Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro.Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor Dickkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN.Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes.Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes. Osteoarthritis (OA) is the leading reason for disability and mainly affects the elderly . RadioloOsteopontin (OPN) is a 44–75 kDa multifunctional phosphoprotein secreted by numerous cell types, including osteoclasts, chondrocytes, synoviocytes, macrophages, and epithelial cells –8. A preThe Wnt/β-catenin signaling pathway can directly affect the bone and bone and synovial tissues to play a role in bone and joint pathology. Wnt/β-catenin proteins affect cell stability by regulating cell proliferation and determining cell fate and the differentiation state , 14. ThePrevious studies have confirmed that OPN is transformed by a variety of Wnt signaling factors, including TCF4 and β-catenin , 23. Hig3 sections. The cartilage tissue was subsequently digested with 5–8 ml of 0.2% collagenase II  for 12–16 h at 37 °C in a constant temperature shaker. Digestion was terminated with 8–10 ml of Dulbecco’s modified Eagle’s medium/F12 . The released cell pellets at the bottom of the centrifuge tube were aspirated and transferred to a culture flask following centrifugation at 1000 rpm for 5 min. Cell pellets were resuspended in 5 ml of DMEM/F12 containing 10% fetal bovine serum  and 1% penicillin/streptomycin solution (Gibco) and incubated for 24 h at 37 °C with 5% CO2 in a plastic culture flask. The nonadherent cells were subsequently washed out. The growth medium was changed every 3 days prior to trypsinization, and cells were then passed to a new 6-well plate at a density of 5 × 105 cells per well. Cells at passages one through two were used for experiments.The concise experimental flowchart is shown in Fig. Total cellular RNA was extracted from cultured normal and osteoarthritic chondrocytes using TRIzol reagent . RNA was further purified using an RNeasy mini kit  according to the manufacturer’s instructions. Preservation of 28S and 18S ribosomal RNA (rRNA) species was used to assess RNA integrity. All samples included in the study had prominent 28S and 18S rRNA components. The total RNA yield was quantified by spectrophotometry. One microgram of total RNA was converted to cDNA using a Revert Aid™ First Strand cDNA Synthesis Kit . First, all components including template RNA (1 μg) and oligo (dT)18 primer (1 μl) were mixed, and nuclease-free water was added to a total volume of 12 μl. The mixture was mixed gently, centrifuged briefly, and incubated at 65 °C for 5 min. The mixture was chilled on ice, spun down, and placed back in a vial on ice. The following components were added, to a volume of 20 μl: 5X reaction buffer (4 μl), RiboLock™ RNase Inhibitor (20 u/μl) (1 μl), 10 mM dNTP Mix (2 μl) and Revert Aid™ M-MuLV Reverse Transcriptase (200 u/μl) (2 μl). This reaction was then incubated for 60 min at 42 °C and terminated by heating at 70 °C for 5 min. The resulting cDNA products were stored in aliquots at − 80 °C until needed.The primers were synthesized by Shanghai Sangon Bioengineering Corporation. All primers used are shown in Table −△△CT values. All quantities were expressed as n-fold relative to a calibrator.Real-time Q-PCR test results: The relative expression level of mRNAs was calculated as the ratio of each mRNA expression level to that of GAPDH mRNA as the reference housekeeping gene. The relative expression levels of genes of interest were calculated and expressed as 2n = 1. No significant variance was observed among the individual wells in each averaged group. After 24 h, total RNA and protein were isolated, and the expression levels of TCF-4 and OPN were detected by real-time Q-PCR and Western blot analyses.siRNAs specific to TCF-4 based on the coding sequence of human TCF-4 were designed and synthesized by GenePharma Corporation . The three shRNA sequences and scrambled shRNA sequences constructed are shown in Table Normal and OA chondrocytes were seeded on six-well plates at 1 × 106 cells/well, and 3 days after seeding, the cells were treated with recombinant DKK-1  for 12, 24, 36, and 48 h; each experiment was conducted with triplicate wells.2 for 4 h. The supernatant was then carefully aspirated, and the formazan reaction products were dissolved in 150 μl dimethylsulfoxide (DMSO)  solution and shaken for 15 min. The spectrophotometric absorbance at 570 nm was measured in an enzyme-linked immunosorbent assay (ELISA) plate reader  or an ELISA reader .Cell viability was determined by a colorimetric 3--2,5-diphenyltetrazolium bromide (MTT) assay. The culture medium was removed, and 20 μl of MTT solution (5 mg/ml in PBS) was added to each well and incubated at 37 °C with 5% COChondrocytes were lysed by using RIPA buffer and a cocktail of protease and phosphatase inhibitors. The protein concentration was quantified by using a BCA Protein Assay Kit  with bovine serum albumin as the standard. Cell lysates from normal and OA chondrocytes were electrophoresed and separated on a 4 to 20% Tris-HCl gel , and the separated proteins (25 μg) were electrotransferred to polyvinylidene fluoride membranes . Nonspecific proteins on the membranes were blocked with 5% skim milk powder in PBS. The membrane was probed with an anti-osteopontin antibody  and an anti-TCF-4 antibody  overnight at 4 °C. A mouse monoclonal anti-actin antibody (Sigma) was used as the loading control. The membranes were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies . Immunoreactive proteins were visualized with western blotting luminol reagent . The polyvinylidene fluoride membranes were then exposed to photographic film, which was scanned, and the intensities of the protein bands were determined with computerized densitometry. The results were normalized by using an anti-GAPDH polyclonal antibody .β-Catenin nuclear accumulation was observed by nucleic acid staining with DAPI. Chondrocytes were treated with or without rDKK 1, and the treated chondrocytes were collected and fixed in 4% paraformaldehyde for 5 min. The fixation solution was discarded, and the cells were rinsed 3 times in 1× PBS. The buffer was discarded, and the cells were incubated in 10 μM DAPI solution in the dark at 37 °C for 15 min. The DAPI solution was removed, and the stained cells were rinsed 3 times in 1× PBS and supplemented with fresh 1× PBS buffer. The stained cells were examined for cellular morphology and nuclear profiles under a Leica laser confocal microscope.t test was used to analyze statistical differences between two groups, and one-way ANOVA was performed to determine the statistical differences among groups. A P value of less than 0.05 was considered statistically significant.All statistical calculations were performed using GraphPad Prism 6.0 . Data are expressed as the means ± standard errors of the mean. Student’s P < 0.01)  .In the present study, we detected the mRNA levels of β-catenin and OPN between chondrocytes isolated from parts of the knee cartilage of OA and non-OA patients. The relative β-catenin mRNA expression in the OA group (6.60 ± 0.31-fold) was significantly higher than that in the normal group (1.08 ± 0.05-fold) (01) Fig. . We also01) Fig. . HoweverP < 0.05)  and one negative control sequence (shRNA NC) were transiently transfected into OA chondrocytes. The transfection efficiency was evaluated with fluorescein by fluorescence microscopy 24 h after transfection. Compared to the same optical field, the fluorescence index value for the transfected cells was nearly 50% Fig. , and the05) Fig. ; therefoP < 0.01) and shRNA NC group (P < 0.05) and that OPN mRNA (0.23 ± 0.01-fold) and protein (0.22 ± 0.02-fold) expression were decreased in the shRNA-1105 group compared to the control group (P < 0.01) and shRNA NC group (P < 0.05)  and protein (0.20 ± 0.01-fold) was decreased in the shRNA-1105 group compared to the control group (05) Fig. .Fig. 4aIn MTT assays, chondrocytes were treated with rDKK-1 at concentrations of 100, 200, 300, 400, and 500 ng/ml and for durations of 12, 24, 36, and 48 h to determine the adverse effects of rDKK-1 on cell viability. Our results showed that the relative cell viability was greater than 90% at 400 ng/ml for 24 h, as shown in Fig. P < 0.05).It is well known that DKK 1 is an inhibitor of the Wnt/β-catenin signaling pathway, an our DAPI staining results indicated that β-catenin nuclear accumulation was decreased in the presence of rDKK-1, as shown in Fig. Cartilage degeneration is the main feature of OA, and OPN is a secreted multifunctional phosphoprotein involved in the molecular pathogenesis of OA by contributing to progressive degeneration of articular cartilage . In the Canonical Wnt signaling is also called Wnt/β-catenin signaling; this pathway is a conserved signaling pathway implicated in the pathogenesis of OA , 20. In Mammals have four members of the TCF/LEF family: TCF1, TCF3, TCF4, and LEF1 . Via altA previous study confirmed that Wnt activation in OA affects the whole joint; that Dkk-1-mediated inhibition of Wnt signaling in bone ameliorates OA in mice; that control of Dkk-1 expression ameliorates chondrocyte apoptosis, cartilage destruction, and subchondral bone deterioration in osteoarthritic knees; and that the protective effect of Dkk-1 appears to be associated with its capacity to inhibit Wnt-mediated expression of catabolic factors , 32, 33.Taken together, our results indicate that the increase in OPN expression might be regulated by the β-catenin/TCF-4 pathway and that the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.


