Excessive mechanical stretch­mediated osteoblasts promote the catabolism and apoptosis of chondrocytes via the Wnt/ß­catenin signaling pathway.

Excessive biomechanical loading is considered an important cause of osteoarthritis. Although the mechanical responses of chondrocytes and osteoblasts have been investigated, their communication during mechanical loading and the underlying molecular mechanisms are not yet fully known. The present study investigated the effects of excessive mechanically stretched osteoblasts on the metabolism and apoptosis of chondrocytes, and also assessed the involvement of the Wnt/ß­catenin signaling pathway. In the present study, rat chondrocytes and osteoblasts were subjected to mechanical tensile strain, and an indirect chondrocyte­osteoblast co­culture model was established. Reverse transcription­quantitative PCR and western blotting were performed to determine the expression levels of genes and proteins of interest. An ELISA was performed to investigate the levels of cytokines, including matrix metalloproteinase (MMP) 13, MMP 3, interleukin­6 (IL­6) and prostaglandin E2 (PG E2), released from osteoblasts. Flow cytometry was performed to detect the apoptosis of chondrocytes exposed to stretched osteoblast conditioned culture medium. The levels of MMP 13, IL­6 and PG E2 increased significantly in the supernatants of stretched osteoblasts compared with the un­stretched group. By contrast, the mRNA expression levels of Collagen 1a and alkaline phosphatase were significantly decreased in osteoblasts subjected to mechanical stretch compared with the un­stretched group. The mRNA expression level of Collagen 2a was significantly decreased, whereas the expression levels of MMP 13 and a disintegrin and metalloproteinase with thrombospondin­like motifs 5 were significantly increased in chondrocytes subjected to mechanical stretch compared with the un­stretched group. In the co­culture model, the results indicated that excessive mechanically stretched osteoblasts induced the catabolism and apoptosis of chondrocytes, which was partly inhibited by Wnt inhibitor XAV­939. The results of the present study demonstrated that excessive mechanical stretch led to chondrocyte degradation and inhibited osteoblast osteogenic differentiation; furthermore, excessive mechanically stretched osteoblasts induced the catabolism and apoptosis of chondrocytes via the Wnt/ß­catenin signaling pathway.

Post-menopausal oestrogen deficiency induces osteoblast apoptosis via regulating HOTAIR/miRNA-138 signalling and suppressing TIMP1 expression.

In this study, we aimed to explore the molecular mechanisms underlying the development of osteoporosis in post-menopausal females. Real-time PCR was conducted to measure the expression of potential lncRNAs involved in the osteoporosis of post-menopausal females. In addition, Western blot and IHC assays were used to study the possible correlation among HOTAIR, miR-138 and TIMP1, while a computational analysis was carried out to predict the 'seed sequence' responsible for the binding between miR-138 and HOTAIR/TIMP1. Furthermore, luciferase reporter assays were conducted to validate the negative regulatory relationship between miR-138 and TIMP1/HOTAIR. To evaluate the effect of oestrogen on the function of HOATIR and its downstream effectors, luciferase activity was measured in cells cotransfected with different vectors or treated with different doses of oestrogen. The results of the luciferase assay were further validated by real-time PCR, Western blot, MTT assay and flow cytometry. Among the candidate lncRNAs, HOTAIR was the only lncRNA down-regulated in post-menopausal females. HOTAIR bound to miR-138 and negatively regulated its expression. Meanwhile, miR-138 could also bind to TIMP1 mRNA and reduce its expression. Furthermore, a dose-dependent up-regulation of HOTAIR was observed in cells treated with oestrogen, and the elevated HOTAIR increased the level of TIMP1 by targeting miR-138. In addition, oestrogen promoted cell viability and suppressed cell apoptosis, and effects of oestrogen were blocked by the silencing of HOTAIR. Therefore, it can be concluded that oestrogen deficiency could induce the apoptosis of osteoblasts and lead to osteoporosis in post-menopausal females via modulation of the HOTAIR/miR-138/TIMP1 signalling axis.

