N-Butanol Extract of Modified You-Gui-Yin Attenuates Osteoclastogenesis and Ameliorates Osteoporosis by Inhibiting RANKL-Mediated NF-κB Signaling.

Postmenopausal Osteoporosis (PMOP) is the most prevalent primary osteoporosis, attributable to an imbalance in osteoblast and osteoclast activity. Modified You-Gui-Yin (MYGY), a traditional Chinese herbal formula, is able to effectively treat PMOP, while the critical components and pharmacological mechanisms of MYGY are still unclear. In this study, we aimed to investigate the therapeutic effects and underlying mechanisms of N-butanol extract of MYGY (MYGY-Nb) in ovariectomized (OVX)-induced osteoporosis mice. Histological staining and micro-computed tomography (µCT) analysis showed that MYGY-Nb was more effective in the suppression of OVX-induced bone loss than MYGY original formula. Subsequently, liquid chromatography and mass spectrometry analysis identified 16 critical compounds of MYGY-Nb and some of them are reported to affect osteoclast functions. Furthermore, in vivo and in vitro experiments demonstrated that MYGY-Nb significantly attenuated osteoclastogenesis by down-regulating RANKL-mediated NF-κB signaling. In conclusion, our study indicated that MYGY-Nb suppresses NF-κB signaling and osteoclast formation to mitigate bone loss in PMOP, implying that MYGY-Nb and its compounds are potential candidates for development of anti-PMOP drugs.

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.

Morroniside ameliorates inflammatory skeletal muscle atrophy via inhibiting canonical and non-canonical NF-κB and regulating protein synthesis/degradation.

No drug options exist for skeletal muscle atrophy in clinical, which poses a huge socio-economic burden, making development on drug interventions a general wellbeing need. Patients with a variety of pathologic conditions associated with skeletal muscle atrophy have systemically elevated inflammatory factors. Morroniside, derived from medicinal herb Cornus officinalis, possesses anti-inflammatory effect. However, whether and how morroniside combat muscle atrophy remain unknown. Here, we identified crucial genetic associations between TNFα/NF-κB pathway and grip strength based on population using 377,807 European participants from the United Kingdom Biobank dataset. Denervation increased TNFα in atrophying skeletal muscles, which inhibited myotube formation in vitro. Notably, morroniside treatment rescued TNFα-induced myotube atrophy in vitro and impeded skeletal muscle atrophy in vivo, resulting in increased body/muscles weights, No. of satellite cells, size of type IIA, IIX and IIB myofibers, and percentage of type IIA myofibers in denervated mice. Mechanistically, in vitro and/or in vivo studies demonstrated that morroniside could not only inhibit canonical and non-canonical NF-κB, inflammatory mediators (IL6, IL-1b, CRP, NIRP3, PTGS2, TNFα), but also down-regulate protein degradation signals (Follistatin, Myostatin, ALK4/5/7, Smad7/3), ubiquitin-proteasome molecules (FoxO3, Atrogin-1, MuRF1), autophagy-lysosomal molecules (Bnip3, LC3A, and LC3B), while promoting protein synthesis signals (IGF-1/IGF-1R/IRS-1/PI3K/Akt, and BMP14/BMPR2/ALK2/3/Smad5/9). Moreover, morroniside had no obvious liver and kidney toxicity. This human genetic, cells and mice pathological evidence indicates that morroniside is an efficacious and safe inflammatory muscle atrophy treatment and suggests its translational potential on muscle wasting.

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.

Cellulose nanofibers coated with silver nanoparticles as a flexible nanocomposite for measurement of flusilazole residues in Oolong tea by surface-enhanced Raman spectroscopy.

Cellulose nanofibers (CNF) coated with inorganic nanoparticles are novel hybrid nanocomposites that have great potential in various areas including agriculture and food science. The objectives of this study were to synthesize nanocomposites consisted of CNF coated with silver nanoparticles (AgNPs), which can be used as a surface-enhanced Raman spectroscopy (SERS) platform for measuring pesticides in Oolong tea. CNF were coated with AgNPs to form uniform CNF-AgNP nanocomposites that were investigated by transmission electron microscopy. Three-dimensional and porous CNF structures were loaded with AgNPs with an average size of 41 nm. CNF-AgNP substrates were applied in characterization and measurement of flusilazole in Oolong tea samples by SERS. A detection limit of 0.5 mg/kg for flusilazole was obtained based on partial least squares (PLS) regression analysis. These results indicate that CNF-AgNP nanocomposites combined with SERS is an accurate, sensitive, and efficient technique for identification and quantification of pesticide residues in Oolong tea.

A spectroscopic approach to detect and quantify phosmet residues in Oolong tea by surface-enhanced Raman scattering and silver nanoparticle substrate.

There have been increasing concerns among consumers about pesticide residues in Oolong tea. This study aimed to establish surface-enhanced Raman scattering (SERS) method for rapid measurement of chemical contaminants in Oolong tea. Synthesis of SERS substrate was achieved by synthesizing silver nanoparticles (AgNPs) via a reduction method. AgNPs were spherical and highly monodispersed, which created remarkable electromagnetic fields during SERS activities to measure phosmet in the methanol-water solution and Oolong tea. Partial least squares regression models were established to predict the concentrations of phosmet in the methanol-water solution (r = 0.934; slope = 0.880; RMSEP = 1.001 mg/L) and Oolong tea samples (r = 0.927; slope = 0.938; RMSEP = 1.157 mg/kg) with the detection limit of 0.1 mg/kg. The results indicate that SERS coupled with silver nanoparticles is a fast, sensitive, and reliable method for detection and characterization of pesticide contaminants in Oolong tea products.

