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The gastroprotective effects of polysaccharides had become a hot topic in the field of functional polysaccharides research. Three polysaccharides, namely HPS-80-1, HPS-80-2, and HPS-80-3 were purified by DEAE-52 column chromatography. The thermodynamic characteristics, scanning electron microscopy, and Congo red experimental results of the above polysaccharides were greatly distinctive. Then a mature GES-1 oxidative stress cell model induced by H2O2 was established to screen out subsequent research subjects. It turned out that HPS-80-1 had a desirable protective effect, which was confirmed by analyses of cell cycle & apoptosis, and oxidative stress-related factors in the cell culture media, and so on. Furthermore, Structural features demonstrated that the backbone of HPS-80-1 appeared to mainly consist of →4)-α-D-Glcp-(1→, →4,6)-β-L-Glcp-(1→, and →6)-α-D-Galp-(1→, with branches at O-1, O-4, and O-6 position consisting of →2,4)-β-D-Rhap-(1→, →1)-α-D-Galp-(4→, and →3,4)-α-D-Manp-(1→. It was speculated that the excellent gastric mucosal protective activity of HPS-80-1 may be due to the high amount of glucose in the backbone. In addition, it was also related to the anti-inflammatory activity and antioxidant bases such as (1 → 4)-Glcp and (1 → 6)-Galp in the structure of HPS-80-1. These findings provide a scientific basis for further utilization of polysaccharides from Radix Hedysari.

No consensus has been reached on the optimal position of PCL tibial tunnel. The purpose of this study was to compare the biomechanical properties of proximal, distal and anatomic tibial tunnel in transtibial posterior cruciate ligament reconstruction. An in-vitro model of transtibial posterior cruciate ligament reconstruction was simulated using porcine tibias and bovine extensor tendons. Two models of biomechanical testing, load-to-failure loading, and cyclic loading, were performed in this study. The load-to-failure loading found that distal tibial tunnel resulted in greater ultimate load and yield load than the anatomic and proximal tunnel group ( p 0.05). The cyclic loading found no differences in the graft displacement at 250, 500, and 1000 cycles among three groups ( p > 0.05). It was found that distal tibial tunnel showed superior ultimate load and yield load in load-to-failure loading testing compared with proximal and anatomic tibial tunnels, whereas no significant difference was found in terms of the mean displacement of the survived grafts in cyclic loading testing among three groups.

The crude Hedysarum polysaccharides (HPS: HPS-50 and HPS-80) obtained from Radix Hedysari exhibited great pharmacological activities in our previous research. This study investigated the effects of HPS on lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute liver injury (ALI) in mice and LPS-induced injury in LO2 cells, as well as the relationship between structural characteristics and hepatoprotective activities. The in vivo results showed that compared with HPS-80, HPS-50 showed stronger hepatoprotection, which improved histopathological changes to normal levels. HPS-50 significantly decreased the levels of ALT, AST, MPO, and MDA, increased the activities of SOD, CAT, and GSH, and suppressed the LPS/D-GalN-triggered production of TNF-α, IL-1β, and IL-6 (p .05). The results in vitro showed that HPS-50-P (HPS-50-1, HPS-50-2, and HPS-50-3) purified from HPS-50 played significant protective roles against LPS-induced injury in LO2 cells by reducing cell apoptosis and relieving cell cycle arrest. HPS-50-2 restored the percentage of normal cells from 54.8% to 94.7%, and reduced the S phase cells from 59.40% to 47.05% (p .01). By analyzing the structure of HPS-50-P, including monosaccharide composition, molecular weight, chain conformation, and surface morphology, we speculated that the best protective effect of HPS-50-2 might be attributed to its beta configuration, highest molecular weight, and high glucose and galactose contents. These findings indicate that HPS-50 might be a promising source of functional foods for the protection and prevention of ALI. PRACTICAL APPLICATIONS: In this study, the protective effect of HPS on ALI was evaluated from multiple perspectives, and HPS-50-2 was screened as a potential active ingredient. This study has two practical applications. First, it provides a new way to improve ALI, and a new option for patients to prevent and treat ALI. Second, this work also complements the pharmacological activity of Radix Hedysari and provides a basis for the development of Radix Hedysari as a functional food.

