Discovery of a Potent Antiosteoporotic Drug Molecular Scaffold Derived from Angelica sinensis and Its Bioinspired Total Synthesis.

Angelica sinensis, commonly known as Dong Quai in Europe and America and as Dang-gui in China, is a medicinal plant widely utilized for the prevention and treatment of osteoporosis. In this study, we report the discovery of a new category of phthalide from Angelica sinensis, namely falcarinphthalides A and B (1 and 2), which contains two fragments, (3R,8S)-falcarindiol (3) and (Z)-ligustilide (4). Falcarinphthalides A and B (1 and 2) represent two unprecedented carbon skeletons of phthalide in natural products, and their antiosteoporotic activities were evaluated. The structures of 1 and 2, including their absolute configurations, were established using extensive analysis of NMR spectra, chemical derivatization, and ECD/VCD calculations. Based on LC-HR-ESI-MS analysis and DFT calculations, a production mechanism for 1 and 2 involving enzyme-catalyzed Diels-Alder/retro-Diels-Alder reactions was proposed. Falcarinphthalide A (1), the most promising lead compound, exhibits potent in vitro antiosteoporotic activity by inhibiting NF-κB and c-Fos signaling-mediated osteoclastogenesis. Moreover, the bioinspired gram-scale total synthesis of 1, guided by intensive DFT study, has paved the way for further biological investigation. The discovery and gram-scale total synthesis of falcarinphthalide A (1) provide a compelling lead compound and a novel molecular scaffold for treating osteoporosis and other metabolic bone diseases.

A Phytomolecule Icariin Protects from Sarcopenia Partially by Suppressing Myosin Heavy Chain Degradation in Orchiectomized Rats.

Treatments are lacking for sarcopenia, which is an age-related disease characterized by loss of skeletal muscle mass, strength, and/or physical performance. Icariin is a phytomolecule from herbal Epimedium, a traditional Chinese medicine widely used to treat musculoskeletal disorders for thousands of years. Here the effects of icariin against sarcopenia are investigated and the underlying mechanism is elucidated. A classic rat model of bilaterally orchiectomized (ORX) is used to induce sarcopenia. After administration for 8 weeks, compared to the control group, the forelimb grip strength, the specific tetanic forces of the soleus (SOL) and extensor digitorum longus muscle (EDL) are higher, and the fiber cross-sectional areas (CSAs) of the gastrocnemius and tibialis anterior muscle are larger in the icariin group. In addition, icariin promotes mRNA and protein expressions of myosin heavy chain (MyHC) both in SOL and EDL. Mechanistically, icariin significantly suppresses the mRNA and protein expressions of FOXO3a, atrogin-1, and MuRF-1, which are related to the degradation of myosin heavy chain. Collectively, icariin protects from sarcopenia in ORX rats characterized by enhancing grip strength and skeletal muscle contraction, as well as increasing skeletal muscle CSA by inhibiting the ubiquitination degradation of the MyHC in skeletal muscle fibers.

PLGA/ß-TCP composite scaffold incorporating cucurbitacin B promotes bone regeneration by inducing angiogenesis.

OBJECTIVES: Vascularization is an essential step in successful bone tissue engineering. The induction of angiogenesis in bone tissue engineering can be enhanced through the delivery of therapeutic agents that stimulate vessel and bone formation. In this study, we show that cucurbitacin B (CuB), a tetracyclic terpene derived from Cucurbitaceae family plants, facilitates the induction of angiogenesis in vitro. METHODS: We incorporated CuB into a biodegradable poly (lactide-co-glycolide) (PLGA) and ß-tricalcium phosphate (ß-TCP) biomaterial scaffold (PT/CuB) Using 3D low-temperature rapid prototyping (LT-RP) technology. A rat skull defect model was used to verify whether the drug-incorporated scaffold has the effects of angiogenesis and osteogenesis in vivo for the regeneration of bone defect. Cytotoxicity assay was performed to determine the safe dose range of the CuB. Tube formation assay and western blot assay were used to analyze the angiogenesis effect of CuB. RESULTS: PT/CuB scaffold possessed well-designed bio-mimic structure and improved mechanical properties. CuB was linear release from the composite scaffold without affecting pH value. The results demonstrated that the PT/CuB scaffold significantly enhanced neovascularization and bone regeneration in a rat critical size calvarial defect model compared to the scaffold implants without CuB. Furthermore, CuB stimulated angiogenic signaling via up-regulating VEGFR2 and VEGFR-related signaling pathways. CONCLUSION: CuB can serve as promising candidate compound for promoting neovascularization and osteogenesis, especially in tissue engineering for repair of bone defects. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study highlights the potential use of CuB as a therapeutic agent and strongly support its adoption as a component of composite scaffolds for tissue-engineering of bone repair.

