Network pharmacology-based mechanism prediction and pharmacological validation of Bushenhuoxue formula attenuating postmenopausal osteoporosis in ovariectomized mice.

BACKGROUND: Bushenhuoxue (BSHX) formula, a ten-compound herbal decoction, is widely used to treat postmenopausal osteoporosis (PMOP) in China. However, the mechanism is not clear yet. METHODS: The underlying biological processes and signaling pathways were predicted by network pharmacology. In vivo experimental study, 24 female C57BL/6 J mice were randomly divided into sham, ovariectomized (OVX) and BSHX formula groups. Mice in the latter two groups were subjected to bilateral ovariectomy, and mice in the BSHX formula group were extra treated by BSHX formula at an oral dosage of 0.2 mL/10 g for 8 weeks. The femur samples were harvested for tissue analyses including µCT assay, histology and immunohistochemical (IHC) staining of VEGF signaling. RESULTS: A total of 218 active ingredients and 274 related targets were identified in BSHX formula. After matching with 292 targets of PMOP, 64 overlapping genes were obtained. GO and KEGG enrichment analyses on these 64 genes revealed that angiogenesis and VEGF signaling were considered as the potential therapeutic mechanism of BSHX formula against PMOP. Animal experiments showed that mice in the BSHX formula-treated group presented increased bone mass, microstructural parameters, blood vessel numbers and an activation of VEGF signaling (VEGF, COX2, eNOS and CD31) compared to the OVX mice. CONCLUSION: This study revealed that BSHX formula exerts anti-PMOP effects possibly through activating VEGF signaling-mediated angiogenesis.

Network pharmacology-based strategy to investigate pharmacological mechanism of Liuwei Dihuang Pill against postmenopausal osteoporosis.

Postmenopausal osteoporosis (PMOP) has became 1 of most prevalent bone disorders with aging population. Liuwei Dihuang (LWDH) Pill, a classical kidney-tonifying prescription, is extensively used to treat PMOP in China. The aim of this study is to explore the pharmacological mechanisms of LWDH Pill against PMOP via network pharmacological strategy. The active ingredients of LWDH Pill were screened out from the Traditional Chinese Medicine System Pharmacology, Encyclopedia of Traditional Chinese Medicine and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine Databases, and their related target genes were fished in the UniProt database. Simultaneously, the GeneCards and DisGeNET databases were used to identify the target genes of PMOP. Through establishing a protein-protein interaction network, the overlapping genes between LWDH Pill and PMOP were identified to analyze their interactions and the hub target genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to predict the underlying biological processes (BP) and signaling pathways, respectively. A total of 64 active ingredients and 653 related target genes were identified in LWDH Pill, and 292 target genes were closely associated with PMOP. After matching the target genes between LWDH Pill and PMOP, 84 overlapping targets were obtained and considered as therapeutically relevant. Through construction of a protein-protein interaction network, we identified 20 hub target genes including IL6, INS, tumor necrosis factor, AKT1, vascular endothelial growth factor A, IGF1, TP53, IL1B, MMP9, JUN, LEP, CTNNB1, EGF, PTGS2, PPARG, CXCL8, IL10, CCL2, FOS and ESR1. Gene Ontology enrichment analysis suggested that LWDH Pill exerted anti-PMOP effects via regulating multiple BP including cell proliferation and apoptosis, oxidative stress, inflammation and angiogenesis. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed several pathways, such as PI3K-AKT pathway, mitogen-activated protein kinase pathway, hypoxia-inducible factors-1 pathway, tumor necrosis factor pathway, interleukin-17 (IL-17) pathway and FoxO pathway that might be involved in modulating the above BP. Through network pharmacological approach, we investigated the potential therapeutic mechanism of LWDH Pill against postmenopausal osteoporosis in a systemic perspective. These identified multi-targets and multi-pathways provide promising directions for further revealing more exact mechanisms.