Subsequent genetic manipulation in vitro studies revealed that Chibby knockdown induced the expression of β-catenin and C-myc, cyclin D1 protein, which promoted cell proliferation and invasiveness. In contrast, overexpression of Chibby decreased β-catenin expression and inhibited the cell proliferation and invasiveness. Our results suggest that low expression of Chibby was associated with advanced tumor-node-metastasis (TNM) stage and poor differentiation. Furthermore, the combination of Chibby and β-catenin can predict poor prognosis in patients with HCC. Chibby inhibited HCC progression by blocking β-catenin signaling in vitro. Chibby is a biomarker and may be a potential therapeutic target for HCC.Chibby is an antagonist of β-catenin and is considered a potential tumor suppressor protein, but the role of Chibby in hepatocellular carcinoma (HCC) has not been characterized. The expression patterns of Chibby and β-catenin in HCC specimens and paired adjacent noncancerous tissues were measured by Western blotting and immunohistochemistry. The correlations between Chibby expression and clinicopathological parameters were analyzed. Then the biological functions of Chibby were analyzed in vitro. The Chibby protein was significantly downexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and is a risk factor for HCC recurrence and shorter survival. Furthermore, we found that in HCC tissues the high expression of β-catenin with low expression of Chibby in the nuclei was an independent predictor for disease-free survival (DFS) ( Hepatocellular carcinoma (HCC) is the second cause of cancer-related mortality worldwide, accounting for an estimated 600,000 deaths annually . AlthougCTNNB1 (the β-catenin gene) have been discovered in around 20%–40% of all HCC cases, and define CTNNB1 as the most frequently mutated gene in HCCs  (n = 18] (n = 59] (n = 66] (p = 0.025). Of them, patients with β-catenin (high)/Chibby (low) had the highest proportion of multiple HCC (50%) than the other three groups.To further investigate the correlation between Chibby and β-catenin and HCC characteristics, we examined β-catenin status by IHC staining. There were three types of β-catenin in HCC tissues: nuclei  . Statist= 0.048) . To furt= 0.048) A: high e (p = 0.001), liver cirrhosis (p = 0.004), TNM stage III and IV (p < 0.001), and HCC with β-catenin (high) and Chibby (low) (p = 0.012) were significantly associated with recurrent HCC (p = 0.021), TNM stage III and IV (p < 0.001), and β-catenin (high) and Chibby (low) (p = 0.005) were significantly associated with mortality  B,C. To erent HCC . In overortality .p < 0.05) (p < 0.05)  to assess whether HCC cell lines endogenously express Chibby by Western blot. We found that higher expression of Chibby in HepG2, Hep3B, J5, and Huh7, compared with PLC, Mahlavu, and SK-Hep-1, which showed relatively lower expression of Chibby A. Then w < 0.05) D and cel < 0.05) E. Moreov < 0.05) F. By Wes < 0.05) G, the twTo further verify the role of Chibby, we overexpressed Chibby in Huh7 cells with an adenovirus carrying Chibby (wild type). The overexpression of the Chibby proteins was confirmed by Western blot analysis A. As shoIn the present study, we first provided evidence that Chibby is downexpressed in HCC samples and correlated with advanced TNM stage, histology grade, postoperative recurrence, and poor survival of patients. The findings suggest that Chibby plays an antioncogenic role in HCC and may constitute a prognostic factor for patients with HCC after resection. Furthermore, we observed that HCC patients with high expression of β-catenin and low expression of Chibby in tumor nuclei had poor outcomes in HCC recurrence and overall survival. To our knowledge, this is the first study using the expression of Chibby to predict outcomes in HCC patients after resection.The Wnt/β-catenin signaling pathway is aberrantly activated in a wide range of HCC patients. Nuclear accumulation of β-catenin is associated with poor prognosis in HCC patients, suggesting that β-catenin promotes tumor proliferation and progression ,9. The eThe present results are consistent with other studies, which documented that overexpression of Chibby is correlated with less aggressive cancer phenotypes in many cancers, such as colorectal cancer, lung cancer, and laryngeal squamous cell carcinoma ,16,17. HIt is worth noting that there is no significance between HCC patients with high and low expression of nuclear Chibby. We further explored the association between β-catenin and Chibby in HCC. The results indicated that high expression of β-catenin and low expression of Chibby in the nuclei in HCC tissues is an independent risk factor for HCC recurrence and overall survival. Taken together, Chibby plays an antioncogenic role in HCC patients with the expression of nuclear β-catenin, especially. However, there are no associations between the expression of Chibby in tumor tissue and gender, age, clinical staging, and tumor differentiated degree in our clinical observations. The factors regulating the expression of Chibby in HCC are still questionable.We manipulated the expression of Chibby in Huh 7 HCC cell lines to see whether Chibby regulates the biological behavior of HCC by targeting the Wnt/β-catenin signaling activity. Our results in vitro were consistent with our clinical findings, which showed that enforced Chibby expression resulted in the attenuation of HCC cell proliferation and invasiveness, while Chibby knockdown promoted cell proliferation and invasiveness. Meanwhile, in these cells with overexpression or knockdown of Chibby, β-catenin is predominantly inversely expressed, and its signaling activity is affected consequently. Xu et al. observed the enforced expression of Chibby effectively suppressed laryngeal carcinoma cell growth by inhibiting Wnt/β-catenin signaling . FischerIn addition to the Huh7 HCC cell line, we also regulated the Chibby expression into the SK-Hep-1, a liver adenocarcinoma cell line, and the results are consistent with the HCC cell lines. Taking all together, we suggested that Chibby can regulate different types of liver tumors, such as HCC and adenocarcinoma. Further clinical studies that enroll different liver tumors, such as cholangiocarcinoma or liver metastatic tumor, need to be further clarified.Although prior studies have documented that overexpression of Chibby in colorectal, lung, and laryngeal squamous cell carcinoma correlate with less aggressive cancer phenotypes ,16,17, wn = 125) of the patients were male. In addition, there was liver cirrhosis, hepatitis B predominance, and TNM stage I and II predominance. The study was performed in accordance with the current Declaration of Helsinki guidelines. The protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan (IRB number:201800049B0). Written informed consent was obtained from each patient.From August 1990 to August 1999, 156 patients who underwent curative hepatic resection for HCC at the Chang Gung Memorial Hospital, Kaohsiung, Taiwan, were recruited into this study. One hundred and fifty-six HCC paraffin tissues after liver resection were obtained from the pathology department. The diagnosis of HCC was based on the criteria of practice guidelines of the European Association for the Study of the Liver (EASL) or the American Association for the Study of Liver Disease (AASLD) . Disease2O2, and counterstained with Harris’s hematoxylin, and mounted with the dePex medium for microscopic observation.The paraffin-embedded tissue blocks were obtained from the department of pathology sectioned for IHC. All slides were incubated overnight at 4 ℃ in a humidified chamber with primary antibodies against Chibby  and β-catenin . After washing with PBST, the sections were then incubated with peroxidase-conjugated secondary antibodies for 30 min. Finally, antibody staining was visualized with 3,3-diaminobenzidine tetrahydrochloride  in 0.1 M Tris pH 7.2, containing 0.01% HThe intensity of Chibby immunostaining in the tumor and non-tumor parts was evaluated by two independent pathologists using a scoring system. The number of positive cells was divided into five grades (percentage scores): 0 (≤10%), 1 (11%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (>75%). The intensity score was graded from 0 to 3 to represent null (0), weak (1), intermediate (2), and strong (3) intensities . The fin−∆∆CT method and normalized to beta-actin. Each PCR reaction was performed in triplicate and averaged. Melting curve analysis was carried out at the end of a 40-cycle amplification to validate the size of the PCR products. The primers were as follows for human Chibby , and for human β-actin .Total RNA was extracted from the cells using the Quick-RNA™ kit . RNA was quantified using a NanoVue spectrophotometer . RNA was reverse-transcribed into cDNA with a PrimeScriptTM RT reagent Kit . The quantitative real-time PCR was performed with SYBR Premix Ex Taq  and a 7500 fast real-time PCR system . Relative mRNA was calculated by the 2g. Protein concentration were determined using a Bio-Rad DC kit . Proteins (25–50 µg/lane) were subjected to immunoblot analysis. Blots were developed using the ECL Detection System . Western blot membranes were incubated using the following primary antibodies: anti-Chibby , anti-c-myc , anti-PCNA , anti-cyclin D1 , and anti-β actin . The reactions were detected with a horseradish peroxidase-conjugated secondary antibody . The primary antibodies were incubated overnight at 4 °C, and the secondary antibodies were incubated at 25 °C for 1 h. Densitometric analysis of bands were performed with Image J software .Western blotting was used to detect the expression of Chibby and β-catenin in transfected and non-transfected Chibby HCC cell lines. Cell extracts of cultured HCC cells were prepared by centrifuging the cells in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor , incubated on ice for 10 min and centrifuged for 10 min at 21,000× Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 5 min, permeabilized with 0.2% Triton X-100 in PBS for 15 min, rinsed three times with PBS, and blocked with normal goat serum diluted 1:20 or with 5% BSA/PBS for 30 min. Staining was performed with primary rabbit polyclonal Ab to Chibby , mouse monoclonal Ab to β-catenin , or rabbit polyclonal Ab to Ki67  at a dilution of 1:100 at 4 °C overnight. Cells were washed five times with PBS and then incubated with a donkey anti-mouse Texas Red –conjugated secondary antibody for 2 h (Molecular Probe) at room temperature. Finally, the slides were mounted by ProLongR Gold antifade reagent with DAPI  and examined using a Zeiss Axiophot microscope .5 cells were plated in a 6-well plate and grown in appropriate media to ~50% confluency for transfection. Then each well was treated with a mixture containing 2 μg plasmid DNA and 6 μL lipofectamine2000. The cells were investigated or harvested 24 h after transfection.The Huh7 and SK-Hep1 cell lines provided by Dr. Ming-Hong Tai  were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum . Transient transfections were carried out using a lipofectamine 2000 transfection reagent (Invitrogen) for HCC cells. Approximately 1–2 × 10Adenoviral vectors encoding human Chibby cDNA (GenBank accession number: NM_015373) with N-terminal hemagglutinin tag (Ad-Chibby) and green fluorescent protein (Ad-GFP) were generated through homologous recombination by calcium phosphate protocol. Cells (80%–90% confluence) were infected with adenoviral vectors at different multiplicity of infection (MOI) in a serum-free medium for 1 h. After 1 h adsorption, the virus-containing media were removed, and cells were incubated with fresh complete media.Cells were transfected with Chibby shRNA plasmid , a pool of three Chibby-specific plasmids, each encoding 19–25 nucleotide long shRNA, or a control shRNA plasmid  encoding a scrambled shRNA sequence. These shRNAs were transfected into Huh7 and SK-HEP1 cells using Lipofectamine 2000 (Invitrogen) in the absence of serum according to the manufacture’s recommendations by addgene. After 48 h of transfection, cells stably expressing shRNA were enriched via puromycin  selection. Then the medium was replaced with DMEM, and cells were cultured for another 24 h. Finally, the transfected cells were harvested to analyze the efficiency of Chibby knockdown.2 for 2 weeks. Then cells were fixed with 4% paraformaldehyde for 10 min and stained with 0.5% crystal violet for 30 min. The numbers of cells were quantified by image analysis using PhotoImpact . Each group was represented in the form of triplicates.After transfection for 24 h, cells were plated into 6-well plates (1000 cells/well) and cultured in normal cultured media at 37 °C and 5% COCell invasion assays were performed on Boyden chamber with 8-μm pore-size filter with a basement membrane matrix . Briefly, cells were trypsinized and resuspended in serum-free media and seeded onto 8-μm polycarbonate membranes coated with a basement membrane matrix. After 4 h incubation, cells migrated through the pores, and the cells attached to the underside of the membrane were stained with Giemsa. Finally, the number of cells was calculated under the microscope. Each group was represented in the form of triplicates.t-test was statistically evaluated to compare the differences between the groups. Curves for recurrence and survival rates were determined by the Kaplan–Meier analysis and compared using the log-rank test. Cox’s proportional hazards model was used to determine the independent factors of recurrence and survival based on the variables selected in the univariate analysis. Statistical analysis was performed with the SPSS software package for Windows . All p-values were two-tailed, and a p-value of less than 0.05 was considered to be statistically significant.Experimental values of continuous variables are expressed as the mean ± standard error of the mean. Two-tailed, unpaired Student’s In conclusion, the study we present here has indicated the high nuclear expression of Chibby predicted good prognosis in HCC patients with high nuclear expression of β-catenin compared with those with low nuclear expression of Chibby, which implies Chibby plays a salvage role in HCC with β-catenin activation. The expression of Chibby and β-catenin in nuclear may be useful as novel prognostic predictors and therapeutic targets for HCC patients.


Downsizing of MnS and encapsulating by conductive N, S-co-doped carbon matrix (MnS@NSC) provide excellent reversible capacity, rate capability, and cycling stability in sodium-based electrolyte.The charge storage mechanism of MnS@NSC was analyzed, showing pseudocapacitive control behavior.The as-fabricated sodium-ion capacitor delivers excellent electrochemical performance.The online version of this article (10.1007/s40820-020-0367-9) contains supplementary material, which is available to authorized users. + reaction kinetics, the present work demonstrated a high-rate MnS-based anode by embedding the MnS nanocrystals into the N, S-co-doped carbon matrix (MnS@NSC). Benefiting from the fast pseudocapacitive Na+ storage behavior, the resulting composite exhibits extraordinary rate capability (205.6 mAh g−1 at 10 A g−1) and outstanding cycling stability without notable degradation after 2000 cycles. A prototype SIC was demonstrated using MnS@NSC anode and N-doped porous carbon (NC) cathode; the obtained hybrid SIC device can display a high energy density of 139.8 Wh kg−1 and high power density of 11,500 W kg−1, as well as excellent cyclability with 84.5% capacitance retention after 3000 cycles. The superior electrochemical performance is contributed to downsizing of MnS and encapsulation of conductive N, S-co-doped carbon matrix, which not only promote the Na+ and electrons transport, but also buffer the volume variations and maintain the structure integrity during Na+ insertion/extraction, enabling its comparable fast reaction kinetics and cyclability with NC cathode.Sodium-ion capacitors (SICs) have received increasing interest for grid stationary energy storage application due to their affordability, high power, and energy densities. The major challenge for SICs is to overcome the kinetics imbalance between faradaic anode and non-faradaic cathode. To boost the NaThe online version of this article (10.1007/s40820-020-0367-9) contains supplementary material, which is available to authorized users. Electrochemical energy storage system is attracting extensive research interest due to the increasing demands of electric/hybrid vehicle, portable electronic devices, and scalable grid storage –4. Curre−1 according to the electromotive force of 1.049 V [Eo(MnS) vs. Na+/Na = 1.049 V] and Gibbs free energy change of − 202.50 kJ mol−1 [+ transport together with improved structural integrity for SICs still remains a challenge.To address this challenge, tremendous efforts are dedicated to investigate advanced anode materials for SIBs, including carbon-based materials –20, metakJ mol−1 , 39, 40.kJ mol−1 , 41, 42.kJ mol−1 , 37. How+ and electrons, but also buffer the accumulated strain resulting from volume variations during Na+ insertion/extraction and maintain the structural integrity, thus displaying high reversibility and rate capability. Owing to these advantages, the MnS@NSC electrode exhibits excellent rate performance (464.3 mAh g−1 at 0.05 A g−1 and maintains 205.6 mAh g−1 at 10 A g−1) and outstanding cycling stability without notable degradation after 2000 cycles. By coupling the anode with a N-doped carbon (NC) cathode, the assembled SIC full cell can deliver energy density as high as 139.8 Wh kg−1 and power density of 11,500 W kg−1. The capacity retention achieved is 84.5% after 3000 cycles, demonstrating the superiority of MnS@NSC as promising anode candidate for SIC.To mitigate these challenges, high-rate MnS-based SIB anode materials were demonstrated in this work by embedding MnS nanocrystals into the N, S-co-doped carbon matrix (MnS@NSC) using polyacrylonitrile (PAN) as both carbon and nitrogen source. The fully encapsulated MnS nanocrystals into the porous and conductive N, S-co-doped carbon matrix not only promote the facile access of Na3COO)2·4H2O were dissolved into 10 mL N,N-dimethylformamide (DMF) under magnetic stirring for 2 h at 60 °C. Then, the as-prepared solution was added dropwise to a mixed solution of ethylene glycol (EG) and ethanol (v:v = 5:2) under vigorous stirring. After finishing the infusion, the resultant mixture was stirred at 180 °C and refluxed for 9 h. When the temperature cooled