An UHPLC-MS/MS method for simultaneous determination of ten sex steroid hormones in ovariectomy-induced osteoporosis rat and its application in discovery of sex steroid hormones regulatory components of Xian-Ling-Gu-Bao capsule.

Sex steroid hormones could directly affect the bone metabolism by regulating cell physiological functions. In female, it inevitably causes the abnormal levels of sex steroid hormones at post-menopause in vivo. Ovariectomized rats and mice are classic animal models of osteoporosis to better understand the action mechanism of anti-osteoporosis drugs. However, it is not clear whether Xian-Ling-Gu-Bao capsule (XLGB), a kidney-tonifying traditional Chinese medicine prescription, treat osteoporosis via regulating multiple sex steroid hormones. In the present study, a reliable method involving ultra high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/TQ-XS-MS) was developed for simultaneous quantitative analysis of ten sex steroid hormones (three estrogens, five androgens and two progestogens) in rat and mouse serum. The results of methodology were acceptable. The validated method was then successfully applied in the determination of the levels of sex steroid hormones in ovariectomy-induced osteoporosis rats, as well as drug (17ß-estradiol and XLGB) intervened rats. As a result, XLGB could not only significantly increase the level of 17ß-estradiol, but also improve the levels of progesterone, 17α-hydroxyprogesterone and androstenedione. Combined with molecular docking results and pharmacokinetic parameters, psoralen, isopsoralen and sweroside were considered as the key effective components of XLGB to activate adenylyl cyclase on promoting the biosynthesis of multiple sex steroid hormones. It is the first time to evaluate the regulatory effect of kidney-tonifying traditional Chinese medicine prescription on the levels of steroids in ovariectomy-induced osteoporosis rat, as well as the potential substance basis and mechanism of steroid hormone regulation.

Inhibition of AXL enhances chemosensitivity of human ovarian cancer cells to cisplatin via decreasing glycolysis.

Anexelekto (AXL), a member of the TYRO3-AXL-MER (TAM) family of receptor tyrosine kinases (RTK), is overexpressed in varieties of tumor tissues and promotes tumor development by regulating cell proliferation, migration and invasion. In this study, we investigated the role of AXL in regulating glycolysis in human ovarian cancer (OvCa) cells. We showed that the expression of AXL mRNA and protein was significantly higher in OvCa tissue than that in normal ovarian epithelial tissue. In human OvCa cell lines suppression of AXL significantly inhibited cell proliferation, and increased the sensitivity of OvCa cells to cisplatin, which also proved by nude mice tumor formation experiment. KEGG analysis showed that AXL was significantly enriched in the glycolysis pathways of cancer. Changes in AXL expression in OvCa cells affect tumor glycolysis. We demonstrated that the promotion effect of AXL on glycolysis was mediated by phosphorylating the M2 isoform of pyruvate kinase (PKM2) at Y105. AXL expression was significantly higher in cisplatin-resistant OvCa cells A2780/DDP compared with the parental A2780 cells. Inhibition of AXL decreased the level of glycolysis in A2780/DDP cells, and increased the cytotoxicity of cisplatin against A2780/DDP cells, suggesting that AXL-mediated glycolysis was associated with cisplatin resistance in OvCa. In conclusion, this study demonstrates for the first time that AXL is involved in the regulation of the Warburg effect. Our results not only highlight the clinical value of targeting AXL, but also provide theoretical basis for the combination of AXL inhibitor and cisplatin in the treatment of OvCa.

PKM2-c-Myc-Survivin Cascade Regulates the Cell Proliferation, Migration, and Tamoxifen Resistance in Breast Cancer.

