Long Non-coding RNA SNHG7 Suppresses Inflammation and Apoptosis of Chondrocytes Through Inactivating of p38 MAPK Signaling Pathway in Osteoarthritis.

This study aims to explore the molecular mechanism of LncRNA SNHG7 in Osteoarthritis (OA). Cartilage tissues of OA patients or patients with trauma or amputation were collected. Compared to normal cartilage tissues, SNHG7 was downregulated while miR-324-3p was upregulated in cartilage tissues of OA patients. IL-1ß was used to induce damage to chondrocytes and treatment with IL-1ß reduced SNHG7 expression in OA chondrocytes. In IL-1ß-treated OA chondrocytes, SNHG7 overexpression reduced the levels of TNF-α and IL-6, inhibited cell apoptosis, and increased cell viability. Additionally, the luciferase reporter assay proved that SNHG7 upregulated dual-specificity phosphatase 1 (DUSP1) by sponging miR-324-3p, thereby inactivating the p38 MAPK signaling pathway by regulating the miR-324-3p/DUSP1 axis. Anisomycin (a p38 MAPK activator) enhanced OA chondrocytes inflammation, promoted cell apoptosis, and reduced cell viability; however, this was reversed by SNHG7 overexpression. This study demonstrates that the SNHG7/miR-324-3p/DUSP1 axis suppresses OA chondrocytes inflammation and apoptosis by inhibiting the p38 MAPK signaling pathway. Thus, this study indicates that SNHG7 is a novel target for OA treatment.

The regulatory role and mechanism of lncTUG1 on cartilage apoptosis and inflammation in osteoarthritis.

BACKGROUND: Long-stranded non-coding RNA TUG1 is lowly expressed in osteoarthritic chondrocytes. This study aimed to elucidate the role of TUG1 in osteoarthritic cartilage damage and the underlying mechanisms. METHODS: Combined database analysis, using primary chondrocytes as well as the C28/I2 cell line, was performed by qRT-PCR, Western blotting, and immunofluorescence to determine the expression of TUG1, miR-144-3p, DUSP1, and other target proteins. Dual luciferase reporter gene and RIP to verify direct interaction of TUG1 with miR-144-3-p and miR-144-3-p with DUSP1, Annexin V-FITC/PI double staining to detect apoptosis. CCK-8 to detect cell proliferation. The biological significance of TUG1, miR-144-3p, and DUSP1 was assessed in vitro experiments using siRNA for TUG1, mimic and repressor for miR-144-3p, and overexpression plasmid for DUSP1. In this study, all data were subjected to a t-test or one-way analysis of variance with a p-value < 0.05 as the cutoff. RESULTS: TUG1 expression was closely associated with osteoarthritic chondrocyte damage, and knockdown of TUG1 significantly promoted chondrocyte apoptosis and inflammation. In the present study, we found that TUG1 inhibited chondrocyte apoptosis and inflammation by competitively binding miR-144-3p, deregulating the negative regulatory effect of miR-144-3p on DUSP1, promoting DUSP1 expression, and inhibiting the p38 MAPK signaling pathway. CONCLUSIONS: In conclusion, our study clarifies the role of the ceRNA regulatory network of TUG1/miR-144-3p/DUSP1/P38 MAPK in OA cartilage injury and provides an experimental and theoretical basis for genetic engineering tools to promote articular cartilage repair.

Long noncoding RNA ZBED3-AS1 induces the differentiation of mesenchymal stem cells and enhances bone regeneration by repressing IL-1ß via Wnt/ß-catenin signaling pathway.

Bone regeneration, as a physiological process of bone formation, is regulated by multiple cytokines. Long noncoding RNAs are involved in the progress of bone formation. The present study investigated role by which ZBED3-AS1 acts to control the differentiation of mesenchymal stem cells (MSCs) and bone regeneration. Bioinformatics prediction and dual luciferase reporter gene assay identified putative ZBED3-AS1 binding sites on the 3'-untranslated region of interleukin-1ß (IL-1ß). Then, RNA immunoprecipitation and chromatin immunoprecipitation assays confirmed that ZBED3-AS1 could regulate the expression of IL-1ß by binding to the transcription factor CREB. Notably, ZBED3-AS1 was shown to negatively regulate IL-1ß expression. After model establishment in rats simulating bone injury, MSCs were isolated and delivered with ZBED3-AS1, Si-ZBED3-AS1, Si-IL-1ß, or DKK (inhibitor of Wnt/ß-catenin signaling pathway) to identify their roles in osteogenic differentiation by evaluating MSC colony formation and proliferation. Then, number of mineralized nodules, alkaline phosphatase (ALP) activity and osteocalcin (OCN) expression, and expression of osteogenesis-related genes were determined. Overexpression of ZBED3-AS1 or silencing of IL-1ß was shown to accelerate ectopic osteogenesis, as reflected by increasing the number of mineralized nodules, ALP activity, and OCN expression, and promoting MSC colony formation and proliferation. Additionally, ZBED3-AS1 activated the Wnt/ß-catenin signaling pathway by negatively regulating IL-1ß. IL-1ß inhibited osteogenic differentiation by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, the effect of ZBED3-AS1 and IL-1ß on osteogenic differentiation was confirmed in vivo. Taken together, upregulation of ZBED3-AS1 could restore differentiation of MSCs and enhance bone regeneration via activation of Wnt/ß-catenin signaling pathway by repressing IL-1ß.

