More Positive or More Negative? Metagenomic Analysis Reveals Roles of Virome in Human Disease-Related Gut Microbiome.

Viruses are increasingly viewed as vital components of the human gut microbiota, while their roles in health and diseases remain incompletely understood. Here, we first sequenced and analyzed the 37 metagenomic and 18 host metabolomic samples related to irritable bowel syndrome (IBS) and found that some shifted viruses between IBS and controls covaried with shifted bacteria and metabolites. Especially, phages that infect beneficial lactic acid bacteria depleted in IBS covaried with their hosts. We also retrieved public whole-genome metagenomic datasets of another four diseases (type 2 diabetes, Crohn's disease, colorectal cancer, and liver cirrhosis), totaling 438 samples including IBS, and performed uniform analysis of the gut viruses in diseases. By constructing disease-specific co-occurrence networks, we found viruses actively interacting with bacteria, negatively correlated with possible dysbiosis-related and inflammation-mediating bacteria, increasing the connectivity between bacteria modules, and contributing to the robustness of the networks. Functional enrichment analysis showed that phages interact with bacteria through predation or expressing genes involved in the transporter and secretion system, metabolic enzymes, etc. We further built a viral database to facilitate systematic functional classification and explored the functions of viral genes on interacting with bacteria. Our analyses provided a systematic view of the gut virome in the disease-related microbial community and suggested possible positive roles of viruses concerning gut health.

Is vitamin K2 a treatment choice for atypical femoral fractures in patients with secondary osteoporosis?

An atypical femoral fracture (AFF) is a rare complication associated with excessive inhibition of osteoclast expression during treatment of osteoporosis. We herein describe a patient who had been treated with alendronate for more than 10 years and subsequently developed an AFF that healed after treatment with vitamin K2 (VK2). We also discuss the potential beneficial effects of VK2 on the healing of AFFs. A 48-year-old Asian man with secondary osteoporosis was treated with alendronate for more than 10 years. The patient underwent surgical treatment for a complete AFF of the right femur. Six months postoperatively, he complained of pain in his left thigh. X-ray examination revealed an incomplete AFF of the left femoral shaft. He was then treated with VK2. After 4 months of VK2 treatment, the patient reported that the pain in his left thigh had decreased, and follow-up X-ray examination demonstrated healing of the left AFF line. This case report indicates that VK2 may be a potential direction for pharmacological treatment of AFFs in future research.

Apelin-13 induces mitophagy in bone marrow mesenchymal stem cells to suppress intracellular oxidative stress and ameliorate osteoporosis by activation of AMPK signaling pathway.

Osteoporosis is characterized by impaired bone metabolism. Current estimates show that it affects millions of people worldwide and causes a serious socioeconomic burden. Mitophagy plays key roles in bone marrow mesenchymal stem cells (BMSCs) osteoblastic differentiation, mineralization, and survival. Apelin is an endogenous adipokine that participates in bone homeostasis. This study was performed to determine the role of Apelin in the osteoporosis process and whether it affects mitophagy, survival, and osteogenic capacity of BMSCs in in vitro and in vivo models of osteoporosis. Our results demonstrated that Apelin was down-regulated in ovariectomized-induced osteoporosis rats and Apelin-13 treatment activated mitophagy in BMSCs, ameliorating oxidative stress and thereby reviving osteogenic function via AMPK-α phosphorylation. Besides, Apelin-13 administration restored bone mass and microstructure as well as reinstated mitophagy, enhanced osteogenic function in OVX rats. Collectively, our findings reveal the intrinsic mechanisms underlying Apelin-13 regulation in BMSCs and its potential therapeutic values in the treatment of osteoporosis.

LightCUD: a program for diagnosing IBD based on human gut microbiome data.