The M2 isoform of pyruvate kinase (PKM2), as a key glycolytic enzyme, plays important roles in tumorigenesis and chemotherapeutic drug resistance. However, the intricate mechanism of PKM2 as a protein kinase regulating breast cancer progression and tamoxifen resistance needs to be further clarified. Here, we reported that PKM2 controls the expression of survivin by phosphorylating c-Myc at Ser-62. Functionally, PKM2 knockdown suppressed breast cancer cell proliferation and migration, which could be rescued by overexpression of survivin. Interestingly, we found that the level of PKM2 expression was upregulated in the tamoxifen resistant breast cancer cells MCF-7/TAMR, and knockdown of PKM2 sensitized the cells to 4-hydroxytamoxifen (4OH-T). In addition, the elevated level of PKM2 correlates with poor relapse-free survival in breast cancer patients treated with tamoxifen. Overall, our findings demonstrated that PKM2-c-Myc-survivin cascade regulated the proliferation, migration and tamoxifen resistance of breast cancer cells, suggesting that PKM2 represents a novel prognostic marker and an attractive target for breast cancer therapeutics, and that PKM2 inhibitor combined with tamoxifen may be a promising strategy to reverse tamoxifen resistance in breast cancer patients.

RSK2 protects human breast cancer cells under endoplasmic reticulum stress through activating AMPKα2-mediated autophagy.

Autophagy can protect stressed cancer cell by degradation of damaged proteins and organelles. However, the regulatory mechanisms behind this cellular process remain incompletely understood. Here, we demonstrate that RSK2 (p90 ribosomal S6 kinase 2) plays a critical role in ER stress-induced autophagy in breast cancer cells. We demonstrated that the promotive effect of RSK2 on autophagy resulted from directly binding of AMPKα2 in nucleus and phosphorylating it at Thr172 residue. IRE1α, an ER membrane-associated protein mediating unfolded protein response (UPR), is required for transducing the signal for activation of ERK1/2-RSK2 under ER stress. Suppression of autophagy by knockdown of RSK2 enhanced the sensitivity of breast cancer cells to ER stress both in vitro and in vivo. Furthermore, we demonstrated that inhibition of RSK2-mediated autophagy rendered breast cancer cells more sensitive to paclitaxel, a chemotherapeutic agent that induces ER stress-mediated cell death. This study identifies RSK2 as a novel controller of autophagy in tumor cells and suggests that targeting RSK2 can be exploited as an approach to reinforce the efficacy of ER stress-inducing agents against cancer.

Magnesium and vitamin C supplementation attenuates steroid-associated osteonecrosis in a rat model.

Magnesium (Mg)-based biometal attracts clinical applications due to its biodegradability and beneficial biological effects on tissue regeneration, especially in orthopaedics, yet the underlying anabolic mechanisms in relevant clinical disorders are lacking. The present study investigated the effect of magnesium (Mg) and vitamin C (VC) supplementation for preventing steroid-associated osteonecrosis (SAON) in a rat experimental model. In SAON rats, 50 mg/kg Mg, or 100 mg/kg VC, or combination, or water control was orally supplemented daily for 2 or 6 weeks respectively. Osteonecrosis was evaluated by histology. Serum Mg, VC, and bone turnover markers were measured. Microfil-perfused samples prepared for angiography and trabecular architecture were evaluated by micro-CT. Primary bone marrow cells were isolated from each group to evaluate their potentials in osteoblastogenesis and osteoclastogenesis. The mechanisms were tested in vitro. Histological evaluation showed SAON lesions in steroid treated groups. Mg and VC supplementation synergistically reduced the apoptosis of osteocytes and osteoclast number, and increased osteoblast surface. VC supplementation significantly increased the bone formation marker PINP, and the combination significantly decreased the bone resorption marker CTX. TNFα expression and oxidative injury were decreased in bone marrow in Mg/VC/combination group. Mg significantly increased the blood perfusion in proximal tibia and decreased the leakage particles in distal tibia 2 weeks after SAON induction. VC significantly elevated the osteoblast differentiation potential of marrow cells and improved the trabecular architecture. The combination supplementation significantly inhibited osteoclast differentiation potential of marrow cells. In vitro study showed promoting osteoblast differentiation effect of VC, and anti-inflammation and promoting angiogenesis effect of Mg with underlying mechanisms. Mg and VC supplementation could synergistically alleviate SAON in rats, indicating great translational potentials of metallic minerals for preventing SAON.

Cinnamaldehyde Inhibits Inflammation of Human Synoviocyte Cells Through Regulation of Jak/Stat Pathway and Ameliorates Collagen-Induced Arthritis in Rats.

