Aucubin Promotes Osteogenic Differentiation and Facilitates Bone Formation through the lncRNA-H19 Driven Wnt/ß-Catenin Signaling Regulatory Axis.

Objectives: Traditional Chinese medicine Cortex Eucommiae has been used to treat bone fracture for hundreds of years, which exerts a significant improvement in fracture healing. Aucubin, a derivative isolated from Cortex Eucommiae, has been demonstrated to possess anti-inflammatory, immunoregulatory, and antioxidative potential. In the present study, our aim was to explore its function in bone regeneration and elucidate the underlying mechanism. Materials and Methods: The effects of Aucubin on osteoblast and osteoclast were examined in mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) and RAW 264.7 cells, respectively. Moreover, the lncRNA H19 and Wnt/ß-catenin signaling were detected by qPCR examination, western blotting, and luciferase activity assays. Using the femur fracture mice model, the in vivo effect of Aucubin on bone formation was monitored by X-ray, micro-CT, histomorphometry, and immunohistochemistry staining. Results: In the present study, Aucubin was found to significantly promote osteogenic differentiation in vitro and stimulated bone formation in vivo. Regarding to the underlying mechanism, H19 was found to be obviously upregulated by Aucubin in MSCs and thus induced the activation of Wnt/ß-catenin signaling. Moreover, H19 knockdown partially reversed the Aucubin-induced osteogenic differentiation and successfully suppressed the activation of Wnt/ß-catenin signaling. We therefore suggested that Aucubin induced the activation of Wnt/ß-catenin signaling through promoting H19 expression. Conclusion: Our results demonstrated that Aucubin promoted osteogenesis in vitro and facilitated fracture healing in vivo through the H19-Wnt/ß-catenin regulatory axis.

Baicalin induces ferroptosis in osteosarcomas through a novel Nrf2/xCT/GPX4 regulatory axis.

BACKGROUND: Osteosarcomas (OS) is a kind of malignant bone tumor which occurs primarily in children and adolescents, and the clinical therapeutics remain disappointing. As a new programmed cell death, ferroptosis is characterized by iron dependent and intracellular oxidative accumulation, which provides a potential alternative intervene for the OS treatment. Baicalin, a major bioactive flavone derived from traditional Chinese medicine Scutellaria baicalensis, has been proved to have anti-tumor properties in OS. Whether ferroptosis participated in the baicalin mediated anti-OS activity is an interesting project. PURPOSE: To explore the pro-ferroptosis effect and mechanisms of baicalin in OS. METHODS/STUDY DESIGN: Pro-ferroptosis effect of baicalin on cell death, cell proliferation, iron accumulation, lipid peroxidation production was determined in MG63 and 143B cells. The levels of glutathione (GSH), oxidized (GSSG) glutathione and malondialdehyde (MDA) were determined by enzyme linked immunosorbent assay (ELISA). The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), Glutathione peroxidase 4 (GPX4) and xCT were detected by western blot in baicalin-mediated ferroptosis regulation. In vivo, a xenograft mice model was adopted to explore the anticancer effect of baicalin. RESULTS: In the present study, it was found that baicalin significantly suppress tumor cell growth in vitro and in vivo. By promoting the Fe accumulation, ROS formation, MDA production and suppressing the ratio of GSH/GSSG, baicalin was found to trigger ferroptosis in OS and ferroptosis inhibitor ferrostatin-1 (Fer-1) successfully reversed these suppressive effects, indicating that ferroptosis participated in the baicalin mediated anti-OS activity. Mechanistically, baicalin physically interacted with Nrf2, a critical regulator of ferroptosis, and influenced its stability via inducing ubiquitin degradation, which suppressed the Nrf2 downstream targets GPX4 and xCT expression, and led to stimulating ferroptosis. CONCLUSIONS: Our findings for the first time indicated that baicalin exerted anti-OS activity through a novel Nrf2/xCT/GPX4-dependent ferroptosis regulatory axis, which hopefully provides a promising candidate for OS treatment.

LncRNA-NEF suppressed oxaliplatin resistance and epithelial-mesenchymal transition in colorectal cancer through epigenetically inactivating MEK/ERK signaling.

