Berberine enhances antidiabetic effects and attenuates untoward effects of canagliflozin in streptozotocin-induced diabetic mice.

The present study aimed at determining whether berberine can enhance the antidiabetic effects and alleviate the adverse effects of canagliflozin in diabetes mellitus. Streptozotocin-induced diabetic mice were introduced, and the combined effects of berberine and canagliflozin on glucose metabolism and kidney functions were investigated. Our results showed that berberine combined with canagliflozin (BC) increased reduction of fasting and postprandial blood glucose, diet, and water intake compared with berberine or canagliflozin alone. Interestingly, BC showed greater decrease in blood urea nitrogen and creatinine levels and lower total urine glucose excretion than canagliflozin alone. In addition, BC showed increased phosphorylated 5' AMP-activated protein kinase (pAMPK) expression and decreased tumor necrosis factor alpha (TNFα) levels in kidneys, compared with berberine or canagliflozin alone. These results indicated that BC was a stronger antidiabetic than berberine or canagliflozin alone with less negative side effects on the kidneys in the diabetic mice. The antidiabetic effect was likely to be mediated by synergically promoting the expression of pAMPK and reducing the expression of TNFα in kidneys. The present study represented the first report that canagliflozin combined with berberine was a promising treatment for diabetes mellitus. The exact underlying mechanisms of action should be investigated in future studies.

Crosslinked Aspartic Acids as Helix-Nucleating Templates.

Described is a facile helix-nucleating template based on a tethered aspartic acid at the N-terminus [terminal aspartic acid (TD)]. The nucleating effect of the template is subtly influenced by the substituent at the end of the side-chain-end tether as indicated by circular dichroism, nuclear magnetic resonance, and molecular dynamics simulations. Unlike most nucleating strategies, the N-terminal amine is preserved, thus enabling further modification. Peptidomimetic estrogen receptor modulators (PERMs) constructed using this strategy show improved therapeutic properties. The current strategy can be regarded as a good complement to existing helix-stabilizing methods.

Suppressive effects of quercetin-3-O-(6″-Feruloyl)-ß-D-galactopyranoside on adipogenesis in 3T3-L1 preadipocytes through down-regulation of PPARγ and C/EBPα expression.

Obesity is a chronic, costly disease, and flavonoids such as quercetin have been proven to play protective roles against it. This study investigated the suppressive effect of quercetin-3-O-(6″-feruloyl)-ß-D-galactopyranoside (QFG) on adipogenesis of 3T3-L1 preadipocytes. Quercetin-3-O-(6″-feruloyl)-ß-D-galactopyranoside and quercetin were both extracted from Psidium guajava (Myrtaceae, commonly known as guava) leaves and were evaluated for their suppressive effect on adipogenesis by means of oil red O staining and triglyceride assay. It was shown that QFG inhibited adipogenesis in a dose- and time-dependent manner, and it exerted a stronger effect than did quercetin at the same concentration. Quantitative real-time polymerase chain reaction and western blotting were conducted to further examine the differentiation expression of marker genes and transcriptional factors. Both mRNA and protein expression of the key adipogenic transcriptional factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT (cytidine-cytidine-adenosine-adenosine-thymidine)/enhancer-binding protein alpha (C/EBPα), were inhibited by QFG. Moreover, the mRNA expression patterns of key participants in the Wnt-ß-catenin pathway were not altered during the QFG-induced adipogenesis inhibition. These results suggest that QFG effectively suppresses adipogenesis and that it exerts its role mainly through the significant down-regulation of PPARγ and C/EBPα and, probably, via a Wnt-ß-catenin independent pathway.

MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling.