down, the product of Mn-PAN complex was collected by filtration, followed by drying at 80 °C in the vacuum oven overnight.0.4 g of polyacrylonitrile (PAN) and 1.376 g of Mn material was prepared using 2 M HCl to etch away the MnS in the MnS@NSC composite. The control samples with different MnS contents were also prepared following the same procedures except with varied refluxing time of 5 and 16 h; the resultant samples are defined as MnS@NSC-5 and MnS@NSC-16, respectively.The as-obtained Mn-PAN complex was first annealed at 700 °C for 2 h under Ar protection, and then, the product and sulfur with a mass ratio of 1:2 were put into two separate sections of ceramic boat with sulfur at the upstream of the furnace. The furnace was heated to 500 °C for 2 h under Ar flow with a heating rate of 2 °C min3N4 was first obtained by annealing urea at the temperature of 550 °C for 2 h under Ar flow. Then, 0.25 g C3N4 was mixed with 50 mL of distilled water and sonicated for 30 min, followed by adding 2 g of wheat flour and 2 g KOH. The prepared mixture was stirred for 30 min and then dried at 80 °C. The resultant dry gel was carbonized at 800 °C for 1, 2, and 3 h under argon atmosphere, which was defined as NC-800-1, NC-800-2, and NC-800-3, respectively. After cooling down, the obtained NC material was washed with 2 M HCl and distilled water, respectively, to remove the impurity and then dried at 80 °C under vacuum for 10 h. For comparison, another NC was prepared by increasing the mass of KOH to 2.5 g with the annealing time of 2 h, which was defined as NC-800-2-2.5.CKa radiation (λ = 1.5406 Å). The X-ray photoelectron spectroscopy (XPS) analysis was carried out on a VGESCALab220i-XL spectrometer with a monochromatic Al Ka radiation (hv = 1486.7 eV). For the ex situ XPS, oxygen plasma etching was conducted to remove any possible SEI layer or oxidation layer on the samples. The nitrogen sorption isotherms and Brunauer–Emmett–Teller (BET) surface area were measured using a Tristar II 3020 instrument at liquid nitrogen temperature. Thermogravimetric analysis (TGA) was performed in air atmosphere from room temperature to 600 °C with a rate of 10 °C min−1 using a TA Q500.The morphology and nanostructures of the samples were recorded by field emission scanning electron microscope  and transmission electron microscope . Raman spectroscopy was conducted on a confocal Raman spectrometer with an excitation wavelength of 633 nm. The detailed crystal structures of the materials were measured by a Shimadzu powder diffractometer with Cu −2. The electrochemical properties were carried out in a CR2032-type coin cells which were assembled in an Ar-filled glove box. Pure sodium metal foil was used as both counter and reference electrodes; a glass microfiber filter was used as the separator, and 1 M NaClO4 in ethylene carbonate and diethyl carbonate  with 5 wt% fluoroethylene carbonate (FEC) as electrolyte. CV curves and EIS were recorded on electrochemical workstation (Autolab PGSTAT30). The galvanostatic charge/discharge and cycle life were performed on Neware battery-testing system. Galvanostatic intermittent titration technique (GITT) was performed by discharging the cells for 1200 s at 0.1 A g−1, followed by 1800 s relaxation. The SIC full cells were also fabricated into coin cells using a pre-sodiated MnS@NSC electrode  as anode, NC as the cathode, and the same separator/electrolyte as half-cell. The energy density and power density of the SICs were calculated based on the total mass of active materials in both anode and cathode. All the electrochemical measurements were tested at room temperature. The voltage of the LEDs is 1.9 V, and 10 LEDs were powered by our SICs for 7 min.The working electrodes were prepared by mixing 75 wt% active material, 15 wt% carbon black, and 10 wt% polyvinylidene fluoride (PVDF) in N-methyl pyrrolidinone (NMP) to form a slurry. The slurry was later casted on current collectors and then dried in vacuum oven at 80 °C for 12 h. The mass loading of the active materials was about 1 mg cmd-spacing of 0.264 nm corresponding to (200) planes of MnS. The scanning transmission electron microscopy (STEM) image and energy-dispersive X-ray spectroscopy (EDX) mapping  of MnS@NSC also confirmed that the MnS nanocrystals were uniformly distributed in the NSC matrix. As shown in Figs. S2 and + diffusion, thus boosting the rate performance. The small crystallite size of MnS is anticipated to provide high electrochemical activity and high utilization of electrode materials, favoring fast Na+ diffusion and releasing local strain. The morphology and nanostructures of the MnS@NSC-5 and MnS@NSC-16 were also investigated using SEM and are displayed in Fig. S3. After etching and dissolving the MnS nanocrystals from the nanocomposite sample, the residue NSC material remains the same morphology and no diffraction peaks of MnS can be detected using XRD, as displayed in Fig. S4a, b. The content of electrochemically active MnS in MnS@NSC composites was quantified with TGA in air atmosphere. As demonstrated in Fig. S5, the initial weight loss of MnS@NSC at about 25–150 °C is ascribed to the evaporation of surface adsorbed water [2O3 in the MnS@NSC composite [2O3 was verified by the XRD pattern  of sample MnS@NSC after TGA test. Thus, the content of MnS in MnS@NSC-5, MnS@NSC, and MnS@NSC-16 can be calculated as 14.8, 39.5, and 51.5 wt%, respectively.The detailed preparation process of the intended MnS@NSC composite is elucidated in the Experimental section. The crystal composition of the samples was first analyzed by X-ray diffraction (XRD). All diffraction peaks can be ascribed to the cubic MnS phase (JCPDS No. 01-1089) without any impurities, indicating that a high purity crystalline phase of MnS has been synthesized, as shown in Fig. ed water , 44. Theomposite , 45. Tra−1 can be observed, which is assigned to intrinsic vibration modes of MnS that are present in both pure MnS and MnS@NSC samples [−1 in MnS@NSC correspond to the D band of disordered carbon and G band of graphitized carbon), respectively [E2g vibrations of graphite [2 g−1, respectively. The small surface area of MnS@NSC sample is tentatively ascribed to the formation of dense carbon nanospheres matrix. The MnS sample has more mesopores with pore sizes below 3 nm, while the MnS@NSC composite shows a hierarchical porous structure with the coexistence of micropores, mesopores, and macropores with pore size ranging from 1 to 90 nm, as shown in Fig. p level , indicating the presence of oxygen in MnS@NSC [s spectrum can be resolved into three peaks centered at 397.8, 398.6, and 400.2 eV, presenting pyridinic-, pyrrolic-, and graphitic-type N atoms, respectively, which further proved that nitrogen is successfully doped into carbon [s spectrum revealed the presence of both C–O/C=O (531.6 eV) and C–OH/C–O–C (532.2 eV) bonds  layer formed on the surface of electrode materials [2S + Mn). The first five galvanostatic charge/discharge (GCD) profiles of MnS@NSC are displayed in Fig. S9. The capacity loss in the first cycle may be ascribed to the electrolyte decomposition, SEI formation, and irreversible phase conversion during the Na+ insertion process [−1 at 0.05 A g−1; the capacity can still retain 205.6 mAh g−1 at 10 A g−1, corresponding to a capacity retention of 44.3%, demonstrating its excellent rate performance. The rate performance of MnS@NSC-5 and MnS@NSC-16 was also tested as shown in Fig. S12, indicating the optimum electrochemical performance of MnS@NSC. The capacity of the composite samples was calculated based on the total weight of composite, including both MnS and NSC. On the other hand, the rate performance of NSC electrode is inferior (317.2 mAh g−1 at 0.05 A g−1 and 93.2 mAh g−1 at 10 A g−1). For the pure MnS, due to the large polarization, the capacity is decreased rapidly with the increased current density. When compared with the previously reported MnS-based sodium-ion anode materials  were first evaluated in SIB half-cells. The first three cyclic voltammetry (CV) scans of MnS@NSC composite within 0.01–3 V at scan rate of 0.2 mV saterials , 43. Thiaterials , 45. Theaterials , 36, 49. process , 22. Thi [x [x@C   and v are the current and the scan rate (mV s−1), and k1 and k2 are the fitting values. A b value close to 0.5 demonstrates that the charge storage is contributed by solid-state diffusion-controlled process, while the b value equal to 1 signifies a capacitive-controlled behavior. As shown in Fig. b values of cathodic peak and anodic peak of MnS@NSC electrode are calculated as 0.82 and 0.91 using Eq. + storage dominates most of the charge storage. To be more accurate, the overall contributions from capacitive processes versus scan rates can be calculated using Eq. −1, respectively. These values are higher than the pure MnS electrode and lower than the NSC electrode  and minimum (352.9) slope values, while the MnS@NSC has a moderate (769.7) slope, indicating that MnS@NSC electrode has higher sodium-ion diffusion coefficient than MnS electrode. The enhanced sodium-ion transport kinetics can be ascribed to downsizing of MnS and encapsulation of NSC, which can shorten the ion diffusion pathway and promote the facile access of Na+/e−. The GITT was also carried out to evaluate the