Exploratory study of sea buckthorn enhancing QiangGuYin efficacy by inhibiting CKIP-1 and Notum activating the Wnt/ß-catenin signaling pathway and analysis of active ingredients by molecular docking.

Background: Sea buckthorn (SBT) is a traditional Chinese medicine (TCM), rich in calcium, phosphorus, and vitamins, which can potentially prevent and treat osteoporosis. However, no research has been conducted to confirm these hypotheses. QiangGuYin (QGY) is a TCM compound used to treat osteoporosis. There is a need to investigate whether SBT enhances QGY efficacy. Objectives: The aim of this study was to explore whether SBT enhances QGY efficacy by inhibiting CKIP-1 and Notum expression through the Wnt/ß-catenin pathway. The study also aimed to explore the active components of SBT. Methods: Experimental animals were divided into control, model, QGY, SBT, SBT + Eucommia ulmoides (EU), and SBT + QGY groups. After treatment, bone morphometric parameters, such as estrogen, PINP, and S-CTX levels, and Notum, CKIP-1, and ß-catenin expression were examined. Screening of SBT active components was conducted by molecular docking to obtain small molecules that bind Notum and CKIP-1. Results: The results showed that all the drug groups could elevate the estrogen, PINP, and S-CTX levels, improve femoral bone morphometric parameters, inhibit Notum and CKIP-1 expression, and promote ß-catenin expression. The effect of SBT + EU and SBT + QGY was superior to the others. Molecular docking identified that SBT contains seven small molecules (folic acid, rhein, quercetin, kaempferol, mandenol, isorhamnetin, and ent-epicatechin) with potential effects on CKIP-1 and Notum. Conclusion: SBT improves bone morphometric performance in PMOP rats by inhibiting CKIP-1 and Notum expression, increasing estrogen levels, and activating the Wnt/ß-catenin signaling pathway. Furthermore, SBT enhances the properties of QGY. Folic acid, rhein, quercetin, kaempferol, mandenol, isorhamnetin, and ent-epicatechin are the most likely active ingredients of SBT. These results provide insight into the pharmacological mechanisms of SBT in treating osteoporosis.

[Mechanism action of Chinese herbal compound and target network pharmacology of Yougui (YG) pill for the treatment of osteoporosis].

OBJECTIVE: To explore compounds, targets and mechanism of Yougui (YG) pill in treating osteoporosis based on systemic pharmacology of traditional Chinese medicine. METHODS: The known effective Chinese herbal compound of YG pill was searched from traditional Chinese medicine integrated database(TCMID). Bioinformatics analysis tool for molecular mechanism of traditional Chinese medicine (BATMAN-TCM) was used to predict target of components;DisGeNET and artificial literature reading were used to obtain targets of osteoporosis and bone remodeling;Cytoscape 3.7.1 software and its plug-ins BiN-GO and ClueGO were used to enrich the GO annotation and pathwaysof the related targets, and validation of the predicted target of YG pill were validated by 87 differentially expressed proteins in postmenopausal osteoporosis and postmenopausal osteoporosis disease models in postmenopausal patients with normal bone mass from the previous serum proteomics data. RESULTS: Totally 392 compounds were retrieved from YG pill, including 83 sovereign drugs (monkshood, cinnamon, deerhorn gelatin), 127 ministerial drugs (prepared rehmannia root, dogwood, wolfberry fruit and Chinese yam) and 182 supplementary drugs (cuscuta chinensis, eucommia ulmoides and Chinese angelica). Among them, there were 4 same compounds between sovereign drug and ministerial drug, 1 same compound between sovereign drug and supplementary drug, and 14 same compounds between ministerial drug and supplementary drug. Totally 2 112 trusted targets were identified, included 775 sovereign drugs, 1 483 ministerial drugs and 1 491 supplementary drugs;227 targets were selected from YG pill for treating osteoporosis, which participate in nearly 20 process of metabolic process, cell differentiation and biology, and data mining revealed that the process involved bone remodeling and bone mineralization. Acting site of cell mainly inclded 9 kinds of cell which had 13 molecular function. Results of KEGG metabolic pathway enrichment analysis showed 137 signal passages were obviously enriched. Among them, classical osteoclast differentiation signal passages and relative estrogen regulates signaling pathways of menopause were widely distributed in 27 signal passages. Sixtargets were screened by target validation, such as AGT, FGA, APOE, DKK3, P4HB and RAB7A. CONCLUSION: The characteristics of multi-targets and multi-pathways of YG pill for the treatment of osteoporosis were clarified, which provided a clear direction for the in-depth research. The pharmacodynamic components of YG pill include 36 compounds, and their main action targets include FGA, AGT, APOE, DKK3, P4HB and RAB7A.