A major cause of oxaliplatin chemoresistance in colorectal cancer (CRC) is acquired epithelial-mesenchymal transition (EMT) in cancer cells, making the cancer cells easy to metastasis and recurrence. LncRNA Neighboring Enhancer of FOXA2 (lncRNA-NEF) has been characterized as a tumor suppressor to mediate cancer metastasis in multiple cancer types. However, whether it mediated the drug resistance remains unknown. In the present study, an oxaliplatin-resistant CRC cell line (SW620R) was established and lncRNA-NEF was obviously down-regulated in this resistant cell line. The further loss and gain-of-function studies demonstrated that this lncRNA suppressed oxaliplatin resistance as well as EMT programme in vitro and inhibited metastasis in vivo. Mechanistically, lncRNA-NEF epigenetically promoted the expression of DOK1 (Downstream of Tyrosine kinase 1), a negative regulator of MEK/ERK signaling, by disrupting DNA methyltransferases (DNMTs)-mediated DNA methylation. DOK1, in turn, induced the inactivation of MEK/ERK signaling, forming the lncRNA-NEF/DOK1/MEK/ERK regulatory axis to mediate oxaliplatin resistance in CRC. Collectively, our work reveals the critical function of lncRNA-NEF in mediating the oxaliplatin chemotherapy resistance in CRC, and provides a promising therapeutic strategy for CRC patients with oxaliplatin resistance.

MLK3 silence suppressed osteogenic differentiation and delayed bone formation via influencing the bone metabolism and disturbing MAPK signaling.

Background: Mixed lineage kinase 3 (MLK3) is a member of a serine/threonine MAP3K family, and it has been demonstrated to play critical roles in various biological activities and disease progression. Previous studies showed that impaired skeletal mineralization and spontaneous tooth fracture in the MLK3-deficient mice, suggesting MLK3 actively participated in the bone formation. However, the detailed function and underlying mechanisms remain obscure. Methods: The MLK3 knockout (KO) mouse was applied in the present study, and multi-omics were performed to compare the metabolites and gene expression between wild type (WT) and KO mice. The bone fracture model was successfully established, and the healing process was evaluated by X-ray, micro-CT examination, histomorphometry and immunohistochemistry (IHC) staining. On the other hand, the effects of MLK3 on osteogenic differentiation were assessed by alkaline phosphatase (ALP) activity, Alizarin red S (ARS) staining and qRT-PCR examination. Finally, the downstream signaling pathways were screened out by RNA-sequencing (RNA-seq) and then validated by Western blotting. Results: In the present study, imbalanced bone metabolism was observed in these MLK3 KO mice, suggesting MLK3 may participate in bone development. Moreover, MLK3 -/- mice displayed abnormal bone tissues, impaired bone quality, and delayed fracture healing. Further investigation showed that the inhibition of MLK3 attenuated osteoblast differentiation in vitro. According to the RNA-seq data, MAPK signaling was screened out to be a downstream pathway, and its subfamily members extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal protein kinase (JNK) were subjected to Western blotting examination. The results revealed that although no differences in their expression were observed between MSCs derived from WT and KO mice, their phosphorylated protein levels were all suppressed in MLK3 -/- MSCs. Conclusion: In conclusion, our results demonstrated that loss of MLK3 suppressed osteoblast differentiation and delayed bone formation via influencing metabolism and disturbing MAPK signaling. The translational potential of this article: The findings based on the current study demonstrated that MLK3 promoted osteogenesis, stimulated new bone formation and facilitated fracture healing, suggesting that MLK3 may serve as a potential therapeutic target for bone regeneration. MLK3 activator therefore may be developed as a therapeutic strategy for bone fracture.

Predicting Collapse in Osteonecrosis of the Femoral Head Using a New Method: Preserved Angles of Anterior and Lateral Femoral Head.

BACKGROUND: Femoral head collapse (FHC) is associated with a poor prognosis in osteonecrosis of the femoral head (ONFH). Preserved angles (PAs), including the lateral preserved angle (LPA), the anterior preserved angle (APA) and the combined preserved angle (CPA), can be used to quantify the extent of femoral head necrosis and predict the risk of femoral head collapse. The purpose of this retrospective cohort study was to assess the efficacy of these preserved angles in the prediction of femoral head collapse using plain radiographs. METHODS: Patients with ONFH treated conservatively between January 2010 and January 2019 were analyzed retrospectively to assess the risk of FHC. A logistic regression model was used to evaluate the independent prognostic factors associated with FHC, including age, sex, etiology, onset of symptom, The Japanese Investigation Committee classification, and PAs (LPA, APA, and CPA). RESULTS: A total of 137 patients, with 180 hips, had follow-up of at least two years and were included. During the follow-up period, FHC occurred in 89 hips (49.44%) after the initial diagnosis. Multivariable analysis indicated that CPA (odds ratio [OR] = 0.95; 95%CI = 0.93-0.97; P < 0.01) was a stronger predictor of femoral head collapse compared with the Japanese Investigation Committee classification (OR = 2.40, 95%CI = 0.92-6.25; P > 0.01). The receiver operating characteristic and survival curve analyses revealed that the predictive cutoff point for the CPA was 118.7° (sensitivity = 96.70%, specificity = 79.78%, log-rank test: P < 0.01). CONCLUSIONS: Assessment of preserved angles on plain radiographs is a simple method to quantify the extent of lateral and anterior necrosis of the femoral head. Specifically, CPA has a potential value in predicting femoral head collapse.