Network pharmacology-based pharmacological mechanism prediction on Eucommia ulmoides against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common chronic autoimmune disease characterized by synovial inflammation and progressive joint destruction. Eucommia ulmoides (EU) is a kidney-tonifying Chinese medicine that has been applied to treat RA for decides. The present study aims to explore pharmacological mechanisms of EU against RA using network pharmacology approach. Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to screen active ingredients of EU, and their relative targets were fished from UniProt database. RA-related targets were screened from GeneCards database and DisGeNET database. The overlapping genes between EU and RA were identified by Venn diagram, and further analyzed for protein-protein interaction (PPI), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG). Fifty active ingredients were identified in EU, and corresponded to 207 targets. Meanwhile, 499 targets were closely associated with RA development. A total of 50 overlapping genes between EU and RA were identified, which were regarded as therapeutically relevant. GO enrichment analysis indicated that EU exerted antiRA effects depending on regulating multiple biological processes including inflammatory response, oxidative stress, cell apoptosis and matrix catabolism. Several key pathways such as TNF pathway, IL-17 pathway, T cell receptor pathway, NOD-like receptor pathway and Toll-like receptor pathway, were involved in the above biological processes. Network pharmacology revealed that EU exerts therapeutic effects on RA through multi-ingredients, multi-targets and multi-pathways, which provides basis for its clinical application and promising directions for subsequent research.

Network pharmacology explores the mechanisms of Eucommia ulmoides cortex against postmenopausal osteoporosis.

ABSTRACT: Postmenopausal osteoporosis (PMOP) has become one of most frequent chronic disease worldwide with aging population. Eucommia ulmoides cortex (EU), a traditional Chinese medicine, has long since been used to treat PMOP. The aim of this study is to explore pharmacological mechanisms of EU against PMOP through using network pharmacology approach.The active ingredients of EU were obtained from Traditional Chinese Medicine System Pharmacology database, and target fishing was performed on these ingredients in UniProt database for identification of their relative targets. Then, we screened the targets of PMOP using GeneCards database and DisGeNET database. The overlapping genes between PMOP and EU were obtained to performed protein-protein interaction, Gene Ontology analysis, Kyoto encyclopedia of genes, and genomes analysis.Twenty-eight active ingredients were identified in EU, and corresponded to 207 targets. Also, 292 targets were closely associated with PMOP, and 50 of them matched with the targets of EU were considered as therapeutically relevant. Gene ontology enrichment analysis suggested that EU exerted anti-PMOP effects via modulating multiple biological processes including cell proliferation, angiogenesis, and inflammatory response. Kyoto encyclopedia of genes and genomes enrichment analysis revealed several pathways, such as PI3K-AKT pathway, mitogen-activated protein kinase pathway, hypoxia-inducible factors-1 pathway, tumor necrosis factor pathway, and interleukin-17 pathway that might be involved in regulating the above biological processes.Through the method of network pharmacology, we systematically investigated the mechanisms of EU against PMOP. The multi-targets and multi-pathways identified here could provide new insights for further determination of more exact mechanisms of EU.

Multi-element processed pyritum mixed to ß-tricalcium phosphate to obtain a 3D-printed porous scaffold: An option for treatment of bone defects.

Bone defects remain a challenging problem for doctors and patients in clinical practice. Processed pyritum is a traditional Chinese medicine that is often used to clinically treat bone fractures. It contains mainly Fe, Zn, Cu, Mn, and other elements. In this study, we added the extract of processed pyritum to ß-tricalcium phosphate and produced a porous composite TPP (TCP/processed pyritum) scaffold using digital light processing (DLP) 3D printing technology. Scanning electron microscopy (SEM) analysis revealed that TPP scaffolds contained interconnected pore structures. When compared with TCP scaffolds (1.35 ± 0.15 MPa), TPP scaffolds (5.50 ± 0.24 MPa) have stronger mechanical strength and can effectively induce osteoblast proliferation, differentiation, and mineralization in vitro. Meanwhile, the in vivo study showed that the TPP scaffold had better osteogenic capacity than the TCP scaffold. Furthermore, the TPP scaffold had good biosafety after implantation. In summary, the TPP scaffold is a promising biomaterial for the clinical treatment of bone defects.

Radix Rehmanniae Praeparata promotes bone fracture healing through activation of TGF-ß signaling in mesenchymal progenitors.

