Exercise promotes osteogenic differentiation by activating the long non-coding RNA H19/microRNA-149 axis.

BACKGROUND: Regular physical activity during childhood and adolescence is beneficial to bone development, as evidenced by the ability to increase bone density and peak bone mass by promoting bone formation. AIM: To investigate the effects of exercise on bone formation in growing mice and to investigate the underlying mechanisms. METHODS: 20 growing mice were randomly divided into two groups: Con group (control group, n = 10) and Ex group (treadmill exercise group, n = 10). Hematoxylin-eosin staining, immunohistochemistry, and micro-CT scanning were used to assess the bone formation-related indexes of the mouse femur. Bioinformatics analysis was used to find potential miRNAs targets of long non-coding RNA H19 (lncRNA H19). RT-qPCR and Western Blot were used to confirm potential miRNA target genes of lncRNA H19 and the role of lncRNA H19 in promoting osteogenic differentiation. RESULTS: Compared with the Con group, the expression of bone morphogenetic protein 2 was also significantly increased. The micro-CT results showed that 8 wk moderate-intensity treadmill exercise significantly increased bone mineral density, bone volume fraction, and the number of trabeculae, and decreased trabecular segregation in the femur of mice. Inhibition of lncRNA H19 significantly upregulated the expression of miR-149 and suppressed the expression of markers of osteogenic differentiation. In addition, knockdown of lncRNA H19 significantly downregulated the expression of autophagy markers, which is consistent with the results of autophagy-related protein changes detected in mouse femurs by immunofluorescence. CONCLUSION: Appropriate treadmill exercise can effectively stimulate bone formation and promote the increase of bone density and bone volume in growing mice, thus enhancing the peak bone mass of mice. The lncRNA H19/miR-149 axis plays an important regulatory role in osteogenic differentiation.

Resveratrol Loaded by Folate-Modified Liposomes Inhibits Osteosarcoma Growth and Lung Metastasis via Regulating JAK2/STAT3 Pathway.

Background: Osteosarcoma is a malignant bone tumor with a high rate of lung metastasis and mortality. It has been demonstrated that resveratrol can inhibit tumor proliferation and metastasis, but its application is limited due to poor water solubility and low bioavailability. In this study, we proposed to prepare folate-modified liposomes loaded with resveratrol to investigate its anti-osteosarcoma effect in vitro and in vivo. Methods: We prepared and characterized resveratrol liposomes modified with folate (denoted as, FA-Res/Lps). The effects of FA-Res/Lps on human osteosarcoma cell 143B proliferation, apoptosis, and migration were investigated by MTT, cell cloning, wound-healing assay, transwell, and flow cytometry. A xenograft tumor and lung metastasis model of osteosarcoma was constructed to study the therapeutic effects of FA-Res/Lps on the growth and metastasis of osteosarcoma in vivo. Results: The FA-Res/Lps were prepared with a particle size of 118.5 ± 0.71 and a small dispersion coefficient of 0.154 ± 0.005. We found that FA-modified liposomes significantly increased resveratrol uptake by osteosarcoma cells 143B in flow cytometric assay, resulting in FA-Res/Lps, which inhibit tumor proliferation, migration and induce apoptosis more effectively than free Res and Res/Lps. The mechanism of action may be associated with the inhibition of JAK2/STAT3 signaling. In vivo imaging demonstrated that FA-modified DiR-modified liposomes significantly increased the distribution of drugs at the tumor site, leading to significant inhibition of osteosarcoma growth and metastasis by FA-Res/Lps. Furthermore, we found that FA-Res/Lps did not cause any adverse effects on mice body weight, liver, or kidney tissues. Conclusion: Taken together, the anti-osteosarcoma effect of resveratrol is significantly enhanced when it is loaded into FA-modified liposomes. FA-Res/Lps is a promising strategy for the treatment of osteosarcoma.

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.

Bidirectional association between metabolic syndrome and osteoarthritis: a meta-analysis of observational studies.

BACKGROUND: Emerging observational studies suggest an association between metabolic syndrome (MetS) and osteoarthritis (OA). This meta-analysis was conducted to examine whether or not there is a bidirectional relationship between MetS and OA. METHODS: The PubMed and Embase databases were searched from their inception to October 2019. We selected studies according to predefined criteria. Random effects were selected to calculate two sets of pooled risk estimates: MetS predicting OA and OA predicting MetS. RESULTS: A total of seven cross-sectional studies and four cohort studies met the criteria for MetS predicting the onset of OA. Another six cross-sectional studies and one cohort study met the criteria for OA predicting the onset of MetS. The pooled odds risk (OR) for OA incidences associated with baseline MetS was 1.45 (95% CI 1.27-1.66). The OR for MetS incidences associated with baseline OA was 1.90 (95% CI 1.11-3.27). In an overall analysis, we found that MetS was associated with prevalent OA in both cross-sectional studies (OR = 1.32, 95% CI 1.21-1.44) and cohort studies (OR = 1.76, 95% CI 1.29-2.42). No indication of heterogeneity was found in the cross-sectional studies (p = 0.395, I2 = 4.8%), whereas substantial heterogeneity was detected in the cohort studies (p = 0.000, I2 = 79.3%). CONCLUSION: Meta-analysis indicated a bidirectional association between MetS and OA. We advise that patients with MetS should monitor their OA status early and carefully, and vice versa.