MicroRNA-425-5p promotes breast cancer cell growth by inducing PI3K/AKT signaling.

MicroRNA-425-5p (miR-425-5p) has been reported to be involved in the tumorigenesis of several tumors, but its function in breast cancer is still unknown. In this study, miR-425-5p was found significantly upregulated in breast cancer cells, and predicted a poor prognosis for breast cancer patients. Overexpression of miR-425-5p could significantly promote breast cancer cell growth. Further studies showed that overexpression of miR-425-5p upregulated the protein levels of Cyclin D1, Cyclin D3, CDK4, and CDK6. However, inhibiting miR-425-5p downregulated their expression and induced cell cycle arrest at G0/G1 phase. In mechanism, overexpression of miR-425-5p increased the phosphorylation of PI3K p85 and AKT, but inhibiting miR-425-5p displayed opposite effects. Moreover, miR-425-5p bound to the 3'UTR of PTEN mRNA, and downregulated the expression levels of PTEN in both mRNA and protein levels in breast cancer cells. Collectively, the results above demonstrated that miR-425-5p was involved in the tumorigenesis of breast cancer by inducing PI3K/AKT signaling and indicated that miR-425-5p could be as a potential target for breast cancer therapy in the future.

Mechanical Stretch Promotes the Osteogenic Differentiation of Bone Mesenchymal Stem Cells Induced by Erythropoietin.

INTRODUCTION: The effects of erythropoietin (EPO) on the behaviors of bone marrow mesenchymal stem cells (BMSCs) subjected to mechanical stretch remain unclear. This study was therefore aimed at establishing the dose-response effect of EPO stimulation on rat BMSCs and investigating the effects of mechanical stretch combined with EPO on the proliferation and osteogenic differentiation of BMSCs. MATERIAL AND METHODS: The proliferation and osteogenic differentiation of rat BMSCs were examined and compared using EPO with different concentrations. Thereafter, BMSCs were subjected to 10% elongation using a Flexcell strain unit, combined with 20 IU/ml EPO. The proliferation of BMSCs was detected by Cell Counting Kit-8, colony formation assay, and cell cycle assay; meanwhile, the mRNA expression levels of Ets-1, C-myc, Ccnd1, and C-fos were detected by reverse transcription and real-time quantitative PCR (qPCR). The osteogenic differentiation of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA expression levels of ALP, OCN, COL, and Runx2 were detected by qPCR. The role of the extracellular signal-regulated kinases 1/2 (ERK1/2) in the osteogenesis of BMSCs stimulated by mechanical stretch combined with 20 IU/ml EPO was examined by Western blot. RESULTS: Our results showed that effects of EPO on BMSCs included a dose-response relationship, with the 20 IU/ml EPO yielding the largest. Mechanical stretch combined with 20 IU/ml EPO promoted proliferation and osteogenic differentiation of BMSCs. The increase in ALP, mineral deposition, and osteoblastic genes induced by the mechanical stretch-EPO combination was inhibited by U0126, an ERK1/2 inhibitor. CONCLUSION: EPO was able to promote the proliferation and osteogenic differentiation of BMSCs, and these effects were enhanced when combined with mechanical stretch. The underlying mechanism may be related to the activation of the ERK1/2 signaling pathway.

Intensity-dependent effect of treadmill running on rat Achilles tendon.