A newly and rapid supercritical fluid chromatography method for the simultaneous determination of 11 active compounds in Radix Hedysari samples has been developed and validated. Optimum separation was achieved on a HSS SB C18 column with a gradient elution at a flow rate of 1.5 mL/min, back pressure of 11.03 Mpa and diode array detector at 260 nm. The results from the quantitative data showed that contents of these 11 active compounds were different from plant regions. Especially the contents of formononetin in the Minxian county are ~6-fold than in wild Radix Hedysari. The chromatographic fingerprint of Radix Hedysari was recorded under the same chromatographic condition. Data analytic procedure was performed to differentiate the 25 batches of Radix Hedysari samples. Data from chromatographic fingerprint were also analyzed using hierarchical cluster analysis. The results showed that 23 batches of Radix Hedysari samples had a high similarity (> 0.90) and overall 25 batches of sample were divided into two clusters. Moreover, according to the comparison contents of active compounds in each Radix Hedysari samples, the cultivated location of Radix Hedysari was successfully distinguished. This method presented good stability, repeatability and precision and would be a useful and reliable approach for the quality control of Radix Hedysari. Moreover, all target compounds were quantified by ultra-high performance liquid chromatography–time-of-flight mass spectrometry.

Calycosin has been reported to have a strong osteogenic activity and a positive correlation with anti-osteoporosis effects. However, its precise mechanism of action remains unclear. Since insulin-like growth factor 1 receptor (IGF1R) signaling and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling have been shown to play a pivotal role in regulating osteogenesis, we hypothesized that the osteogenic activity of calycosin is mediated by these signaling pathways. Rat calvarial osteoblasts (ROBs) were cultured in osteogenic medium containing calycosin with or without GSK1904529A (GSK) or LY294002 (LY) (inhibitors of IGF1R and PI3K, respectively). The effects on cell proliferation, alkaline phosphatase (ALP) activity, calcified nodules, mRNA or protein expression of osteogenic genes [alkaline phosphatase (Alpl), collagen type I (Col1a1), runt-related transcription factor 2 (Runx2), Osterix, and bone morphogenetic protein 2 (Bmp2)], and phosphorylation of IGF1R and Akt were examined. The present results showed that calycosin enhanced cell proliferation, ALP activity and Alizarin Red-S staining in a dose-dependent manner in the range of 10-8 -10-6 M, while an inhibitory effect was observed at 10-5 M. Treatment at the optimal concentration (10-6 M, a physiologically achievable concentration) increased mRNA levels of osteogenic genes and phosphorylation of IGF1R and Akt. Furthermore, treatment with GSK or LY partly reversed the effects of calycosin on ROBs, as indicated by the decreases in calycosin-induced ALP activity, calcified nodules and osteogenic gene expression. These results suggest that the osteogenic effect of calycosin partly involves the IGF1R/PI3K/Akt signaling pathway.

The dry root of Hedysarum polybotrys Hand.-Mazz., commonly known as "Hong Qi", has a variety of health benefits. The present study was undertaken to explore the anti-gastric ulcer potential effect of Hedysarum polysaccharides (HPS; HPS-50, HPS-80), the principal active fraction of Radix Hedysari (RH). The anti-gastric ulcer effects of HPS were evaluated using an animal model of ulcerative lesions induced by acetic acid. The effects of antioxidant factors, anti-inflammatory cytokines, and mucosal blood flow regulatory factor levels in the gastric tissue homogenate of rats were analyzed for the bioactivities of HPS. The results showed that, compared with the acetic acid-induced ulcerated group, the ulcer inhibition rate of HPS-treated rats was significantly increased. The pathological findings suggested that mucosal regeneration, cell migration, and inflammatory cell infiltration were decreased, and collagen fibers were significantly reduced. Extensive granulation tissue proliferation indicated the healing stage was initiated, suggesting a good prognosis. The oxidative stress status of the gastric ulcer rats was improved, the levels of TNF-α and IL-6 were significantly decreased, and the levels of PGE-2 and NO were increased (P < 0.05). HPS-80-H may be a promising ingredient for incorporation into functional foods or nutritional supplements for the prevention of gastric ulcers.