Baicalein mediates the anti-tumor activity in Osteosarcoma through lncRNA-NEF driven Wnt/ß-catenin signaling regulatory axis.

Background: Osteosarcoma (OS) is a common type of malignant bone tumor in adolescents with high risk of metastasis. However, the clinical management still remains unsatisfactory. Traditional Chinese medicine (TCM) has been widely considered as an alternative treatment, and their extracts have proved to possess great potential for drug discovery. Baicalein (BA), the active pharmaceutical ingredient of rhizoma coptidis, was proved to have anti-tumor properties in OS, but the mechanism remains poorly understood. Methods: The potential anti-cancer effects on cell growth, cell cycle, apoptosis and migration were examined in OS cells. Moreover, the lncRNA-Neighboring Enhancer of FOXA2 (lncRNA-NEF) and Wnt/ß-catenin signaling were detected by qPCR and Western blotting assays. The in vivo effect of GA on tumor growth was investigated using a xenograft mice model. Results: In the present study, BA was found to significantly suppress tumor growth in vitro and in vivo. And it was also found to inhibit the invasion and metastasis as well. As for the mechanism investigation, lncRNA-NEF was obviously upregulated by BA in OS cells, and thus induced the inactivation of Wnt/ß-catenin signaling. Moreover, lncRNA-NEF knockdown partially reversed the BA-induced anti-cancer activities; and successfully compensated the suppressive effect on Wnt/ß-catenin signaling. We therefore suggested that BA induced the inactivation of Wnt/ß-catenin signaling through promoting lncRNA-NEF expression. Conclusions: In conclude, our results demonstrated that BA suppressed tumor growth and metastasis in vitro and in vivo through an lncRNA-NEF driven Wnt/ß-catenin regulatory axis, in which lncRNA-NEF was upregulated by BA, and thus induced the inactivation of Wnt/ß-catenin signaling. The Translational potential of this article: The findings derived from this study validates the anti-cancer activity of BA in OS and provides a novel underlying mechanism, which suggest that BA may be a potential candidate to develop the effective drug for OS patients.

Finite element modeling of proximal femur with quantifiable weight-bearing area in standing position.

BACKGROUND: The positional distribution and size of the weight-bearing area of the femoral head in the standing position as well as the direct active surface of joint force can directly affect the result of finite element (FE) stress analysis. However, the division of this area was vague, imprecise, and un-individualized in most studies related to separate FE models of the femur. The purpose of this study was to quantify the positional distribution and size of the weight-bearing area of the femoral head in standing position by a set of simple methods, to realize individualized reconstruction of the proximal femur FE model. METHODS: Five adult volunteers were recruited for an X-ray and CT examination in the same simulated bipedal standing position with a specialized patented device. We extracted these image data, calculated the 2D weight-bearing area on the X-ray image, reconstructed the 3D model of the proximal femur based on CT data, and registered them to realize the 2D weight-bearing area to 3D transformation as the quantified weight-bearing surface. One of the 3D models of the proximal femur was randomly selected for finite element analysis (FEA), and we defined three different loading surfaces and compared their FEA results. RESULTS: A total of 10 weight-bearing surfaces in 5 volunteers were constructed, and they were mainly distributed on the dome and anterolateral of the femoral head with a crescent shape, in the range of 1218.63-1,871.06 mm2. The results of FEA showed that stress magnitude and distribution in proximal femur FE models among three different loading conditions had significant differences, and the loading case with the quantized weight-bearing area was more in accordance with the physical phenomenon of the hip. CONCLUSION: This study confirmed an effective FE modeling method of the proximal femur, which can quantify the weight-bearing area to define a more reasonable load surface setting without increasing the actual modeling difficulty.

Polydatin improves osteogenic differentiation of human bone mesenchymal stem cells by stimulating TAZ expression via BMP2-Wnt/ß-catenin signaling pathway.