It is understood that mechanical loading may affect tendon properties. However, how different mechanical loading conditions may affect tendons remains unknown. The present study aimed to investigate the effect of treadmill running at various intensities on rat Achilles tendon. A total of 18 male Wistar rats were randomly assigned to one of three groups: Control (CON), medium-intensity running (MIR), and high-intensity running (HIR). Following 8 weeks of treadmill running protocols, all Achilles tendons were harvested for histological observation and gene expression analysis. Significant morphological changes were observed with regular and large diameter collagen fibrils in the MIR group, whereas irregular and small diameter collagen fibrils were observed in the HIR group. Collagen type I was significantly upregulated in the MIR group compared with the CON group, and downregulated in the HIR group compared with the CON or MIR groups (P<0.05). However, collagen type III was significantly upregulated in the HIR group in comparison with the CON or MIR groups (P<0.05). Furthermore, the expression of matrix metallopeptidase-13 was significantly increased in the MIR and HIR groups compared with the CON group (P<0.05). The expression of tissue inhibitor of metalloproteinases-1 was increased in the MIR group compared with the CON group, but decreased in the HIR group compared with the CON and MIR groups (P<0.05). Additionally, decorin expression was significantly higher in the MIR group compared with the CON group, and significantly decreased in the HIR group compared with the CON or MIR groups (P<0.05). A converse pattern of changes in biglycan expression was identified among the three groups. Aggrecan expression was significantly higher in the HIR group compared with the CON or MIR groups (P<0.05). These findings indicated that moderate exercise may induce increased collagen synthesis and organize regular and large collagen fibers, thus benefiting the Achilles tendon. However, overuse during exercise may result in collagen degradation and disturbance, which predisposes individuals to injury.

Intensity­dependent effect of treadmill running on differentiation of rat bone marrow stromal cells.

The effect of running on bone mass depends on its intensity. However, the underlying molecular mechanism that associates running intensity with bone mass is unclear. The current study examined the effects of treadmill running at different intensities on bone mass and osteogenic differentiation of bone marrow stromal cells (BMSCs) in a rat model. A total of 24 male Wistar rats were randomly divided into groups and subjected to no running (Con group), low­intensity running (LIR group), moderate­intensity running (MIR group), and high­intensity running (HIR group). Histological, immunohistochemistry and micro­CT examinations were performed on the femora harvested after 8 weeks of treadmill running. The study demonstrated that treadmill running affected trabecular bone mass in an intensity­dependent manner. In addition, such an intensity­dependent effect was also demonstrated on the osteogenic and adipogenic differentiation and proliferation of BMSCs. Furthermore, the Wnt/ß­catenin signaling pathway may be involved in the running­induced increase in bone mass in rats in the MIR group. There appears to be a biomechanical 'window', in which running­induced strain signals can increase the number of BMSCs and progenitor cells (specific to the osteoblast lineage) causing upregulation of osteogenesis and downregulation of adipogenesis of BMSCs. This finding may provide insight into the molecular and cellular mechanisms responsible for bone homeostasis.

Response of decorin to different intensity treadmill running.

Decorin is widely understood to affect collagen fibrillogenesis. However, little is understood about its response to various mechanical loading conditions. In the present study, 36 Wistar rats were randomly divided into control (CON), moderate treadmill running (MTR) and strenuous treadmill running (STR) groups. Animals in the MTR and STR groups were subjected to a 4­ or 8­week treadmill running protocol. Subsequently, all Achilles tendons were harvested to perform histological and biochemical analyses. Decorin expression was markedly increased in the MTR group compared with the CON group at 4 and 8 weeks. Conversely, decorin expression was markedly decreased in the STR group compared with the CON and MTR group at 4 and 8 weeks. Furthermore, between the two time points, decorin expression levels were significantly increased in the MTR group, whereas they were markedly decreased in the STR group. These results suggested that MTR exercise may induce increased decorin expression via a balance of MMP­2 and TIMP­2, improving tendon structure and function. However, STR exercise may result in degradation of decorin due to an imbalance of MMP­2 and TIMP­2, with a bias to MMP­2, resulting in a predisposition to tendinopathy.

Effects of treadmill running with different intensity on rat subchondral bone.

Subchondral bone (SB) is recognized as a key factor in normal joint protection, not only does it provide a shock absorbing and supportive function for the cartilage, but it may also be important for cartilage metabolism. Mechanical loading is considered to be a critical regulator of skeletal homeostasis, including bone and cartilage. It is suggested that both cartilage and bone may respond to mechanical loading in an intensity-dependent manner. In this report, we have discovered that the subchondral plate became thicker with higher bone mineral density (BMD) and lower porosity, while trabecular bone became more plate-like and denser with higher BMD in high-intensity running (HIR) group. Further, HIR led to highly remodeled, less mineralized, and stiffer subchondral plate and trabecular bone. On the contrary, low-intensity running and moderate-intensity running failed to result in considerable changes in microstructure, composition and hardness. Our findings suggested that running affects SB in an intensity-dependent manner. In addition, HIR may induce change in organization and composition of SB, and consequently alter its mechanical properties. HIR-induced "brittle and stiff" SB may adversely affect the overlying articular cartilage.