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.

Delivery of curcumin by directed self-assembled micelles enhances therapeutic treatment of non-small-cell lung cancer.

BACKGROUND: It has been widely reported that curcumin (CUR) exhibits anticancer activity and triggers the apoptosis of human A549 non-small-cell lung cancer (NSCLC) cells. However, its application is limited owing to its poor solubility and bioavailability. Therefore, there is an urgent need to develop a new CUR formulation with higher water solubility and better biocompatibility for clinical application in the future. MATERIALS AND METHODS: In this study, CUR-loaded methoxy polyethylene glycol-polylactide (CUR/mPEG-PLA) polymeric micelles were prepared by a thin-film hydration method. Their characteristics and antitumor effects were evaluated subsequently. RESULTS: The average size of CUR/mPEG-PLA micelles was 34.9±2.1 nm with its polydispersity index (PDI) in the range of 0.067-0.168. The encapsulation efficiency and drug loading were 90.2%±0.78% and 9.1%±0.07%, respectively. CUR was constantly released from the CUR/mPEG-PLA micelles, and its cellular uptake in A549 cells was significantly increased. It was also found that CUR/mPEG-PLA micelles inhibited A549 cell proliferation, increased the cell cytotoxicity, induced G2/M stage arrest and promoted cell apoptosis. Moreover, the CUR/mPEG-PLA micelles suppressed the migration and invasion of A549 cells more obviously than free CUR. Additionally, CUR/mPEG-PLA micelles inhibited human umbilical vein endothelial cells migration, invasion and corresponding tube formation, implying the antiangiogenesis ability. Its enhanced antitumor mechanism may be related to the reduced expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, MMP-9 and Bcl-2 as well as the increased expression of Bax. CONCLUSION: The mPEG-PLA copolymer micelles can serve as an efficient carrier for CUR. The CUR/mPEG-PLA micelles have promising clinical potential in treating NSCLC.

MiR-451 suppresses proliferation, migration and promotes apoptosis of the human osteosarcoma by targeting macrophage migration inhibitory factor.

Previous studies have shown that MiR-451 plays an important role in human osteosarcoma carcinogenesis, but the underlying mechanism by which MiR-451 affects the osteosarcoma has not been fully understood. This study intends to uncover the mechanism by which MiR-451 functions as a tumor suppressor. The expression of MiR-451 in osteosarcoma tissues and osteosarcoma cell lines was monitored by real-time PCR. The proliferation ability was examined by MTT and cell cycle assay. The migration and apoptosis of cells were monitored by migration assay and flow cytometry, respectively. Moreover, the angiogenesis of HUVEC cells transfected with MiR-451 mimics was examined by tube formation assay. The effect of MiR-451 on MIF was determined by luciferase assays and Western blot assay. The results showed that MiR-451 expression level was significantly reduced in the osteosarcoma compared with normal bone tissues. Overexpression of MiR-451 significantly attenuated the proliferation and migration, and induced the apoptosis of osteosarcoma cells. Furthermore, the angiogenesis of HUVEC cells transfected with MiR-451 mimics was assayed and the decreased angiogenic ability was detected compared to the controls. Finally, we demonstrated that MiR-451 overexpression inhibited the malignant behavior of osteosarcoma by downregulating MIF. These findings suggest that MiR-451 may act as a tumor suppressor in osteosarcoma. MiR-451 inhibited cell proliferation, migration and angiogenesis and promoted apoptosis of human osteosarcoma cells, at least partially, by inhibiting the expression of MIF. MiR-451/MIF may be a novel therapeutic target in treatment of osteosarcoma.

miR-451 inhibits cell growth, migration and angiogenesis in human osteosarcoma via down-regulating IL 6R.