sodiation/desodiation ion diffusion kinetics, as shown in Fig. + insertion into bulk MnS that induced size reduction and more active sites exposure. This sodium-ion diffusion coefficients of MnS@NSC and bulk MnS in the second discharge/charge cycles are also quantified as shown in Fig. S18. The sodium-ion diffusion coefficients of MnS@NSC in the second cycle are higher than those of the pure MnS.To further understand the kinetics of MnS@NSC composite, the electrochemical impedance spectroscopy (EIS) of MnS@NSC, MnS, and NSC was performed after three cycles; the Nyquist plots are illustrated in Fig. p spectra of bulk MnS at different charge/discharge states in the first and second cycles were collected by ex situ XPS, as shown in Fig. S19. During the first discharge process to 0.01 V, an obvious downshift of the binding energies can be clearly seen. The Mn 2p3/2 peaks located at 641.6 eV of the pristine MnS have downshifted to 640.7 eV in the first discharge to 0.01 V, revealing the reduction of manganese oxidation state from 2+. A new peak located at 642.6 eV was deconvoluted upon discharge to 0.75 V and was absent after further discharge to 0.01 V, demonstrating the formation of intermediate NaxMnS (where x < 1). Ex situ XRD and TEM were also conducted to reveal the electrochemical mechanism of MnS. As shown in Fig. S20a–c, the presence of Na2S and Mn can be clearly detected in the sample of MnS discharged to 0.01 V. The above result clearly shows that MnS undergoes combined intercalation and conversion process during discharge. When being recharged back to 3 V, the deconvoluted peaks in Mn spectra restored to higher binding energies, indicating the increased oxidation states of Mn that is possibly correlated with reformation of MnS after desodiation. After discharge to 0.01 V in the second cycle, the binding energy of Mn was again reduced, repeating the discharge process in the first cycle. This result is in good agreement with the CV curves shown in Fig. S15a; the first two cathodic peaks are related to the Na+ intercalation reaction, leading to the formation of intermediate NaxMnS phase with reduced oxidation states. The third reduction peak is assigned to conversion reaction, with notably reduced binding energies.In order to study the electrochemical mechanism, the Mn 23N4 and biomass wheat flour as nitrogen and carbon source , respectively. The annealing time and mass ratio of wheat flour to KOH were optimized . The NC material with a carbonation time of 2 h and a wheat flour/KOH ratio of 1:1 demonstrated the highest performance. As displayed in Fig. S25a, the NC material can demonstrate a highest capacity of 108.8 mAh g−1 at 0.1 A g−1 and maintains 62.3 mAh g−1 at 10 A g−1, implying its outstanding rate performance and fast ion adsorption/desorption from the large surface area . Furthermore, after 5000 cycles at current of 3 A g−1, the capacity of NC shows minor loss and coulombic efficiency retains nearly 100% , demonstrating the highly reversible adsorption/desorption process.In addition, N-doped 3D porous carbon (NC) cathode material was synthesized using C−1 in a Na half-cell to harness high efficiency. Considering the charge balance between anode and cathode, the mass ratio of the active materials in two electrodes is fixed to 1:3. A suitable voltage window of 0.5–4 V of the SIC is controlled to avoid the possible electrolyte decomposition and side reactions. The CV curves of the SIC ranging from 2 to 10 mV s−1 were measured and are displayed in Fig. vs. power) is presented in Fig. −1 at power density of 230 W kg−1. The energy density can still maintain 36.4 Wh kg−1 at a high power density up to 11,500 W kg−1, demonstrating its superior rate performance. In comparison, our SIC also outperforms other previously reported SIC systems, such as Fe1-xS//NCN (87.8 Wh kg−1) [2O5@C/RGO//AC (76 Wh kg−1) [2Ti2O5-x//RGO/AC (70 Wh kg−1) [−1) [2/G//AC (82 Wh kg−1) [2/G//CNT (86 Wh kg−1) [−1, with high coulombic efficiency of nearly 100%  , Nb2O5@CWh kg−1) , Na2Ti2O−1) [−1) , MoSe2/GWh kg−1) , and SnOWh kg−1) . Further−1 and 11,500 W kg−1, respectively), along with outstanding cycle life with 84.5% capacity maintaining after 3000 cycles. This study provides a new route to design high-performance metal sulfide-based electrode for application in high-power and energy density SICs.In summary, we have demonstrated a high-rate SIB anode material by encapsulating MnS nanocrystals into the N, S-co-doped carbon matrix. The N, S-co-doped carbon can improve not only the conductivity of the hybrid material, but also can buffer the volume changes and agglomeration of MnS nanocrystals. The downsized MnS can expose more active sites for charge storage, enhance the diffusion kinetics, and promote pseudocapacitive behavior. Thus, the well-designed nanostructured MnS@NSC electrode displays a large specific capacity, excellent rate capability, and long-term stability as anode for SIB. A prototype SIC with MnS@NSC as anode and NC as cathode could deliver high energy and power densities Below is the link to the electronic supplementary material.


Panax notoginseng saponins (PNS) is a Traditional Chinese Medicine that is commonly used to treat a range of angiogenic diseases. However, the mechanisms whereby PNS alters angiogenesis in endothelial progenitor cells (EPCs) have yet to be clarified.Distraction osteogenesis (DO) is an effective treatment in craniomaxillofacial surgery. However, the issue of sufficient blood supply at the regeneration tissue has limited its wide application. EPCs were identified by immunofluorescence, confirmed by their uptake of fluorescently labeled Dil-ac-LDL and FITC-UEA-1. EPCs were treated with different concentrations of PNS, and the effects of PNS on cell proliferation were measured on the optimal concentration of PNS determined. The effects of PNS on angiogenesis and migration, angiogenic cytokines mRNA expression and the proteins of the Wnt pathway were investigated. Then knocked down β-catenin in EPCs and treated with the optimum concentrational PNS, their angiogenic potential was evaluated in tube formation and migration assays. In addition, the expression of cytokines associated with angiogenesis and Wnt/β-catenin was then assessed via WB and RT-qPCR.We were able to determine the optimal concentration of PNS in the promotion of cell proliferation, tube formation, and migration to be 6.25 mg/L. PNS treatment increased the mRNA levels of VEGF, bFGF, VE-Cadherin, WNT3a, LRP5, β-catenin, and TCF4. After knocked down β-catenin expression, we found that PNS could sufficient to partially reverse the suppression of EPC angiogenesis.Overall, 6.25 mg/L PNS can promote EPC angiogenesis via Wnt/β-catenin signaling pathway activation. Reconstruction of bone deformities and defects caused by congenital malformation, injury, or tumorectomy represent significant clinical challenges in oral and maxillofacial surgery . In contPanax notoginseng saponins (PNS) is a TCM compound that is isolated from Panax notoginseng (Burk.) F. H. Chen [The application of Traditional Chinese medicine (TCM) to bone regeneration and angiogenesis has stimulated considerable interest in recent years.  H. Chen . Histori H. Chen . In addi H. Chen . Moreove H. Chen . There i H. Chen , with th H. Chen . HoweverRecently, endothelial progenitor cells (EPCs), isolated from the peripheral blood, cord blood, and the bone marrow , have be2 humidified incubator. Cells were cultured until 80–90% confluent, at which time they were passaged. Following two such passages, cells were utilized for downstream experiments.Two-week-old male Beagle dogs were purchased from the Experimental Animal Center of Guangxi Medical University . All animal experimental procedure was approved by the Animal Care and Use Committee of Guangxi Medical University. The isolation procedure was performed as described in previous studies . Mononuc2. Cells were fixed with 4% paraformaldehyde and incubated for 1 h with 10 μg/ml FITC-UEA-1  at room temperature. Nuclei were labeled with DAPI . The incorporation of Dil-Ac-LDL and binding of FITC-UEA-1 were assessed and photos were taken in an Intelligent Full-Automatic Fluorescence Microscopy Imaging System .EPCs were washed with phosphate-buffered saline (PBS) three times, and incubated in medium containing 20 μg/ml Dil-ac-LDL  for 4 h at 37 °C, 5% CO4/well) until 80% confluent, at which time they were fixed for 15 min using 4% paraformaldehyde (PFA), then permeabilized with 0.1% Triton X-100 for 20 min, and blocked for 30 min using 10% goat serum. These cells were then incubated overnight with mouse monoclonal anti-CD34 , goat polyclonal anti-VEGFR2 , rat monoclonal anti-CD133 , and/or mouse monoclonal anti-Beta-Catenin  at 4 °C. Samples were then washed, probed for 1 h with secondary antibodies at 37 °C, and Nuclei were labeled with DAPI . Samples were then combined with 100 μl of anti-fluorescence attenuating mount, after which they were evaluated with an Intelligent Full-Automatic Fluorescence Microscopy Imaging System .Immunofluorescent staining for CD34, CD133, VEGFR2, and β-catenin was conducted using EPCs after two passages. Briefly, these cells were plated in 12-well plates . To determine the optimal concentrations of PNS for the subsequent experiments, EBM