This study linked natural plant materials and nanomaterials; reporting an environmentally friendly, non-toxic and efficient method for the green synthesis of gold nanoparticles (AuNPs) using an ethyl acetate extract of Radix Hedysari (EAR). The components of the extract were identified using HPLC and it was found that formononetin accounted for more than 90% of the total contents. We predicted that formononetin in EAR plays a crucial role in green synthesis. Thus, formononetin was used as a standard reductant to synthesize AuNPs, and the result confirmed our prediction. The synthetic mechanism was also discussed in detail in the article. Moreover, EAR–AuNPs realized the sensitive and selective colorimetric detection of ferrous ions (Fe2+) among other metal ions, and were applied to spiked tap water with a low detection limit of 1.5 μM in a wide range from 10 μM to 500 μM. EAR–AuNPs were green synthesized using Radix Hedysari extract for the first time and were successfully applied in real sample detection.

Ulcerative colitis (UC) is a chronic inflammatory disease with an unknown precise etiology. This study proves that Radix Hedysari (RH) ameliorates UC. Four RH extracts were used to ameliorate UC induced by 2,4-Dinitrobenzenesulfonic acid by 7 days intervention in agreement to preliminary studies. Compared to treatment with RH extracts, the RH ethanol extract (EE) was found to be more effective in ameliorating UC. With EE, the DAI were significantly decreased. Macroscopic and histopathological assessments suggest that the colon mucosa was repaired, the organizational structure of the colon had been rebuilt. The levels of MPO, TNF-α, IL-1β, and MDA were significantly decreased (p < .01), the levels of T-SOD and CAT were significantly increased (p < .01). Moreover, the compounds in EE were analyzed by HPLC. The results show that EE can ameliorate UC, and its anti-inflammatory capability probably plays an important role. RH can act as a functional food and ameliorate UC. PRACTICAL APPLICATIONS: In this work, the ameliorative effect of RH on UC was evaluated from multiple angles. There are two practical applications of this work. On the one hand, a new approach to ameliorating UC is provided by this work. In addition, UC patients have a new option for improving their symptoms. On the other hand, this work also provides information on how best to process RH for therapeutic use. In addition, we can utilize some compounds of RH that were once considered useless and reduce the waste of natural resources.

A new, more effective and environmentally friendly method involving a complex enzyme combined with ultrasonication was employed to extract and isolate three novel polysaccharides (HPS-MCs: HPS-MC, HPS-MC (50%) and HPS-MC (80%)) of Radix Hedysari. Compared with polysaccharides obtained using a traditional extraction method (hot water extraction, HPS-R), the yields and total carbohydrate contents of HPS-MCs were significantly higher. HPS-MC (80%) exhibited relatively strong immunomodulatory activity and a concentration-dependent dose-response relationship under cyclophosphamide (CP)-induced immunosuppressive conditions in mice models. To more comprehensively investigate the relationships between structural characteristics and immunomodulatory activity, HPS-MC (80%) was fractionated into three major homogeneous polysaccharide fractions (HPS-MC (80%)s: HPS-MC (80%)-1, HPS-MC (80%)-2, and HPS-MC (80%)-3). These three homogeneous polysaccharides had different mass percentages of monosaccharides species (rhamnose, arabinose, mannose, glucose, and galactose) by gas chromatography (GC) and different molecular weights and chain conformations by high-performance gel permeation chromatography coupled with multi-angle laser light scattering (HPGPC-MALLS), and promoted macrophage and splenocyte proliferation to different degrees. These findings indicated that HPS-MC (80%) had a prominent potential immune response, especially HPS-MC (80%)-2 and HPS-MC (80%)-3, and might be suitable candidates for functional foods or potential novel immunomodulators.

