Hapidin, Hermizi
(2019)
Expression of bone-specific genes and proteins in human fetal osteoblast cell
line (hfob 1.19) treated with semi-purified fraction of quercus infectoria.
Expression of bone-specific genes and proteins in human fetal osteoblast cell line (hfob 1.19) treated with semi-purified fraction of quercus infectoria.
Abstract
The study aim to investigate the effect of established Quercus infectoria (QI) semi-purified
fractions (Qlsm-F) and combination of established Qlsm-F fraction with readily available
osteoporotic drug (pamidronate) on proliferation, differentiation and mineralisation of osteoblast
as well as to delineate the molecular mechanism of Qlsm-F that enhanced bone formation by
employing hFOB 1.19 cell model. A total of 13 Qlsm-F were isolated by chromatographic
technique and tested in series of bio-guided assay by MTT assay to determine the most potent
fraction. Three most potent fractions; Fraction A (FA), Fraction B (FB) and Fraction C (FC) were
used throughout this study. Polyphenolic content of each Qlsm-F were identified by LCMS. The
cells were treated on day 1, day 3 and day 7 for assessments of mineralisation by Alizarin Red
S staining [calcium (Ca) depositions] and von Kossa staining [phosphate (P) depositions] and
also evaluation of cellular morphology by using microscope. Detailed assessment of bone
specific markers were conducted by RT-PCR, western blot and immunofluorescence staining.
The LCMS analysis of each FA, FB and FC reveals that each fraction consists of mainly
polyphenolic compounds (gallic acid, digallate, ellagic acid, syringic acid and theogallin). The
mineral deposition per viable cell increases with treatment of FA, FB and FC as well as
combined treatment of FA, FB and FC with pamidronate on hFOB1 .19 cells in a time-dependent
manner. Interestingly, from day 3 until day 7; quantification of investigated bone specific genes
(Runx2, Osx, BMP-2, BSPll, BGLAP, and TGF-J31) and proteins (Runx2, Osx and BSPll) on hFOB1.19 cells were highest in cells treated with combined treatment of FA, FB and FC with
pamidronate and at peak in cells treated with combined treatment of FC with pamidronate
compared to hFOB 1.19 cells treated with single individual treatment. Protein analysis results •
were consistent with investigation of immunofluorescence staining. Therefore, these finding
demonstrated that polyphenols presence in each FA, FB and FC enhanced mineral deposition
along with expression of investigated bone marker as well as acknowledge the potential
therapeutic effects when combining each Qlsm-F with pamidronate through increasing
efficiency of pamidronate acting on osteoblast cells.
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