The chemical constituents and hypoglycemic activity of alcoholic extract of sea buckthorn leaves / 药学学报

The purpose of this research is to identify the chemical constituents of sea buckthorn leaves extract (SBLE) and explore its hypoglycemic biological activity. SBLE was prepared by hot reflux extraction with 65% ethanol, and its chemical composition was analyzed by ultra-high-performance liquid chromatography-photodiode array-mass spectrometry/mass spectrometry (UHPLC-PDA-MS/MS) system. The animal experiments were compliant with ethical principles for animal use and had been approved by the Animal Experiment Ethics Committee of Jinan University. Mice were injected with streptozocin (STZ) to establish a hyperglycemic animal model, and SBLE (1.5 g·kg-1) was administered by gavage for 5 weeks. The fasting blood glucose (FBG) and oral glucose tolerance were detected. Normal mice were given SBLE (1.5 g·kg-1) by intragastric administration for 10 days, and blood was collected from the tail vein to detect the changes in blood glucose within 120 min after sucrose or starch loading. The mucous membrane of the small intestine of mice was taken to detect the activity of α-glucosidase (AG), and the activity of yeast-derived AG incubated with SBLE was evaluated. The glucose uptake by Caco-2 cells treated with SBLE was detected by fluorescence microscopy and cytometry, and the gene expression of sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) in Caco-2 cells were detected by real-time quantitative PCR (qPCR). A total of 18 compounds were identified, mainly including tannins and flavonoids. SBLE reduced FBG and increased oral glucose tolerance in STZ hyperglycemic mice. SBLE effectively inhibited the increase of blood glucose caused by starch intake in normal mice. SBLE exerted good inhibitory activity on yeast-derived AG (IC50 = 16.94 μg·mL-1) and small intestinal mucosa AG with an inhibition rate of 15.48%. SBLE (25-100 μg·mL-1) dose-dependently inhibited glucose uptake by Caco-2 cells, and SBLE significantly reduced the mRNA level of SGLT1 without changing the expression of GLUT2. In conclusion, the UHPLC characteristic fingerprint of SBLE is established with 18 chemical components identified by mass spectrometry, and SBLE exerts hypoglycemic effect by inhibiting the activity of AG and the absorption of glucose by intestinal epithelial cells.

Effect of platelet reactivity on clinical events in patients using bivalirudin in selective percutaneous coronary intervention / 中华心血管病杂志

Objective: To investigate the effect of platelet reactivity and other clinical factors on the postoperative 1-year adverse clinical events in patients who underwent selective percutaneous coronary intervention (PCI) anticoagulated with bivalirudin. Methods: This is a multicenter, retrospective and observational study, enrolling 632 patients at high risk of bleeding adjudicated by operators who underwent selective PCI anticoagulated with bivalirudin and had preoperative thrombelastography (TEG) test results in Fuwai Hospital, Northern Theater General Hospital and Xinxiang Central Hospital between January 2017 and August 2018. Platelet reactivity was tested by TEG and adenosine-induced maximal amplitude (MAADP) was recorded. According to MAADP patients were divided into three groups: low on-treatment platelet reactivity (LTPR) group (MAADP<31 mm, n=229), normal on-treatment platelet reactivity (NTPR) group (31 mm≤MAADP≤47 mm, n=207) and high on-treatment platelet reactivity (HTPR) group (MAADP>47 mm, n=196). The endpoints consisted of major adverse cardiovascular and cerebrovascular events (MACCE) and bleeding events. The definition of MACCE was the composite of all-cause mortality, myocardial infarction, intrastent thrombosis, stroke and revascularization. Bleeding events were defined by bleeding academic research consortium (BARC) type 2, 3 and 5 bleeding. Using multivariate Cox regression to analyze the factors of MACCE and bleeding events in patients underwent selective PCI anticoagulated with bivalirudin. Results: A total of 632 patients were finally enrolled in the study with age of (68.3±10.0) years and there were 423 (66.9%) males. All of 632 patients finished one-year follow-up, and 48 (7.6%) patients occurred MACCE and 11 (1.7%) patients occurred bleeding events. There was not statistically significant difference in the incidence of MACCE (8.3% (19/229) vs. 6.3% (13/207) vs.8.2% (16/196), P=0.68) and bleeding events (1.8% (4/229) vs. 2.9% (6/207) vs. 0.5% (1/196), P=0.17) in LTPR, NTPR and HTPR group. Multivariate Cox regression showed that HTPR was not the independent factor of MACCE (HR=1.25, 95%CI 0.67-2.30, P=0.49), and the history of peripheral vessel disease was the independent risk factor of MACCE (HR=2.47, 95%CI 1.19-5.11, P=0.02). LTPR was not the independent factor of bleeding events (HR=1.35, 95%CI 0.39-4.66, P=0.64), and the independent factors of bleeding events were history of peripheral vessel disease (HR=3.95, 95%CI 1.03-15.22, P=0.05) and hemoglobin (HR=0.96, 95%CI 0.93-0.99, P=0.01). Conclusions: In patients undergoing selective PCI anticoagulated with bivalirudin, there is no significant association between platelet reactivity and postoperative 1-year MACCE or bleeding events. History of peripheral vessel disease is an independent risk factor of MACCE, and history of peripheral vessel disease and decreased hemoglobin are independent risk factors of bleeding events.