Correction for ‘Formononetin in Radix Hedysari extract-mediated green synthesis of gold nanoparticles for colorimetric detection of ferrous ions in tap water’ by Xinyue Chen et al., RSC Adv., 2020, 10, 32897–32905, DOI: 10.1039/D0RA05660J.

We reported an eco-friendly, rapid and easy method for the green synthesis of gold nanoparticles (AuNPs) using Hedysarum polysaccharides (HPS) extracted from Radix Hedysari. To deeply explore the synthetic mechanism, the monosaccharides that made up HPS and other Radix Hedysari extract were also used as reductant to synthesize AuNPs. A series of techniques were conducted to confirm the formation of HPS-AuNPs. Moreover, as a sensitive optical detector, the large-sized HPS-AuNPs was used to selectively detect iron ion (Fe3+) among the other metal irons and was successfully applied in the tap water detection with a wide range of 1 μM to 10 mM, a low detection limit of 0.57 μM. Besides, the catalytic activity of HPS-AuNPs was also proved to be well in the reduction of 4-nitrophenol to 4-aminophenol by NaBH4. This is the first report for the green synthesis of HPS-AuNPs, providing a sensitive detector and a well-active catalyst.

An extremely sensitive and highly selective sensing assay for histidine (His) in biological sample is demonstrated through the N-acetyl-cysteine (NAC) functionalized gold nanoparticles (AuNPs). To improve the detection efficiency, the external conditions of temperature and pH value are optimized. We assume that three main forces of hydrogen bond, electrostatic attraction, and hydrophobic force between His and NAC cause the formation of close crosslinking, resulting in the aggregation of AuNPs accompanied by an obvious color change. Thus, the assay is highly sensitive for His with a fast readout within 10 s and the detection limit of 176 pM which is the lowest one from what's been reported in the colorimetric detection. Furthermore, due to the special imidazole ring in His structure, the assay exhibits the high selectivity of His sensing among 18 common amino acids. Besides, the system has been successfully used for His detection in spiked urine and serum samples. This novel colorimetric sensor significantly improves the analytical merit of His sensing and applies in the biological sample detection.

Background: BMPs play important roles in regulating stem cell proliferation and differentiation. Using adenovirus-mediated expression of the 14 types of BMPs we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of mesenchymal stem cells (MSCs), which was undetected in the early studies using recombinant BMP9 proteins. Endogenous BMPs are expressed as a precursor protein that contains an N-terminal signal peptide, a prodomain and a C-terminal mature peptide. Most commercially available recombinant BMP9 proteins are purified from the cells expressing the mature peptide. It is unclear how effectively these recombinant BMP9 proteins functionally recapitulate endogenous BMP9. Methods: A stable cell line expressing the full coding region of mouse BMP9 was established in HEK-293 cells by using the piggyBac transposon system. The biological activities and stability of the conditioned medium generated from the stable line were analyzed. Results: The stable HEK-293 line expresses a high level of mouse BMP9. BMP9 conditioned medium (BMP9-cm) was shown to effectively induce osteogenic differentiation of MSCs, to activate BMP-R specific Smad signaling, and to up-regulate downstream target genes in MSCs. The biological activity of BMP9-cm is at least comparable with that induced by AdBMP9 in vitro. Furthermore, BMP9-cm exhibits an excellent stability profile as its biological activity is not affected by long-term storage at -80oC, repeated thawing cycles, and extended storage at 4oC. Conclusions: We have established a producer line that stably expresses a high level of active BMP9 protein. Such producer line should be a valuable resource for generating biologically active BMP9 protein for studying BMP9 signaling mechanism and functions.

