Geniposide reduces cholesterol accumulation and increases its excretion by regulating the FXR-mediated liver-gut crosstalk of bile acids.

Hypercholesterolemia is the main risk factor to threaten human health and geniposide has been found to have hypolipidemic functions. However, its underlying mechanism is not clear. In this study, we firstly confirmed the hypolipidemic functions of geniposide in C57BL/6 and ApoE-/- mice (i.p, 50 mg/kg/d). Then hepatic or arterial lipid accumulation was analyzed through histomorphology. Moreover, the effects of geniposide on the bile acid metabolism were analyzed by the hepatic RNA-seq and biological molecular analysis. Mechanistically, GW4064, an FXR agonist, was carried out to verify the mechanisms of geniposide in human HepG2 and Caco2 cells. As expected, geniposide decreased the lipid accumulations both in plasma and liver. Morever, the atherosclerotic plaque shrank in HCD-fed ApoE-/- mice with geniposide treatment. The molecular analysis revealed that geniposide accelerated the hepatic synthesis of bile acids through inactivating the negative feedback regulation of bile acids mediated by FXR, led to the enhancive reverse cholesterol transport and cholesterol catabolism. What's more, geniposide reduced ileal FXR-mediated reabsorption of bile acids, resulting in the increasing excretion of bile acids. Our study pointed out the regulatory functions of geniposide on FXR-mediated liver-gut crosstalk of bile acids and geniposide might be a novel strategy for maintaining cholesterol homeostasis.

l-Arabinose Inhibits Colitis by Modulating Gut Microbiota in Mice.

l-Arabinose is a monosaccharide extracted from plants or fibers, which is known to have a variety of functional properties. In this study, we aim to investigate whether l-arabinose could inhibit colitis by modulating gut microbiota. l-Arabinose was administered in mice daily in a dextran sodium sulfate (DSS)-induced colitis model. The histological analysis, disease index, and the expression of inflammatory genes were measured. 16S-rRNA sequence analysis was performed to investigate gut microbiota. Intriguingly, we found that l-arabinose could repress DSS-induced colitis and inhibit p38-/p65-dependent inflammation activation. Besides that, our data revealed that l-arabinose-modulated DSS-induced gut microbiota were disturbed. Additionally, the perturbed gut microbiota was responsible for the suppressive effects of l-arabinose on DSS-induced colitis treated with antibiotics. Lastly, Caco-2 cells were used to confirm the protective effects of l-arabinose in colitis or inflammatory bowel disease. As expected, the protein expression levels in Caco-2 cells of pro-inflammatory genes, which were treated with l-arabinose and incubated with or without tumor necrosis factor alpha. Our work suggested that l-arabinose exerts anti-inflammation effects in DSS-induced colitis. These beneficial effects have correlations with the composition, diversity, and abundance of the gut microbiota regulated by l-arabinose. l-Arabinose could be a remarkable candidate as a functional food or novel therapeutic strategy for intestinal health.

Phosphorylation and Enzymatic Hydrolysis with Alcalase and Papain Effectively Reduce Allergic Reactions to Gliadins in Normal Mice.

Gliadins are major allergens responsible for wheat allergies. Food processing is an effective strategy to reduce the allergenicity of gluten. In the present study, we determined the secondary and tertiary structures of gluten and gliadins treated by chemical, physical, and enzymatic means through FTIR, surface hydrophobicity, intrinsic fluorescence spectra, and UV absorption spectra. The results showed that the three treatments of phosphorylation and alcalase and papain hydrolyses significantly changed the conformational structures of gliadins, especially the secondary structure. Then, the potential allergenicity of the phosphorylated and alcalase and papain hydrolyzed gliadins were further characterized, and we observed a significant decrease in the allergenicity through the results of the index of spleen, serum total IgE, gliadin-specific IgE, histamine, and serum cytokine concentrations. An elevation of Th17 cells, the absence of Treg cells, and an imbalance in Treg/Th17 are associated with allergy. On the basis of the expression levels of related cytokines and key transcription factors, we also confirmed that phosphorylation and alcalase and papain hydrolysis could effectively reduce the allergenicity of gliadins by improving the imbalance of both Th1/Th2 and Treg/Th17 in the spleens of sensitized mice. This study suggested that the changes in conformational structure contribute to gliadin hyposensitization and that phosphorylation and alcalase and papain hydrolysis may be promising strategies for the production of wheat products with low allergenicity.

Geniposide Improves Glucose Homeostasis via Regulating FoxO1/PDK4 in Skeletal Muscle.