BACKGROUND: Radix Rehmanniae Praeparata (RR), the steamed roots of Rehmannia glutinosa, is a traditional Chinese medicine with the function of kidney-nourishing, and it has been safety used for centuries to treat bone-related disorders. The aim of this study is to investigate the positive effect and underlying mechanism of RR enhancing bone fracture healing in mouse model. METHODS: Ten-week-old C57BL/6J mice were subjected to a unilateral open transverse tibial fracture and provided a daily treatment of RR. Bone samples were harvested for tissue analyses including x-ray, µCT, histology, histomorphometry, biomechanical testing, immunohistochemical (IHC) and quantitative gene expression analysis. To determine the role of TGF-ß in accelerating fracture healing effect of RR, aforementioned experiments were performed on Gli1-CreER; Tgfbr2 flox/flox (Tgfbr2Gli1ER) conditional knockout mice. RESULTS: RR promoted bone fracture healing and strengthened bone intensity in wild-type and Cre- mice with the activation of TGF-ß/Smad2 signaling, on the contrary, RR failed to accelerating fracture healing in Tgfbr2Gli1ER mice. CONCLUSION: RR promotes bone fracture healing by intensify the contribution of Gli1+ cells on bone and cartilage formation mainly in TGF-ß-dependent manner. RR is an alternative option for clinical treatment of fracture.

Bushenhuoxue formula accelerates fracture healing via upregulation of TGF-ß/Smad2 signaling in mesenchymal progenitor cells.

BACKGROUND: Although Bushenhuoxue formula (BSHXF) is successfully used as a non-traumatic therapy in treating bone fracture in China, the molecular mechanism underlying its effects remains poorly understood. PURPOSE: The present study aims to explore the therapeutic effects of BSHXF on fracture healing in mice and the underlying mechanism. METHODS: We performed unilateral open transverse tibial fracture procedure in C57BL/6 mice which were treated with or without BSHXF. Fracture callus tissues were collected and analyzed by X-ray, micro-CT, biomechanical testing, histopathology and quantitative gene expression analysis. Tibial fracture procedure was also performed in Cre-negative and Gli1-CreER; Tgfbr2flox/flox conditional knockout (KO) mice (Tgfbr2Gli1ER) to determine if BSHXF enhances fracture healing in a TGF-ß-dependent manner. In addition, scratch-wound assay and cell counting kit-8 (CCK-8) assay were used to evaluate the effect of BSHXF on cell migration and cell proliferation in C3H10T1/2 mesenchymal stem cells, respectively. RESULTS: BSHXF promoted endochondral ossification and enhanced bone strength in wild-type (WT) or Cre- control mice. In contrast, BSHXF failed to promote bone fracture healing in Tgfbr2Gli1ER conditional KO mice. In the mice receiving BSHXF treatment, TGF-ß/Smad2 signaling was significantly activated. Moreover, BSHXF enhanced cell migration and cell proliferation in C3H10T1/2 cells, which was strongly attenuated by the small molecule inhibitor SB525334 against TGF-ß type I receptor. CONCLUSION: These data demonstrated that BSHXF promotes fracture healing by activating TGF-ß/Smad2 signaling. BSHXF may be used as a type of alternative medicine for the treatment of bone fracture healing.

Bushenhuoxue formula promotes osteogenic differentiation of growth plate chondrocytes through ß-catenin-dependent manner during osteoporosis.

BACKGROUND: Bushenhuoxue formula (BSHXF) has shown excellent clinical effects on the treatment of osteoporosis in China. The aim of this study is to determine the anti-osteoporosis effects and precise molecular mechanisms of BSHXF on mouse models. METHODS: Ten-week-old female C57BL/6 J mice were subjected to ovariectomy and provided a daily treatment of BSHXF. At 8 weeks post-surgery, the femurs were harvested for tissue analyses including µCT, histology, qRT-PCR and immunohistochemical (IHC) staining of ß-catenin, ALP and FABP4. To investigate the role of ß-catenin in the anti-osteoporosis effects of BSHXF, relative experiments mentioned above were performed in ß-catenin conditional knockout mice. RESULTS: Ovariectomized (OVX) mice presented severe bone loss and excessive fat accumulation in the chondro-osseous junction underneath the growth plate, with decreased expression of ALP and increased expression of FABP4. BSHXF significantly recovered the OVX-induced abnormal osteogenesis and adipogenesis with the activation of ß-catenin in growth plate chondrocytes. Further, we generated growth plate chondrocyte-specific ß-catenin knockout (ß-cateninGli1ER) mice that exhibited bone loss and fat accumulation in the chondro-osseous junction, similar to the OVX mice. However, BSHXF failed to rescue the osteoporosis-like phenotype in ß-cateninGli1ER mice, indicating the anti-osteoporosis effects of BSHXF act mainly through ß-catenin signaling. No significant restoration of ALP and FABP4 was observed in ß-cateninGli1ER mice after the treatment of BSHXF. CONCLUSIONS: BSHXF attenuates osteoporosis by promoting osteogenic differentiation of growth plate chondrocytes mainly in ß-catenin-dependent manner. BSHXF is considered as a new candidate for the treatment of osteoporosis.