CTHRC1 mediates IL­1ß­induced apoptosis in chondrocytes via JNK1/2 signaling.

Osteoarthritis (OA), also known as degenerative joint disease or degenerative arthritis, is characterized by chondrocyte apoptosis. The aim of the present study was to investigate the effects of collagen triple helix repeat containing 1 (CTHRC1) and the c­Jun N­terminal kinase (JNK) 1/2 inhibitor SP600125 on rat chondrocytes cultured in vitro with interleukin (IL)­1ß. Chondrocytes were treated with different doses of IL­1ß and cell viability and CTHRC1 expression were assessed using Cell Counting Kit­8 and western blot assays, respectively. In separate experiments, chondrocytes were treated with CTHRC1­expressing constructs (pLVX­Puro­CTHRC1) and/or SP600125, or IL­1ß with either CTHRC1 short hairpin (sh)RNA constructs (shNRA­CTHRC1) or SP600125. The expression of CTHRC1, B­cell lymphoma (Bcl)­2, Bcl­2­associated X protein (Bax), cleaved caspase­3, poly ADP ribose polymerase (PARP)­1 and matrix metalloproteinase (MMP)­13 was measured using reverse transcription­quantitative polymerase chain reaction and western blotting assays. A Cell Counting Kit­8 assay was performed to examine cell viability. Annexin V/propidium iodide staining and flow cytometry assays were used to detect chondrocyte apoptosis. The expression of JNK1/2 and phosphorylated JNK1/2 was measured using western blotting. CTHRC1 was highly expressed in patients with OA compared with normal controls. IL­1ß treatment (5, 10 and 20 ng/ml) increased the protein expression of CTHRC1 in a dose­dependent manner and decreased the viability of chondrocytes in a time­dependent manner. pLVX­Puro­CTHRC1 mimics the effect of IL­1ß on chondrocyte apoptosis and JNK1/2 activity, and this is reversed by SP600125 treatment. However, transfection with shRNA­CTHRC1 or treatment with SP600125 inhibited IL­1ß­induced cell apoptosis and JNK1/2 activation. These results indicate that CTHRC1 downregulation may protect chondrocytes from IL­1ß­induced apoptosis by inactivating the JNK1/2 pathway.

TRPV4 functions in flow shear stress induced early osteogenic differentiation of human bone marrow mesenchymal stem cells.

Mechanical cues have been shown to induce osteogenic differentiation of bone marrow stromal cells (MSCs). The TRPV4 channel, a Ca2+-permeable membrane ion channel, is implicated in the transduction of external mechanical stimulation into specific intracellular responses in a wide variety of bone cells. However, the role of TRPV4 in transducing and regulating the differentiation of human MSCs in response to flow shear stress (FSS) is unclear. In this study, using FSS and calcium imaging, we demonstrated that FSS activated early osteogenic differentiation, as shown by the early osteogenic differentiation marker osterix (Osx) and alkaline phosphatase (ALP) staining. Increases in intracellular Ca2+ and in the percentage of responding cells were induced by FSS. However, the late osteogenic differentiation marker Ocn and in vitro mineralization were unchanged after FSS stimulation. TRPV4 channels mediated the FSS-induced Ca2+ influx and osteogenic differentiation of MSCs, which were inhibited by a selective TRPV4 blocker HC-067047 and specific Trpv4 siRNA. Ca2+ influx through TRPV4 promoted NFATc1 nuclear localization. These results identify an essential role of TRPV4 in FSS-induced early osteogenic differentiation of human MSCs.

Gremlin-1 suppression increases BMP-2-induced osteogenesis of human mesenchymal stem cells.

Previous research focusing on rodent cells and animal models has demonstrated that gremlin-1 antagonizes bone morphogenetic proteins (BMPs) in order to suppress osteogenesis. However, the impact of gremlin­1 on osteogenesis in human bone marrow-derived mesenchymal stem cells (MSCs) remains unknown. The aim of the present study was to test the effects of gremlin-1 on viability and in vitro BMP-2-induced osteogenic differentiation of human bone marrow­derived mesenchymal stem cells (MSCs). Gremlin­1­specific small interfering RNA (siRNA) inhibited gremlin­1 mRNA and protein expression in human MSCs. The mRNA expression levels of osteoblastic genes were analyzed using reverse transcription-quantitative polymerase chain reaction, and calcification and enzymatic alkaline phosphatase (ALP) activity assessed the BMP­2­induced osteogenic differentiation of human MSCs. The results indicated that gremlin­1 suppression significantly increased human MSC metabolism and DNA content. The expression levels of osteoblastic genes were also significantly increased by gremlin­1 inhibition. In the gremlin­1­inhibited group, enzymatic ALP activity was significantly increased. In addition, due to BMP­2­inducing osteoblasts, gremlin­1 inhibition increased calcium deposits. The present study indicated that gremlin­1 inhibited the cell viability and osteogenic differentiation of human MSCs and that the suppression of gremlin­1 expression suppressed can increase the cell viability and osteogenic differentiation of human MSCs induced by BMP-2.