It is well-known that imbalance state of glucose metabolism triggers many metabolic diseases and glucose uptake in skeletal muscle accounts for 90% of body weight. Geniposide is one of the major natural bioactive constituents of gardenia fruit, and the regulation of geniposide on glucose metabolism in skeletal muscle has not yet been investigated. Here, on the basis of microarray analysis, we discovered that geinposide decreased pyruvate dehydrogenase kinase 4 (PDK4) expression in skeletal muscle of mice and subsequently found that geniposide inhibited the expressions of forkhead box O1 (FoxO1), PDK4, and phosphorylated pyruvate dehydrogenase in vitro and in vivo. Moreover, geniposide promoted a switch of slow-to-fast myofiber type and glucose utilization, suggesting that geniposide improved glucose homeostasis. In addition, mechanistic studies revealed that geniposide played above roles by regulating FoxO1/PDK4, which controlled fuel selection via pyruvate dehydrogenase. Meanwhile, effects of geniposide mentioned above could be reversed by FoxO1 overexpression. Together, these results establish that geniposide confers controls on fuel usage and glucose homeostasis through FoxO1/PDK4 in skeletal muscle.

Effects of functional ß-glucan on proliferation, differentiation, metabolism and its anti-fibrosis properties in muscle cells.

Skeletal muscles plays a crucial role in metabolism and exercise. Fuctional ß-glucan is polysaccharide that is found in the cell walls of cereal, which is known to reduce cholesterol and lipid, prevent diabetes, cancer and cardiovascular diseases. In an attempt to identify ß-glucan that could promote skeletal muscle function, we analyzed the proliferation, differentiation, metabolism and anti-fibrotic properties of ß-glucan in C2C12 muscle cells. Treatment of ß-glucan in C2C12 myoblasts led to increased proliferation and differentiation. Besides that, we found that C2C12 myotubes treated with ß-glucan displayed a fast-to-slow muscle fiber conversion and improved oxidative metabolism. Further study revealed that ß-glucan treatment could prevent myotubes from becoming myofibroblasts. Together, our study suggests that functional ß-glucan might have a therapeutic potential to improve skeletal muscle function, which might contribute to the development of ß-glucan.

Effects of Geniposide from Gardenia Fruit Pomace on Skeletal-Muscle Fibrosis.

Geniposide is the main bioactive constituent of gardenia fruit. Skeletal-muscle fibrosis is a common and irreversibly damaging process. Numerous studies have shown that geniposide could improve many chronic diseases, including metabolic syndrome and tumors. However, the effects of geniposide on skeletal-muscle fibrosis are still poorly understood. Here, we found that crude extracts of gardenia fruit pomace could significantly decrease the expression of profibrotic genes in vitro. Moreover, geniposide could also reverse profibrotic-gene expression induced by TGF-ß and Smad4, a regulator of skeletal-muscle fibrosis. In addition, geniposide treatment could significantly downregulate profibrotic-gene expression and improve skeletal-muscle injuries in a mouse model of contusion. These results together suggest that geniposide has an antifibrotic effect on skeletal muscle through the suppression of the TGF-ß-Smad4 signaling pathway.

Characteristic expression of MSX1, MSX2, TBX2 and ENTPD1 in dental pulp cells.

Dental pulp cells (DPCs) are a promising source of transplantable cells in regenerative medicine. However, DPCs have not been fully characterized at the molecular level. The aim of the present study was to distinguish DPCs from various source-derived mesenchymal stem cells (MSCs), fibroblasts (FBs) and other cells by the expression of several DPC-characteristic genes. DPCs were isolated from human pulp tissues by the explant method or the enzyme digestion method, and maintained with media containing 10% serum or 7.5% platelet-rich plasma. RNA was isolated from the cells and from dental pulp tissue specimens. The mRNA levels were determined by DNA microarray and quantitative polymerase chain reaction analyses. The msh homeobox 1, msh homeobox 2, T-box 2 and ectonucleoside triphosphate diphosphohydrolase 1 mRNA levels in DPCs were higher than that of the levels identified in the following cell types: MSCs derived from bone marrow, synovium and adipose tissue; and in cells such as FBs, osteoblasts, adipocytes and chondrocytes. The enhanced expression in DPCs was consistently observed irrespective of donor age, tooth type and culture medium. In addition, these genes were expressed at high levels in dental pulp tissue in vivo. In conclusion, this gene set may be useful in the identification and characterization of DPCs in basic studies and pulp cell-based regeneration therapy.

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2.

BACKGROUND AIMS: Human bone marrow mesenchymal stromal cells are useful in regenerative medicine for various diseases, but it remains unclear whether the aging of donors alters the multipotency of these cells. In this study, we examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25-81 years old), including patients with or without systemic vascular diseases. METHODS: All stem cell lines were expanded with fibroblast growth factor-2 and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining. RESULTS: Systemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes. CONCLUSIONS: The aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells. This information will be useful in stromal cell-based therapy for cartilage-related diseases.