Ubiquitylomes Analysis of the Whole blood in Postmenopausal Osteoporosis Patients and healthy Postmenopausal Women.

OBJECTIVES: To determine the mechanisms of ubiquitination in postmenopausal osteoporosis and investigate the ubiquitinated spectrum of novel targets between healthy postmenopausal women and postmenopausal osteoporosis patients, we performed ubiquitylome analysis of the whole blood of postmenopausal women and postmenopausal osteoporosis patients. METHODS: To obtain a more comprehensive understanding of the postmenopausal osteoporosis mechanism, we performed a quantitative assessment of the ubiquitylome in whole blood from seven healthy postmenopausal women and seven postmenopausal osteoporosis patients using high-performance liquid chromatography fractionation, affinity enrichment, and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). To examine the ubiquitylome data, we performed enrichment analysis using an ubiquitylated amino acid motif, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. RESULTS: Altogether, 133 ubiquitinated sites and 102 proteins were quantified. A difference of more than 1.2 times is considered significant upregulation and less than 0.83 significant downregulation; 32 ubiquitinated sites on 25 proteins were upregulated and 101 ubiquitinated sites on 77 proteins were downregulated. These quantified proteins, both with differently ubiquitinated sites, participated in various cellular processes, such as cellular processes, biological regulation processes, response to stimulus processes, single-organism and metabolic processes. Ubiquitin conjugating enzyme activity and ubiquitin-like protein conjugating enzyme activity were the most highly enriched in molecular function of upregulated sites with corresponding proteins, but they were not enriched in downregulated in sites with corresponding proteins. The KEGG pathways analysis of quantified proteins with differentiated ubiquitinated sites found 13 kinds of molecular interactions and functional pathways, such as glyoxylate and decarboxylate metabolism, dopaminergic synapse, ubiquitin-mediated proteolysis, salivary secretion, coagulation and complement cascades, Parkinson's disease, and hippo signaling pathway. In addition, hsa04120 ubiquitin-mediated proteolysis was the most highly enriched in proteins with upregulated sites, hsa04610 complement and coagulation cascades was the most highly enriched in proteins with downregulated ubiquitinated sites, and hsa04114 Oocyte meiosis was the most highly enriched among all differential proteins. CONCLUSION: Our study expands the understanding of the spectrum of novel targets that are differentially ubiquitinated in whole blood from healthy postmenopausal women and postmenopausal osteoporosis patients. The findings will contribute toward our understanding of the underlying proteostasis pathways in postmenopausal osteoporosis and the potential identification of diagnostic biomarkers in whole blood.

Discovery and Identification of Serum Succinyl-Proteome for Postmenopausal Women with Osteoporosis and Osteopenia.

OBJECTIVE: For the purpose of providing evidence for the treatment of osteoporosis and osteopenia, this study retrospectively identified succinylation-modified sites and proteins in postmenopausal women, and bioinformatics analysis were performed. METHODS: From January 2016 to June 2018, a total of 30 postmenopausal women aged from 55 to 70 years old were assigned to three groups: 10 cases with osteoporosis; 10 cases with osteopenia; and 10 cases with normal bone mass. Subsequently, the serum samples were collected from all cases for succinyl-proteome. Measures comprised label-free quantitative analysis, succinylation enrichment techniques, the liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) methods, and bioinformatics. RESULTS: A total of 113 succinylation sites on 35 proteins were identified based on quantitative information. The variation of the different multiple folds were more than 1.2 times as a significant increase for up-regulated and less than 1/1.2 times as a significant decrease for down-regulated. Among the quantified succinylation sites, 66 were up-regulated and 11 down-regulated in the Osteopenia/Normal comparison group, 24 were up-regulated and 44 down-regulated in the Osteoporosis/Osteopenia comparison group, 45 were up-regulated and 32 down-regulated in the Osteoporosis/Normal comparison group. Among the quantified succinylation proteins, 24 were up-regulated and 7 down-regulated in the Osteopenia/Normal comparison group, 15 were up-regulated and 20 down-regulated in the Osteoporosis/Osteopenia comparison group, 20 were up-regulated and 17 down-regulated in the Osteoporosis/Normal comparison group. The percentage of proteins differed in immune response, signaling pathway, proteolysis, lymphocyte, leukocyte, and cell activation. Four differentially expressed proteins (apolipoprotein A-I, apolipoprotein A-II, hemoglobin subunit alpha, and haptoglobin) contained quantitative information; they were mediated with receptors, factors, mechanisms, that related to bone metabolism. Hemoglobin subunit alpha was screened for diagnosis of osteopenia. CONCLUSIONS: The succinyl-proteome experimental data indicated that apolipoprotein A-I, apolipoprotein A-II, hemoglobin subunit alpha, and haptoglobin were valuable for diagnosis and treatment in postmenopausal women with osteoporosis and osteopenia.