BACKGROUND: The diagnosis of inflammatory bowel disease (IBD) and discrimination between the types of IBD are clinically important. IBD is associated with marked changes in the intestinal microbiota. Advances in next-generation sequencing (NGS) technology and the improved hospital bioinformatics analysis ability motivated us to develop a diagnostic method based on the gut microbiome. RESULTS: Using a set of whole-genome sequencing (WGS) data from 349 human gut microbiota samples with two types of IBD and healthy controls, we assembled and aligned WGS short reads to obtain feature profiles of strains and genera. The genus and strain profiles were used for the 16S-based and WGS-based diagnostic modules construction respectively. We designed a novel feature selection procedure to select those case-specific features. With these features, we built discrimination models using different machine learning algorithms. The machine learning algorithm LightGBM outperformed other algorithms in this study and thus was chosen as the core algorithm. Specially, we identified two small sets of biomarkers (strains) separately for the WGS-based health vs IBD module and ulcerative colitis vs Crohn's disease module, which contributed to the optimization of model performance during pre-training. We released LightCUD as an IBD diagnostic program built with LightGBM. The high performance has been validated through five-fold cross-validation and using an independent test data set. LightCUD was implemented in Python and packaged free for installation with customized databases. With WGS data or 16S rRNA sequencing data of gut microbiome samples as the input, LightCUD can discriminate IBD from healthy controls with high accuracy and further identify the specific type of IBD. The executable program LightCUD was released in open source with instructions at the webpage http://cqb.pku.edu.cn/ZhuLab/LightCUD/ . The identified strain biomarkers could be used to study the critical factors for disease development and recommend treatments regarding changes in the gut microbial community. CONCLUSIONS: As the first released human gut microbiome-based IBD diagnostic tool, LightCUD demonstrates a high-performance for both WGS and 16S sequencing data. The strains that either identify healthy controls from IBD patients or distinguish the specific type of IBD are expected to be clinically important to serve as biomarkers.

Degradation of subchondral bone collagen in the weight-bearing area of femoral head is associated with osteoarthritis and osteonecrosis.

BACKGROUND: The aim of the study was to evaluate the change of subchondral bone collagen and trabecular bone in the weight-bearing area of femoral head from patients with osteoarthritis (OA) or osteonecrosis of femoral head (ONFH), and discuss the effect of collagen degradation on OA and ONFH. METHODS: Femoral heads from patients with femoral neck fracture (FNF) were collected as control group. All collected samples were divided into OA group (N = 10), ONFH group (N = 10), and FNF group (N = 10). Differences of subchondral bone collagen were compared through scanning electron microscope (SEM) observation, immunohistochemistry staining, and Masson's trichrome staining. Alteration of subchondral bone was displayed through hematoxylin and eosin (H&E) staining and gross morphology. RESULTS: SEM results showed that collagen fibers in OA and ONFH group appeared to be thinner, rougher, sparser, and more wizened. Immunohistochemistry and Masson's trichrome staining results demonstrated that the content of collagen fibers in the OA and ONFH group was obviously less than the FNF group. H&E staining results showed that trabecular bone in OA and ONFH group appeared to be thinner and ruptured. Gross morphology results showed that the degeneration and destruction of cartilage and subchondral bone in OA and ONFH group were severer than FNF group. The characteristics mentioned above in ONFH group were more apparent than OA group. CONCLUSIONS: This study revealed that degradation of collagen fibers from subchondral bone in the weight-bearing area of femoral head was associated with OA and ONFH, which may help to find new therapeutic strategies of the diseases.

Proanthocyanidins-Mediated Nrf2 Activation Ameliorates Glucocorticoid-Induced Oxidative Stress and Mitochondrial Dysfunction in Osteoblasts.

The widespread use of therapeutic glucocorticoids has increased the frequency of glucocorticoid-induced osteoporosis (GIOP). One of the potential pathological processes of GIOP is an increased level of oxidative stress and mitochondrial dysfunction, which eventually leads to osteoblast apoptosis. Proanthocyanidins (PAC) are plant-derived antioxidants that have therapeutic potential against GIOP. In our study, a low dose of PAC was nontoxic to healthy osteoblasts and restored osteogenic function in dexamethasone- (Dex-) treated osteoblasts by suppressing oxidative stress, mitochondrial dysfunction, and apoptosis. Mechanistically, PAC neutralized Dex-induced damage in the osteoblasts by activating the Nrf2 pathway, since silencing Nrf2 partly eliminated the protective effects of PAC. Furthermore, PAC injection restored bone mass and promoted the expression of Nrf2 in the distal femur of Dex-treated osteoporotic rats. In summary, PAC protect osteoblasts against Dex-induced oxidative stress and mitochondrial dysfunction via the Nrf2 pathway activation and may be a promising drug for treating GIOP.