Selection of common markers for bone marrow stromal cells from various bones using real-time RT-PCR: effects of passage number and donor age.

Bone marrow stromal cells (BMSCs) are valuable in tissue engineering and cell therapy, but the quality of the cells is critical for the efficacy of therapy. To test the quality and identity of transplantable cells, we identified the molecular markers that were expressed at higher levels in BMSCs than in fibroblasts. Using numerous BMSC lines from tibia, femur, ilium, and jaw, together with skin and gum fibroblasts, we compared the gene expression profiles of these cells using DNA microarrays and low-density array cards. The differentiation potential of tibia and femur BMSCs was similar to that of iliac BMSCs, and different from jaw BMSCs, but all BMSC lines had many common markers that were expressed at much higher levels in BMSCs than in fibroblasts; several BMSC markers showed discrete expression patterns between jaw and other BMSCs. The common markers are probably useful in routine tests, but their efficacy may depend upon the passage number or donor age. In our study the passage number markedly altered the expression levels of several markers, while donor age had little effect on them. Considering the effects of in vivo location of BMSCs and passage, magnitude of increase in expression levels, and interindividual differences, we identified several reliable markers -- LIF, IGF1, PRG1, MGP, BMP4, CTGF, KCTD12, IGFBP7, TRIB2, and DYNC1I1 -- among many candidates. This marker set may be useful in a routine test for BMSCs in tissue engineering and cell therapy.

Molecular markers distinguish bone marrow mesenchymal stem cells from fibroblasts.

To characterize mesenchymal stem cells (MSC), we compared gene expression profiles in human bone marrow MSC (11 lines) and human fibroblasts (4 lines) by RT-PCR and real time PCR. Messenger RNA levels of MHC-DR-alpha, MHC-DR-beta, MHC-DR-associated protein CD74, tissue factor pathway inhibitor-2, and neuroserpin were much higher in MSC than in fibroblasts, even in the presence of large interindividual variations. Those of adrenomedullin, apolipoprotein D, C-type lectin superfamily member-2, collagen type XV alpha1, CUG triplet repeat RNA-binding protein, matrix metalloproteinase-1, protein tyrosine kinase-7, and Sam68-like phosphotyrosine protein/T-STAR were lower in MSC than in fibroblasts. FACS analysis showed that cell surface expression of MHC-DR was also higher in MSC than in fibroblasts. MHC-DR expression decreased after osteogenic differentiation, whereas the expression of adrenomedullin-a potent stimulator of osteoblast activity-along with collagen XV alpha1 and apolipoprotein D increased after osteogenic differentiation. The marker genes identified in this study should be useful for characterization of MSC both in basic and clinical studies.

A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix.

Mesenchymal stem cells (MSC) show a very short proliferative life span and readily lose the differentiation potential in culture. However, the growth rate and the proliferative life span of the stem cells markedly increased using tissue culture dishes coated with a basement membrane-like extracellular matrix, which was produced by PYS-2 cells or primary endothelial cells. Furthermore, the stem cells expanded on the extracellular matrix, but not those on plastic tissue culture dishes, retained the osteogenic, chondrogenic, and adipogenic potential throughout many mitotic divisions. The extracellular matrix had greater effects on the proliferation of MSC and the maintenance of the multi-lineage differentiation potential than basic fibroblast growth factor. Mesenchymal stem cells expanded on the extracellular matrix should be useful for regeneration of large tissue defects and repeated cell therapies, which require a large number of stem or progenitor cells.

Anti-membrane-bound transferrin-like protein antibodies induce cell-shape change and chondrocyte differentiation in the presence or absence of concanavalin A.

Membrane-bound transferrin-like protein (MTf), a glycosylphosphatidylinositol-anchored protein, is expressed at high levels in many tumors and in several fetal and adult tissues including cartilage and the intestine, as well as in the amyloid plaques of Alzheimer's disease, although its role remains unknown. MTf is one of the major concanavalin A-binding proteins of the cell surface. In this study, we examined the effects of anti-MTf antibodies and concanavalin A on cell shape and gene expression, using cultures of chondrocytes and MTf-overexpressing ATDC5 and C3H10T1/2 cells. In cultures expressing MTf at high levels, concanavalin A induced cell-shape changes from fibroblastic to spherical cells, whereas no cell-shape changes were observed with wild-type ATDC5 or C3H10T1/2 cells expressing MTf at very low levels. The cell-shape changes were associated with enhanced proteoglycan synthesis and expression of cartilage-characteristic genes, including aggrecan and type II collagen. Some anti-MTf antibodies mimicked this action of concanavalin A, whereas other antibodies blocked the lectin action. The findings suggest that the crosslinking of MTf changes the cell shape and induces chondrogenic differentiation. MTf represents the first identification of a plant lectin receptor involved in cell-shape changes and the differentiation of animal cells.