, and miR-375 could bind to SIRT1. Circ_0001103 overexpression increased the expression of SIRT1 by suppressing miR-375. Rescue experiments suggested that miR-375 restoration reversed the effects of circ_0001103 overexpression, and SIRT1 knockdown overturned the effects of miR-375 inhibition. CONCLUSION: Circ_0001103 governed the miR-375/SIRT1 axis to ameliorate IL-1ß-induced chondrocyte injuries, implying that circ_0001103 was a promising biomarker in OA pathogenesis.

Serotonergically dependent antihyperalgesic and antiallodynic effects of isoliquiritin in a mouse model of neuropathic pain.

Chronic neuropathic pain poses a significant health problem worldwide, for which effective treatment is lacking. The current work aimed to investigate the potential analgesic effect of isoliquiritin, a flavonoid from Glycyrrhiza uralensis, against neuropathic pain and elucidate mechanisms. Male C57BL/6J mice were subjected to chronic constriction injury (CCI) by loose ligation of their sciatic nerves. Following CCI surgery, the neuropathic mice developed pain-like behaviors, as shown by thermal (heat) hyperalgesia in the Hargreaves test and tactile allodynia in the von Frey test. Repetitive treatment of CCI mice with isoliquiritin (p.o., twice per day for two weeks) ameliorated behavioral hyperalgesia to thermal (heat) stimuli and allodynia to tactile stimuli in a dose-dependent fashion (5, 15 and 45 mg/kg). The isoliquiritin-triggered analgesia seems serotonergically dependent, since its antihyperalgesic and antiallodynic actions were totally abolished by chemical depletion of spinal serotonin by p-chlorophenylalanine, whereas potentiated by 5-HTP (a precursor of 5-HT). Consistently, isoliquiritin-treated neuropathic mice showed escalated levels of spinal monoamines especially 5-HT, with depressed monoamine oxidase activity. Moreover, isoliquiritin-evoked antihyperalgesia and antiallodynia were preferentially counteracted by the 5-HT1A receptor antagonist WAY-100635 delivered systematically or spinally. Of notable benefit, isoliquiritin was able to correct co-morbid behavioral symptoms of depression and anxiety evoked by neuropathic pain. Collectively, these findings demonstrate, for the first time, the therapeutic efficacy of isoliquiritin on neuropathic hypersensitivity, and this effect is dependent on the spinal serotonergic system and 5-HT1A receptors.

Identification of microRNA­363­3p as an essential regulator of chondrocyte apoptosis in osteoarthritis by targeting NRF1 through the p53­signaling pathway.

Osteoarthritis (OA) is a degenerative joint disease that affects the physical, and mental health of middle­aged and elderly people. The aims of the present study were to determine the biological function and molecular mechanisms of miR­363­3p in chondrocyte apoptosis. Exploration of the molecular mechanisms of OA may be helpful in the understand of the causes, and facilitating the prevention and treatment of OA. In the present study, the expression of nuclear respiratory factor1 (NRF1) was downregulated in the articular cartilage of OA rats in vivo and lipopolysaccharide (LPS)­treated chondrocytes in vitro. MicroRNAs (miRNA) are regulators of gene expression in the progression of OA. TargetScan software was used to predict that NRF1 was a potential target for miRNA (miR)­363, and this was confirmed in subsequent experiments. The expression of miR­363­3p was negatively correlated with the expression of NRF1, and its expression was significantly upregulated in OA model rats and in LPS­induced chondrocytes compared with the expression in the respective controls. In addition, the overexpression of miR­363­3p increased the levels of interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α in vivo, and was demonstrated to promote chondrocyte injury and apoptosis by Safranin O staining and TUNEL. Moreover, the inhibition of miR­363­3p expression increased the expression of NRF1 and protected chondrocytes from apoptosis in vitro and in vivo, whereas the overexpression of miR­363­3p downregulated NRF1 expression and promoted LPS­induced chondrocyte apoptosis through the p53 pathway in vitro. The results of this study suggested that miR­363­3p­mediated inhibition of NRF1may be associated with chondrocyte apoptosis in OA.