Detection and quantification of carbendazim in Oolong tea by surface-enhanced Raman spectroscopy and gold nanoparticle substrates.

The contamination of pesticide residues in Oolong tea has raised much concern in recent years. The objective of this study was to synthesize gold nanoparticles (AuNPs) and develop surface-enhanced Raman spectroscopy (SERS) methods for detection and quantification of pesticides in Oolong tea. Facile synthesis of spherical and monodispersed AuNPs with an average diameter of 15 nm was achieved, which induced strong electromagnetic fields in SERS analysis. AuNP substrates were employed for rapid detection and quantification of carbendazim in Tieguanyin Oolong tea. Partial least squares (PLS) analysis and leave-one-out cross validation were utilized in spectral data analysis. The PLS results for Oolong tea samples were obtained: R value = 0.964; the detection limit = 100 µg/kg. These results demonstrate that SERS coupled with gold nanoparticle substrate is a simple, rapid, and sensitive analytical tool for measurement and quantification of carbendazim residues in Oolong tea.

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.

Essence of "Shen (Kidney) Controlling Bones": Conceptual Analysis Based on Hypothalamic-Pituitary-Adrenal-Osteo-Related Cells Axis.

As a traditional concept of Chinese medicine (CM), the theory of "Shen (Kidney) controlling bones" has been gradually proven. And in modern allopathic medicine, the multiple mechanisms of bone growth, development and regeneration align with the theory. Shen deficiency as a pathological condition has a negative effect on the skeleton of body, specifically the disorder of bone homeostasis. Present studies indicate that Shen deficiency shares a common disorder characterized by dysfunction of hypothalamic-pituitary-adrenal (HPA) axis. HPA axis may be an important regulator of bone diseases with abnormal homeostasis. Therefore, we posit the existence of hypothalamic-pituitary-adrenal-osteo-related cells axis: cells that comprise bone tissue (osteo-related cells) are targets under the regulation of HPA axis in disorder of bone homeostasis. Chinese herbs for nourishing Shen have potential in the development of treatments for disorder of bone homeostasis.

Ablation of Dnmt3b in chondrocytes suppresses cell maturation during embryonic development.

DNA methylation is a major mode of epigenetic regulation in the mammalian genome and is essential for embryonic development. The three catalytic DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, catalyze the methylation of cytosine. Dnmt3b is highly expressed in chondrocytes and global knockout of Dnmt3b led to skeletal deformations and embryonic lethality, suggesting an essential role of Dnmt3b in endochondral bone formation. To further define the role of Dnmt3b in skeletal development, Dnmt3b was deleted in Col2 positive chondrocyte lineage cells. Both axial and appendicular skeletal size were reduced and bone mineralization was delayed in Col2Cre+ ;Dnmt3bf/f (Dnmt3bCol2 ) mice at E14.5 and E18.5. While Alcian Blue Hematoxylin/Orange G (ABH/OG) staining showed normal chondrocyte columns in control growth plates, the length of hypertrophic chondrocyte zone and type X collagen expression were decreased in E18.5 growth plates from Dnmt3bCol2 mice. TUNEL and PCNA staining demonstrated that the delay in chondrocyte maturation observed in the Dnmt3bCol2 growth plates was not secondary to altered chondrocyte apoptosis or proliferation. Complementary in vitro experiments were performed on primary sternal chondrocytes isolated from control and Dnmt3bCol2 mice. Gene expression studies confirmed delayed terminal maturation as Mmp13 and Col10a1 expression was down-regulated in Dnmt3bCol2 chondrocytes. In addition, alkaline phosphatase (ALP) and Alizarin Red staining confirmed that Dnmt3b deletion in chondrocytes delays in vitro chondrocyte hypertrophic differentiation and matrix mineralization. Mechanistically, Dnmt3b gene deletion resulted in decreased BMP signaling through reduction of Smad1 phosphorylation. These findings show that epigenetic factor, Dnmt3b is necessary for normal chondrocyte hypertrophic maturation and limb development.

Effect of Auricular Point Acupressure on Axial Neck Pain After Anterior Cervical Discectomy and Fusion: A Randomized Controlled Trial.

Objectives: To evaluate the effect of auricular point acupressure (APA) on axial neck pain after anterior cervical discectomy and fusion (ACDF) surgery. Design: A prospective randomized controlled trial was performed. Subjects and setting: Twenty-nine participants were randomly divided into two groups, real or sham APA. Participants were enrolled from Shaoxing Hospital of Traditional Chinese Medicine, affiliated with Zhejiang Chinese Medical University. Methods: Eligible participants received a four-week real or sham APA treatment according to their assigned groups. The clinical outcomes were assessed by the criteria of Hosono et al., the Brief Pain Inventory Short Form (BPI), and the 36-item Short Form Health Survey (SF-36). In addition, plasma interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α were analyzed. Results: Patients with severe or moderate axial neck pain accounted for 28.6% and 35.7% in the real APA group at the end of treatment and one-month follow-up. BPI scores were decreased in the real APA group at the end of treatment and one-month follow-up. The total mean score of SF-36 was improved in the real APA group and significantly higher than in the sham APA group. Additional, the levels of IL-1ß, IL-6, and TNF-α were decreased in the real APA group. Conclusions: The findings supported the therapeutic effect of APA treatment on axial neck pain after ACDF surgery, and they exert the possible therapeutic effect on downregulating the levels of plasma IL-1ß, IL-6, and TNF-α.