Cinnamaldehyde (Cin), a bioactive cinnamon essential oil from traditional Chinese medicine herb Cinnamomum cassia, has been reported to have multipharmacological activities including anti-inflammation. However, its role and molecular mechanism of anti-inflammatory activity in musculoskeletal tissues remains unclear. Here, we first investigated the effects and molecular mechanisms of Cin in human synoviocyte cells. Then in vivo therapeutic effect of Cin on collagen-induced arthritis (CIA) also studied. Cell Counting Kit CCK-8 assay was performed to evaluate the cell cytotoxicity. Proinflammatory cytokine expression was evaluated using quantitative polymerase chain reaction and ELISA. Protein expression was measured by western blotting. The in vivo effect of Cin (75 mg/kg per day) was evaluated in rats with CIA by gavage administration. Disease progression was assessed by clinical scoring, radiographic, and histologic examinations. Cin significantly inhibited interleukin (IL)-1ß-induced IL-6, IL-8, and tumor necrosis factor-α release from human synoviocyte cells. The molecular analysis revealed that Cin impaired IL-6-induced activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), and STAT3 signaling pathway by inhibiting the phosphorylation of JAK2, STAT1, and STAT3, without affecting NF-κB pathway. Cin reduced collagen-induced swollen paw volume of arthritic rats. The anti-inflammation effects of Cin were associated with decreased severity of arthritis, joint swelling, and reduced bone erosion and destruction. Furthermore, serum IL-6 level was decreased when Cin administered therapeutically to CIA rats. Cin suppresses IL-1ß-induced inflammation in synoviocytes through the JAK/STAT pathway and alleviated collagen-induced arthritis in rats. These data indicated that Cin might be a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug. SIGNIFICANCE STATEMENT: In this study, we found that cinnamaldehyde (Cin) suppressed proinflammatory cytokines secretion in rheumatology arthritis synoviocyte cells by Janus kinase/signal transducer and activator of transcription pathway. The in vivo results showed that Cin ameliorated collagen-induced arthritis in rats. These findings indicate that Cin is a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug.

Regulation of Inflammatory Response in Human Osteoarthritic Chondrocytes by Novel Herbal Small Molecules.

In this study, 34 Traditional Chinese Medicine (TCM) compounds were screened for potential anabolic and anti-inflammatory properties on human osteoarthritic (OA) chondrocytes. The anabolic effects were assessed by measuring the glycosaminoglycan (GAG) relative to the DNA content using a 3D pellet culture model. The most chondrogenic compounds were tested in an inflammatory model consisting of 3 days of treatment with cytokines (IL-1ß/TNF-α) with or without supplementation of TCM compounds. The anti-inflammatory effects were assessed transcriptionally, biochemically and histologically. From the 34 compounds, Vanilic acid (VA), Epimedin A (Epi A) and C (Epi C), 2''-O-rhamnosylicariside II (2-O-rhs II), Icariin, Psoralidin (PS), Protocatechuicaldehyde (PCA), 4-Hydroxybenzoic acid (4-HBA) and 5-Hydroxymethylfurfural (5-HMF) showed the most profound anabolic effects. After induction of inflammation, pro-inflammatory and catabolic genes were upregulated, and GAG/DNA was decreased. VA, Epi C, PS, PCA, 4-HBA and 5-HMF exhibited anti-catabolic and anti-inflammatory effects and prevented the up-regulation of pro-inflammatory markers including metalloproteinases and cyclooxygenase 2. After two weeks of treatment with TCM compounds, the GAG/DNA ratio was restored compared with the negative control group. Immunohistochemistry and Safranin-O staining confirmed superior amounts of cartilaginous matrix in treated pellets. In conclusion, VA, Epi C, PS, PCA, 4-HBA and 5-HMF showed promising anabolic and anti-inflammatory effects.

Identification of a Novel Bcl-2 Inhibitor by Ligand-Based Screening and Investigation of Its Anti-cancer Effect on Human Breast Cancer Cells.