Yougui pills exert osteoprotective effects on rabbit steroid-related osteonecrosis of the femoral head by activating ß-catenin.

OBJECTIVE: To investigate the effect and underlying mechanism of Yougui pills (YGPs) on steroid-related osteonecrosis of the femoral head (SONFH). METHODS: Male New Zealand white rabbits were divided into three groups: control group, SONFH group and YGPs group. Rabbit SONFH was induced by methylprednisolone (MPS) combined with lipopolysaccharide (LPS). At 6 weeks post induction, the femoral heads were harvested for tissue analyses, including histopathology, mechanical test of femoral heads, micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry for osteocalcin (OCN), vascular endothelial growth factor (VEGF) and ß-catenin. Protein levels of cathepsin K (CTSK), phospho-glycogen synthase kinase-3 beta (p-Ser9 GSK-3ß) and total glycogen synthase kinase-3 beta (GSK-3ß) in femoral heads were also detected. Additionally, the serum TRAP activity was measured using enzyme-linked immunosorbent assay (ELISA). Finally, the effects of YGPs treatment on osteoclast differentiation and osteoblast formation were evaluated in vitro. RESULTS: The ratio of empty lacuna was markedly lower in YGPs group than SONFH group. Micro-CT evaluation indicated that YGPs has a preventive effect on bone loss in rabbit SONFH. YGPs treatment could suppress bone resorption by reducing TRAP+ osteoclast and serum TRACP5b levels in necrotic femoral heads. Moreover, YGPs treatment could promote bone formation by up-regulating the expression of OCN, VEGF and ß-catenin, while increasing load-bearing capacity of femoral heads. Interestingly, p-Ser9 GSK-3ß downregulation, and CTSK upregulation in necrotic femoral head could be reversed by YGPs treatment, which also effectively inhibited RANKL-induced osteoclast differentiation and promoted osteoblast formation in vitro. CONCLUSION: YGPs could suppress osteoclastogenesis and promote bone formation during SONFH in rabbits by activating ß-catenin.

Bushenhuoxue Formula Facilitates Articular Cartilage Repair and Attenuates Matrix Degradation by Activation of TGF-ß Signaling Pathway.

OBJECTIVE: To investigate the effect and underlying mechanism of Bushenhuoxue (BSHX) formula on articular cartilage repair. METHODS: Twenty-four full-thickness cartilage defect rats were divided into two groups: model group and BSHX group (treated with BSHX formula). Macroscopic observation and histopathological study were conducted after 4- and 8-week treatment. Additionally, we also evaluated chondrocyte proliferation, extracellular matrix (ECM) deposition, cartilage degradation, and chondrocyte hypertrophy-related genes expression in chondrogenic ATDC5 cells cultured in BSHX formula-mediated serum. Moreover, we assessed aforementioned genes expression and pSMAD2/3 protein level in Tgfßr2 siRNA transfected chondrogenic ATDC5 cells in order to address whether BSHX formula exerts cartilage repairing effect through TGF-ß signaling. RESULTS: Neocartilage regeneration promotion effect was observed in cartilage defect rats after BSHX formula treatment, with increases in Col2 and pSMAD2 and decreases in Mmp13 and Runx2. Moreover, cell proliferation, the elevated Col2a1, Aggrecan and pSMAD2/3, reduced Mmp13, Adamts5, Col10a1, and Runx2 expression were also observed in chondrogenic ATDC5 cells cultured in BSHX formula-mediated serum. Besides, the expression alteration of ECM deposition, cartilage degradation, chondrocyte hypertrophy-related genes, and pSMAD2/3 protein levels presented in Tgfßr2 downregulated chondrogenic ATDC5 cells couldn't be adjusted by BSHX formula treatment. CONCLUSION: By activation of TGF-ß signaling, BSHX formula can promote articular cartilage repair by accelerating chondrocyte proliferation and maintaining chondrocyte phenotype, upregulate ECM accumulation, and inhibit matrix degradation.