α-Mangostin (α-M) is a commonly used traditional medicine with various biological and pharmacological activities. Our study aimed to explore the effects and mechanism of α-M in regulating apoptosis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). α-M of 10 to 100 μM was used to treat RA-FLS for 24 hours, followed by measuring cell viability and apoptosis. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases was detected. Treatment of α-M promoted apoptosis and reduced viability of RA-FLS in a dose-dependent manner. The mitochondrial membrane potential in RA-FLS was remarkably reduced by α-M treatment, accompanied by the cytochrome c accumulation in the cytosol and increased activities of caspase-3 and caspase-9. Moreover, we found that α-M treatment promoted ROS production and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The proapoptotic activity of α-M in RA-FLS was markedly reversed by the co-induction with the ERK1/2 inhibitor LY3214996 or ROS scavenger N-acetyl-l-cysteine. In conclusion, our studies found that α-M had remarkable proapoptotic activities in RA-FLS, which is regulated by the induction of ROS accumulation and ERK1/2 phosphorylation. α-M may thus have potential therapeutic effects for rheumatoid arthritis.

Calycosin has been reported to have a strong osteogenic activity and a positive correlation with anti-osteoporosis effects. However, its precise mechanism of action remains unclear. Since insulin-like growth factor 1 receptor (IGF1R) signaling and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling have been shown to play a pivotal role in regulating osteogenesis, we hypothesized that the osteogenic activity of calycosin is mediated by these signaling pathways. Rat calvarial osteoblasts (ROBs) were cultured in osteogenic medium containing calycosin with or without GSK1904529A (GSK) or LY294002 (LY) (inhibitors of IGF1R and PI3K, respectively). The effects on cell proliferation, alkaline phosphatase (ALP) activity, calcified nodules, mRNA or protein expression of osteogenic genes [alkaline phosphatase (Alpl), collagen type I (Col1a1), runt-related transcription factor 2 (Runx2), Osterix, and bone morphogenetic protein 2 (Bmp2)], and phosphorylation of IGF1R and Akt were examined. The present results showed that calycosin enhanced cell proliferation, ALP activity and Alizarin Red-S staining in a dose-dependent manner in the range of 10

Osteosarcoma (OS) is the most common primary malignant tumor of bone with a high propensity for lung metastasis. Despite significant advances in surgical techniques and chemotherapeutic regimens over the past few decades, there has been minimal improvement in OS patient survival. There is an urgent need to identify novel antitumor agents to treat human OS. Repurposing the clinically-used drugs represents a rapid and effective approach to the development of new anticancer agents. The anthelmintic drug niclosamide has recently been identified as a potential anticancer agent in human cancers. Here, we investigate if niclosamide can be developed as an anti-OS drug. We find that niclosamide can effectively inhibit OS cell proliferation and survival at low micromolar concentrations. Cell migration and wounding closure are significantly inhibited by niclosamide. Niclosamide induces cell apoptosis and inhibits cell cycle progression in OS cells. Analysis of niclosamide's effect on 11 cancer-related signal pathway reporters reveals that three of them, the E2F1, AP1, and c-Myc-responsive reporters, are significantly inhibited. To a lesser extent, the HIF1α, TCF/LEF, CREB, NFκB, Smad/TGFβ, and Rbpj/Notch pathway reporters are also inhibited, while the NFAT and Wnt/β-catenin reporters are not significantly affected by niclosamide treatment. We demonstrate that the expression of c-Fos, c-Jun. E2F1, and c-Myc in OS cells is effectively inhibited by niclosamide. Furthermore, niclosamide is shown to effectively inhibit tumor growth in a mouse xenograft tumor model of human osteosarcoma cells. Taken together, these results strongly suggest that niclosamide may exert its anticancer activity in OS cells by targeting multiple signaling pathways. Future investigations should be directed to exploring the antitumor activity in clinically relevant OS models and ultimately in clinical trials.