Vitamin K2 Can Rescue the Dexamethasone-Induced Downregulation of Osteoblast Autophagy and Mitophagy Thereby Restoring Osteoblast Function In Vitro and In Vivo.

Chronic long-term glucocorticoids (GC) use is associated with glucocorticoid-induced osteoporosis (GIOP) by inhibiting the survival and impairing the functions of osteoblasts. Autophagy and mitophagy play key roles in osteoblast differentiation, mineralization and survival, and mounting evidence have implicated osteoblast autophagy and mitophagy as a novel mechanism in the pathogenesis of GIOP. Vitamin K2 (VK2) is an essential nutrient supplement that have been shown to exert protective effects against osteoporotic bone loss including GIOP. In this study, we showed that the glucocorticoid dexamethasone (Dex) deregulated osteoblast autophagy and mitophagy by downregulating the expression of autophagic and mitophagic markers LC3-II, PINK1, Parkin. This consequently led to inhibition of osteoblast differentiation and mineralization function in vitro. Interestingly, co-treatment with VK2 significantly attenuated the Dex-induced downregulation of LC3-II, PINK1, Parkin, thereby restoring autophagic and mitophagic processes and normal osteoblastic activity. In addition, using an established rat model of GIOP, we showed that VK2 administration can protect rats against the deleterious effects of Dex on bone by reinstating autophagic and mitophagic activities in bone tissues. Collectively, our results provide new insights into the role of osteoblast autophagy and mitophagy in GIOP. Additionally, the use of VK2 supplementation to augment osteoblast autophagy/mitophagy may significantly improve clinical outcomes of GIOP patients.

Surface Functionalization with Proanthocyanidins Provides an Anti-Oxidant Defense Mechanism That Improves the Long-Term Stability and Osteogenesis of Titanium Implants.

PURPOSE: Aseptic loosening is a major complication after total joint replacement. Reactive oxygen species generated by local tissue cells and liberated from implant surfaces have been suggested to cause implant failures. Surface modification of titanium (Ti)-based implants with proanthocyanidins (PAC) is a promising approach for the development of anti-oxidant defense mechanism to supplement the mechanical functions of Ti implants. In this study, a controlled PAC release system was fabricated on the surface of Ti substrates using the layer-by-layer (LBL) assembly. MATERIALS AND METHODS: Polyethyleneimine (PEI) base layer was fabricated to enable layer-by-layer (LBL) deposition of hyaluronic acid/chitosan (HA/CS) multi-layers without or with the PAC. Surface topography and wettability of the fabricated HA/CS-PAC substrates were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR) and contact angle measurement. PAC release profiles were investigated using drug release assays. MC3T3-E1 pre-osteoblast cells were used to assess the osteo-inductive effects of HA/CS-PAC substrates under conditions H2O2-induced oxidative stress in vitro. A rat model of femoral intramedullary implantation evaluated the osseo-integration and osteo-inductive potential of the HA/CS-PAC coated Ti implants in vivo. RESULTS: SEM, AFM, FTIR and contact angle measurements verified the successful fabrication of Ti surfaces with multi-layered HA/CS-PAC coating. Drug release assays revealed controlled and sustained release of PAC over 14 days. In vitro, cell-based assays showed high tolerability and enhanced the osteogenic potential of MC3T3-E1 cells on HA/CS-PAC substrates when under conditions of H2O2-induced oxidative stress. In vivo evaluation of femoral bone 14 days after femoral intramedullary implantation confirmed the enhanced osteo-inductive potential of the HA/CS-PAC coated Ti implants. CONCLUSION: Multi-layering of HA/CS-PAC coating onto Ti-based surfaces by the LBL deposition significantly enhances implant osseo-integration and promotes osteogenesis under conditions of oxidative stress. This study provides new insights for future applications in the field of joint arthroplasty.