Bcl-2 family protein is an important factor in regulating apoptosis and is associated with cancer. The anti-apoptotic proteins of Bcl-2 family, such as Bcl-2, are overexpression in numerous tumors, and contribute to cancer formation, development, and therapy resistance. Therefore, Bcl-2 is a promising target for drug development, and several Bcl-2 inhibitors are currently undergoing clinical trials. In this study, we carried out a QSAR-based virtual screening approach to develop potential Bcl-2 inhibitors from the SPECS database. Surface plasmon resonance (SPR) binding assay was performed to examine the interaction between Bcl-2 protein and the screened inhibitors. After that, we measured the anti-tumor activities of the 8 candidate compounds, and found that compound M1 has significant cytotoxic effect on breast cancer cells. We further proved that compound M1 downregulated Bcl-2 expression and activated apoptosis by inducing mitochondrial dysfunction. In conclusion, we identified a novel Bcl-2 inhibitor by QSAR screening, which exerted significant cytotoxic activity in breast cancer cells through inducing mitochondria-mediated apoptosis.

Effect and mechanism of psoralidin on promoting osteogenesis and inhibiting adipogenesis.

BACKGROUND: Psoralidin (PL), a prenylated coumestrol, is isolated from Psoralea corylifolia L. (Fabaceae), which is frequently used for treatment of osteoporosis. PURPOSE: This study was designed to investigate the dual effects and potential mechanism of PL on promoting osteogenesis and inhibiting adipogenesis. METHODS: Bone marrow mesenchymal stem cells (BMSCs) were used to investigate the effect of PL on stimulating osteogenesis and inhibiting adipogenesis, while preosteoblast MC3T3-E1 cells and preadipocyte 3T3-L1 cells were employed to explore the potential mechanisms. Estradiol (E2) and ICI 182,780 (ICI) were used as the specific agonist and antagonist of classical estrogen receptors (ER), respectively, to interfere with classical ER signaling. Meanwhile, G-1 and G-15 were introduced as the selective agonist and antagonist of G protein coupled receptor 30 (GRP30, a membrane ER) to further clarify if membrane ER involved in PL mediating osteogenesis and adipogenesis RESULTS: PL not only promoted mineralization, but also inhibited adipocytes formation of BMSCs. In terms of osteogenesis, PL enhanced calcium nodule formation, alkaline phosphatase activity and osteocalcin levels in MC3T3-E1 cells. As for adipogenesis, PL decreased adipocyte formation in 3T3-L1 cells through down-regulating several mRNA expressions and protein synthesis of adipogenesis related factors. ICI completely blocked the effect of PL in promoting osteogenesis, but only partially suppressed its effect in inhibition of adipogenesis, while G-15 partially suppressed the effect of PL on promoting mineralization and inhibiting oil drop formation. Furthermore, during suppression of adipocyte differentiation, PL regulated protein kinase B / glycogen synthase kinase 3ß / ß-catenin signaling pathway. CONCLUSION: PL promoted osteogenesis via mediating classical ER pathway, and inhibited adipocytes formation by regulating combined classical and membrane ER pathways. PL might be a potential candidate for the treatment of postmenopausal osteoporosis by modulating the competitive relationship between osteogenesis and adipogenesis of bone marrow mesenchymal stem cells.

Eukaryotic elongation factor-2 kinase regulates the cross-talk between autophagy and pyroptosis in doxorubicin-treated human melanoma cells in vitro.

Eukaryotic elongation factor-2 kinase (eEF-2K), a negative regulator of protein synthesis, has been shown to play an important role in modulating autophagy and apoptosis in tumor cells under various stresses. In this study, we investigated the regulatory role of eEF-2K in pyroptosis (a new form of programmed necrosis) in doxorubicin-treated human melanoma cells. We found that doxorubicin (0.5-5 µmol/L) induced pyroptosis in melanoma cell lines SK-MEL-5, SK-MEL-28, and A-375 with high expression of DFNA5, but not in human breast cancer cell line MCF-7 with little expression of DFNA5. On the other hand, doxorubicin treatment activated autophagy in the melanoma cells; inhibition of autophagy by transfecting the cells with siRNA targeting Beclin1 or by pretreatment with chloroquine (20 µmol/L) significantly augmented pyroptosis, thus sensitizing the melanoma cells to doxorubicin. We further demonstrated that doxorubicin treatment activated eEF-2K in the melanoma cells, and silencing of eEF-2K blunted autophagic responses, but promoted doxorubicin-induced pyroptotic cell death. Taken together, the above results demonstrate that eEF-2K dictates the cross-talk between pyroptosis and autophagy in doxorubicin-treated human melanoma cells; suppression of eEF-2K results in inhibiting autophagy and augmenting pyroptosis, thus modulating the sensitivity of melanoma cells to doxorubicin, suggesting that targeting eEF-2K may reinforce the antitumor efficacy of doxorubicin, offering a new insight into tumor chemotherapy.