Osteogenic differentiation of mesenchymal stem cells (MSCs) is a complex process, which is regulated by various factors including microRNAs. Our preliminary data showed that the expression of endogenous miR-20a was increased during the course of osteogenic differentiation. Simultaneously, the expression of osteoblast markers and regulators BMP2, BMP4, Runx2, Osx, OCN and OPN was also elevated whereas adipocyte markers PPARγ and osteoblast antagonist, Bambi and Crim1, were downregulated, thereby suggesting that miR-20a plays an important role in regulating osteoblast differentiation. To validate this hypothesis, we tested its effects on osteogenic differentiation by introducing miR-20a mimics and lentiviral-miR20a-expression vectors into hMSCs. We showed that miR-20a promoted osteogenic differentiation by the upregulation of BMP/Runx2 signaling. We performed bioinformatics analysis and predicted that PPARγ, Bambi and Crim1 would be potential targets of miR-20a. PPARγ is a negative regulator of BMP/Runx2 signaling whereas Bambi or Crim1 are antagonists of the BMP pathway. Furthermore, we confirmed that all these molecules were indeed the targets of miR-20a by luciferase reporter, quantitative RT-PCR and western blot assays. Similarly to miR-20a overexpression, the osteogenesis was enhanced by the silence of PPARγ, Bambi or Crim1 by specific siRNAs. Taken together, for the first time, we demonstrated that miR-20a promoted the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARγ, Bambi and Crim1, the negative regulators of BMP signaling.

Aqueous extracts of Fructus Ligustri Lucidi enhance the sensitivity of human colorectal carcinoma DLD-1 cells to doxorubicin-induced apoptosis via Tbx3 suppression.

Chemoresistance has imposed a great challenge for cancer therapy. Fructus Ligustri Lucidi (FLL) is one of the commonest Chinese herbs that has been used for thousand years. This study shows that the aqueous extract of FLL (AFLL) enhanced the sensitivity of DLD-1 colon cancer cells to doxorubicin-induced apoptosis. Furthermore, Tbx3 expression was found to be suppressed by AFLL when the expression of tumor suppressor genes p14 and p53 were activated. Therefore, reduction of Tbx3 rescued the dysregulated P14(ARF)-P53 signaling, which in turn contributed to the sensitivity of DLD-1 cells to doxorubicin-induced apoptosis. As a conclusion, the findings suggest that FLL has a potential of being an appealing agent for auxiliary chemotherapy in treatment of human colorectal carcinoma.

Enhanced peroxisomal ß-oxidation metabolism in visceral adipose tissues of high-fat diet-fed obesity-resistant C57BL/6 mice.

This study aimed to investigate the potential mechanisms of natural resistance to high-fat diet-induced obesity. Four-week-old C57BL/6 mice were fed a high-fat diet for 6 weeks and were then designated as high-fat diet-fed obesity-prone (HOP) and obesity-resistant (HOR) animals. Their blood biochemistry was evaluated, and visceral adipose tissue samples were subjected to proteomic, Western blot and quantitative real-time PCR (q-PCR) analyses. The HOR mice showed reduced visceral fat weight and size, as well as lowered serum lipid and leptin levels. Proteomic analysis showed that enoyl coenzyme A hydratase 1, peroxisomal (Ech1) expression was significantly increased in their visceral adipose tissues. Moreover, other proteins, such as α-tropomyosin, myosin light chain, urine-nucleoside phosphorylase and transgelin, were also significantly increased. Furthermore, q-PCR analysis showed that the expression of acyl-CoA oxidase 1 palmitoyl, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase and 3-oxoacyl-CoA thiolase responsible for peroxisomal ß-oxidation was also up-regulated in the visceral adipose tissues of the HOR mice. The expression of peroxisome proliferator-activated receptor α (PPARα) was increased in the HOR mice as shown by Western blot analysis. Obesity-resistant animals show enhanced peroxisomal ß-oxidation metabolism and reduced fat accumulation in visceral adipose tissues by up-regulating the expression of Ech1, peroxisomal or other related peroxisomal ß-oxidation marker genes, which may be driven or enhanced by the up-regulation of the expression of PPARα. However, further validation in future studies is required.

[A novel spectral surface plasmon resonance 2-D sensing technique and its applications in DNA microarrays].