OBJECTIVES: Polydatin (PD), extracted from Polygonum cuspidatum, has shown potential therapeutic applications due to its antiosteoporotic and anti-inflammatory activities. Our previous study suggested that PD promotes the osteogenesis of human bone marrow stromal cells (hBMSCs) via the BMP2-Wnt/ß-catenin pathway. The aim of our present study was to further explore the role of PD-mediated regulation of Tafazzin (TAZ), a transcriptional coactivator with a PDZ-binding motif, in osteogenesis. MATERIALS AND METHODS: hBMSCs were isolated and treated with PD at various concentrations. Alizarin red staining and RT-qPCR were performed to identify calcium complex deposition in hBMSCs as well as the expression of specific osteoblast-related markers, respectively, in each group. Next, TAZ-silenced hBMSCs were generated by lentivirus-produced TAZ shRNA. After treatment with PD, the osteogenic abilities of the TAZ-silenced and control hBMSCs were estimated by ALP activity assay, and expression of the TAZ protein was detected by Western blot analysis and immunofluorescence staining. In vitro, an ovariectomized (OVX) mouse model was established and used to evaluate the effect of PD on bone destruction by micro-CT, immunohistochemistry, and ELISA. RESULTS: In vitro, 30 µM PD significantly improved the proliferation and calcium deposition of hBMSCs and markedly stimulated the expression of the mRNAs RUNX2, Osteopontin, DLX5, ß-catenin, TAZ, and Osteocalcin (OCN). Osteogenic differentiation induced by PD was blocked by lentivirus-mediated TAZ shRNA. Furthermore, Noggin (a regulator of bone morphogenic protein 2 (BMP2)) and DKK1 (an inhibitor of the Wnt/ß-catenin pathway) were found to inhibit the increase in TAZ expression induced by PD. In vivo, PD prevented estrogen deficiency-induced bone loss in the OVX mouse model. CONCLUSION: Taken together, our findings suggest that PD improved the osteogenic differentiation of hBMSCs and maintained the bone matrix in the OVX mouse model through the activation of TAZ, a potential target gene of the BMP2-Wnt/ß-catenin pathway.

Polydatin promotes the osteogenic differentiation of human bone mesenchymal stem cells by activating the BMP2-Wnt/ß-catenin signaling pathway.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a refractory disease induced by glucocorticoids. Marrow mesenchymal stem cells (MSCs) differentiate into multiple bone matrix cells and have been used as cell-based therapies to treat ONFH. However, the osteogenesis of MSCs isolated from patients with SONFH is significantly decreased. Polydatin has been widely used in traditional Chinese remedies due to its multiple pharmacological actions. As shown in our previous study, Polydatin protects from oxidative stress and promotes BMSC migration. However, little is known about its role in BMSC (Bone marrow mesenchymal stem cells) osteogenesis; therefore, we further investigated the effect and mechanism of Polydatin in hBMSC osteogenesis. The ability of Polydatin to promote the proliferation and osteogenic differentiation of hBMSCs was determined using the MTT assay, ALP staining and the ALP activity assay. Next, qPCR and western blotting were performed to measure the levels of genes and proteins related to the osteogenesis of hBMSCs. Then, the effect of Polydatin on the nuclear translocation of ß-catenin was determined using immunofluorescence staining. Polydatin (30 µM) markedly enhanced the proliferation of hBMSCs and alkaline phosphatase (ALP) activity. Additionally, it also significantly upregulated the expression of osteogenic genes (Runx2, osteopontin, DLX5, osteocalcin, collagen type I and BMP2) and components of the Wnt signaling pathway (ß-catenin, Lef1, TCF7, c-jun, c-myc and cyclin D). These osteogenesis-potentiating effects of Polydatin were blocked by Noggin, an inhibitor of the BMP pathway, and DKK1, an inhibitor of the Wnt/ß-catenin pathway. However, DKK1 did not affect Polydatin-induced BMP2 expression. Based on our results, Polydatin promotes the proliferation and osteogenic differentiation of hBMSCs through the BMP2-Wnt/ß-catenin signaling pathway.

Plasma C-terminal cross-linking telopeptide of type II collagen as a biomarker in advanced stages of femoral head osteonecrosis.

AIM: The aim of this study is to investigate the potential role of C-terminal cross-linking telopeptide of type II collagen (CTX-II) in the development of femoral head osteonecrosis (FHN).Materials and methodsOne hundred and thirty-two patients diagnosed with FHN and 66 age- and sex-matched healthy subjects were included in this cross-sectional case-control study between May 2016 and November 2016. Bone histomorphology, cartilaginous immunohistochemistry, Western blotting, and level of plasma CTX-II, as well as the receiver operating characteristic (ROC) curve, were evaluated. ResultsImmunohistochemistry and Western blotting showed that CTX-II was abundantly detected in the cartilage of FHN samples in stages III & IV. Plasma CTX-II levels were significantly higher in FHN patients in the advanced stages (III & IV) than was the level in the control group (164.81% and 198.15% higher in stages III & IV, respectively). However, the levels of CTX-II did not differ significantly among the FHN patients with different etiology or other clinical data. ConclusionOur result suggests that the degeneration of cartilage has already begun in stage III, even though a relatively normal articular gap can be found in the X-ray. Plasma CTX-II level may be a biomarker of the development of FHN.