Enhancement of local bone formation on titanium implants in osteoporotic rats by biomimetic multilayered structures containing parathyroid hormone (PTH)-related protein.

Osteoporosis is a severe health problem causing bone fragility and consequent fracture. Titanium (Ti) implants, used in patients with osteoporotic fractures, are prone to failure because of the decreased bone mass and strength. Therefore, it is of utmost importance to fabricate implants possessing osteogenic properties to improve implant osseointegration. To improve the long-term survival rate of Ti implants in osteoporotic patients, hyaluronic acid/ϵ-polylysine multilayers containing the parathyroid hormone (PTH)-related protein (PTHrP) were deposited on Ti implants by a layer-by-layer (LBL) electro assembly technique. The murine pre-osteoblast cell line MC3T3-E1, possessing a high potential of osteoblast differentiation, was used to evaluate the osteo-inductive effects of Ti-LBL-PTHrP in vitro. In addition, the performance of the Ti (Ti-LBL-PTHrP) implant was evaluated in vivo in a femoral intramedullary implantation in Sprague Dawley rats. The Ti-LBL-PTHrP implant regulated the release of the loaded PTHrP to increase bone formation in the early stage of implantation. The in vitro results revealed that cells on Ti-LBL-PTHrP did not show any evident proliferation, but a high level of alkaline phosphatase activity and osteoblast-related protein expression was found, compared to the uncoated Ti group (p < 0.05). In addition, in vivo micro-CT and histological analysis demonstrated that the Ti-LBL-PTHrP implants could significantly promote the formation and remodeling of new bone in osteoporotic rats at 14 d after implantation. Overall, this study established a profound and straightforward methodology for the manufacture of biofunctional Ti implants for the treatment of osteoporosis.

Imperatorin promotes osteogenesis and suppresses osteoclast by activating AKT/GSK3 ß/ß-catenin pathways.

Osteoporosis is caused by disturbance in the dynamic balance of bone remodelling, a physiological process, vital for maintenance of healthy bone tissue in adult humans. In this process, a new bone is formed by osteoblasts and the pre-existing bone matrix is resorbed by osteoclasts. Imperatorin, a widely available and inexpensive plant extract with antioxidative and apoptotic effects, is reported to treat osteoporosis. However, the underlying mechanism and specific effects on bone metabolism have not been elucidated. In this study, we used rat bone marrow-derived mesenchymal stem cells and found that imperatorin can activate RUNX2, COL1A1 and osteocalcin by promoting the Ser9 phosphorylation of GSK3ß and entry of ß-catenin into the nucleus. Imperatorin also enhanced the production of phospho-AKT (Ser473), an upstream factor that promotes the Ser9 phosphorylation of GSK3ß. We used ipatasertib, a pan-AKT inhibitor, to inhibit the osteogenic effect of imperatorin, and found that imperatorin promotes osteogenesis via AKT/GSK3ß/ß-catenin pathway. Next, we used rat bone marrow-derived monocytes, to check whether imperatorin inhibits osteoclast differentiation via AKT/GSK3ß/ß-catenin pathway. Further, we removed the bilateral ovaries of rats to establish an osteoporotic model. Intragastric administration of imperatorin promoted osteogenesis and inhibited osteoclast in vivo. Our experiments showed that imperatorin is a potential drug for osteoporosis treatment.

PPR-Meta: a tool for identifying phages and plasmids from metagenomic fragments using deep learning.

