Antiadipogenesis of Oroxylum indicum (L.) Kurz Extract via PPARγ2 in 3T3-L1 Adipocytes

Oroxylum indicum is regarded as a traditional food with medicinal properties and is used widely throughout Asia. It has previously been demonstrated that O. indicum extract (OIE) was able to suppress the differentiation of 3T3-L1 preadipocytes to adipocytes. However, the mechanism underlying the antiadipogenesis of this plant has not been fully investigated. The present study aimed to explore the impact of OIE at 50 to 200 μg mL−1 on the molecular mechanism involved in the antiadipogenic activity in 3T3-L1 cells at day 0 of their differentiation to adipocytes. The morphology and biochemistry of the cells on day 12 were investigated and compared to the relevant controls. Adiponectin was measured using enzyme-linked immunosorbent assay (ELISA). The mRNA expression of peroxisome proliferator-activated receptor-gamma 2 (PPARγ2), sterol regulatory element-binding proteins 1c (SREBP-1c), fatty acid synthetase (FAS), glucose transporter (GLUT4), and leptin in adipocytes was determined by real-time PCR. The results demonstrated that the OIE at 200 μg mL−1 exhibited strongest suppression on intracellular lipid accumulation. The levels of adiponectin were dramatically increased in the untreated adipocytes, whereas significantly decreased in the 200 μg mL−1 OIE-treated adipocytes (P<0.05). Expression of the mRNAs revealed that OIE-treated adipocytes at 200 μg mL−1 significantly inhibited the expression of PPARγ2 and SREBP-1c and lowered the level of expression of GLUT4, FAS, and leptin compared to the control (P<0.05). These findings suggest that OIE inhibits adipocyte differentiation along with the downregulation of PPARγ2, SREBP-1c, and GLUT4, leading to the decrease in the expression of FAS and adipokine (leptin and adiponectin). Thus, OIE might be developed for hyperlipidemia and obesity prevention

Enhanced Chondrogenic Differentiation of Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stem Cells by GSK-3 Inhibitors.

Articular cartilage is an avascular, alymphatic, and aneural system with very low regeneration potential because of its limited capacity for self-repair. Mesenchymal stem cells (MSCs) are the preferred choice for cell-based therapies. Glycogen synthase kinase 3 (GSK-3) inhibitors are compounds that can induce the Wnt signaling pathway, which is involved in chondrogenesis and cartilage development. Here, we investigated the influence of lithium chloride (LiCl) and SB216763 synergistically with TGF-β3 on chondrogenic differentiation in human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs). hWJ-MSCs were cultured and chondrogenic differentiation was induced in monolayer and pellet experiments using chondrogenic medium, chondrogenic medium supplemented with LiCl, or SB216763 for 4 weeks. After in vitro differentiation, cultured cells were examined for the expression of Sox9, ACAN, Col2a1, and β-catenin markers. Glycosaminoglycan (GAG) accumulation was also examined by Alcian blue staining. The results indicated that SB216763 was more effective than LiCl as evidenced by a higher up-regulation of the expression of cartilage-specific markers, including Sox9, ACAN, Col2a1 as well as GAG accumulation. Moreover, collagen type II expression was strongly observed in cells cultured in the chondrogenic medium + SB216763 as evidenced by western blot analysis. Both treatments appeared to mediate the Wnt signaling pathway by up-regulating β-catenin gene expression. Further analyses showed that all treatments suppressed the progression of chondrocyte hypertrophy, determined by decreased expression of Col10a1 and Runx2. These results indicate that LiCl and SB216763 are potential candidates for further in vivo therapeutic trials and would be of great importance for cartilage regeneration

Characterization of hWJ-MSCs.

<p><b>(A)</b> Immunophenotype of MSCs, immunofluorescent micrographs staining expression of MSC markers (CD73, 90, and 105), Nuclei were counterstains with DAPI (blue). Cells were negative for hematopoietic marker (CD34). Scale bar = 20 μm. <b>(B)</b> Differentiation of hWJ-MSCs to mesodermal linage cells. The cells were induced to undergo adipogenic, osteogenic, and condrogenic differentiation.</p

The collagen type II protein after 4 weeks of inductions examined by western blot analysis.

<p>β-actin was used as an internal control.</p

The toxicity effect of LiCl and SB216763 on hWJ-MSC viability.

<p>hWJ-MSCs were cultured with 0–20 mM LiCl <b>(A)</b>, 0–5 μM SB216763 <b>(B)</b>, or 0–0.5% DMSO <b>(C)</b>, for 72 hrs in 96-well plate. Then, the viability was detected by MTT assay. **DMSO without chemical was use as vehicle control. **Data were exposed as mean ± SD. <i>*P<0</i>.<i>05</i>.</p

Accumulation of GAGs was stained by alcian blue.

<p><b>(A-D)</b> Photographs of monolayer expanded cells cultured for 2 weeks. Scale bar = 10 μm. <b>(E-H)</b> Pellets culture at 4 weeks after differentiation. Scale bar = 20 μm. <b>(I)</b> Morphology of pellet culture at 4 weeks after differentiation.</p

qPCR analysis for chondrogenic gene expressions after 4 weeks of inductions.

<p><b>(A)</b><i>Col2a1</i>, <b>(B)</b><i>ACAN</i>, <b>(C)</b><i>Sox9</i> and <b>(D)</b> <i>β-catenin</i>. Gene expression was normalized to coresponding <i>GAPDH</i> and calculated by relative expression compared to control cells. The experiments were perfromed three times. **Data were expressed as mean±SD, *<i>P<0</i>.<i>05</i>.</p

Immunofluorescent staining of cartilage specific type collagen.

<p><b>(A)</b> immunofluorescent staining for collagen type II in monolayer expanded cultured on 3 weeks after differentiation. Scale bar = 20 μm. <b>(B)</b> Collagen type II and X expressions in pellet experiment on 4 weeks after differentiation. Scale bar = 20 μm.</p

Primers used for qPCR analysis.

<p>Primers used for qPCR analysis.</p