containing different concentrations of PNS was used. The EPCs were seeded in 96-well plates at a density of 3× 10To knock down β-catenin, we purchased lentiviruses encoding short hairpin RNAs (shRNAs) specific for this gene or control shRNAs from GeneChem (China). The following three candidate shRNAs were used in these analyses: GCACCATGCAGAATACAAA (shRNA-β-catenin1), GGACCTACACTTACGAGAA (shRNA-β-catenin2), and GCTGCTTTATTCTCCCATT (shRNA-β-catenin3). Cells were transduced with these lentiviruses under appropriate conditions, after which β-catenin knockdown was confirmed via western blotting and RT-PCR, with the shRNA that achieved maximal knockdown being used for downstream studies.The impact of PNS on EPC angiogenesis was assessed by treating cells with a range of different conditions. Control (CTRL) cells were untreated and were not lentivirally transduced, while negative control (NC) EPCs were untreated and were transduced with a lentivirus encoding a control scrambled shRNA. In addition, cells in the shRNA-β-catenin group were transduced with an shRNA-β-catenin lentivirus, while cells in the CTRL + PNS group were treated with 6.25 mg/L PNS, those in the NC + PNS group were treated with 6.25 mg/L and were transduced with a control lentivirus, and those in the shRNA-β-catenin + PNS group were treated with 6.25 mg/L and were transduced with an shRNA-β-catenin lentivirus. To prevent any confounding results associated with the supplements in the EGM-2 media, cells were instead grown in basal EBM media containing 10% FBS  following passage. PNS treatment was conducted for 72 h in all appropriate groups.-△△Ct approach was used to assess relative gene expression, with GAPDH being used as a normalization control. The primer sequences are listed in Table Trizol  was used to extract cellular RNA, which was then used to generate cDNA with a RevertAid First Strand cDNA Synthesis Kit (Invitrogen) based on provided instructions. All primers were synthesized by Sangon Biological Engineering Technology & Services Co., Ltd. . A QuantStudio-5 system (Applied Biosystems) was used for RT-qPCR reactions with the following thermocycler settings: 50 °C for 2 min; 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min; 72 °C for 1 min. Each reaction well contained 10 μl of 2×PowerUp SYBR Green Master Mix (Invitrogen), 2 μl of each primer (800 nM),2 μl of cDNA (10 ng/μl), and 4 μl ddH2O. The 2EPCs were harvested in PBS and spun for 5 min at 12,000×g at 4 °C. Total protein extracts and nuclear protein extracts were then prepared from these samples using RIPA lysis buffer and a Nuclear and Cytoplasmic Protein Extraction kit , respectively. In both cases, 1 mM PMSF (Beyotime) was added to solutions, and an Enhanced BCA kit (Beyotime) was used to quantify protein levels in isolated samples.https://imagej.nih.gov/ij/) being used for densitometric analyses. All experiments were repeated a minimum of three times.Protein samples isolated as above were used for standard Western blotting experiments. Briefly, 12% SDS-PAGE was used to separate equal protein samples (30 μg each), which were then transferred to PVDF membranes . Blots were next blocked for 1 h using 5% non-fat milk in TBST, prior to incubation overnight with antibodies specific for Dishevelled (DVL) (Invitrogen), β-catenin (ThermoFisher), glycogen synthase kinase-3b (GSK-3β) , (phospho-S9)-GSK-3b (BIOSS), VEGF-A (ThermoFisher), bFGF , VE-Cadherin (AVIVA SYSTEMS BIOLOGY), histone H3 , and β-actin (Beyotime)  at 4 °C. Blots were then washed thrice using TBST (5 min/wash) prior to an additional 1 h room temperature incubation with biotin-conjugated secondary antibodies (1:1000 in TBST) (Beyotime). A BeyoECL Plus kit (Beyotime Bio) was then used to detect all protein samples based on provided directions, with ImageJ  and cells were returned to the incubator for 6 h. Cells were then imaged with an inverted microscope (100 ×) to assess tube formation. For each sample, at least five micrographs were taken at different positions. The ability of EPCs to form tube-like structures was assessed by counting the number of network circles in each image.EPC tube formation was assessed in Matrigel as previously described . One day3 cells/well suspended in EBM were seeded in the upper chamber of a modified Boyden chamber , and PNS was added to the lower chamber to induce EPC migration. After 72 h incubation at 37 °C and 5% CO2, the microporous membranes were washed with PBS, fixed in 4% paraformaldehyde for 15 min, and stained with 0.5% crystal violet solution. Non-migrating cells on the top side were removed with cotton-tipped swabs and the number of migrated cells was counted manually in five random microscopic fields (100 ×) in each insert.Cell migration was measured in culture inserts containing microporous membranes . Briefly, 5× 10p < 0.05 was the significance threshold. *p-values < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, or ##p < 0.01 were considered as statistically significant. All experiments were repeated at least three times to confirm reproducibility.SPSS 23.0 was used for all statistical analyses. Data were compared via Student’s t-tests and one-way ANOVAs, as appropriate. All data were presented as means ± standard deviation, and EPCs were isolated from canine bone marrow by density gradient centrifugation. As shown in Fig. p < 0.05). After culturing for 72 h, the migration rate of EPCs exposed to PNS was significantly greater than that of the control group . These data suggested that PNS promotes proliferation, migration, and angiogenesis of EPCs, functions that are essential for bone regeneration.The proliferation of EPCs was higher after treatment with different concentrations of PNS compared to control group at every time point. Notably, the results demonstrated optimal proliferation at the 6.25 mg/L dose . Meanwhile, the mRNA levels of LRP5, β-catenin and TCF4 were also elevated . These findings indicated that the canonical Wnt signaling pathway may participate in this process. To further elucidate the cellular localization of β-catenin, immunofluorescence staining analysis was performed, which confirmed that the presence of β-catenin in EPCs. PNS clear promoted the expression of β-catenin, and it can be seen that β-catenin was largely transferred into the cell nucleus in the PNS groups, a significantly different situation from that in the control group where it barely entered the cell nucleus  and WB  but decreased in the β-catenin inhibitor and β-catenin-inhibitor+PNS groups . No significant difference in angiogenesis was found between the CTRL and NC groups nor between the CTRL + PNS and NC + PNS groups . The results also clearly demonstrated that the angiogenetic capability was partially rescued in EPCs treated with PNS after inhibition of β-catenin . For the migration assay, the results were very similar to the tube formation experiment. The migration of EPCs transfected with the β-catenin inhibitor was significantly decreased compared with the CTRL and NC groups, while treatment with PNS could reverse the effect of the β-catenin inhibitor on migration ability (Fig. In comparison with the CTRL and NC groups, angiogenesis increased significantly in the PNS + CTRL and PNS + NC groups (Fig. ity Fig. d-e. ThesBone defects are a great challenge to oral and maxillofacial surgery. DO is a valuable and promising technique that has been widely used for bone regeneration in deformities and defects caused by tumor resection, trauma, congenital abnormality, and infection, amongst others . HoweverOver the last decade, TCM and cell-based therapeutics have gained attention for the treatment of DO in both fundamental and clinical studies. PNS is a TCM treatment that has been employed for centuries in China to treat a variety of diseases and conditions. Many different PNS preparations including injections, powders, and capsules are regularly used , 29 and To the best of our knowledge, this study is the first to investigate the safety and efficiency of PNS by evaluating EPC proliferation, migration, and angiogenesis in vitro. Firstly, the CCK-8 assay, transwell assay, and tube formation assay were performed to confirm the pro-angiogenic effects of PNS on EPCs. Furthermore, qRT-PCR and WB indicated that PNS promotes the expression of VEGF, bFGF and VE-cadherin in EPCs at both the mRNA and the protein level. Based on these results, it is suggested that PNS not only promotes the proliferation, migration and tube formation of EPCs but also enhances the expression of angiogenic-related cytokines and proteins in vitro, making it a prerequisite for DO callus tissue repair and regeneration. Since angiogenesis plays a pivotal role in the process of new bone formation and bone repair during DO, the beneficial effects of PNS on osteogenesis and consolidation during DO shown in our previous study  may alsoThere are several limitations to the present study. For one, PNS is known to be an active mixture of compounds and contains multiple dammarane-type saponins, including ginsenoside-Rh1, ginsenoside Rg1, ginsenoside Re, notoginsenoside R1, ginsenoside Rb1, and ginsenoside Rb1  As such,In summary, our results reveal that 6.25 mg/L PNS promotes EPC angiogenesis via activation of the Wnt/β-catenin pathway (Fig.