Semaphorin 3A-hypoxia inducible factor 1 subunit alpha co-overexpression enhances the osteogenic differentiation of induced pluripotent stem cells-derived mesenchymal stem cells in vitro / 中华医学杂志(英文版)

BACKGROUND@#Mesenchymal stem or stromal cells (MSCs) derived from the induced pluripotent stem cells (iPSCs) have uniform biological activity, which makes the clinical application of MSCs in bone repair possible. Culturing the iPSC-MSCs onto osteoconductive materials is a promising tissue engineering-based strategy in bone regeneration. The aim of this work was to evaluate the effects of semaphorin 3A (Sema3A) and hypoxia inducible factor 1 subunit alpha (HIF1α) co-overexpression on the survival and osteogenic differentiation of iPSC-MSCs.@*METHODS@#Sema3A and HIF1α were linked together with the three (GGGGS; G, glycine; S, serine) peptide fragment, and their co-expression in iPSC-MSCs was mediated by a lentiviral vector. The fusion protein retained the immune reactivity for both Sema3A and HIF1α as determined with Western blotting. iPSC-MSCs were infected with overexpression lentivirus (oeLenti) as negative control, oeLenti-Sema3A, oeLenti-HIF1α or oeLenti-Sema3A-HIF1α lentiviruses.@*RESULTS@#Sema3A overexpression alone promoted the osteogenic differentiation of iPSC-MSCs (the activity and/or expression of osteoblast markers, such as alkaline phosphatase, osteopontin, and osteocalcin, were upregulated), and suppressed cell survival. The Sema3A-HIF1α fusion protein showed a comparable osteoconductive effect to that of Sema3A without reducing cell survival. We further seeded iPSC-MSCs modified by SemaA-HIF1α overexpression onto hydroxyapatite (HA) scaffolds, and evaluated their growth and differentiation on this three-dimensional material. Additional data indicated that, as compared to iPSC-MSCs cultured in ordinary two-dimensional dishes, cells cultured in HA scaffolds grew (blank vs. HA scaffolds: 0.83 vs. 1.39 for survival) and differentiated better (blank vs. HA scaffolds: 11.29 vs. 16.62 for alkaline phosphatase activity).@*CONCLUSION@#Modifying iPSC-MSCs with pro-osteogenic (Sema3A) and pro-survival (HIF1α) factors may represent a promising strategy to optimize tissue engineering-based strategy in bone repair.

Construction and preliminary applications of a Saccharomyces cerevisiae detection plasmid using for screening promoter elements / 药学学报

Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of DsRed-Monomer gene. The successful construction of pGBT9Red lays the foundation for further analysis of promoter activity and screening of promoter element libraries.