The effect of bone morphogenetic protein 2 composite materials combined with cannulated screws in treatment of acute displaced femoral neck fractures.

The risk of avascular necrosis (AVN) and nonunion after treatment of displaced femoral neck fractures is increased in patients aged <60 years. Therefore we established a new protocol for closed reduction and internal fixation (CRIF) using cannulated screws combined with bone morphogenetic protein 2 (BMP-2) composite materials to treat acute femoral neck fractures.This study enrolled 78 patients with acute femoral neck fractures between April 2014 and September 2016. We treated 46 patients with a mean age of 43.8 years in study group. These patients were treated by CRIF combined with BMP-2 composite materials. In control group, there were 32 patients with a mean age of 42.09 years. The patients were treated by CRIF without BMP-2. The duration between presentation and surgery, operative time, Harris score and complications were recorded.In study group, 43 patients were followed up with an average of 31.3 months. One patient suffered nonunion and three patients presented AVN. In control group, 28 patients were followed up with an average of 32.3 months, the rate of AVN and fracture nonunion were 25% (7/28) and 21.4% (6/28) respectively, significantly higher than those in study group (P < .05).Acute displaced femoral neck fractures can be treated with CRIF and BMP-2 composite materials in a minimally invasive manner. This technique was reproducible and had fewer complications.

Up-regulation of P21-activated kinase 1 in osteoarthritis chondrocytes is responsible for osteoarthritic cartilage destruction.

Osteoarthritis is mainly caused by a degenerative joint disorder, which is characterized by the gradual degradation of articular cartilage and synovial inflammation. The chondrocyte, the unique resident cell type of articular cartilage, is crucial for the development of osteoarthritis. Previous studies revealed that P21-activated kinase-1 (PAK1) was responsible for the initiation of inflammation. The purpose of the present study was to determine the potential role of PAK1 in osteoarthritis. The level of PAK1 expression was measured by Western blot and quantitative real-time PCR in articular cartilage from osteoarthritis model rats and patients with osteoarthritis. In addition, the functional role of aberrant PAK1 expression was detected in the chondrocytes. We found that the expression of PAK1 was significantly increased in chondrocytes treated with osteoarthritis-related factors. Increased expression of PAK1 was also observed in knee articular cartilage samples from patients with osteoarthritis and osteoarthritis model rats. PAK1 was found to inhibit chondrocytes proliferation and to promote the production of inflammatory cytokines in cartilages chondrocytes. Furthermore, we found that PAK1 modulated the production of extracellular matrix and cartilage degrading enzymes in chondrocytes. Results of the present studies demonstrated that PAK1 might play an important role in the pathogenesis of osteoarthritis.

Contralateral monoarthritis exacerbated chronic constriction injury-induced pain hypersensitivity through upregulating inducible nitric oxide synthase.

INTRODUCTION: High comorbidity of osteoarthritis (OA) and neuropathic pain has been reported in aged patients. Evidence shows that central sensitization of pain processing occurs in late-phase OA and may facilitate the development of neuropathic pain. Few studies reveal whether acute monoarthritis (MA) aggravates neuropathic pain on the opposite side of the body from the injury. METHODS: To address whether neuropathic pain is affected by contralateral MA through distinct inflammatory pathway, MA was induced by intra-articular injection of complete Freund's adjuvant (CFA) into the right tibiotarsal joint, and neuropathic pain was established by chronic constriction injury (CCI) of the left sciatic nerve. RESULTS: We observed that MA aggravated mechanical allodynia and thermal hyperalgesia in CCI rats. Furthermore, MA affected the other side of the spinal cord in multiple aspects, including the upregulation of iNOS mRNA and the enhancement of forskolin-induced facilitation of excitatory synaptic transmission in the spinal cord dorsal horn substantia gelatinosa neurons. DISCUSSION: Interestingly, intrathecal injection of 1400W, an antagonist of iNOS, attenuated intensity of pain behaviors in CCI rats with contralateral MA to similar levels in CCI rats without MA, and also normalized the facilitatory effect of forskolin on excitatory synaptic transmission in the spinal cord dorsal horn neurons in contralateral MA rats. Therefore, contralateral MA worsened CCI-induced pain hypersensitivity probably through upregulating iNOS and enhancing the facilitation of synaptic transmission following CCI. CONCLUSION: Inhibiting the iNOS might be a potential therapeutic strategy for concurrent OA and neuropathic pain.