Most studies have focused on the functions and influence of ncRNAs, but seldom on their maturation. DEAD box genes are a family of RNA-binding proteins that may influence the development of ncRNAs, which attracted our attention. By combining a small sample for high-throughput gene microarray screening with large samples of The Cancer Genome Atlas (TCGA) data and our cohort, we aimed to find some gastric cancer-related genes. We evaluated the clinical significance and prognostic value of candidate gene DDX18, which is overexpressed in gastric cancer tissues. To provide a theoretical basis for the development of new therapeutic targets for the treatment of gastric cancer, we investigated its effect on the malignant biological behavior of gastric cancer in vitro. A transwell assay was also performed to examine the migration and invasion of gastric cancer cells. Cell apoptosis was analyzed by using a fluorescein isothiocyanate–annexin V/propidium iodide double-staining assay. To identify the role of DDX18 in the tumorigenic ability of gastric cancer cells in vivo, we also established a subcutaneous gastric cancer xenograft model. Coimmunoprecipitation, small RNAseq, and western blotting were performed to explore the mechanism of action of DDX18 in gastric cancer. A patient-derived xenograft (PDX) model was used to confirm the effect of DDX18 in gastric cancer tissues.(i) The differential profiling of mRNA expression in five pairs of gastric cancer and adjacent normal tissues was studied by Arraystar Human mRNA Microarray. By combining this with TCGA data and our cohort, we finally filtered out DDX18, which was upregulated in gastric cancer tissues, for further investigation. (ii) The protein expression of DDX18 was detected by immunohistochemistry staining. Then the relationship between the DDX18 expression level and the clinicopathological data and prognosis was analyzed. (iii) A CCK-8 assay and colony formation assay were used to evaluate the effect of DDX18 on cell growth and proliferation in vivo and in vitro. (iii) DDX18 could promote the maturation of microRNA-21 through direct interaction with Drosha, decreasing PTEN, which could upregulate the AKT signaling pathway. (iv) The PDX model showed that DDX18 could promote the proliferation of gastric cancer tissues by means of the PTEN–AKT signaling pathway.(i) DDX18 was upregulated in gastric cancer tumor tissues from a TCGA database and our cohort. The expression of DDX18 was also closely related to tumor volume, Borrmann classification, degree of tumor differentiation, cancer embolus, lymph node metastasis, and TNM stage. (ii) DDX18 could promote cell proliferation, migration, and invasion and inhibit cell apoptosis (i) DDX18 can be treated as a molecular marker to assess the prognosis of patients with gastric cancer. (ii) DDX18 could be a potential therapeutic target in gastric cancer. As the third most deadly cancer worldwide, gastric cancer is a serious threat to human health . While eAn increasing number of studies have shown that noncoding RNAs (ncRNAs) such as microRNAs and long noncoding RNAs (lncRNAs) play an important role in the occurrence and development of gastric cancer. However, these studies have mainly focused on the regulation and influence of microRNAs and lncRNAs on downstream genes. How ncRNAs are regulated is relatively unknown.The DDX (DEAD-box gene) family is a family of RNA helicases that was first proposed by Patrick Linder in 1989 , 3. The The expression levels of mature microRNAs are mainly determined by a critical rate-limiting step: the processing of premature microRNAs (pri-microRNAs and pre-microRNAs) . The DroIn this study, we first used gene chip technology to explore the differentially expressed genes in gastric cancer. Further analysis of the clinical and pathological data identified the high expression of DDX18 in gastric cancer tissues. We performed cell function experiments to study the role of DDX18 in gastric cancer. We also explored its molecular mechanism by means of molecular cytology technology and animal experiments to elucidate DDX18 and to determine its role in the occurrence and development of gastric cancer.Thirty-seven pairs of fresh gastric cancer and control normal gastric tissue specimens were obtained during surgery carried out on 37 patients from January 2013 to August 2014. All 37 patients underwent resection of primary gastric cancer at Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. Resected cancer tissues and paired noncancerous tissues were immediately cut and frozen in liquid nitrogen, and kept at –80°C until RNA and DNA extraction for quantitative real-time polymerase chain reaction (PCR).For the assessment of immunoreactivity and the prognostic value of DDX18 in gastric cancers, the inclusion criteria for patients with gastric cancer were as follows: (i) a distinct pathological diagnosis of gastric adenocarcinoma; (ii) no radiotherapy, chemotherapy, or other anticancer therapies prior to surgery; (iii) primary tumor resection, including radical gastrectomy and palliative gastrectomy; and (iv) availability of complete clinicopathological and follow-up data.A total of 585 paraffin-embedded tissue samples that met the above criteria were collected from patients with gastric cancer at the Department of Gastrointestinal Surgery, Renji Hospital, from January 2006 to December 2011 for tissue microarray construction and immunohistochemistry (IHC) staining. The overall survival (OS) is calculated from the date of tumor resection until death.2 at 37°C.Cells from the human gastric cancer cell lines SGC-7901, NCI-N87, HGC27, MGC-803, BGC-823, and AGS were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. All the cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium . The medium contained 10% fetal bovine serum and 1% penicillin/streptomycin. All cells were incubated in a cell incubator under 5% COGenomeditech  assisted in the design and production of DDX18 short hairpin RNA (shRNA).Six-well plates were prepared and inoculated with the appropriate gastric cancer cells, with the adherent cells occupying about 50% of the total area of the plates. The appropriate amount of lentivirus was added to each well according to the multiplicity of infection value of gastric cancer cells. The cells were screened with antibiotics recommended by Genomeditech, and the transfection efficiency was measured by fluorescence quantitative PCR or western blot. The target sequences of DDX18 were: sh-1, sense: 5′–3′, GCAGCGGAACCTAAAGTTT; sh-2, sense: 5′–3′, GCATACCTATGGCTTGATA; sh-control, sense: 5′–3′, TTCTCCGAACGTGTCACGT.–ΔΔCt method. The primer sequences were: DDX18: 5′–3′, F-ATGTCACACCTGCCGATGAAA, R-CCCTGAAACTTTAGGTTCCGC; GAPDH: 5′–3′, F-GGAGCGAGATCCCTCCAAAAT, R-GGCTGTTGTCATACTTCTCATGG.Total RNA was extracted with Trizol and reverse-transcribed into complementary DNA (cDNA) by PrimeScript™ . Using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal reference, real-time PCR analysis was conducted using an Applied Biosystems 7500 Real-Time PCR System , and the relative expression level of the target genes was calculated by the 2Gastric cancer cells were lysed by radioimmunoprecipitation assay (RIPA) buffer  and protease inhibitors . Then, 10% sodium dodecyl sulfate glue was used for electrophoresis and electrorotation to transfer the protein to the nitrocellulose (NC) membrane. The NC membrane was blocked for 1 hour at room temperature in Tris-buffered saline containing 5% skimmed milk. After incubation with primary and secondary antibodies, electrochemiluminescence was used to obtain bands .We linked three labels  to the indicated proteins. The prepared cells transfected with FLAG-DDX18, HA-Drosha, and His-DGCR8 plasmids were collected for nuclear protein extraction followed by coimmunoprecipitation. A nuclear protein extraction kit  was used according to the manufacturer’s protocol. In brief, alternate vortexing and centrifugation combined with the extraction kit were used to separate the total proteins into nuclear and cytoplasmic proteins. At the same time as the cell nuclear proteins were prepared, protein A/G magnetic beads  were preincubated on a spinning wheel at 4°C for 30–60 minutes and washed three times with PBS. The antibody complex was then suspended in the nuclear protein solution. After the protein solution was fully combined with the magnetic bead–antibody complex, the extraction buffer was washed three times. Magnetic separation was performed by heating. The immunoprecipitate was collected and western blotting was performed.The proliferation capacity of gastric cancer cells was determined by cell count kit 8 . After transfection with DDX18 shRNA, gastric cancer cells were inoculated in 96-well plates, with about 2000 cells per well. Under