Study on Zoledronic Acid Reducing Acute Bone Loss and Fracture Rates in Elderly Postoperative Patients with Intertrochanteric Fractures.

OBJECTIVE: To observe the effect of zoledronic acid on the reduction of acute bone loss and fracture rate in elderly postoperative patients with intertrochanteric fracture. METHODS: From August 2012 to January 2015, a total of 482 patients with senile osteoporotic femoral intertrochanteric fracture, who accepted proximal femoral intramedullary fixation under anesthesia were analysed. The patients were divided into two groups. Treatment group (353 cases) were treated with 100 mL/5 mg of zoledronic acid injection in 1 week after operation, as well as orally taken 600 mg/d of calcium carbonate and active vitamin D3 400 IU/d. Control group (129 cases) were given the same dose of calcium carbonate and active vitamin D3 orally. Efficacy evaluation were conducted during different periods of medication RESULTS: Compared with pre-medication, indexes of bone metabolism (TARP-5b, CTX) in the treatment group were brought down, especially significantly statistically different after 12 months of medication. The treatment group performed superior to control group in alleviating the pain of back and posture changing (P < 0.05), improving bone density (P < 0.05), depressing re-fracture rate (P < 0.01) after 24 months of medication. In addition, BP, PF and MH dimension scores were demonstrated with statistical significance (P < 0.05). CONCLUSIONS: The application of zoledronic acidin elderly postoperative patients with intertrochanteric fracture can not only relieve acute bone loss, reduce the incidence rate of re-fracture, alleviate osteoporosis pain and the pain from osteoporotic fracture, but also improve bone metabolism and quality of life, which may offer an acceptable clinical opinion.

Effect of Traditional Chinese Medicine Product, QiangGuYin, on Bone Mineral Density and Bone Turnover in Chinese Postmenopausal Osteoporosis.

Introduction. The aim of this study was to investigate the efficacy of herbal formula QiangGuYin (QGY) in postmenopausal women. Materials and Methods. A total of 240 participants from six clinical centers were randomly to receive alendronate 70 mg/week, QGY granules 20 g/day, and placebo. Primary end points were BMD changes over 6 and 12 months; secondary end points were bone turnover markers changes at 3, 6, 9, and 12 months. Safety was monitored by clinical adverse events reported during the follow-up. Results. Of 240 women recruited, 218 completed the study. Significant BMD increases from baseline were observed over 6 and 12 months at each observed part both in QGY and alendronate compared with placebo (p < 0.01). Alendronate-treated subjects had significant decreases in ß-CTX compared to QGY-treated subjects at each time point assessed (p < 0.01). Reduction in t-P1NP was only observed in the QGY group at 3 and 6 months (-23.81% and -3.07%, resp.). No significant difference was observed in the overall incidence of clinical adverse events among the alendronate group and the QGY group (5.0% versus 7.5%, p = 0.513). Conclusion. 1-Year treatment with QGY demonstrated a safe statistical increase in BMD and new balance may be rebuilt after 9 months. This trail is registered with ChiCTR-POC-16008026.

Intravenous Zoledronic Acid 5 mg on Bone Turnover Markers and Bone Mineral Density in East China Subjects with Newly Diagnosed Osteoporosis: A 24-month Clinical Study.