Anti-inflammatory effect of hesperidin enhances chondrogenesis of human mesenchymal stem cells for cartilage tissue repair.

BACKGROUND: Articular cartilage diseases are considered a major health problem, and tissue engineering using human mesenchymal stem cells (MSCs) have been shown as a promising solution for cartilage tissue repair. Hesperidin is a flavonoid extract from citrus fruits with anti-inflammatory properties. We aimed to investigate the effect of hesperidin on MSCs for cartilage tissue repair. MSCs were treated by hesperidin, and colony formation and proliferation assays were performed to evaluate self-renewal ability of MSCs. Alcian blue staining and Sox9 expression were measured to evaluate chondrogenesis of MSCs. Secretion of pro-inflammatory cytokines IFN-γ, IL-2, IL-4 and IL-10, and expression of nuclear factor kappa B (NF-κB) subunit p65 were also assessed. RESULTS: Hesperidin improved self-renewal ability and chondrogenesis of MSCs, inhibited secretion of pro-inflammatory cytokines IFN-γ, IL-2, IL-4 and IL-10, and suppressed the expression of p65. Overexpression of p65 was able to reverse the hesperidin inhibited secretions of pro-inflammatory cytokines, and abolish the enhancing effect of hesperidin on chondrogenesis of MSCs. CONCLUSION: Hesperidin could serve as a therapeutic agent to effectively enhance chondrogenesis of human MSCs by inhibiting inflammation to facilitate cartilage tissue repair.

MiRNA-221 from tissue may predict the prognosis of patients with osteosarcoma.

This study was aimed to investigate the relationship between miR-221 expression and prognosis in patients with osteosarcoma.miR-221 expression in 69 osteosarcoma specimens and corresponding noncancer tissues were characterized by quantitative reverse transcription polymerase chain reaction. The associations of miR-221 expression with clinicopathologic factors and prognosis in patients with osteosarcoma were statistically analyzed.miR-221 expression in patients with osteosarcoma was significantly higher than in the corresponding noncancer tissues (P < .01). miR-221 overexpression was significantly associated with tumor stage, metastatic status, and response to chemotherapy pretreatment. Cox regression analysis revealed that miR-221expression, metastasis, and response to chemotherapy were independent prognostic indicators for osteosarcoma.miR-221 upregulation may predict clinical outcomes in patients with osteosarcoma.

Relationship between osteoporosis and expression of Bcl-2 and CXCL12.

The changes of expression of anti-apoptotic factor B-cell lymphoma 2 (Bcl-2) and chemokine C-X-C motif ligand 12 (CXCL12) in the pathological process of osteoporosis (OP) were investigated, to provide new ideas for the diagnosis and treatment of OP. A total of 60 postmenopausal women who needed to undergo hip replacement surgery were enrolled and divided into osteoporosis group (OP, n=32) and control group (CK, n=28) according to the results of dual-energy X-ray bone density measure; after operation, cancellousbone from the femoral head or femoral neck was removed, and osteoblasts and osteoclasts were isolated and cultured in vitro. The proliferation and apoptosis in the two groups of osteoblasts and osteoclasts were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetry and Annexin V/PI double staining method, respectively. The expression levels of Bcl-2 and CXCL12 mRNA and protein in the two groups of osteoblasts and osteoclasts were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The analysis of cell proliferation and apoptosis showed that compared with the CK group, osteoblast proliferation was significantly inhibited and apoptosis rate was distinctly increased in the OP group, compared with the CK group, osteoclast proliferation was distinctly enhanced and apoptosis rate was remarkably reduced in the OP group. The results of RT-qPCR and western blot analysis displayed that Bcl-2 and CXCL12 mRNA and protein levels in osteoblasts of the OP group were significantly lower than those of the CK group, while mRNA and protein levels of Bcl-2 and CXCL12 in osteoclast of the OP group were distinctly increased compared to those in the CK group. The incidence of OP is closely associated with the bone balance maintained by osteoblasts and osteoclasts, and this mechanism may be achieved by inhibiting osteoblast proliferation and osteoclast apoptosis via regulating Bcl-2 and CXCL12 gene expression changes.

