Epimedium-Curculigo herb pair enhances bone repair with infected bone defects and regulates osteoblasts through LncRNA MALAT1/miR-34a-5p/SMAD2 axis.

Infected bone defects (IBDs) are the common condition in the clinical practice of orthopaedics. Although surgery and anti-infective medicine are the firstly chosen treatments, in many cases, patients experience a prolonged bone union process after anti-infective treatment. Epimedium-Curculigo herb pair (ECP) has been proved to be effective for bone repair. However, the mechanisms of ECP in IBDs are insufficiency. In this study, Effect of ECP in IBDs was verified by micro-CT and histological examination. Qualitative and quantitative analysis of the main components in ECP containing medicated serum (ECP-CS) were performed. The network pharmacological approaches were then applied to predict potential pathways for ECP associated with bone repair. In addition, the mechanism of ECP regulating LncRNA MALAT1/miRNA-34a-5p/SMAD2 signalling axis was evaluated by molecular biology experiments. In vivo experiments indicated that ECP could significantly promote bone repair. The results of the chemical components analysis and the pathway identification revealed that TGF-ß signalling pathway was related to ECP. The results of in vitro experiments indicated that ECP-CS could reverse the damage caused by LPS through inhibiting the expressions of LncRNA MALAT1 and SMAD2, and improving the expressions of miR-34a-5p, ALP, RUNX2 and Collagen type І in osteoblasts significantly. This research showed that ECP could regulate the TGF-ß/SMADs signalling pathway to promote bone repair. Meanwhile, ECP could alleviate LPS-induced bone loss by modulating the signalling axis of LncRNA MALAT1/miRNA-34a-5p/ SMAD2 in IBDs.

The miRNA-144-5p/IRS1/AKT axis regulates the migration, proliferation, and mineralization of osteoblasts: A mechanism of bone repair in diabetic osteoporosis.

Diabetic osteoporosis (DOP) is a disorder of bone metabolism induced by multiple mechanisms. Previous studies have revealed that microRNAs (miRNAs) play crucial roles in bone metabolism. MiRNA-144-5p has been proven to participate in the regulation of osteoblast activities; however, its specific mechanism in DOP has not been elucidated. This study investigated whether high glucose (HG) inhibited osteoblasts by regulating miRNA-144-5p. Our results showed that HG inhibited bone formation not only in vivo but also in vitro. We observed that HG severely hindered the migration, proliferation and mineralization of osteoblasts, while miRNA-144-5p was upregulated by way of the cell counting kit-8 assay, wound healing assay, alkaline phosphatase (ALP) activity assay and alizarin red staining. Double luciferase reporter experiments showed that miRNA-144-5p directly targeted insulin receptor substrate 1 (IRS1). The IRS1/AKT signaling pathway is closely related to osteoblasts' migration, proliferation, and mineralization. Silencing miRNA-144-5p promoted the mRNA, and protein expression of IRS1, thereby letting the expression of total AKT down, and then preventing phosphorylation of AKT into the nucleus to regulate migration, proliferation, and mineralization genes of osteoblasts. In conclusion, this study indicated that HG regulated the migration, proliferation, and mineralization of osteoblasts via the miRNA-144-5p/IRS1/AKT axis, which suggested a possible mechanism for DOP pathology.

Prediction of allergic reaction risk of Shenmai injection based on real-world evidence coupled with UPLC-Q-TOF-MS.

The allergic reaction (AR) of Chinese herbal injection (CHI) has become one of the most noticeable focuses of public health in China. However, it still remains a considerable controversy as to whether low-molecular-weight components in CHI have potential sensitization. In this study, the relationship between AR and low-molecular-weight component profile of Shenmai injection was explored by an interdisciplinary technology integrating real-world evidence and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectroscopy (UPLC-Q-TOF-MS). The AR information of hospitalized patients was obtained by comprehensively analyzing real-world evidence from January 2015 to June 2019 at two Chinese hospitals. The UPLC-Q-TOF-MS was exploited to systematically investigate the low-molecular-weight component profile with 50-1500 m/z mass range, and 3725 MS1 peaks were detected. The optimized partial least squares discriminant analysis model was established to map the influence of low-molecular-weight components on AR. The results of this study showed that high levels of organic acids administered intravenously might be a potential risk factor for inducing AR. By using this method, Shenmai injection with high AR risk could be recognized precisely with 100% accuracy before clinical use.

