THE mRNA EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN KELOID USING TOCOTRIENOL-RICH FRACTION IN PRIMARY HUMAN EPIDERMAL KERATINOCYTES AND PRIMARY HUMAN DERMAL FIBROBLAST CULTURES
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Date
2012
Authors
SITI MAHIRAH, YUSUF
Journal Title
Journal ISSN
Volume Title
Publisher
Pusat Pengajian Sains Perubatan Universiti Sains Malaysia
Abstract
Keloid is characterized by excess collagen deposition that can damage
healthy tissue. With the widespread use of Vitamin E- tocopherols based product and
the discovery of tocotrienol (T3) potential to treat various skin injuries; there are
possibilities of tocotrienol-rich fraction (TRF) to intercede normal, hypertrophic, or
keloid scarring during the inflammatory process. However, there is a lack of
scientific evidence to validate the efficacy and the therapies of TRF in scar
prevention. Thus, this study was carried out to evaluate the beneficial effects of TRF
in wound healing and its possible stimulation towards mRNA expression of inducible
nitric oxide synthase (iNOS) in keloid human skin keratinocytes and fibroblasts.
Primary human epidermal keratinocytes (pHEK) and primary human dermal
fibroblasts (pHDF) were successfully established using cell dissociation method.
Verification of pHEK and pHDF was conducted using Cytokeratin-6 (CK6),
Involucrin, Heat Shock Protein-47 (HSP47) and Fibroblast Surface Protein (FSP)
markers using imunocytochemistry analysis. The effect of TRF on pHEK and pHDF
were determined by using MTT assay. The mRNA expression of iNOS in primary
normal human dermal fibroblasts (pNHDF) and primary keloid human dermal
fibroblasts (pKHDF) treated with TRF was evaluated using real-time PCR. Primary
normal human epidermal keratinocytes (pNHEK) achieved higher cellular growth
rate compared to primary keloid human epidermal keratinocytes (pKHEK). Whilst,
pKHDF exhibited linear growth and sustained higher cellular growth rate compared
to pNHDF. pHEK cultures were positive for the presence of CK6 and Involucrin
whereas HSP47 and FSP were found in pHDF cultures. TRF ranged from 2.85 μg/
mL to180 μg/ mL. TRF (45 μg/ mL to 180 μg/ mL) was found to inhibit the growth
of pHEK while for pHDF was at 90 ug/mL at 72 hours. At lower concentrations
(2.8-22.5 μg/mL), TRF increased pHEK cell growth at 24 and 48 hours of incubation
but have no significant effect in pHDF viability at all time intervals. TRF at 2.8
μg/mL has been found to reduce the mRNA expression of iNOS at 24 hours, which
may in turn suppress the production of nitric oxide (NO) by pKHDF. The observed
suppression and growth inhibition effect in pHEK and pHDF cultures may be caused
by the antiproliferative and antioxidant activities of TRF. Whereas, the reduction of
iNOS mRNA level in pKHDF may suggest that TRF possess antioxidant and
antifibrogenic effect. This finding showed that TRF may play a role in keloid
intervention by suppressing the mRNA expression of iNOS.
Description
Keywords
Human Genome