BACKGROUND: Phages and plasmids are the major components of mobile genetic elements, and fragments from such elements generally co-exist with chromosome-derived fragments in sequenced metagenomic data. However, there is a lack of efficient methods that can simultaneously identify phages and plasmids in metagenomic data, and the existing tools identifying either phages or plasmids have not yet presented satisfactory performance. FINDINGS: We present PPR-Meta, a 3-class classifier that allows simultaneous identification of both phage and plasmid fragments from metagenomic assemblies. PPR-Meta consists of several modules for predicting sequences of different lengths. Using deep learning, a novel network architecture, referred to as the Bi-path Convolutional Neural Network, is designed to improve the performance for short fragments. PPR-Meta demonstrates much better performance than currently available similar tools individually for phage or plasmid identification, while testing on both artificial contigs and real metagenomic data. PPR-Meta is freely available via http://cqb.pku.edu.cn/ZhuLab/PPR_Meta or https://github.com/zhenchengfang/PPR-Meta. CONCLUSIONS: To the best of our knowledge, PPR-Meta is the first tool that can simultaneously identify phage and plasmid fragments efficiently and reliably. The software is optimized and can be easily run on a local PC by non-computer professionals. We developed PPR-Meta to promote the research on mobile genetic elements and horizontal gene transfer.

Characteristic dysbiosis of gut microbiota of Chinese patients with diarrhea-predominant irritable bowel syndrome by an insight into the pan-microbiome.

BACKGROUND: Irritable bowel syndrome (IBS) is reported associated with the alteration of gut microbial composition termed as dysbiosis. However, the pathogenic mechanism of IBS remains unclear, while the studies of Chinese individuals are scarce. This study aimed to understand the concept of dysbiosis among patients with Chinese diarrhea-predominant IBS (IBS-D), as a degree of variance between the gut microbiomes of IBS-D population and that of a healthy population. METHODS: The patients with IBS-D were recruited (assessed according to the Rome III criteria, by IBS symptom severity score) from the Outpatient Department of Gastroenterology of Peking University Third Hospital, and volunteers as healthy controls (HCs) were enrolled, during 2013. The 16S rRNA sequences were extracted from fecal samples. Ribosomal database project resources, basic local alignment search tool, and SparCC software were used to obtain the phylotype composition of samples and the internal interactions of the microbial community. Herein, the non-parametric test, Wilcoxon rank-sum test was carried out to find the statistical significance between HC and IBS-D groups. All the P values were adjusted to q values to decrease the error rate. RESULTS: The study characterized the gut microbiomes of Chinese patients with IBS-D, and demonstrated that the dysbiosis could be characterized as directed alteration of the microbiome composition leading to greater disparity between relative abundance of two phyla, Bacteroidetes (Z = 4.77, q = 1.59 × 10) and Firmicutes (Z = -3.87, q = 5.83 × 10). Moreover, it indicated that the IBS symptom features were associated with the dysbiosis of whole gut microbiome, instead of one or several certain genera even they were dominating. Two genera, Bacteroides and Lachnospiracea incertae sedis, were identified as the core genera, meanwhile, the non-core genera contribute to a larger pan-microbiome of the gut microbiome. Furthermore, the dysbiosis in patients with IBS-D was associated with a reduction of network complexity of the interacted microbial community (HC vs. IBS-D: 639 vs. 154). The disordered metabolic functions of patients with IBS-D were identified as the potential influence of gut microbiome on the host (significant difference with q < 0.01 between HC and IBS-D). CONCLUSIONS: This study supported the view of the potential influence of gut microbiome on the symptom of Chinese patients with IBS-D, and further characterized dysbiosis in Chinese patients with IBS-D, thus provided more pathological evidences for IBS-D with the further understanding of dysbiosis.

The fast degradation of ß-TCP ceramics facilitates healing of bone defects by the combination of BMP-2 and Teriparatide.

Accumulating evidence suggests that the degradation and resorption of calcium phosphate ceramics is always relatively slow, which may inhibit calcium phosphate ceramics' replacement by new bone tissues and the ultimate bone defect repair. Bone morphogenetic proteins (BMPs) and Teriparatide (PTH) are extensively applied in the treatment of bone pathologies, while their effects on the degradation of calcium phosphate ceramics is limited. In this study, we tested the effects of BMP and PTH on degradation of ß-tricalcium phosphate (ß-TCP) ceramics and bone formation on ß-TCP in ovariectomized (OVX) rat models. After establishment of femur defect model on OVX rats, the BMP + PTH group's rats were injected Teriparatide (30 µg/kg) subcutaneous every other day, while rats of control group and group BMP were injected equal-to-group volume sterilized saline water. Twelve weeks after femur surgery, all rats were sacrificed for Micro-CT scanning and histology tests. The results showed that BMP facilitated degradation of ß-TCP and new bone formation on ß-TCP ceramics. And PTH showed an additional effect on degradation of ß-TCP when combined with BMP. In addition, the results explained that PTH promoted the remodeling of the bone callus occurred during repair.