One of the greatest obstacles to current cancer treatment efforts is the development of drug resistance by tumors. Despite recent advances in diagnostic practices and surgical interventions, many neoplasms demonstrate poor response to adjuvant or neoadjuvant radiation and chemotherapy. As a result, the prognosis for many patients afflicted with these aggressive cancers remains bleak. The insulin-like growth factor (IGF) signaling axis has been shown to play critical role in the development and progression of various tumors. Many basic science and translational studies have shown that IGF pathway modulators can have promising effects when used to treat various malignancies. There also exists a substantial body of recent evidence implicating IGF signaling dysregulation in the dwindling response of tumors to current standard-of-care therapy. By better understanding both the IGF-dependent and -independent mechanisms by which pathway members can influence drug sensitivity, we can eventually aim to use modulators of IGF signaling to augment the effects of current therapy. This review summarizes and synthesizes numerous recent investigations looking at the role of the IGF pathway in drug resistance. We offer a brief overview of IGF signaling and its general role in neoplasia, and then delve into detail about the many types of human cancer that have been shown to have IGF pathway involvement in resistance and/or sensitization to therapy. Ultimately, our hope is that such a compilation of evidence will compel investigators to carry out much needed studies looking at combination treatment with IGF signaling modulators to overcome current therapy resistance.

Radix Hedysari polysaccharides (HPS) is the principal active fraction of Radix Hedysari (RH). The information about HPS3d, the main fraction of HPS3, and its effect on bone is still unknown. In the present study, the purified HPS3d was obtained by anion-exchange column. It consisted of 94.38% polysaccharide, 3.40% protein and 13.30% uronic acid. The molecular weight was measured to be 84.6kDa. The backbone consisted of galactopyranose and galacturonopyranose, and the side chains were composed of glucopyranose, rhamnopyranose and arabinofuranose. The FT-IR and elemental analysis showed that HPS3d was the sulfated polysaccharide. HPS3d upregulated alkaline phosphatase (ALP) activity and the expression of other osteogenic marker genes in osteoblast. In addition, HPS3d increased the expression and transcriptional activity of Runt-related transcription factor 2 (Runx-2) and Osterix, the two master genes of osteoblast differentiation. These findings suggest that HPS3d stimulates osteoblast differentiation by activation of Runx-2 and Osterix.

This study provided a comprehensive component analysis and structure identification of active substances for the anti-gastric ulcer effects of Radix Astragali. The data were generated by organically combining the results from in vivo pharmacodynamic experiments, a cell growth-promoting assay, structure identification, content determination, fingerprinting, and correlation analyses. The fingerprints from high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) and from HPLC coupled with evaporative light scattering detectors (ELSD) from 95% ethanol extracts of Radix Astragali (ERA) were determined using HPLC-DAD-ELSD. The structures of 16 compounds were identified using ultra-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). The contents of these 16 compounds were simultaneously determined in a single run using HPLC-DAD-ELSD. The strength of the anti-ulceration effect of each of the 16 compounds was correlated to its content in the HPLC spectrum using gray relation statistics. The sequence of the contribution from each of the 16 compounds to the anti-gastric ulcer effect was determined. The results showed that ononin, astragalosideIII, and astragalosideIV contributed most to the observed anti-gastric ulcer effects and that these three compounds also exhibited strong growth-promoting effects in cultured GES-1 cells. The results of this study can be used to evaluate the quality of Radix Astragali and to provide a theoretical foundation for its further study.

Background Consistent reference data used for anatomic posterior cruciate ligament (PCL) reconstruction is not well defined. Quantitative guidelines defining the location of PCL attachment would aid in performing anatomic PCL reconstruction. The purpose was to characterize anatomic parameters of the PCL tibial attachment based on magnetic resonance imaging (MRI) in a large population of adult knees. Methods The PCL tibial attachment site was examined in 736 adult knees with an intact PCL using 3.0-T proton density–weighted sagittal MRI. The outcomes measured were the anterior-posterior diameter (APD) of the tibial plateau; angle between the tibial plateau and the posterior tibial ‘shelf’ (the slope where the PCL tibial attachment site was) (PTS); length of the PTS; proximal, central, and distal PCL attachment positions as well as the width of the PCL attachment site; and vertical dimension of the PCL attachment site inferior from the tibial plateau. Results The average APD of the tibia plateau was 33.6 ± 3.5 mm, yielding significant differences between males (35.5 ± 3.0 mm) and females (31.6 ± 2.7 mm), P .05). The proximal, central positions and width of the PCL attachment site were 13.4 ± 3.0 mm, 17.8 ± 3.0 mm and 9.6 ± 2.4 mm along the PTS, with significant differences between males and females (P .05). Conclusions This study provides reference data of the tibial PCL attachment based on MRI in the sagittal orientation. In analysis of retrospective data from a large population of adult patients, the quantitative values can be used as references to define the inserted angle and depth of the drill guide, and the exact position and size of the tibial PCL tunnel for performing arthroscopic anatomic PCL reconstruction.