The authors have previously proposed a novel refractive index two-dimensional sensing technique named "parallel scan spectral surface plasmon resonance imaging". In the technique, with a line-shaped light illumination, an image acquired with CCD detector could provide both SPR wavelength information and one-dimensional spatial distribution, and then provide one-dimensional distribution of refractive index with further calculation. Thus, two-dimensional distribution of refractive index of the entire sensing area can be obtained with one-dimensional optical line parallel scan. The technique offers advantages of both high sensitivity and high throughput, and could have potential applications in microarray analysis. In the present paper, the authors improve the data processing methods of the technique. The authors use the refractive index of air as a reference to get over the problem of precision of the incident angle. The authors also sense a manually dotted Legionella pneumophila mip DNA probe array with this technique and prove the feasibility of sensing microarrays by this highly sensitive and label-free technique. The relation between the equivalent refractive indices and the concentrations of the dotted Legionella pneumophila mip DNA probes is obtained, which has important reference value for further study.

Total flavonoids of Herba Epimedii improves osteogenesis and inhibits osteoclastogenesis of human mesenchymal stem cells.

INTRODUCTION: In China Herba Epimedii is one of the most common herbs that could be prescribed for treating osteoporosis. It is known to increase the overall mineral content, therefore, to promote bone formation and to increase lumbar bone mineral density (BMD). The present study was aimed at investigating the effect of flavonoids of Herba Epimedii (HEF) on osteogenesis in human MSCs. METHODS: The human bone marrow-derived MSCs (BM-MSCs) were isolated and their osteogenic differentiation was evaluated by their alkaline phosphatase (ALP) activities and level of mineralization. After treating with total flavonoids during osteogenic differentiation process, differential mRNA expression was examined by RT-PCR. RESULTS: The total time needed for osteogenic differentiation of BM-MSCs was significantly shortened by adding HEF. Up-regulation of mRNA expression by HEF was observed for several marker genes and osteogenic regulators. HEF was also found to inhibit osteoclastogenesis of MSCs by enhancing the ratio OPG/RANKL. CONCLUSIONS: Our study demonstrated that the HEF could improve osteogenic differentiation and inhibit the osteoclast differentiation of BM-MSCs concurrently.

[Study on morphology of surface atoms conformation of nanogold-based genechip].

Conformations of surface atoms in various stages of nanogold-based genechip testing were scanned by the atomic force microscope based on the scanning tunneling microscope. The findings were: First, the surface atoms of genechip slide (formylphenyl glass) were in a regular porous-arrangement; Second, after combination with probe, the regular porous arrangement changed to be irregular; Third, after hybridization with the target nucleic acid, the surface atoms were once again in a cable-like arrangement which was relatively structured and intensively cross-parallel. However, after the silver staining, the surface atoms showed a larger block structure with serious unevenness. From these results we can intuitively know the process and differences in probe combination, nucleic acid hybridization, and silver staining. Moreover, the relevant experiment was verified at the micro-level.

[A study on surface plasmon resonance-based gene chip].

The surface plasmon resonance (SPR)-based gene chip was prepared according to the following processes: First, a film of nanogold, which was synthesized by using Frens' method, was plated on chip by Chlorauric acid/hydroxylamine method. Then probes were fixed on nanogold film by Self-assembled monolayer (SAM) technology. Subsequently, the fixing time and concentration of probes, the sensitivity and the specificity of the chip were optimized. Our results suggested that the chip plated with 2.5 nm nanogold film has a better SPR reflection, and when fixed by probes for 4.5 h at the concentration of 1 500 nmol/L, the gene chip also shows a fine performance of detection and can identify accurately the mismatch between bases in SPR detection system. The gene chip constructed in the research can be used for SPR sensor detection.

[Interleukin-8 inhibits clonal expansion of 3T3-L1 preadipocyte during differentiation].