Preparation and application of polyvinyl alcohol-decorated cell membrane chromatography for screening anti-osteoporosis components from Liuwei Dihuang decoction-containing serum.

Cell membrane chromatography is a powerful tool for screening active components from complex matrices. New cell membrane carriers need to be developed to increase the coverage of cell membranes on the surface of stationary phases, thereby improving cell membrane chromatographic retention. In this work, we prepared polyvinyl alcohol-poly dimethyl diallyl ammonium chloride modified silica gel as a cell membrane carrier. Osteoblast cell was used as cell membrane source, which was widely used to evaluate the osteogenic activity of drugs. The new cell membrane chromatographic stationary phase was used to screen anti-osteoporosis components in Liuwei Dihuang decoction-containing serum. The chemical structures of the new modified materials were characterized by scanning electronic microscopy, Fourier transform infrared spectroscopy, and elemental analysis characterization. Compared with the common cell membrane column, the cell membrane coverage of this modified material was increased by 30%, and thus provided a stronger retention effect in positive drugs. Nineteen metabolites in rat serum samples were retained on the cell membrane chromatography and identified by ultra-high-performance liquid chromatography/time-of-flight mass spectrometry. Among those, four components (morroniside, catalpol, loganin, and acteoside) were selected for in vitro pharmacodynamics validation. They significantly increased the osteoblast proliferation. The new modified material was successfully applied to screen anti-osteoporosis components from Liuwei Dihuang Decoction-containing serum.

A nanocomposite consisting of an amorphous seed and a molecularly imprinted covalent organic framework shell for extraction and HPLC determination of nonsteroidal anti-inflammatory drugs.

A novel nanocomposite consisting of an amorphous seed and a molecularly imprinted covalent organic framework shell was prepared via a heterogeneous nucleation and growth method. By using ibuprofen as the dummy template, a molecularly imprinted covalent organic framework (MICOF) with a large surface area was prepared from 1,3,5-triformylbenzene and 4,4'-diaminobiphenyl. It was placed on the surface of monodisperse amorphous seeds. Owing to strong π-interaction, the MICOF@SiO2 nanocomposite displays fast binding kinetics, large adsorption capacities and selectivity for nonsteroidal anti-inflammatory drugs (NSAIDs). Following desorption from the MICOF@SiO2 with methanol containing 1% ammonium hydroxide, the NSAIDs ketoprofen, ibuprofen, diclofenac, indomethacin, flurbiprofen and naproxen were quantified by HPLC with UV detection. Under optimized conditions, the method exhibits good linearity within the range of 0.002-1.0 µg mL-1, low limits of detection (0.38-2.92 µg L-1), and acceptable repeatability. The recoveries of NSAIDs at three spiking levels range from 77 to 112%, and the RSDs are <9.4%. The method was successfully applied to the analysis of NSAIDs in spiked environmental water samples. Graphical abstract A molecular imprinted covalent organic framework nanocomposite (MICOF@SiO2) was prepared by heterogeneous nucleation and growth method. It was explored as a sorbent for the solid phase extraction of nonsteroidal anti-inflammatory drugs before determination by HPLC with UV detection.

Effects of alkaloids from Sophora flavescens on osteoblasts infected with Staphylococcus aureus and osteoclasts.

Chronic osteomyelitis is primarily caused by infection with Staphylococcus aureus (S. aureus). Antibiotics are commonly administered; however, it is a challenge to promote bone healing. The aim of this study was to investigate the in vitro effects of alkaloids from the herbal remedy Sophora flavescens (ASF) on rat calvarial osteoblasts (ROBs) infected with S. aureus and healthy osteoclasts. Cell proliferation and alkaline phosphatase, interleukin-6, and tumour necrosis factor-α activity was measured in infected ROBs; tartrate-resistant acid phosphatase was evaluated in osteoclasts via enzyme-linked immunosorbent assay. The mRNA and protein expression levels of bone morphogenetic protein 2, runt-related transcription factor 2, osteoprotegerin, and receptor activator of nuclear factor kappa-B ligand were assessed in infected ROBs through reverse transcription-polymerase chain reaction and western blotting analysis, respectively. Results indicated that ASF increased the viability of uninfected ROBs and infected ROBs treated with vancomycin via regulation of bone morphogenetic protein 2, runt-related transcription factor, osteoprotegerin, and receptor activator of nuclear factor kappa-B ligand mRNA and protein expression levels. In addition, the secretion of the inflammatory factor tumour necrosis factor-α was decreased and alkaline phosphatase activity was increased, inhibiting the viability of osteoclasts and tartrate-resistant acid phosphatase activity. Therefore, the herbal remedy ASF has potential as a new treatment for chronic osteomyelitis.