Detection of serum PICP and PINP and COL1alpha1 DNA sequence analysis in patients with arsenic poisoning in Dazhuyuan Town, Ankang area in southern Shaanxi Province / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate type I collagen metabolism in patients with arsenic poisoning in Dazhuyuan town, Ankang area in southern Shaanxi province known for high-level exposure to arsenic (As), fluoride (F) and selenium (Se).</p><p><b>METHODS</b>This case-control study involved 29 patients with arsenic poisoning from Dazhuyuan, a town in Ankang area of Shaanxi province affected by coal-borne endemic arsenicosis. Thirty individuals without arsenic poisoning from Chang'an county of Xi'an city were included as the control group. The serum concentrations of As and Se were determined using a graphite furnace atomic absorption spectrophotometer, and the levels of F detected with a fluoride ion-selective electrode. Enzyme-linked immunosorbent assay (ELISA) was used for assessing serum contents of PICP and PINP, and the sequence of COL1alpha1 gene was analyzed with ABI sequence analyzer.</p><p><b>RESULTS</b>The contents of PICP and PINP were similar between the two groups, which had also identical COL1alpha1 DNA sequences. After controlling for all the other factors, an inverse correlation was noted between PICP and serum F contents (r=-0.4, P<0.05) and between PINP and hair F content (r=-0.5, P<0.05), but a positive correlation was found between PINP and hair Se content (r=0.5, P<0.05).</p><p><b>CONCLUSION</b>Hair F and Se levels are the major factors affecting PINP synthesis, and increased hair F level is associated with elevated PINP content, whereas hair Se level is inversely correlated to PINP content. But in the patients with arsenic poisoning from Dazhuyuan town, the PICP, PINP contents and COL1alpha1 DNA sequence show no significant alterations, suggesting that hair Se increment abolishes the effect of hair F increment in inducing the decrease in PINP content.</p>

Serum hyaluronic acid, tumor necrosis factor -alpha, vascular endothelial growth factor, NO, and Se levels in adult patients with Kashin-Beck disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the association of serum levels of hyaluronic acid (HA), tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF), NO, and Se with the clinical manifestations in adult patients with Kashin-Beck disease (KBD).</p><p><b>METHODS</b>Total 216 adults were selected for KBD screening from the KBD-prevalent areas in Yongshou county and the non-KBD areas of Chang'an county, Xi'an city, ShaanXi Province. According to the National Diagnostic Criteria of Kashin-Beck Disease in China, the diagnoses of KBD was established in 25 adult patients (11 men and 14 women, average age of 47.88+/-11.16 years), and 20 healthy control subjects from the KBD areas (8 men and 12 women, average age of 47.85+/-12.05 years) and 20 from the non-KBD areas (8 men and 12 women, average age of 47.45+/-11.24 years) were also selected to serve as controls. There was no significant difference in the average age and gender distribution between the 3 groups. The serum levels of HA, TNF-alpha, VEGF, NO and Se were measured by enzyme-linked immunosorbent assay, nitrate reductase method and griphite furnace atomic absorption spectrometry.</p><p><b>RESULTS</b>Serum NO level was significantly higher in KBD group (41.7+/-21.89 micromol/L) than in the health controls from KBD areas (17.1+/-13.01 micromol/L) and non-KBD areas (17.58+/-11.48 micromol/l, F=13.11, df=2, P<0.001). Serum TNF-alpha level in KBD group (32.7+/-3.55 pg/ml) was significantly higher than that in the control subjects from the non-KBD areas (30.95+/-2.22 pg/ml, F=3.672, df=2, P=0.031), but similar with the control subjects from the KBD areas (32.7+/-3.55 pg/ml). Serum TNF-alpha and NO levels were identified as the indices that differed between adult KBD patients and the controls from both KBD and non-KBD areas by differential analysis (the function of differentiation was 0.062xNO+0.173xTNF -7.218).</p><p><b>CONCLUSION</b>Serum TNF-alpha and NO levels are significantly increased in adult KBD patients and are associated with the clinical manifestations of KBD.</p>