Cardioprotective effects of arjunolic acid in LPS-stimulated H9C2 and C2C12 myotubes via the My88-dependent TLR4 signaling pathway.

CONTEXT: Arjunolic acid (AA) is a triterpenoid saponin found in Terminalia arjuna (Roxb.) Wight & Arn. (Combretaceae). It exerts cardiovascular protective effects as a phytomedicine. However, it is unclear how AA exerts the effects at the molecular level. OBJECTIVE: This study investigates the cardioprotective effects of arjunolic acid (AA) via MyD88-dependant TLR4 downstream signaling marker expression. MATERIALS AND METHODS: The MTT viability assay was used to assess the cytotoxicity of AA. LPS induced in vitro cardiovascular disease model was developed in H9C2 and C2C12 myotubes. The treatment groups were designed such as control (untreated), LPS control, positive control (LPS + pyrrolidine dithiocarbamate (PDTC)-25 µM), and treatment groups were co-treated with LPS and three concentrations of AA (50, 75, and 100 µM) for 24 h. The changes in the expression of TLR4 downstream signaling markers were evaluated through High Content Screening (HCS) and Western Blot (WB) analysis. RESULTS: After 24 h of co-treatment, the expression of TLR4, MyD88, MAPK, JNK, and NF-κB markers were upregulated significantly (2-6 times) in the LPS-treated groups compared to the untreated control in both HCS and WB experiments. Evidently, the HCS analysis revealed that MyD88, NF-κB, p38, and JNK were significantly downregulated in the H9C2 myotube in the AA treated groups. In HCS, the expression of NF-κB was downregulated in C2C12. Additionally, TLR4 expression was downregulated in both H9C2 and C2C12 myotubes in the WB experiment. DISCUSSION AND CONCLUSIONS: TLR4 marker expression in H9C2 and C2C12 myotubes was subsequently decreased by AA treatment, suggesting possible cardioprotective effects of AA.

Novel discovery of Averrhoa bilimbi ethanolic leaf extract in the stimulation of brown fat differentiation program in combating diet-induced obesity.

BACKGROUND: Brown adipocytes are known to promote energy expenditure and limit weight gain to combat obesity. Averrhoa bilimbi, locally called belimbing buluh (DBB), is mainly used as an ethnomedicine in the treatment of metabolic disorders including diabetes mellitus, hypertension and obesity. The present study aims to investigate the browning activity on white adipocytes by A. bilimbi leaf extract and to evaluate the potential mechanisms. METHODS: Ethanolic leaf extract of A. bilimbi was exposed to Myf5 lineage precursor cells to stimulate adipocyte differentiation. Protein expressions of brown adipocyte markers were determined through high content screening analysis and validated through western blotting. Mito Stress Test assay was conducted to evaluate the cellular oxygen consumption rate upon A. bilimbi treatment. RESULTS: A. bilimbi ethanolic leaf extract exhibited an adipogenesis effect similar to a PPARgamma agonist. It also demonstrated brown adipocyte differentiation in myoblastic Myf5-positive precursor cells. Expression of UCP1 and PRDM16 were induced. The basal metabolic rate and respiratory capacity of mitochondria were increased upon A. bilimbi treatment. CONCLUSIONS: The findings suggest that Averrhoa bilimbi ethanolic leaf extract induces adipocyte browning through PRDM16 activation and enhances mitochondria activity due to UCP1 up-regulation.

Potent PPARγ Ligands from Swietenia macrophylla Are Capable of Stimulating Glucose Uptake in Muscle Cells.

Numerous documented ethnopharmacological properties have been associated with Swietenia macrophylla (Meliaceae), with its seed extract reported to display anti-hypoglycemic activities in diabetic rats. In the present study, three compounds isolated from the seeds of S. macrophylla were tested on a modified ELISA binding assay and showed to possess PPARγ ligand activity. They were corresponded to PPARγ-mediated cellular response, stimulated adipocyte differentiation but produced lower amount of fat droplets compared to a conventional anti-diabetic agent, rosiglitazone. The up-regulation of adipocytes was followed by increased adipocyte-related gene expressions such as adiponectin, adipsin, and PPARγ. The S. macrophylla compounds also promoted cellular glucose uptake via the translocation of GLUT4 glucose transporter.

Dextran sulfate provides a quantitative and quick microarray hybridization reaction.

Microarray technology is a powerful tool to speed up genomics study, yet many technical aspects need to be improved. The hybridization reaction of microarray experiments is carried out for 16h or overnight in order to obtain reasonably strong signals for analysis in the presence of high salt buffer, like SSC. However, the quantitative aspect of microarray hybridization has seldom been investigated. In this study, we showed that higher overall signals from hybridization were achieved in a buffer system containing dextran sulfate, which can accelerate the kinetics of reaction by increasing the local concentration of the reactants. The dextran sulfate containing hybridization solution increases the reaction 4-fold (median) for cDNA microarray and 29-fold for oligonucleotide microarray. More importantly, the solution also provides a quantitative hybridization reaction, where the hybridization signals are proportional to the abundance of transcript added. The enhancement in the kinetics of hybridization is due to both dextran sulfate and formamide present in the solution, but the effect is not due to the higher temperature used during the reaction. With a slightly longer reaction time the hybridization reaction with the solution allows the detection of hybridization signals from rare transcripts that is not possible with regular hybridization buffers. With appropriate washing, the enhancement of kinetics by the solution does not increase the background signals at all, allowing higher signal-to-noise ratios to be achieved.

Synergistic effects of epoxy- and amine-silanes on microarray DNA immobilization and hybridization.

Most microarray slides are manufactured or coated with a layer of poly(L-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.

Linear amplification of catalyzed reporter deposition technology on nylon membrane microarray.

The application of microarray analysis to gene expression from limited tissue samples has not been very successful because of the poor signal qualityfrom the genes expressed at low levels. Here we discussed the use of catalyzed reporter deposition (CARD) technology to amplify signals from limited RNA samples on nylon membrane cDNA microarray. When the input RNA level was greater than 10 microg, the genes expressed at high levels did not amplify in proportion to those expressed at low levels. Compared to conventional colorimetric detection, the CARD method requires less than 10% of the total RNA used for amplification of signal displayed onto a nylon membrane cDNA microarray. Total RNA (5-10 microg), as one can extract from a limited amount of specimen, was determined to produce a linear correlation between the colorimetric detection and CARD methods. Beyond this range, it can cause a nonlinear amplification of highly expressed and low-abundance genes. These results suggest that when amplification is needed for any applications using the CARD method, including DNA microarray experiments, precaution has to be taken in the amount of RNA used to avoid skew amplification and thus misleading conclusions.