Osteoblast cell membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry for screening specific active components from traditional Chinese medicines.

A method using osteoblast membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry was developed to recognize and identify the specific active components from traditional Chinese medicines. Primary rat osteoblasts were used for the preparation of the stationary phase in the cell chromatography method. Retention components from the cell chromatography were collected and analyzed by liquid chromatography with time-of-flight mass spectrometry. This method was applied in screening active components from extracts of four traditional Chinese medicines. In total, 24 potentially active components with different structures were retained by osteoblast cell chromatography. There were five phenolic glucosides and one triterpenoid saponin from Curculigo orchioides Gaertn, two organic acids and ten flavonoids from Epimedium sagittatum Maxim, one phthalide compound and one organic acid from Angelica sinensis Diels, and two flavonoids and two saponins from Anemarrhena asphodeloides Bunge. Among those, four components (icariin, curculigoside, ferulaic acid, and timosaponin BII) were used for in vitro pharmacodynamics validation. They significantly increased the osteoblast proliferation, alkaline phosphatase activity, levels of bone gla protein and collagen type 1, and promoted mineralized nodule formation. The developed method was an effective screening method for finding active components from complex medicines that act on bone diseases.

A Network Pharmacology Approach to Determine the Active Components and Potential Targets of Curculigo Orchioides in the Treatment of Osteoporosis.

BACKGROUND Osteoporosis is a complex bone disorder with a genetic predisposition, and is a cause of health problems worldwide. In China, Curculigo orchioides (CO) has been widely used as a herbal medicine in the prevention and treatment of osteoporosis. However, research on the mechanism of action of CO is still lacking. The aim of this study was to identify the absorbable components, potential targets, and associated treatment pathways of CO using a network pharmacology approach. MATERIAL AND METHODS We explored the chemical components of CO and used the five main principles of drug absorption to identify absorbable components. Targets for the therapeutic actions of CO were obtained from the PharmMapper server database. Pathway enrichment analysis was performed using the Comparative Toxicogenomics Database (CTD). Cytoscape was used to visualize the multiple components-multiple target-multiple pathways-multiple disease network for CO. RESULTS We identified 77 chemical components of CO, of which 32 components could be absorbed in the blood. These potential active components of CO regulated 83 targets and affected 58 pathways. Data analysis showed that the genes for estrogen receptor alpha (ESR1) and beta (ESR2), and the gene for 11 beta-hydroxysteroid dehydrogenase type 1, or cortisone reductase (HSD11B1) were the main targets of CO. Endocrine regulatory factors and factors regulating calcium reabsorption, steroid hormone biosynthesis, and metabolic pathways were related to these main targets and to ten corresponding compounds. CONCLUSIONS The network pharmacology approach used in our study has attempted to explain the mechanisms for the effects of CO in the prevention and treatment of osteoporosis, and provides an alternative approach to the investigation of the effects of this complex compound.

Flavonoids of Herba Epimedii Enhances Bone Repair in a Rabbit Model of Chronic Osteomyelitis During Post-infection Treatment and Stimulates Osteoblast Proliferation in Vitro.

Flavonoids are the active component of the Herba Epimedii (H. Epimedii), which is commonly used in Asia. This study is to investigate the effect of H. Epimedii on bone repair after anti-infection treatment in vivo. The bioactive-composition group of H. Epimedii (BCGE) contained four flavonoids with the total content of 43.34%. Rabbits with chronic osteomyelitis in response to injection with Staphylococcus aureus were treated with BCGE of 242.70 mg/kg/day intragastrically after vancomycin-calcium sulphate treatment. Micro-computerd tomography (CT), morphology, blood biochemistry and osteocalcin levels were assessed for effect evaluation. In addition, the rat calvarial osteoblasts infected with S. aureus were treated with vancomycin and BCGE. Cell viability, alkaline phosphatase activity, bone morphogenetic protein 2, Runt-related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor-κB ligand mRNA levels and protein expression were assessed. Our results indicated that BCGE promoted bone repair via increasing the bone mass, the volume of bone, promoting osteocalcin secretion after vancomycin-calcium sulfate treatment. BCGE enhanced the cell proliferation, by regulating bone morphogenetic protein 2, runt-related transcription factor 2, and osteoprotegerin/receptor activator of nuclear factor κ-B ligand mRNA and protein expression to maintain the balance between bone formation and bone resorption. Therefore, BCGE is a potential adjuvant herbal remedy for the post-infection treatment of chronic osteomyelitis. Copyright © 2016 John Wiley & Sons, Ltd.