OBJECTIVE: This randomized, double-blind, placebo-controlled study assessed the necessity of early intervention, safety and efficacy of intravenous zoledronic acid 5 mg/year in East China women with newly diagnosed osteoporosis at high risk of fracture during a 24-month treatment period. METHODS: Subjects (57 [52-62] years old) were randomized 3:2 to zoledronic acid versus placebo (randomized at baseline, zoledronic acid [175 cases], placebo-zoledronic acid [110 cases]). The bone mineral density of the lumbar spine and total hip was measured every 6 months with the use of dual-energy X-ray absorptiometry. Serum procollagen I N-terminal pro-peptide (PINP) and serum C-telopeptide of type I collagen (CTX) levels were measured every 6 months. The primary end point was the rate of change in the bone mineral density at the posteroanterior spine. RESULTS: For subjects with measurements at 24 months, zoledronic acid significantly increased bone mineral density (BMD) at the lumbar spine (mean percent change ± SD, zoledronic acid 5.390% ± 0.854% versus placebo-zoledronic acid -1.038% ± 0.599%), the total hip (zoledronic acid 1.900% ± 0.262% versus placebo-zoledronic acid -1.631% ± 0.649%). Serum procollagen I N-terminal pro-peptide (PINP) and CTX decreased rapidly with zoledronic acid 5 mg treatment (P < 0.001 versus placebo at 6 month and 24 months) and changed from baseline in the zoledronic acid 5 mg and placebo-zoledronic acid 5 mg at 6 months by a mean of -66.348% and -75.375%, respectively (P < 0.001), and at 24 months by -49.950% and -52.325%, respectively (P < 0.001). No cases of serious adverse events were observed in two groups. Headache, pyrexia and myalgia occurred more commonly within the first 3 days after infusion with zoledronic acid 5 mg than with placebo (13.7% versus 2.1%, P = 0.0018; 28.0% versus 3.2%, P < 0.001; 21.7% versus 4.2%, P < 0.001, respectively). CONCLUSIONS: These data show that early application of zoledronic acid 5 mg/year was well stimulated and tolerated for bone mass in newly diagnosed east china subjects with osteoporosis in a 24-month treatment.

Identification of human serum protein targets of Qianggu Decoction () in primary type I osteoporosis based on tandem mass tag labeling and liquid chromatography-tandem mass spectrometry technology.

OBJECTIVE: To investigate the serum protein targets of Qianggu Decoction (, QGD) on treating osteoporosis by the proteomics analysis using tandem mass tag (TMT) and liquid chromatographytandem mass spectrometry (LC-MS/MS). METHODS: Twenty serum protein samples were recruited (10 patients with primary type I osteoporosis before and after QGD treatment) and the high abundance ratios protein was removed, two serum samples were extracted and labeled with TMT reagent. Then, mass spectrometric detection, identification of differentially expressed proteins and bioinformatics analysis of differentially expressed proteins were carried out. RESULTS: A total of 60 proteins were identified, within a 99% confidence interval, to be differentially regulated of which, 34 proteins were up-regulated and 26 proteins were down-regulated. Differentially expressed proteins analyzed by Gene Ontology (GO) annotation mainly get involved in 12 different biological processes, 7 types of cellular components, and 6 kinds of molecular functions. Angiotensinogen (AGT), stromelysin-1 (MMP3), heparanase (HPSE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were screened as candidate protein targets of QGD treatment, which were related to metabolic mechanism of bone remodeling and/or bone collagen of osteoporosis. By the utilization of the protein-protein interaction network analysis tool named STRING10.0, it showed that AGT, MMP3, HPSE and GAPDH were located in the key node of the protein-protein interactions network. Furthermore, AGT, MMP3, HPSE and GAPDH were found to be directly related to BMP, MAPK, Wnt, SMAD and tumor necrosis factor ligand superfamily member 11 (TNFSF11) families. CONCLUSIONS: The proteomics analysis by using TMT combined with LC-MS/MS was a feasible method for screening the potential therapeutic targets associated with QGD treatment. It suggests that AGT, MMP3, HPSE and GAPDH may be candidate protein targets of QGD treatment which can be used as therapeutic effect monitor and early diagnosis of primary type I osteoporosis.

[Effect and mechanism of total flavone of epimedium on primary callus formation in ovariectomized rats with fractures].