Tubeimoside-1, a triterpenoid saponin, induces cytoprotective autophagy in human breast cancer cells in vitro via Akt-mediated pathway.

Autophagy, a form of cellular self-digestion by lysosome, is associated with various disease processes including cancers, and modulating autophagy has shown promise in the treatment of various malignancies. A number of natural products display strong antitumor activity, yet their mechanisms of action remain unclear. To gain a better understanding of how traditional Chinese medicine agents exert antitumor effects, we screened 480 natural compounds for their effects on autophagy using a high content screening assay detecting GFP-LC3 puncta in HeLa cells. Tubeimoside-1 (TBMS1), a triterpenoid saponin extracted from Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), was identified as a potent activator of autophagy. The activation of autophagy by TBMS1 was evidenced by increased LC3-II amount and GFP-LC3 dots, observation of autophagosomes under electron microscopy, and enhanced autophagic flux. To explore the mechanisms underlying TBMS1-activated autophagy, we performed cheminformatic analyses and surface plasmon resonance (SPR) binding assay that showed a higher likelihood of the binding between Akt protein and TBMS1. In three human breast cancer cell lines, we demonstrated that Akt-mTOR-eEF-2K pathway was involved in TBMS1-induced activation of autophagy, while Akt-mediated downregulations of Mcl-1, Bcl-xl, and Bcl-2 led to the activation of apoptosis of the breast cancer cells. Inhibition of autophagy enhanced the cytotoxic effect of TBMS1 via promoting apoptosis. Our results demonstrate the role and mechanism of TBMS1 in activating autophagy, suggesting that inhibition of cytoprotective autophagy may act as a therapeutic strategy to reinforce the activity of TBMS1 against cancers.

Herbal Fufang Xian Ling Gu Bao prevents corticosteroid-induced osteonecrosis of the femoral head-A first multicentre, randomised, double-blind, placebo-controlled clinical trial.

BACKGROUND/OBJECTIVE: This is a multicentre, randomised, double-blind, placebo-controlled clinical trial to investigate the safety and efficacy of Chinese herbal Fufang Xian Ling Gu Bao (XLGB) with antiadipogenic compounds for the prevention of corticosteroid (CS)-induced osteonecrosis of femoral head (ONFH). METHODS: Patients of both genders, aged between 18 and 65 years, with diseases such as systemic lupus erythematosus, nephrosis, dermatosis and rheumatoid arthritis indicated for CS treatment and who did not show magnetic resonance imaging of ONFH at baseline were recruited into the study and then randomised into either XLGB group (n = 129) with daily oral administration of XLGB or placebo group (n = 146). RESULTS: Magnetic resonance imaging revealed a total of 30 ONFH cases at 6 months after CS treatment, with 6.98% (9 of 129 cases) and 14.4% (21 of 146 cases) in the XLGB group and placebo group, respectively, (p < 0.05), i.e., a 2-fold significantly less ONFH identified in the XLGB treatment group. Blood tests suggested that XLGB significantly inhibited the elevation of activated protein C resistance induced by CS treatment. CONCLUSION: This is the first multicentre clinical study to demonstrate that the antiadipogenic compounds-rich herbal Fufang (formula) XLGB is effective in preventing CS-associated ONFH in patients with immune-inflammatory diseases under CS treatment. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The translation potential of this clinical trial is that the initially officially approved clinical indication for XLGB for treatment of osteoporosis has been now also proven to be effective for a new clinical application.

Steroid-associated osteonecrosis animal model in rats.