Combined treatment with vitamin K2 and PTH enhanced bone formation in ovariectomized rats and increased differentiation of osteoblast in vitro.

Osteoporosis is accompanied by insufficient osteogenic capacity. Several lines of evidence suggested that solutions to enhance osteoblastogenesis were important strategies for osteoporotic bone defect repair. This study investigated the effect of combined treatment with vitamin K2 and PTH on bone formation in calvarial bone defect in osteoporotic rats and its influence on osteoblast in vitro. Bilateral ovariectomy was used in SPF Sprague Dawley rats to generate an osteoporosis model. Subsequently, a calvarial defect model was established and all osteoporotic rats were randomly assigned to the following groups: control, VK (vitamin K2, 30 mg/kg everyday), PTH (recombinant human PTH (1-34), 60 µg/kg, three times a week) or VK + PTH (vitamin K2, 30 mg/kg everyday plus PTH, 60 µg/kg three times a week) for 8 weeks. In vitro, bone marrow-derived stem cells (BMSCs) were cultured and treated with vitamin K2, PTH or vitamin K2+PTH. ALP staining and western blot were performed to observe the influence of combined treatment on BMSCs. Bone formation within calvarial defect were assessed by serum γ-carboxylated osteocalcin (Gla-OC), micro-CT, histological and immunofluorescent labeling. In this study, combined treatment of PTH and vitamin K2 showed positive effects on preventing bone loss in femurs in OVX rats. Combined treatment increased serum Gla-OC and promoted bone formation in osteoporotic calvarial bone defects. Immunohistochemistry showed that OCN and RUNX2 were more highly expressed in the VK + PTH group than in the control groups. In vitro studies results suggested that combined treatment with PTH and vitamin K2 increased expression of ALP, BMP2 and RUNX2 in BMSCs. Our data suggested that the combination of vitamin K2 and PTH increased differentiation of osteoblast and had a synergistic effect on bone formation in osteoporotic calvarial bone defect.

Effects of combined menaquinone-4 and PTH1-34 treatment on osetogenesis and angiogenesis in calvarial defect in osteopenic rats.

PURPOSE: The aim of this study was to evaluate the effect of combining human parathyroid hormone (1-34) (PTH1-34; PTH) and menaquinone-4 (MK-4) on calvarial bone defect repair in osteopenic rats. METHODS: Fourteen week olds were subject to craniotomy for the establishment of osteopenic animal models fed through a chronically low-protein diet. After that, critical calvarial defect model was established and all rats were randomly divided into four groups: sham, MK-4, PTH, and PTH + MK-4. The animals received MK-4 (30 mg/kg/day), PTH1-34 (60 µg/kg, three times a week), or PTH1-34 (60 µg/kg, three times a week) plus MK-4 (30 mg/kg/day) for 8 weeks, respectively. Serum γ-carboxylated osteocalcin (Gla-OC) levels, histological and immunofluorescent labeling were employed to evaluate the bone formation and mineralization in calvarial bone defect. In addition, Microfil perfusion, immunohistochemical, and micro-CT suggested enhanced angiogenesis and bone formation in calvarial bone healing. RESULTS: In this study, treatment with either PTH1-34 or MK-4 promoted bone formation and vascular formation in calvarial bone defects compared with the sham group. In addition, combined treatment of PTH1-34 plus MK-4 increased serum level of Gla-OC, improved vascular number and vascular density, and enhanced bone formation in calvarial bone defect in osteopenic conditions as compared with monotherapy. CONCLUSIONS: In summary, this study indicated that PTH1-34 plus MK-4 combination therapy accelerated bone formation and angiogenesis in calvarial bone defects in presence of osteopenia.

Combined treatment with Cinnamaldehyde and ß-TCP had an additive effect on bone formation and angiogenesis in critical size calvarial defect in ovariectomized rats.