Exercise affects biological characteristics of mesenchymal stromal cells derived from bone marrow and adipose tissue.

Both bone marrow mesenchymal stromal cells (BMSCs) and adipose-derived mesenchymal stromal cells (ADSCs) are good sources for tissue engineering. To maximize therapeutic efficacy of MSCs, an appropriate source of MSCs should be selected according to their own inherent characteristics for future clinical application. Hence, this study was conducted to compare proliferative, differential and antiapoptosis abilities of both MSCs derived from exercised and sedentary rats under normal and hypoxia/serum deprivation conditions (H/SD). Our results showed that exercise may enhance proliferative ability and decrease adipogenic ability of BMSCs and ADSCs. However, positive effect of exercise on osteogenesis was only observed for BMSCs in either environment. Little effect was observed on the antiapoptotic ability of both MSC types. It was also suggested that biological characteristics of both types were partly changed. It is therefore believed that BMSCs derived from exercised rat on early passage may be a good cell source for bone tissue engineering.

Strenuous Treadmill Running Induces a Chondrocyte Phenotype in Rat Achilles Tendons.

BACKGROUND Although tendinopathy is common, its underlying pathogenesis is poorly understood. This study aimed to investigate the possible pathogenesis of tendinopathy. MATERIAL AND METHODS In this study, a total of 24 rats were randomly and evenly divided into a control (CON) group and a strenuous treadmill running (STR) group. Animals in the STR group were subjected to a 12-week treadmill running protocol. Subsequently, all Achilles tendons were harvested to perform histological observation or biochemical analyses. RESULTS Histologically, hypercellularity and round cells, as well as disorganized collagen fibrils, were presented in rat Achilles tendon sections from the STR group. Furthermore, our results showed that the expression of aggrecan, collagen type II (Col II), and Sex-Determining Region Y Box 9 (Sox 9) were markedly increased in the STR group compared with that in the CON group. Additionally, the mRNA expression of bone morphogenetic protein-2 (BMP-2) and biglycan was significantly up-regulated in the STR group in contrast to that in CON group. CONCLUSIONS These results suggest that a 12-week strenuous treadmill running regimen can induce chondrocyte phenotype in rat Achilles tendons through chondrogenic differentiation of tendon stem cells (TSCs) by BMP-2 signaling.

Periosteal osteosarcoma and Marfan's syndrome: A case report and literature review.

Periosteal osteosarcoma (POS) is a rare primary malignant bone tumor arising from the surface of long bones. In addition, Marfan's syndrome (MFS) is an infrequent hereditary autosomal dominant connective tissue disorder with high penetrance and variable phenotypes, which primarily affects the ocular, skeletal and cardiovascular systems. The present study reported a case of POS and MFS co-occurring in a child. A 6-year-old girl with MFS presented with pain, swelling and deformity in the right thigh following a fall. The patient was diagnosed with a right femoral shaft fracture and underwent open internal fixation surgery at a local hospital. At 2 weeks following surgery, the patient's parents observed increased swelling in the right thigh and thus, revisited the clinic. X-ray examination revealed extensive osteotylus around the fracture site and the clinician decided to remove the internal fixation. Following removal of the implant, aggravated swelling and superficial venous engorgement were observed. The patient was then admitted to Nanfang Hospital, where magnetic resonance imaging was performed, which identified symptoms of an abnormal periosteal reaction with bone erosion, indicating POS. The patient underwent a wide resection of the tumor and the histopathological examination confirmed the diagnosis of POS. No recurrence was identified at 9 months postoperatively. In conclusion, the present case report may result in increased awareness of the possibility of malignant bone tumors in a hereditary patient with osteotylus overgrowth following fracture surgery; in addition, the present case indicated a possible correlation between POS and MFS.