A water-soluble sulfated glucan, SHG, was isolated from Hedysarum polybotrys Hand.-Mazz using anion-exchange and gel-permeation chromatography. Elemental analysis indicated that SHG was a sulfated polysaccharide containing small amount of sulfate groups (1.47 mass%). The molecular weight was estimated to be 1.72 × 10 5 Da by high-performance gel permeation chromatography (HPGPC) and size exclusion chromatography–multi angle laser light scattering (SEC–MALLS). SHG took random coil compact conformation in 0.1 M NaNO 3 . Compositional analysis revealed that SHG was composed of glucose only. On the base of partial acid hydrolysis, methylation analysis, gas chromatography–mass spectrometry (GC–MS), Fourier transform infrared (FT-IR) spectra, nuclear magnetic resonance (NMR) spectroscopy of 1 H, 13 C, β-correlation spectroscopy (β-COSY), total correlation spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC), the results showed that SHG was composed of α- d -(1 → 4)-linked glucopyranosyl residues, with branches at C-6 consisting of non-reducing terminal approximately every eight residues. Sulfate groups may attach to the backbone at O-6, occasionally occurring per thirty-eight anhydrous glucose units.

Ovarian cancer is the most lethal gynecologic malignancy with an overall cure rate of merely 30%. Most patients experience recurrence within 12–24 months of cure and die of progressively chemotherapy-resistant disease. Thus, more effective anti-ovarian cancer therapies are needed. Here, we investigate the possibility of repurposing antibiotic monensin as an anti-ovarian cancer agent. We demonstrate that monensin effectively inhibits cell proliferation, migration and cell cycle progression and induces apoptosis of human ovarian cancer cells. Monensin suppresses multiple cancer-related pathways including Elk1/SRF, AP1, NFκB and STAT and reduces EGFR expression in ovarian cancer cells. Monensin acts synergistically with EGFR inhibitors and oxaliplatin to inhibit cell proliferation and induce apoptosis of ovarian cancer cells. Xenograft studies confirm that monensin effectively inhibits tumor growth by suppressing cell proliferation through targeting EGFR signaling. Our results suggest monensin may be repurposed as an anti-ovarian cancer agent although further preclinical and clinical studies are needed.

The intervertebral disc (IVD) is a fibrocartilaginous joint between two vertebral bodies. An IVD unit consists of a gelatinous central nucleus pulposus, encased by the annulus fibrosus, which is sandwiched between cartilaginous endplates (EP). The IVD homeostasis can be disrupted by injuries, ageing, and/or genetic predispositions, leading to degenerative disc disorders and subsequent low back pain. The complex structure and distinct characteristics of the IVDs warrant the establishment of robust in vitro IVD organ culture for studying the etiology and treatment of disc degeneration. Here, we isolate mouse lumbar IVDs and culture the minimal IVD units in submersion or suspension medium supplemented with 2% bovine serum or 10% fetal bovine serum (FBS). We find the minimal IVD units remain healthy up to 14 days when cultured in submersion culture supplemented with 10% FBS. New bone formation in the EPs of the cultured IVDs can be assessed with calcein labeling. Furthermore, the cultured IVDs can be effectively transduced by recombinant adenovirus, and transgene expression lasts for two weeks. Thus, our findings demonstrate that the optimized intervertebral disc organ culture system can be used to study intervertebral disc biology and screen for biological factors that may prevent, alleviate, and/or treat disc degeneration.