Monosaccharide composition analysis of immunomodulatory polysaccharides by on-line hollow fiber microextraction with high-performance liquid chromatography.

The monosaccharide compositions of functional polysaccharides are essential for structure elucidation and biological activity determination. A sensitive method based on on-line hollow-fiber liquid-phase microextraction with high-performance liquid chromatography has been established for the analysis of ten monosaccharide compositions (two uronic acids, two amino sugars and six neutral sugars) of the immunomodulatory polysaccharides. After derivatization, the sample was injected into the lumen of a hollow fiber immersed in butyl ether and separated by liquid chromatography. Under optimized conditions, the calibration curves were linear (r ≥ 0.9996) in the range of 10-2000 µmol L(-1) . The limits of detection were in the range of 0.04-1.58 µmol L(-1) , and the recoveries were in the range of 92.1-99.6%, which shows that the method is applicable to the analysis of the monosaccharide composition of various polysaccharides.

Metabolomics Study of Type 2 Diabetes Mellitus and the AntiDiabetic Effect of Berberine in Zucker Diabetic Fatty Rats Using Uplc-ESI-Hdms.

The present study aimed to evaluate the pathogenesis of type 2 diabetes mellitus (T2DM) and the anti-diabetic effect of berberine in Zucker diabetic fatty (ZDF) rats. A urinary metabolomics analysis was performed with ultra-performance liquid chromatography/electrospray ionization synapt high-definition mass spectrometry. Pattern recognition approaches were integrated to discover differentiating metabolites. We identified 29 ions (13 in negative mode and 16 in positive mode) as 'differentiating metabolites' with this metabolomic approach. A functional pathway analysis revealed that the alterations were mainly associated with glyoxylate and dicarboxylate metabolism, pentose and glucuronate interconversions and sphingolipid metabolism. These results indicated that the dysfunctions of glycometabolism and lipometabolism are involved in the pathological process of T2DM. Berberine could decrease the serum levels of glycosylated hemoglobin, total cholesterol and triglyceride and increase the secretion of insulin. The urinary metabolomics analysis showed that berberine could reduce the concentrations of citric acid, tetrahydrocortisol, ribothymidine and sphinganine to a near-normal state. These results suggested that the anti-diabetic effect of berberine occurred mainly via its regulation of glycometabolism and lipometabolism and activation of adenosine 5'-monophosphate-activated protein kinase. Our work not only provides a better understanding of the anti-diabetic effect of berberine in ZDF rats but also supplies a useful database for further study in humans and for investigating the pharmacological actions of drugs. Copyright © 2016 John Wiley & Sons, Ltd.

Urinary Metabolomic Profiling in Zucker Diabetic Fatty Rats with Type 2 Diabetes Mellitus Treated with Glimepiride, Metformin, and Their Combination.

Type 2 diabetes mellitus (T2DM) is a high incidence metabolic disease. Glimepiride, metformin, and their combination are the most commonly used therapeutics for T2DM in the clinic, but little is known about the metabolic responses of these therapies. In this study, ultrahigh-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOF-MS)-based metabolomics was applied to detect changes in the urinary metabolomic profile of Zucker diabetic fatty (ZDF) rats in response to these treatments. Additionally, standard biochemical parameters (e.g., fasting plasma glucose, glycosylated hemoglobin, oral glucose tolerance, urinary glucose, triglyceride, total cholesterol, and insulin) and liver histopathology were monitored and observed. Six metabolites, including 3-galactosyl lactose, citric acid, sphingosine, phytosphingosine, ribothymidine, and succinoadenosine, were found significantly reverted to the normal level after these therapies. The present study is the first to present citric acid and sphinganine as the potential markers of T2DM, which could be used as indicators to observe the anti-diabetic effects of glimepiride, metformin, and their combination treatments.

Effects of Sanguis Draconis on Perforator Flap Survival in Rats.