dark conditions, each chamber was incubated for 1 hour with 10 µl CCK-8 reagent and the optical density was measured at a wavelength of 450 nm with a SpectraMax Plus 384 (Molecular Devices). Six-well plates were used to lay about 500 gastric cancer cells in each well, and then the cells were allowed to grow for about 2 weeks. After washing twice with phosphate-buffered saline (PBS), cells were fixed in 4% paraformaldehyde for 15 minutes, and 0.5% crystal violet staining was performed for a further 15 minutes. The number of gastric cancer cell clones in different groups was calculated.5 cells per well were cultured under serum deprivation in six-well plates. Adherent cells were detached with 0.25% trypsin without ethylenediaminetetraacetic acid in 1 × PBS. Cells were harvested in complete RPMI 1640 and centrifuged at 1000 r.p.m. for 5 minutes. Each batch of cells was washed with 1 × PBS and stained with 50 mg/ml propidium iodide (PI) and annexin V–fluorescein isothiocyanate according to the manufacturer’s instructions. The percentage of annexin V (+) and PI (–) cells was analyzed by flow cytometry.For the cell apoptosis assay, 20 × 105 cells. The culture medium containing 20% serum was used in the lower layer. After 24 hours, the chamber was washed twice with PBS, 4% paraformaldehyde was used to fix the cells for 15 minutes, crystal violet was used for staining for 15 minutes, and the number of cells passing through the chamber was counted under a microscope.A transwell chamber was prepared with 600 µl serum-free RPMI 1640 medium added to each well, followed by 5 × 10The DDX18 gene, PTEN gene 3′-untranslated region (UTR) target clone (CmiT010099-MT05), and negative control clone (CmiT000001-MT05) were purchased from GengCopia Company, USA. For the secretory double luciferase reporter gene analysis, 293T cells were inoculated in six-well plates and cotransfected when the cell confluence reached 50%. The culture medium was changed to whole medium 24 hours after transfection and incubation continued. The supernatant of the cell culture solution was carefully collected 48 hours after transfection and packed into two tubes, each containing 200 μL, which was used for the detection of Gaussia luciferase (GLuc) and secretory alkaline phosphatase (SeAP), respectively. The supernatant used to detect SeAP was placed in a water bath at 65°C for 15 minutes; the working solution of 1 × glucose was prepared and incubated in the dark at room temperature for 25 minutes; the working solution of 1 × SeAP was prepared and incubated in the dark at room temperature for 10 minutes; 10 µl of supernatant and 100 μl of the corresponding working solution were added to each well of a 96-well plate, and multiples of three were used in each group. After incubation at room temperature for 1–10 minutes, the fluorescence was determined using a microplate reader. For data processing, with the SeAP fluorescence reading as the internal reference, the ratio of the GLuc fluorescence reading to the SeAP fluorescence reading was compared among the groups, and the mean values of three reading plates and three complex holes were taken.Paraffin sections were dewaxed with xylene, rehydrated by fractional ethanol, and antigens were extracted. The sections were blocked with 10% bovine serum albumin (BSA), incubated with primary antibody for 1 hour, and then incubated with secondary antibody for 30 minutes at room temperature. DAPI  was used to stain the nucleus. An automatic fluorescence microscope (Nikon) was used for image observation and analysis. The tissue sections were assessed and graded by two independent investigators who were unaware of the clinicopathological factors. The staining intensity was 0 (negative), 1 (weak), 2 (medium), and 3 (strong).The degree of staining was stratified as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%), defined as the percentage of positive staining area in the total tumor invasion area. The final score of DDX18 expression was 0–7. The samples were divided into two groups: low DDX18 expression  and high DDX18 expression .After immersion in 100% alcohol, alcohol lamp burning, and ultraviolet irradiation, a glass slide was placed into a six-well plate, and gastric cancer cells were spread on the glass slide. The cells were allowed to grow to 30–50% of the area of the glass slide on the second day. Cells were fixed with 4% paraformaldehyde for 15 minutes and treated with 0.5% Triton X-100 for 1 minute. Nonspecific binding sites were blocked by 1% BSA. The primary antibody was incubated for 1 hour at room temperature and the secondary antibody for 30 minutes, and then treated with DAPI for 30 minutes for nuclear staining. Observation and analysis were conducted by automatic fluorescence microscope (Nikon).6 gastric cancer cells were injected into the left axilla of each nude mouse. After 4 weeks, the nude mice were killed, the subcutaneous tumor was removed, and its weight and volume were measured; 4% paraformaldehyde was used to store the tumor. All animal experiments were approved by the Ethics Committee of Renji Hospital.To construct the subcutaneous tumor model of nude mice, 5 × 10t-test and the chi-squared test were used in the study. The Cox proportional hazard model was used for univariate and multivariate analysis to understand the factors affecting survival. P < 0.05 was considered statistically significant.SPSS 22.0  was used to analyze and calculate all the data, and the value was the mean ± SD. Student’s P < 0.05 was used for standard screening. In all the genes upregulated, five genes with the highest fold change were obtained, which were TWIST2, MET, HOXA13, DDX18, and PRND . Similar results were verified in the TCGA database  and the DDX18 low-expression group , which were divided by DDX18 immunohistochemistry. The OS rates of the patients in the low-expression group were 90.6%, 76.7%, and 67.8%. The OS rates of the patients with high expression were 88.9%, 63.1%, and 54.7%. The difference between the two groups in the first stage of gastric cancer was not significant, but in stage II and stage III gastric cancer significant differences were observed (P < 0.05) , lymph node metastasis (N stage), and DDX18 expression level had significant effects on the OS rate of 585 patients with gastric cancer, and the difference was significant ( < 0.05) Table 1.450 values of each group were detected for five consecutive days. After 5 days, DDX18 knockdown significantly decreased the proliferation rate of tumor cells by 63.0% (P < 0.001) (P < 0.001) (We detected the expression of DDX18 in gastric cancer cell lines (P < 0.01) (We found that the interference group’s ability to form clones (82.3 ± 5.31) was significantly lower than that of the control group (289.4 ± 11.2) (P < 0.01) after stable knockdown of DDX18 gene expression (The transwell results showed that the number of penetrating cells in the control group was 83.6 ± 12.5 cells/high-power field (HPF), which was significantly higher than that of the interference group (26.6 ± 4.3 cells/HPF). The migration of the AGS cells was significantly weaker than that of the control group , suggesting that interference with the DDX18 gene in the AGS cells significantly reduced the antiapoptotic ability (P < 0.01), suggesting that the antiapoptotic ability of the SGC-7901 cells was significantly enhanced after overexpression of the DDX18 gene  and confirmed the direct effect of miR-21-3p and the PTEN 3′-UTR  and the interference group (0.56 ± 0.31 g). DDX18 interference strongly inhibited the AGS cell subcutaneous tumorigenic ability in vivo, which was consistent with the results of the in vitro experiments  models. PDX models with low DDX18 expression were implanted in the mice. From the third day, group 1 was injected with PBS every day, while the corresponding group 2 was injected with the PTEN inhibitor SF1670. After 21 days, we found that the injection of SH1670 could effectively promote tumor growth  family have been defined as cofactors to the Drosha complex, which controls the post-transcriptional maturation of a specific group of microRNAs. Drosha is an RNase III enzyme, which binds and cleaves double-stranded RNA without any sequence specificity . It is kin vivo and in vitro experiments to detect the function of DDX18 in gastric cancer. Our findings showed that DDX18 promoted tumor growth. Knockdown of DDX18 also substantially decreased gastric cancer cell migration and invasion. Further studies showed that DDX18 could regulate the maturation of microRNA-21 and then affect the function of the downstream PTEN/AKT signaling pathway. Both microRNA-21 and the PTEN/AKT signaling pathway have already been proved to play an important role in gastric cancer .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.