Accumulating evidence suggests that improvements in osteogenesis and angiogenesis play an important role in repairing osteoporotic bone defects. Cinnamomum cassia (C. cassia), a traditional Chinese medicinal herb, is reported to show anabolic effects on osteoblasts. However, whether C. cassia could actually repair bone defects in osteoporotic conditions remains unknown. The purpose of this study was to evaluate the effect of combined treatment with Cinnamaldehyde (main oil isolated from the C. cassia) and ß-tricalcium phosphate (ß-TCP) on bone formation and angiogenesis in critical size calvarial defects in ovariectomized (OVX) rats. Using a previously established OVX model, 5 mm critical size calvarial defect was established in OVX rats. All OVX rats were then randomly divided into OVX group (OVX rats + empty defect), TCP group (OVX rats + ß-TCP), and CTCP group (Cinnamaldehyde 75 mg/kg/day for 12 weeks + ß-TCP). Twelve weeks after treatment, according to Micro-CT and HE staining, combination of Cinnamaldehyde and ß-TCP had an additive effect on bone regeneration compared with other groups (p < 0.05). Based on dynamic fluorochrome-labelling analysis, Cinnamaldehyde+ß-TCP continuously promoted new bone mineralization compared with other groups at each time point (p < 0.05). Microfil perfusion suggested that CTCP group showed more neovascularization compared with other groups (p < 0.05). Immunohistochemical assay supported the findings that Cinnamaldehyde+ß-TCP enhanced expression of OCN, VEGF and CD31. The present study demonstrated that combined treatment with Cinnamaldehyde and ß-TCP promoted bone formation and angiogenesis in osteoporotic bone defects, which provides a promising new strategy for repairing bone defects in osteoporotic conditions.

Combined treatment with cinnamaldehyde and PTH enhances the therapeutic effect on glucocorticoid-induced osteoporosis through inhibiting osteoclastogenesis and promoting osteoblastogenesis.

The study was to investigate the effect of combining treatment with cinnamaldehyde and parathyroid hormone (1-34) (PTH) on glucocorticoid-induced osteoporosis (GIO) and compare with monotherapy. Forty Sprague-Dawley male rats with GIO were divided into four groups randomly: control group (CON group, N = 10); group that intragastric administration with cinnamaldehyde (CIN group, N = 10); group that subcutaneous injection with PTH, three times per week(PTH group, N = 10); both administration with cinnamaldehyde and PTH (CIN + PTH group, N = 10). Distal femurs were harvested for hematoxylin and eosin (H&E) staining, micro-CT scanning and immunohistochemical analysis. Murine mesenchymal stem cells were cultured and dealt with the presence of dexamethasone(DEX group), DEX + cinnamaldehyde(DEX + CIN group), DEX + PTH(DEX + PTH group) or DEX + cinnamaldehyde + PTH(DEX + CIN + PTH group). Alkaline phosphatase (ALP) staining was performed subsequently. The results showed that bone formation in CIN + PTH group was notably promoted compared with other groups. And the expression of tartrate-resistant acid phosphatase (trap) and runt-related transcription factor 2 (runx2) in CIN + PTH group were down-regulated and up-regulated respectively compared with PTH group. In vitro study revealed that ALP-positive cell number in DEX + CIN + PTH group was obviously enhanced compared with other groups. The study revealed that combined treatment with cinnamaldehyde and PTH enhances the therapeutic effect on GIO through inhibiting osteoclastogenesis and promoting osteoblastogenesis.

Administration of cinnamaldehyde promotes osteogenesis in ovariectomized rats and differentiation of osteoblast in vitro.