Sanguis draconis, a resin known to improve blood circulation, relieve pain, stimulate tissue regeneration, and heal wounds, is widely used in clinical practice. In this study, we prepared an ethanol extract of sanguis draconis (EESD) containing 75.08 mg/g of dracorhodin. The experiment was carried out on 20 rats that were divided into two groups, a control group (n = 10) and an EESD group (n = 10). All the rats underwent a perforator flap surgery, after which post-operative abdominal compressions of EESD were given to the EESD group for seven days, while the control group received saline. Flap survival percentages were determined after seven days, and were found to be significantly higher in the EESD group than in the control group. Results of laser Doppler flowmetry (LDF) showed that perforator flaps in the EESD group had higher perfusion values than those of the control group. The flap tissues were stained with hematoxylin and eosin, followed by immunohistochemical evaluation. Superoxide dismutase (SOD) expression and micro-vessel development markedly increased in the EESD group, while malondialdehyde (MDA) levels decreased. This is the first study to investigate the effect of sanguis draconis on perforator flap survival. Our results demonstrate that sanguis draconis can improve perforator flap survival in rats by promoting microvessel regeneration and blood perfusion.

Ultra high performance liquid chromatography with synapt high-definition mass spectrometry and a pattern recognition approach to characterize chemical constituents and rat metabolites after the oral administration of Phellinus igniarius.

Phellinus igniarius has antibacterial, antiviral, antioxidative, antitumor, and antimutagenic effects. In this study, an integrative pattern recognition approach using principal component analysis and orthogonal partial least squares discriminant analysis was successfully applied for the rapid analysis of natural compounds in traditional Chinese medicine. An ultra high performance liquid chromatography with synapt high-definition mass spectrometry method and MassLynx software was used. This method employed gradient elution to rapidly analyze and characterize chemical constituents and metabolites after the oral administration of a P. igniarius ethanol extract. There were 24 peaks within 10 min of the analysis time and 20 of these were identified or tentatively characterized on the basis of their fragmentation behaviors. In the S-plot of the orthogonal partial least squares discriminant analysis, 27 ions were extracted to make the serum. Among them, nine absorbed the prototype components and 18 metabolites were identified in vivo. Glucuronidation, oxidation, and methylation were the major metabolic reactions. This study is the first systematic analysis and characterization of the chemical constituents and metabolites in an ethanol extract of P. igniarius. This method can be applied to the rapid analysis and characterization of constituents in rat serum after the oral administration of other compounds used in traditional Chinese medicines.

[Study on HPLC fingerprint of ethanol extract of Phellinus igniarius].

OBJECTIVE: To establish the HPLC fingerprint of ethanol extract of Phellinus igniarius ( EEPI), and to evaluate its quality. METHODS: Adopted 0.2% phosphoric acid -methanol as mobile phase with a SHISEIDO CAPCELL PAK C18 column (250 mm x 4.6 mm, 5 microm), the flow rate was 1.0 mL/min,and the detection wavelength was set at 395 nm. Inoscavin A was used as reference peak to establish common mode of characteristic absorption peaks of EEPI, and its similarity was evaluated by peaks overlap rate calculation,similarity evaluation system and cluster analysis. RESULTS: Under the chromatographic conditions, HPLC fingerprint of EEPI was established, and nine common peaks were selected. The similarities was between 0.652 and 0.995. According to cluster analysis, 10 batches of Phellinus igniarius were divided into three groups. CONCLUSION: This method is simple, accurate and repeatable with good separation. It can provide basis for the comprehensive quality evaluation of Phellinus igniarius.

miRNA-seq analysis of high glucose induced osteoblasts provides insight into the mechanism underlying diabetic osteoporosis.

The present study aims to explore the etiology of Diabetic osteoporosis (DOP), a chronic complication associated with diabetes mellitus. Specifically, the research seeks to identify potential miRNA biomarkers of DOP and investigated role in regulating osteoblasts. To achieve this, an animal model of DOP was established through the administration of a high-sugar and high-fat diet, and then injection of streptozotocin. Bone microarchitecture and histopathology analysis were analyzed. Rat calvarial osteoblasts (ROBs) were stimulated with high glucose (HG). MiRNA profiles of the stimulated osteoblasts were compared to control osteoblasts using sequencing. Proliferation and mineralization abilities were assessed using MTT assay, alkaline phosphatase, and alizarin red staining. Expression levels of OGN, Runx2, and ALP were determined through qRT-PCR and Western blot. MiRNA-sequencing results revealed increased miRNA-702-5p levels. Luciferase reporter gene was utilized to study the correlation between miR-702-5p and OGN. High glucose impaired cell proliferation and mineralization in vitro by inhibiting OGN, Runx2, and ALP expressions. Interference with miR-702-5p decreased OGN, Runx2, and ALP levels, which were restored by OGN overexpression. Additionally, downregulation of OGN and Runx2 in DOP rat femurs was confirmed. Therefore, the miRNA-702-5p/OGN/Runx2 signaling axis may play a role in DOP, and could be diagnostic biomarker and therapeutic target for not only DOP but also other forms of osteoporosis.