Osteosarcoma (OS) has become one of the most common primary malignant tumors in the children and adolescents with a poor prognosis owing to its high malignant and metastatic potential. Although increasing evidence indicates that miR-451 could inhibit the growth and metastasis of OS, its effect on angiogenesis in OS is still very poor. What is more, the mechanism by which miR-451 affects the OS has not been fully elucidated. In the present study, miR-451 was reduced in human osteosarcoma tissues compared with the adjacent bone tissues, and the introduction of miR-451 dramatically inhibited the growth, migration and angiogenesis in OS. Additionally, it was suggested that IL 6R is a direct target gene of miR-451. Silencing of IL 6R suppressed the growth, migration and angiogenesis of OS, which was consistent with the effect of overexpression of miR-451. In conclusion, our data demonstrate that miR-451 may function as a potential suppressor of tumor growth, migration and angiogenesis in OS via down-regulating IL 6R, suggesting a promising therapeutic avenue for managing OS.

Different responses of articular cartilage to strenuous running and joint immobilization.

OBJECTIVE: To compare the pathological changes in cartilage derived from rats that developed osteoarthritis either by joint immobilization or by strenuous treadmill running in order to better understand their respective pathomechanism. METHOD: A total of 24 male Wistar rats were randomly assigned to three groups: sedentary control (CON), immobilization (IM), and strenuous running (SR). For rats in the IM group, unilateral knee joint was immobilized in flexion. Rats in the SR group underwent treadmill running with high intensity. Eight weeks later, all animals were sacrificed. Femoral condyles were collected to take histological observation for cartilage characteristic and immunohistochemistry for collagen type II. In addition, cartilage samples were obtained to assess gene expression of aggrecan, collagen type II, biglycan, and fibromodulin by quantitative RT-PCR. RESULTS: Gross and histological observation showed osteoarthritic changes in groups SR and IM; however, more severe cartilage degradation was revealed in the latter. Proteoglycan and collagen II content decreased in groups SR and IM in comparison to group CON, with more loss in group IM. In group SR, mRNA levels in femoral cartilage were found to be unaltered for all the molecules measured. On the contrary, these molecules were significantly downregulated in group IM. CONCLUSION: Differences in gross observation, histological characteristics, and gene expression of proteoglycans and collagen II suggest that both knee immobilization and strenuous running would lead to degenerative change of cartilage, but at different stages of the degenerative process.

[Effects of plastic mulch on soil moisture and temperature and limiting factors to yield increase for dryland spring maize in the North China].

Four treatments, including ridge tillage with plastic mulch (RP), ridge tillage without mulch (RB), flat tillage with plastic mulch (FP) and flat tillage without mulch (FB), were carried out to examine the tillage type and mulch on the effects of soil moisture and temperature, yield and water use efficiency (WUE) of dry land spring maize in the North China. Results showed that the average soil temperature was increased by 1-3 °C and the accumulated soil temperature was increased by 155.2-280.9 °C from sowing to tasseling by plastic mulch, and the growing duration was extended by 5.9-10.7 d. The water conservation effect of plastic mulch was significant from sowing to the seedling establishment, with WUE being increased by 81.6%-136.4% under mulch as compared with that without mulch. From the seedling to jointing stage, which coincided with the dry period in the region, soil water utilization by the maize under mulch could reach the depth of 80-100 cm, and its WUE was about 17.0%-21.6% lower than the maize without mulch, since the latter was affected by dry stress. With the coming of rainy season around the trumpeting stage, soil water in each treatment was replenished and maintained at relative high level up to harvest. Yield of maize was increased by 9.5% under RP as compared with RB. However, yield was reduced by 5.0% under FP, due to the plastic film under flat tillage prevented the infiltration of rainfall and waterlogging occurred. No significant difference in yield was found between RB and FB. Higher yield of spring maize was limited because of the mismatching in water supply and demand characterized by soil water shortage before the rainy season and abundant soil water storage after the rainy season.

Intensity-dependent effect of treadmill running on knee articular cartilage in a rat model.

OBJECTIVE: To understand the changes of femoral cartilage in response to treadmill running with different intensities in the hope of differentiating "moderate" and "strenuous" running in a rat model. METHOD: A total of 24 male Wistar rats were randomly assigned into groups of sedentary (SED), low-intensity running (LIR), medium-intensity running (MIR), and high-intensity running (HIR). Rats in LIR, MIR, and HIR groups underwent 8 weeks' treadmill running programs. After sacrificed, femoral condyles were collected to take histomorphometric analysis and immunohistochemistry for collagen II. RESULTS: Gross and histological observation showed osteoarthritic changes in group HIR. In comparison to SED group, there was significant increase in cartilage thickness, number of chondrocytes, and GAG content in groups LIR and MIR. Conversely, decrease in cartilage thickness, chondrocyte number, and GAG content was found in rats of HIR group, without significant difference though. In addition, in comparison to SED group, HIR group exhibited disorganization of collagen fibril and significantly lower content of collagen type II. CONCLUSION: An intensity-dependent effect was suggested on the articular cartilage. Our results also demonstrated that running with low-to-medium intensity applied in the present study should be regarded as "moderate" running, whereas high-intensity running as "strenuous" running.