Effects of five treatment regimens on blood loss and blood transfusion in total knee arthroplasty: a preliminary study in China
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OBJECTIVE: Our study is aimed to explore effects of five treatment regimens on blood loss and blood transfusion rate in total knee arthroplasty (TKA) patients. METHODS: 191 TKA patients were divided into the rivaroxaban, nadroparin, and tranexamic acid groups (n = 37 each) as well as into the affected-limb-position and tourniquet group (n = 40 each). A 3-month follow-up after operation was needed for all patients. The total blood loss, hidden blood loss, and dominant blood loss were recorded, and hemoglobin and red blood cell changes, pain and knee swelling degrees, hospital for special surgery (HSS), and American knee society (KSS) knee scores were observed. RESULTS: When compared with the rivaroxaban, nadroparin, and tourniquet groups, TKA patients' dominant blood loss, hidden blood loss, total blood loss, rate and volume of blood transfusion in the tranexamic acid and affected-limb-position groups were significantly decreased. While 7 days after operation, the hemoglobin and red blood cells in the tranexamic acid and affected-limb-position groups were significantly increased. At 1 month and 3 months after operation, when compared with the rivaroxaban, nadroparin, and tourniquet groups, the HSS and KSS scores in the tranexamic acid and affected-limb-position groups were all increased. In comparison with the rivaroxaban, nadroparin, and tourniquet groups, the D-Dimers after operation in the tranexamic acid and affected-limb-position groups were significantly lower. CONCLUSION: These results demonstrated that for TKA patients, the tranexamic acid and affected-limb-position could obviously reduce the blood loss and blood transfusion rate.
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A predictive model to estimate the pretest probability of metastasis in patients with osteosarcoma.

Osteosarcomas (OSs) represent a huge challenge to improve the overall survival, especially in metastatic patients. Increasing evidence indicates that both tumor-associated elements but also on host-associated elements are under a remarkable effect on the prognosis of cancer patients, especially systemic inflammatory response. By analyzing a series prognosis of factors, including age, gender, primary tumor size, tumor location, tumor grade, and histological classification, monocyte ratio, and NLR ratio, a clinical predictive model was established by using stepwise logistic regression involved circulating leukocyte to compute the estimated probabilities of metastases for OS patients. The clinical predictive model was described by the following equations: probability of developing metastases = ex/(1 + ex), x = -2.150 +  (1.680 × monocyte ratio) + (1.533 × NLR ratio), where is the base of the natural logarithm, the assignment to each of the 2 variables is 1 if the ratio >1 (otherwise 0). The calculated AUC of the receiver-operating characteristic curve as 0.793 revealed well accuracy of this model (95% CI, 0.740-0.845). The predicted probabilities that we generated with the cross-validation procedure had a similar AUC (0.743; 95% CI, 0.684-0.803). The present model could be used to improve the outcomes of the metastases by developing a predictive model considering circulating leukocyte influence to estimate the pretest probability of developing metastases in patients with OS.

Inhibition of the p38-MAPK signaling pathway suppresses the apoptosis and expression of proinflammatory cytokines in human osteoarthritis chondrocytes.

This study aims to investigate the effects of p38-MAPK signaling pathway on the apoptosis and expression of proinflammatory cytokines in human osteoarthritis (OA) chondrocytes. Human articular cartilage specimens were obtained from 57 OA patients and 31 patients with lower extremity traumatic amputations. The expressions of p38-MAPK pathway-related proteins in cartilage tissue were detected by immunochemistry. Cultured chondrocytes isolated from human OA cartilage were assigned into the blank group, the IL-1ß group, the PD (PD980959, ERK pathway inhibitors)+IL-1ß group, the SB (SB203580, p38 pathway inhibitors)+IL-1ß group, and the SP (SP600125, JNK signaling pathway inhibitors)+IL-1ß group. Cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Western blotting was used to detect the expressions of MAPK pathway-related proteins. The mRNA expressions of IL-1, IL-6, and TNF-α were detected by qRT-PCR. The positive rates of p-p38, p-JNK and p-ERK in OA cartilage were higher than those in normal cartilage. Compared with the blank group, cell proliferation rate was decreased, cell apoptotic rate was increased, the mRNA expressions of IL-1, IL-6, TNF-α and the expressions of p-p38, p-JNK and p-ERK were increased in the IL-1ß group, while opposite trends were observed in the PD+IL-1ß, SB+IL-1ß, and SP+IL-1ß groups. Our study provides evidence that inhibition of the p38-MAPK signaling pathway could suppress the apoptosis and expression of proinflammatory cytokines in human OA chondrocytes.