[Transcriptomic characteristics analysis of bone from chronic osteomyelitis].

OBJECTIVE: To explore the molecular mechanism of chronic osteomyelitis and to clarify the role of MAPK signal pathway in the pathogenesis of chronic osteomyelitis, by collecting and analyzing the transcriptional information of bone tissue in patients with chronic osteomyelitis. METHODS: Four cases of traumatic osteomyelitis in limbs from June 2019 to June 2020 were selected, and the samples of necrotic osteonecrosis from chronic osteomyelitis (necrotic group), and normal bone tissue (control group) were collected. Transcriptome information was collected by Illumina Hiseq Xten high throughput sequencing platform, and the gene expression in bone tissue was calculated by FPKM. The differentially expressed genes were screened by comparing the transcripts of the Necrotic group and control group. Genes were enriched by GO and KEGG. MAP3K7 and NFATC1 were selected as differential targets in the verification experiments, by using rat osteomyelitis animal model and immunohistochemical analysis. RESULTS: A total of 5548 differentially expressed genes were obtained by high throughput sequencing by comparing the necrotic group and control group, including 2701 up-regulated and 2847 down-regulated genes. The genes enriched in MAPK pathway and osteoclast differentiation pathway were screened, the common genes expressed in both MAPK and osteoclast differentiation pathway were (inhibitor of nuclear factor κ subunit Beta, IκBKß), (mitogen-activated protein kinase 7, MAP3K7), (nuclear factor of activated t cells 1, NFATC1) and (nuclear factor Kappa B subunit 2, NFκB2). In rat osteomyelitis model, MAP3K7 and NFATC1 were highly expressed in bone marrow and injured bone tissue. CONCLUSION: Based on the transcriptome analysis, the MAPK signaling and osteoclast differentiation pathways were closely related to chronic osteomyelitis, and the key genes IκBKß, MAP3K7, NFATC1, NFκB2 might be new targets for clinical diagnosis and therapy of chronic osteomyelitis.

Explorating the mechanism of Epimedii folium-Rhizoma drynariae herbal pair promoted bone defects healing through network pharmacology and experimental studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Bone defects are difficult to treat and have a high incidence of nonunion. The Epimedii folium-Rhizoma drynariae herbal pair (EDP) is a traditional Chinese medicine (TCM) used for treating bone diseases. However, the mechanisms by which EDP promotes osteogenesis or bone formation remain largely unclear. AIM OF THE STUDY: This study aimed to investigate the mechanism of EDP promoted bone formation in bone defects using network pharmacology and experiments. MATERIALS AND METHODS: The chemical components of EDP were analyzed by UHPLC-MS. The hub target and pathway enrichment analysis was conducted using molecular docking or network pharmacology. The pharmacological actions of EDP were determined by µCT and histopathology examination using a bone defect rat model. The effects of EDP on the mRNA expression of Bmp2, Smad2/5, Runx2, and Alp genes were measured by RT-PCR, while changes in the protein expressions of BMP2, COL1A1, SPP1, ALP, and RUNX2in the tibia tissues of the rats in response to EDP were analyzed by immunohistochemical staining or Western blot. We also performed cell viability assays, Alizarin Red and ALP staining assays, and RT-PCR to better understand how EDP affected osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). RESULTS: Identified 14 key compounds and 47 hub targets of EDP that may be involved in promoting osteogenesis to repair bone defects. And the BMP/Smad/Runx2 pathway was likely the key pathway through which EDP promoted bone defects repairing. The results of in vivo rat experiments indicated that EDP effectively promoted tibia repair in the model rats and activated the BMP/Smad/Runx2 pathway in the tibia tissue, with upregulating Bmp2, Bmpr1α, Smad2/5, Runx2, and Alp genes, and increased the protein expression of BMP2, COL1A1, RUNX2, and ALP. In vitro, EDP was found to increase the proliferation, differentiation, and mineralization in BMSCs- and also up-regulated the expression of key genes in the BMP/Smad/Runx2 pathway. CONCLUSION: This study highlighted the ability of EDP to promote the osteogenic differentiation to enable bone repair by activating the BMP/Smad/Runx2 pathway.