To explore the effect of cinnamaldehyde on the distal femur in ovariectomized rats and its influence on osteoblast in vitro. Female Sprague-Dawley rats which underwent either bilateral ovariectomy or sham operation were divided into five groups randomly: group OVX (OVX, N = 10) and group sham (SHAM, N = 10) received normal saline (NS) by gavage at a dose of 50 ml/kg·d; group low dose, group middle dose and group high dose received cinnamaldehyde by gavage at a dose of 25 mg/kg·d (OLD, N = 10), 50 mg/kg·d (OMD, N = 10), and 75 mg/kg·d (OHD, N = 10) respectively. Distal femurs were harvested for hematoxylin and eosin (HE) staining, micro-ct scanning and immunohistochemical analysis. Murine mesenchymal stem cells were cultured and dealt with the presence of either cinnamaldehyde at a dose of 15ug/ml (OLD), 30ug/ml (OMD), 60ug/ml (OHD) or vehicle. ALP staining and western blot were performed to observe the influence of cinnamaldehyde on the differentiation of osteoblast. HE and micro-ct results indicated that osteogenesis were promoted with the treatment of cinnamaldehyde. Immunohistochemical results showed that cinnamaldehyde increased the number of osteoblast and decreased the number of osteoclast. In vitro studies indicated that cinnamaldehyde promoted expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and collagen type Iɑ1 (COL1ɑ1). The treatment effect behaved as dose-dependently. Thus, cinnamaldehyde inhibits osteoclastogenesis and promotes osteoblastogenesis, and may plays an important role in the treatment of osteoporosis clinically.

Hydroxysafflor yellow A (HSYA) targets the NF-κB and MAPK pathways and ameliorates the development of osteoarthritis.

The inflammatory environment has been demonstrated to be strongly associated with the progression of osteoarthritis (OA). HSYA, the main active component in the medical and edible dual purpose plant safflower, has previously showed significant anti-inflammatory effects in several diseases. In the current study, the protective effects of HSYA in the inhibition of OA development and its underlying mechanism were examined by both in vitro and in vivo experiments. Our data indicated that interleukin-1 beta (IL-1ß) induced over-production of pro-inflammatory cytokines, such as nitric oxide (NO) and prostaglandin E2 (PGE2); also, the expression of cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) were all inhibited by pretreatment with HSYA in a dose-dependent manner (2.5 to 40 µM). Furthermore, HSYA attenuated IL-1ß-induced degradation of the extracellular matrix (ECM) by decreasing the expression of metalloproteinases (MMPs) and thrombospondin motifs 5 (ADAMTS5). Mechanistically, HSYA suppressed IL-1ß-induced activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) cascades. Meanwhile, molecular docking studies revealed that HSYA has excellent binding abilities to p65, extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK). In addition, the protective effects of HSYA were observed in a surgically induced mouse OA model. In summary, this study provides evidence that HSYA can be applied as a potential therapeutic agent in the treatment of OA.

Inhibition of Dll4/Notch1 pathway promotes angiogenesis of Masquelet's induced membrane in rats.

The Masquelet's induced membrane technique for repairing bone defects has been demonstrated to be a promising treatment strategy. Previous studies have shown that the vessel density of induced membrane is decreased in the late stage of membrane formation, which consequently disrupts the bone healing process. However, relatively little is known about certain mechanisms of vessel degeneration in the induced membrane tissue and whether promotion of angiogenesis in induced membranes can improve bone regeneration. Here, we showed that the Delta-like ligand 4/ Notch homolog 1 (Dll4/Notch1) pathway was relatively activated in the late stage of induced membrane, especially at the subcutaneous site. Then, DAPT, a classical γ-secretase inhibitor, was applied to specifically inhibit Notch1 activation, followed by up-regulation of vascular endothelial growth factor receptor 2 (VEGFR2) and CD31 expression. DAPT-modified induced membranes were further confirmed to contribute to bone regeneration after autogenous bone grafting. Finally, in vitro experiments revealed that knocking down Notch1 contributed to the functional improvement of endothelial progenitor cells (EPCs) and that DAPT-treated induced membrane tissue was more favorable for angiogenesis of EPCs compared with the vehicle group. In conclusion, the present findings demonstrate that Dll4/Notch1 signaling is negatively associated with the vessel density of induced membrane. Pharmacological inhibition of Notch1 attenuated the vessel degeneration of induced membrane both in vitro and in vivo, which consequently improved bone formation at the bone defect site and graft resorption at the subcutaneous site.