OBJECTIVE: Established preclinical disease models are essential for not only studying aetiology and/or pathophysiology of the relevant diseases but more importantly also for testing prevention and/or treatment concept(s). The present study proposed and established a detailed induction and assessment protocol for a unique and cost-effective preclinical steroid-associated osteonecrosis (SAON) in rats with pulsed injections of lipopolysaccharide (LPS) and methylprednisolone (MPS). METHODS: Sixteen 24-week-old male Sprague-Dawley rats were used to induce SAON by one intravenous injection of LPS (0.2 mg/kg) and three intraperitoneal injections of MPS (100 mg/kg) with a time interval of 24 hour, and then, MPS (40 mg/kg) was intraperitoneally injected three times a week from week 2 until sacrifice. Additional 12 rats were used as normal controls. Two and six weeks after induction, animals were scanned by metabolic dual energy X-ray absorptiometry for evaluation of tissue composition; serum was collected for bone turnover markers, Microfil perfusion was performed for angiography, the liver was collected for histopathology and bilateral femora and bilateral tibiae were collected for histological examination. RESULTS: Three rats died after LPS injection, i.e., with 15.8% (3/19) mortality. Histological evaluation showed 100% incidence of SAON at week 2. Dual energy X-ray absorptiometry showed significantly higher fat percent and lower lean mass in SAON group at week 6. Micro-computed tomography (Micro-CT) showed significant bone degradation at proximal tibia 6 weeks after SAON induction. Angiography illustrated significantly less blood vessels in the proximal tibia and significantly more leakage particles in the distal tibia 2 weeks after SAON induction. Serum amino-terminal propeptide of type I collagen and osteocalcin were significantly lower at both 2 and 6 weeks after SAON induction, and serum carboxy-terminal telopeptide was significantly lower at 6 weeks after SAON induction. Histomorphometry revealed significantly lower osteoblast surface and higher marrow fat fraction and oedema area in SAON group. Hepatic oedema appeared 2 weeks after SAON induction, and lipid accumulation appeared in the liver of SAON rats 6 weeks after SAON induction. CONCLUSION: The present study successfully induced SAON in rats with pulsed injection of LPS and MPS, which was well simulating the clinical feature and pathology. Apart from available large animal models, such as bipedal emus or quadrupedal rabbits, our current SAON small model in rats could be a cost-effective preclinical experimental model to study body metabolism, molecular mechanism of SAON and potential drugs developed for prevention or treatment of SAON. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The present study successfully induced SAON in a small animal model in rats with pulsed injection of LPS and MPS. The evaluation protocols with typical histopathologic ON features and advanced evaluation approaches to identify the metabolic disorders of SAON could be used in future rat SAON studies. The SAON rat model is a suitable and cost-effective animal model to study molecular mechanism of SAON and potential drugs developed for prevention and treatment of SAON.

Simultaneous Quantification of Multiple Representative Components in the Xian-Ling-Gu-Bao Capsule by Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Xian-Ling-Gu-Bao capsule (XLGB), a famous traditional Chinese medicine prescription, is extensively used for the treatment of osteoporosis in China. However, few studies on the holistic quality control of XLGB have been reported. In this study, a reliable method using 18 representative components in XLGB was successfully established and applied to evaluate 34 batches of XLGB samples by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The choice of quantitative markers mostly followed four principles, i.e., absorbed components in plasma, bioactive compounds with in vitro anti-osteoporosis activity, those derived from multiple individual medicinal herbs in XLGB with multiple representative structure types, and quantitative chemical markers in the Chinese Pharmacopoeia. The results showed chemical consistency was good except for individual batches. Multivariate statistical analysis indicated that asperosaponin VI from Radix Dipsaci, epimedin C, magnoflorine, and icariin from Herba Epimedii as well as timosaponin BII from Rhizoma Anemarrhenae varied significantly in multiple samples, which hinted an assay for these four components should be completed during all of the manufacturing processes. Taken together, this study provided a feasible method for holistic quality control of XLGB by multiple chemical markers, which could play a vital role in guaranteeing the safety, effectiveness, and controllability of administering the capsules as a medication in clinics.

Differential response of bone and kidney to ACEI in db/db mice: A potential effect of captopril on accelerating bone loss.