Celastrol attenuates pain and cartilage damage via SDF-1/CXCR4 signalling pathway in osteoarthritis rats.

OBJECTIVES: Celastrol has attracted wide interests for its anticancer and anti-inflammation properties, and studies have demonstrated that celastrol negatively modulates the stromal cell-derived factor-1 (SDF-1) and receptor C-X-C chemokine receptor type 4 (CXCR4) signalling. We aim in this study to investigate the effects of celastrol in osteoarthritis (OA) in vivo and explored the underlying molecular mechanisms. METHODS: We established a monoiodoacetate (MIA)-induced rat OA model and evaluated the joint pain and cartilage damage with or without celastrol treatments. We further assessed the alterations of the SDF-1/CXCR4 pathway and cartilage-specific genes, at both mRNA and protein levels. KEY FINDINGS: Celastrol significantly attenuated the joint pain and cartilage damage induced by MIA in OA rats and suppressed the upregulation of SDF-1/CXCR4 and associated genes caused by MIA injections. Furthermore, MIA induced a decrease in cartilage-specific genes which was also prevented by celastrol treatments. CONCLUSIONS: Celastrol ameliorate OA in vivo as evidenced by the attenuated joint pain and less cartilage damage in OA rats given celastrol treatments, an effect mediated via suppression of the SDF-1/CXCR4 pathway.

Artesunate Decreases ß-Catenin Expression, Cell Proliferation and Apoptosis Resistance in the MG-63 Human Osteosarcoma Cell Line.

BACKGROUND: This study aims to determine the effects of artesunate on proliferation, apoptosis and ß-catenin expression in the human osteosarcoma cell line MG-63. METHODS: MG-63 cells in the logarithmic growth phase were collected and cultured with different concentrations of artesunate (12.5 µg/mL, 25 µg/mL and 50 µg/mL) for 24 h, 48 h and 72 h. The total number of MG-63 cells and the morphological changes were observed under an inverted microscope. The MTT assay was adopted to test the inhibition rate (IR) of cell growth. The apoptosis rate was detected using annexin V/propidium iodide (PI) staining. Cell cycle distribution was identified by flow cytometry (FCM), and the expression levels of ß-catenin, cyclins and cyclin dependent kinases (CDKs) were measured using Western blotting. RESULTS: The results of the MTT assay indicated that artesunate could remarkably inhibit MG-63 cell proliferation compared with the rates in the untreated control group (0 µg/mL artesunate), and the inhibitory effect was dose-dependent. The apoptosis rate of MG-63 cells was elevated as the concentration of artesunate increased, and all the rates were significantly higher than that in the control group. Additionally, as the artesunate concentration increased, the proportion of MG-63 cells in G0/G1 phase gradually declined whereas that of cells in the G2/M and S phases increased. Western blotting confirmed that a higher concentration of artesunate reduced the expression levels of ß-catenin, cyclin A, cyclin D1 and CDK1 and increased the expression levels of cyclin B1; however, artesunate had no impact on CDK2 expression in MG-63 cells. CONCLUSION: These results demonstrated that artesunate can inhibit ß-catenin expression and cell proliferation as well as promote cell apoptosis in MG-63 cells, which indicates that artesunate may serve as a promising drug in the clinical treatment of osteosarcoma.