Hepatitis B surface antigen seroclearance in patients with chronic hepatitis B infection: a clinical study.

OBJECTIVE: To investigate the clinical characteristics of hepatitis B surface antigen (HBsAg) seroclearance in patients with chronic hepatitis B virus (HBV) infection. METHODS: Patients with chronic HBV infection who achieved sustained virological response (SVR) within 6 years of ceasing formal antiviral treatment were assessed for HBsAg seroclearance (defined as loss of serum HBsAg on repeated testing for a period of >6 months), using enzyme immunoassays. Phase of HBV infection and liver function (serum alanine aminotransferase [ALT] and aspartate aminotransferase [AST] levels) and HBV DNA levels were also assessed. RESULTS: In total, 272 patients with chronic HBV and SVR were included; HBsAg seroclearance was achieved in 42 patients and not achieved in 230 patients. Serum HBsAg and ALT levels, ratios of HBsAg to HBV DNA and ratios of AST to ALT were significantly different between patients achieving, and not achieving, HBsAg seroclearance. The area under the receiver operating characteristic (ROC) curve of HBsAg levels for predicting the likelihood of HBsAg seroclearance was 0.85; the cut-off value was 203.86 IU/ml. CONCLUSIONS: These data demonstrate that HBsAg seroclearance was independently associated with host immunity, serum HBsAg level, serum ALT level, serum HBsAg to HBV DNA ratio and timing of drug therapy within the course of chronic HBV infection.

Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo.

AIM: To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS: We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad-p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS: The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was >or= 50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G(0)/G(1), while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION: p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G(0)/G(1). Therefore, p27mt can induce apoptosis in colorectal cells.

Impact of p27mt gene on transplantation model of human colorectal cancer in nude mice.

AIM: To investigate the inhibitory and anti-metastatic effect of mutant p27 gene (p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism. METHODS: Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intra-tumoral injection of Ad-p27mt in a pre-established transplantation model of human colorectal cancer in nude mice. Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor. Expression of MMP-9 in tumor tissue was detected by immunohistochemistry. RESULTS: The average sizes of transplantation tumors were 1.94 +/- 0.67 cm(3), 2.75 +/- 0.83 cm(3) and 3.01 +/- 0.76 cm(3) in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.05). The average proliferation rates were 37.34% +/- 1.45%, 53.16% +/- 3.27% and 54.48% +/- 2.43%, in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.05). The average apoptosis rates were 19.79% +/- 3.32%, 6.38% +/- 4.91% and 7.25% +/- 5.20% in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.01). The average MMP-9 expression rates were 20%, 75% and 66.7% in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.01). CONCLUSION: p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells, as well as decrease transpl-anted tumor metastasis.

Antitumor bioactivity of adenovirus-mediated p27mt in colorectal cancer cell line SW480.

AIM: To explore the antitumor bioactivity of adenovirus-mediated mutant type p27(kip1) gene in a colorectal cancer cell line SW480. METHODS: We constructed recombinant adenovirus vector expressing a mutant type p27(kip1) gene (ad-p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), and transduced into SW480 cells. Then we detected expression of p27, Bcl-2 and Bax protein in the transductants by Western blotting, cell cycle of transductants by a digital flow cytometric system, migrating potential with Boyden Chamber and SW480 tumor cell growth inhibition in vitro and in vivo. RESULTS: We found that a recombinant adenovirus vector of expressing ad-p27mt, with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) has potent inhibition of SW480 tumor cell growth in vitro and in vivo. Furthermore, ad-p27mt induced cell apoptosis via regulating bax and bcl-2 expressions, and G(1)/S arrest in SW480 cells and inhibited cell migration. CONCLUSION: ad-p27mt has a strong anti-tumor bioactivity and has the potential to develop into new therapeutic agents for colorectal cancer.