OBJECTIVE: To explore the effects and related mechanisms of total flavone of epimedium treatment(TFE)on primary callus for mation in ovariectomized rats. METHODS: Forty male SD rats weighted from 209 to 246 g and aged 6 to 8 weeks were selected. Six weeks after ovariectomy a femur fracture model with middiaphyseal segment fracture was established, estimated and randomly divided into TFE group (150 mg·kg⁻¹·d⁻¹) and control group(received saline). HE staining was used to evaluate the morphologic difference of primary callus during the bone callus healing between these two groups. The relative expression of Runt-related transcription factor 2(Runx2) mRNA in the callus was identified by real-time polymerase chain reaction. Immunohistochemical technique was used to observe the Casein kinase 2-interacting protein 1(CKIP-1) protein level in the callus of the two groups. Maximum fracture load was tested by three point bend test. RESULTS: The BMD, primary callus volume, trabecular member(Tb.N) and trabecular thickness(Tb.Th) were higher in TFE group than that in control group(P<0.001). The Tb.N and Tb.Th of primary callus were higher in TFE group than control group (P=0.001). The volume and bone volume/tissue volume of primary callus were in TFE group than control group(P<0.01). The trabecular separation(Tb.Sp) of primary callus were in control group higher than TFE group(P<0.01). The HE staining of the 6 week slices showed that the degree of cartilage ossification in callus of the TFE group was significantly higher than that in control group under high magnification. Real-time PCR revealed that the comparative expression of Runx2 mRNA in control group was higher than that in TFE group(P<0.001); the positive number of CKIP-1 was less in TFE group than that in control group (P<0.001). TFE could increase the maximum load of the primary callus (P<0.001). CONCLUSIONS: TFE can promote the cartiage ossification of callus in ovariectomized rats, enhancing the bone strength and bone quality in the process of fracture healing via the CKIP-1/Runx2 pathway.

Weak cation exchange magnetic beads coupled with matrix-assisted laser desorption ionization-time of flight-mass spectrometry in screening serum protein markers in osteopenia.

The present study aimed at investigating the weak cation magnetic separation technology and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) in screening serum protein markers of osteopenia from ten postmenopausal women and ten postmenopausal women without osteopenia as control group, to find a new method for screening biomarkers and establishing a diagnostic model for primary type I osteoporosis. Serum samples were collected from postmenopausal women with osteopenia and postmenopausal women with normal bone mass. Proteins were extracted from serum samples by weak cation exchange magnetic beads technology, and mass spectra acquisition was done by MALDI-TOF-MS. The visualization and comparison of data sets, statistical peak evaluation, model recognition, and discovery of biomarker candidates were handled by the proteinchip data analysis system software(ZJU-PDAS). The diagnostic models were established using genetic arithmetic based support vector machine (SVM). The SVM result with the highest Youden Index was selected as the model. Combinatorial Peaks having the highest accuracy in distinguishing different samples were selected as potential biomarker. From the two group serum samples, a total of 133 differential features were selected. Ten features with significant intensity differences were screened. In the pair-wise comparisons, processing of MALDI-TOF spectra resulted in the identification of ten differential features between postmenopausal women with osteopenia and postmenopausal women with normal bone mass. The difference of features by Youden index showed that the highest features had a mass to charge ratio of 1699 and 3038 Da. A diagnosis model was established with these two peaks as the candidate marker, and the specificity of the model is 100 %, the sensitivity was 90 % by leave-one-out cross validation test. The two groups of specimens in SVM results on the scatter plot could be clearly distinguished. The peak with m/z 3038 in the SVM model was suggested as Secretin by TagIdent tool. To provide further validation, the secretin levels in serum were analyzed using enzyme-linked immunosorbent assays that is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of secretin in human serum.

[Clinical observation of improved proximal femoral locking plate in treating osteoporotic intertrochanteric fractures].

OBJECTIVE: To investigate the effects of proximal femoral locking plate (PFP) in treating osteoporotic intertrochanteric fractures and to analyze the failure cases. METHODS: Totally 32 patients with osteoporotic intertrochanteric fractures of Evans I and II were treated with improved locking PFP, including 17 males and 15 females with an average age of 77.3 years old ranging from 70 to 86 years old. After operation, according to Harris hip scores, the hip function and therapeutic effects were evaluated. RESULTS: The observed 32 patients' operative time was (60.5±15.7) min, intraoperative blood loss was (128.8±73.6) ml;perioperative blood transfusion was (224.0±72.7) ml. Hospitalization time was from 14 to 20 d with an average of 17.2 d. All patients were followed up from 6 to 18 months with an average of 14.1 months. The fracture healing time was from 3 to 6 months with an average of 3.1 months. One patient occurred internal fixation loosening and screw backward, 4 cases occurred urinary tract infection, 1 patient died of cardiovascular disease for 6 months postoperative, 2 patients died of a stroke for 1 year postoperative. No incision deep infection, peri internal fixation fractures, lower extremity deep venous thrombosis, internal fixation breakage, nonunion, severe coax vara and coax valgus occurred. The final Harris score was 89.74±6.84, the result was excellent in 10 cases, good in 16 cases, fair in 4 cases, and poor in 2 cases. CONCLUSIONS: Locking PFP can provide relative stable fixation to proximal end of osteoporotic femoral fractures, which is a good choice for the treatment of intertrochanteric fractures. It could provide stableness of fractures and bone union, even avoid screws loose or slide out.