The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited.

Bone cells respond to various mechanical stimuli including fluid shear stress (FSS) in vitro. Induction of cyclooxygenase-2 (COX-2) is thought to be important for the anabolic effects of mechanical loading. Recently, extracellular-signal-regulated kinase 5 (ERK5) has been found to be involved in multiple cellular processes. However, the relationship between ERK5 and the induction of COX-2 is still unknown. Here, we investigated the potential involvement of ERK5 in the response of pre-osteoblastic MC3T3-E1 cells upon FSS. MC3T3-E1 cells were subjected to 12 dyn/cm(2) FSS. Then, we established a ERK5 small interfering RNA (siRNA) transfected cell line using the MC3T3-E1 cells. After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction and Western blotting, the expression of COX-2, cAMP response element-binding protein (CREB), and nuclear factor kappa B cells (NF-κB) were assayed for downstream effectors of activated ERK5 under FSS by Western blotting. Our results showed that FSS could stimulate COX-2 activity, and induce the phosphorylation of ERK5, CREB, and NF-κB. When the MC3T3-E1 cells were transfected using siRNA before exposure to FSS, COX-2 activity was suppressed, and the phosphorylation of CREB and NF-κB was significantly downregulated. In summary, we demonstrated that ERK5 pathway is essential in the induction of COX-2 gene.

RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.

The aim of this study was to determine the role of the mitogen-activated protein kinase kinase (MEK) 5/extracellular signal-regulated kinase (ERK) 5 pathway in osteoblast differentiation promoted by intermittent fluid shear stress (FSS). MC3T3-E1 osteoblastic cells were subjected to 12 dyn/cm(2) intermittent FSS, and the phenotypic markers for osteoblast differentiation, such as alkaline phosphatase (ALP) activity and expression of osteopontin (OPN) and osteocalcin (OCN), were then examined. The results showed that intermittent FSS could stimulate ERK5 phosphorylation, ALP activity and the expression of OPN and OCN. When the MEK5/ERK5 pathway was selectively inhibited by BIX02189, ALP activity was suppressed, and the expression of OPN and OCN was downregulated. Intermittent FSS induce the expression of Runt-related transcription factor-2 (Runx-2), which is involved in osteoblast differentiation by promoting the transcription of the above genes. Furthermore, the expression of Runx-2 was also reduced after treatment with BIX02189. Finally, we found that intermittent FSS was a more intense stimulus than steady FSS for promoting osteoblast differentiation. In summary, our results suggest that the MEK5/ERK5 pathway mediates osteoblast differentiation promoted by intermittent FSS, which was more effective than steady FSS in the differentiation process. The MEK5/ERK5 pathway also mediates FSS-induced Runx-2 expression in osteoblast differentiation.

A systematic review and meta-analysis based on randomized controlled trials (RCTs) were conducted to evaluate the efficiency and safety of periarticular multimodal drug injection in total knee arthroplasty (TKA). Periarticular injection with the use of multimodal drugs is an efficient alternative for postoperative analgesia in TKA. A systematical electronic search was performed to identify the eligible RCTs in the databases of PubMed, Embase, Cochrane Central Register of Controlled Trials, Web of Science and the Chinese Biomedical Literature Database. Two independent reviewers completed data collection and assessment of methodological quality. The quality of evidence of outcomes was judged using GRADE criteria. Meta-analysis was performed for the outcomes of pain, straight leg raise, operating time, hospital stay and complications. Ten RCTs including eight studies with 1,216 TKAs in 835 patients met the inclusion criteria. Periarticular injection with multimodal drugs in TKA was associated with short-term benefits in terms of pain relief, straight leg raise, narcotic consumption, and the rates of nausea, vomiting, rash and pruritus. There were no statistically significant differences in operating time, hospital stay, wound complications and deep vein thrombosis between both groups. The current evidence suggests that periarticular multimodal drug injection in TKA provides short-term advantages in pain relief, straight leg raise and postoperative complications. Therapeutic study, Level I.