The components of renin-angiotensin system (RAS) are expressed in the kidney and bone. Kidney disease and bone injury are common complications associated with diabetes. This study aimed to investigate the effects of an angiotensin-converting enzyme inhibitor, captopril, on the kidney and bone of db/db mice. The db/db mice were orally administered by gavage with captopril for 8weeks with db/+ mice as the non-diabetic control. Serum and urine biochemistries were determined by standard colorimetric methods or ELISA. Histological measurements were performed on the kidney by periodic acid-schiff staining and on the tibial proximal metaphysis by safranin O and masson-trichrome staining. Trabecular bone mass and bone quality were analyzed by microcomputed tomography. Quantitative polymerase chain reaction and immunoblotting were applied for molecular analysis on mRNA and protein expression. Captopril significantly improved albuminuria and glomerulosclerosis in db/db mice, and these effects might be attributed to the down-regulation of angiotensin II expression and the expression of its down-stream profibrotic factors in the kidney, like connective tissue growth factor and vascular endothelial growth factor. Urinary excretion of calcium and phosphorus markedly increased in db/db mice in response to captopril. Treatment with captopril induced a decrease in bone mineral density and deterioration of trabecular bone at proximal metaphysis of tibia in db/db mice, as shown in the histological and reconstructed 3-dimensional images. Even though captopril effectively reversed the diabetes-induced changes in calcium-binding protein 28-k and vitamin D receptor expression in the kidney as well as the expression of RAS components and bradykinin receptor-2 in bone tissue, treatment with captopril increased the osteoclast-covered bone surface, reduced the osteoblast-covered bone surface, down-regulated the expression of type 1 collagen and transcription factor runt-related transcription factor 2 (markers for osteoblastic functions), and up-regulated the expression of carbonic anhydrase II (marker for bone resorption). Captopril exerted therapeutic effects on renal injuries associated with type 2 diabetes but worsened the deteriorations of trabecular bone in db/db mice; the latter of which was at least in part due to the stimulation of osteoclastogenesis and the suppression of osteogenesis by captopril.

Identification, bioactivity evaluation and pharmacokinetics of multiple components in rat serum after oral administration of Xian-Ling-Gu-Bao capsule by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

The Xian-Ling-Gu-Bao capsule (XLGB) is a famous traditional Chinese medicine prescription (TCMP), which has proven effective in osteoporosis treatment. However, due to the lack of a dynamic XLGB profile, the in vivo pharmacokinetics of multiple bioactive components within this medicine remains unknown. In the present study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) identified a total of eighteen prototypes (using reference standards) in rat serum after oral administration of XLGB. These prototypes were subsequently evaluated to ascertain their effects on the proliferation and alkaline phosphatase activity of UMR106 cells and the adipogenesis of 3T3-L1 cells. Furthermore, a rapid and sensitive UPLC/Q-TOF-MS method was developed and validated for simultaneous quantitative analysis of 11 prototypes in rat serum. Chromatographic separation was achieved using a Waters Acquity BEH C18 column (2.1×100mm, 1.7µm) and linear gradient elution employing a mobile phase consisting of water and acetonitrile (both containing 0.1% formic acid). All calibration curves showed excellent linearity (r2>0.99) within the sampling ranges considered. The assay was accurate, precise and reproducible, as demonstrated by the obtained intra- and inter-day precisions (less than 12.3%) and accuracies (between -12.7% and 11.0%), and the matrix effects, extraction recoveries and stabilities were all satisfactory. Moreover, pharmacokinetic parameters were calculated from the plasma concentration-time data. Compared to single-compound dosing, significantly enhanced responses were obtained when several analytes were administered simultaneously, indicating possible drug-drug interactions among the complex ingredients of TCMP. This work provides an experimental baseline regarding the clinical applications and medicinal effectiveness of XLGB in the treatment of osteoporosis.

Discovery of a New Class of Cathepsin K Inhibitors in Rhizoma Drynariae as Potential Candidates for the Treatment of Osteoporosis.

Rhizoma Drynariae (RD), as one of the most common clinically used folk medicines, has been reported to exert potent anti-osteoporotic activity. The bioactive ingredients and mechanisms that account for its bone protective effects are under active investigation. Here we adopt a novel in silico target fishing method to reveal the target profile of RD. Cathepsin K (Ctsk) is one of the cysteine proteases that is over-expressed in osteoclasts and accounts for the increase in bone resorption in metabolic bone disorders such as postmenopausal osteoporosis. It has been the focus of target based drug discovery in recent years. We have identified two components in RD, Kushennol F and Sophoraflavanone G, that can potentially interact with Ctsk. Biological studies were performed to verify the effects of these compounds on Ctsk and its related bone resorption process, which include the use of in vitro fluorescence-based Ctsk enzyme assay, bone resorption pit formation assay, as well as Receptor Activator of Nuclear factor κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis using murine RAW264.7 cells. Finally, the binding mode and stability of these two compounds that interact with Ctsk were determined by molecular docking and dynamics methods. The results showed that the in silico target fishing method could successfully identify two components from RD that show inhibitory effects on the bone resorption process related to protease Ctsk.