AIM: To investigate the effect of interleukin-8 (IL-8) on the differentiation and clonal expansion of 3T3-L1 preadipocyte during the differentiation period. METHODS: The morphological changes of 3T3-L1 cells during differentiation after the treatment of IL-8 was observed by Oil-Red O staining. Glycerol-3-phosphate dehydrogenase (GPDH) activity was measured by a spectrophotometric method. MTT method and 3H-TdR incorporation were applied to examine the changes of cell proliferation and DNA synthesis in clonal expansion of 3T3-L1 cells. Cell cycle analysis was taken by flow cytometry. RESULTS: IL-8 could inhibit the differentiation and GDPH activity in a dose dependent manner. IL-8 decreased the cell proliferation and DNA synthesis in clonal expansion after induction. Also, the proportion of cells in G1 phase was increased and that of cells in S and G2 phase was declined after the treatment of IL-8. CONCLUSION: IL-8 inhibits the differentiation of 3T3-L1 preadipocytes by decreasing the clonal expansion of the cells.

Macrostemonoside A promotes visfatin expression in 3T3-L1 cells.

The newly identified fat cell-secreted factor, visfatin, is insulin-mimetic and play a positive role in attenuating insulin resistance and diabetes. Natural steroidal saponins including tigogenin saponins have been reported to provide anti-diabetic activity and modulate glucose metabolism, but the mechanism remains unknown. In this study, we examined the effect of macrostemonoside A, a tigogenin steroidal saponin isolated from the bulbs of Allium macrostemon Bung on the expression of visfatin in differentiated 3T3-L1 adipocytes. It was found that macrostemonoside A markedly enhanced the synthesis and secretion of visfatin protein in 3T3-L1 adipocytes, and increased visfatin mRNA in a dose and time dependent manner as well. Moreover, visfatin promoter-driven luciferase expression in cells was elevated by macrostemonoside A, which was blocked by SB-203580, a specific inhibitor of p38 MAPK pathway. Lastly, we found that macrostemonoside A did not affect the expression of PPARgamma and its DNA-binding ability to visfatin promoter. These results indicate that macrostemonoside A could potently stimulate visfatin expression in 3T3-L1 adipocytes, which occurs at the transcriptional level and is mediated at least partially via p38 MAPK signaling pathway. Its regulation on visfatin in adipocytes may constitute an important element in its improvement of insulin resistance and diabetes.

Effect of WeiJia on carbon tetrachloride induced chronic liver injury.

AIM: To study the effect of WeiJia on chronic liver injury using carbon tetrachloride (CCl(4)) induced liver injury animal model. METHODS: Wistar rats weighing 180-220g were randomly divided into three groups: normal control group (Group A), CCl(4) induced liver injury control group (Group B) and CCl(4) induction with WeiJia treatment group (Group C). Each group consisted of 14 rats. Liver damage and fibrosis was induced by subcutaneous injection with 40% CCl(4) in olive oil at 3 mL/kg body weight twice a week for eight weeks for Groups B and C rats whereas olive oil was used for Group A rats. Starting from the third week, Group C rats also received daily intraperitoneal injection of WeiJia at a dose of 1.25 microg/kg body weight. Animals were sacrificed at the fifth week (4 male, 3 female), and eighth week (4 male, 3 female) respectively. Degree of fibrosis were measured and serological markers for liver fibrosis and function including hyaluronic acid (HA), type IV collagen (CIV), gamma-glutamyl transferase (gamma-GT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Alpha smooth muscle actin (alpha-SMA) and proliferating cell nuclear antigen (PCNA) immunohistochemistry were also performed. RESULTS: CCl(4) induction led to the damage of liver and development of fibrosis in Group B and Group C rats when compared to Group A rats. The treatment of WeiJia in Group C rats could reduce the fibrosis condition significantly compared to Group B rats. The effect could be observed after three weeks of treatment and was more obvious after eight weeks of treatment. Serum HA, CIV, ALT, AST and gamma-GT levels after eight weeks of treatment for Group C rats were 58+/-22 microg/L (P<0.01), 57+/-21 microg/L (P<0.01), 47+/-10 U/L (P<0.01), 139+/-13 U/L (P<0.05) and 52+/-21 U/L (P>0.05) respectively, similar to normal control group (Group A), but significantly different from CCl(4) induced liver injury control group (Group B). An increase in PCNA and decrease in alpha-SMA expression level was also observed. CONCLUSION: WeiJia could improve liver function and reduce liver fibrosis which might be through the inhibition of stellate cell activity.