Pathobiol Aging Age Relat Dis. 2012;2. doi: 10.3402/pba.v2i0.11905. Epub 2012 Feb 22.
Investigation of the biological properties of Cinnulin PF in the context of diabetes: mechanistic insights by genome-wide mRNA-Seq analysis.
Rafehi H1, Ververis K, Balcerczyk A, Ziemann M, Ooi J, Hu S, Kwa FA, Loveridge SJ, Georgiadis GT, El-Osta A, Karagiannis TC.
Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, Melbourne, VIC., Australia.
The accumulating evidence of the beneficial effects of cinnamon (Cinnamomum burmanni) in type-2 diabetes, a chronic age-associated disease, has prompted the commercialisation of various supplemental forms of the spice. One such supplement, Cinnulin PF(®), represents the water soluble fraction containing relatively high levels of the double-linked procyanidin type-A polymers of flavanoids. The overall aim of this study was to utilize genome-wide mRNA-Seq analysis to characterise the changes in gene expression caused by Cinnulin PF in immortalised human keratinocytes and microvascular endothelial cells, which are relevant with respect to diabetic complications. In summary, our findings provide insights into the mechanisms of action of Cinnulin PF in diabetes and diabetic complications. More generally, we identify relevant candidate genes which could provide the basis for further investigation.
cinnulin PF; diabetes; diabetic complications; diabetic ulcer; mRNA-Seq
10.3402/pba.v2i0.11905
Double-linked type-A procyanidin polymer (trimer) (A). Comprised of the flavanoid catechin (B), which is made up of two substituted benzene rings, and a substituted dihydropyran ring.
Investigation of the biological properties of Cinnulin PF in the context of diabetes: mechanistic insights by genome-wide mRNA-Seq analysis
Pathobiol Aging Age Relat Dis. 2012;2:10.3402/pba.v2i0.11905.
The different factors which lead to impaired wound healing in diabetes.
Cell viability and apoptosis. Graphs showing the relative survival and EC50 (µg/mL) values following 24 hour incubations with CPF (0, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024 µg/mL), in HMEC-1 (A) and keratinocytes (B) using the CellTiter-Blue® Assay. The results of the ApoOne homogeneous 3/7® assay indicating caspase activity in HMEC-1 (C) and keratinocytes (D) cells treated with various concentrations of CPF for 24hrs. The data is representative of at least two independent experiments and is presented as the mean±SD of triplicate determination from a single experiment. *indicates p<0.05, compared to the control.
Graph showing the average% of cells in the different phases of the cell cycle in HMEC-1 cells (A) and keratinocyte cells (B). Cells were incubated with CPF for 24 hours, fixed and stained with propidium iodide. The proportion of cells in the different stages of the cell cycle was determined by FACS analysis. The data is representative of two independent experiments and is expressed as the mean±SD from a single experiment performed in duplicate. C–E show graphs of the cell cycle distribution of keratinocyte cells and F–H HMEC-1 cells treated with CPF at 0 (C, F), 20 (D, G) and 100 (E, H) µg/mL.
CPF promotes cell migration at low concentrations. Images are of keratinocytes (top) and HMEC-1 cells (bottom) treated with various concentrations of CPF (0, 10, 20 and 40 µg/mL) for 1 hour. Scratches were performed immediately following the incubation and images were taken at 0 hrs (representative images shown) and 24 hrs post scratching; magnification x4. Data is representative of five independent experiments for keratinocytes and two independent experiments for HMEC-1.
(A) The% of transcripts up/down regulated for the three treatment groups. HMEC-1 cells were incubated with 20 µg/mL CPF and keratinocytes were incubated with 20 and 100 µg/mL for 24 hours. Significant gene changes were based on a fold change of ±1.5 compared to the relevant untreated control. (B) Venn diagrams showing the number of genes up and down regulated in HMEC-1 and keratinocyte cells treated with CPF (20 µg/mL) for 24 hours (i) and in keratinocytes treated with 20 and 100 µg/mL CPF for 24 hours (ii).
RT-PCR (white, left) was used to validate the mRNA-Seq results (grey, right). Keratincoye (A) and HMEC-1 (B) cells were treated with 20 µg/mL CPF for 24 hours. RNA was then isolated from the cells, and the RNA of a selected number of genes was then amplified by RT-PCR. The results are expressed as the fold change in mRNA expression compared to untreated cells. RT-PCR results represent the mean±SD from a single experiment performed in triplicate.