Beneficial effect and mechanism of natural resourced polysaccharides on regulating bone metabolism through intestinal flora: A review.

Bone metabolism is an important biological process for maintaining bone health. Polysaccharides of natural origin exert beneficial effects on bone metabolism. Polysaccharide molecules often have difficulty passing through the intestinal cell membrane and are directly absorbed in the gastrointestinal tract. Therefore, polysaccharides may affect intestinal flora and play a role in disease treatment. We performed a comprehensive review of the relevant literature published from 2003 to 2023. We found that several polysaccharides from traditional Chinese medicines, including Astragalus, Achyranthes bidentata and Eucommia ulmoides, and the polysaccharides from several dietary fibers mainly composed of inulin, resistant starch, and dextran could enrich the intestinal microbiota group to regulate bone metabolism. The promotion of polysaccharide decomposition by regulating the Bacteroides phylum is particularly critical. Studies on the structure-activity relationship showed that molecular weight, glycosidic bonds, and monosaccharide composition may affect the ability of polysaccharides. The mechanism by which polysaccharides regulate intestinal flora to enhance bone metabolism may be related to the regulation of short-chain fatty acids, immunity, and hormones, involving some signaling pathways, such as TGF-ß, Wnt/ß-catenin, BMP/Smads, and RANKL. This paper provides a useful reference for the study of polysaccharides and suggests their potential application in the treatment of bone metabolic disorders.

[Dispersive solid-phase extraction combined with high-performance liquid chromatography for determination of seven anesthetics in aquatic products].

Nowadays, anesthetics are widely used in fishery production processes, such as fish breeding, surgery, and fresh aquatic product transportation. Because of the widespread application of anesthetic drugs in aquatic products, there is an increasing demand for the rapid and sensitive detection of anesthetic drugs in aquatic products. The complex aquatic product matrix contains a variety of interfering substances, such as proteins, fats, and phospholipids, along with anesthetic drug residues at very low concentrations; therefore, it is necessary to adopt appropriate pretreatment methods for improving the sensitivity of detection. In this study, a dispersive solid-phase extraction (DSPE) method, combined with high-performance liquid chromatography, was established for the simultaneous detection of seven anesthetic drugs in aquatic products, viz. procaine, oxybuprocaine, tricaine, eugenol, methyl eugenol, isoeugenol, and methyl isoeugenol. For the DSPE step, pretreatment conditions, such as extraction solvent, extraction time, adsorbent amount, and DMSO dosage, were optimized. Sample pretreatment is a three-step process. First, in ultrasound-assisted extraction, 2.0 g samples were extracted using 10.0 mL 1.0% formic acid in acetonitrile under ultrasound conditions for 10 min. Then, DSPE was performed with mixed adsorbents: the solvent extracts were cleaned using 20 mg poly(styrene-glycidylmethacrylate) microspheres (PS-GMA), 50 mg primary secondary amines (PSA), and 10 mg C18, followed by separation by centrifugation. Finally, DMSO-assisted concentration was applied: the organic layer was collected and was dried at 40 ℃ in a N2 stream with 100 µL DMSO. Water was added to the residue to obtain a final volume of 1.0 mL for HPLC analysis. The seven anesthetic drugs were separated on a Welch welchrom C18 column (250 mm×4.6 mm, 5 µm) by gradient elution using methanol and 0.05% formic acid in 5 mmol/L ammonium acetate aqueous solution as mobile phases. The detection wavelengths were 235, 260, and 290 nm. Two matrix matching standard curves for fish and shrimp were applied for quantitative analysis. Under optimized conditions, the seven target anesthetics showed good linear relationships in their respective concentration ranges (R2>0.999), with the limit of detection (LOD) ranging from 0.011 to 0.043 mg/kg. In fish samples, the mean recoveries obtained at three concentration levels were between 79.7% and 109%, with relative standard deviations (RSDs) being less than 7.2%. In shrimp samples, mean recoveries were 78.0%-99.9%, with RSDs being less than 8.3%. This simple, rapid, accurate, and sensitive method can be applied to the detection of three kinds of aminobenzoic acid esters and four kinds of eugenol anesthetic drugs in aquatic products.