Active components to regulate lipid synthesis and inflammatory cascade in cultivated human SZ95 sebocytes

The aim of this work was to search for new active compounds which regulate lipid
synthesis in vitro in SZ95 sebocytes. More than 200 extracts and fractions derived
from plants, microorganisms, Bothrops moojeni snake venom as well as peptides
were tested in a newly established screening model to identify active ingredients,
which act on neutral and polar lipid synthesis in SZ95 sebocytes. The clinical
background for this work was the fact that during the ageing process sebocytes
reduce lipid production. The relationships between ageing effects and lipid reduction
on a molecular level and ways to influence them are not fully identified. For this
reason, after identification of active lipid regulating compounds, this work further
focused on lipid stimulation in SZ95 sebocytes by Bothrops moojeni snake venom gel
filtration fractions (Botmo GF).
Botmo GF increased lipid synthesis in SZ95 sebocytes without apparent toxic or
apoptotic effects in applied concentrations. Partly purified Botmo GF fractions were
identified as fraction with phospholipase (PLA2) activity (Botmo GF 11-117) and
another fraction without enzymatic PLA2 activity (Botmo GF 11-101). Botmo GF 11-
101 (1 μg/ml) enhanced neutral lipid synthesis by up to 150% and polar lipid
synthesis by up to 120%. The enzymatically active PLA2 Botmo GF 11-117 (1 μg/ml)
increased synthesis of neutral lipids by up to 310% and polar lipid synthesis by up to
120% compared to untreated SZ95 sebocytes. The present data surprisingly indicate
that lipid synthesis stimulation by Botmo GF 11-101 and Botmo GF 11-117 was
independent of PLA2 enzymatic activity in Botmo GF 11 subfractions.
It is hypothesized that SZ95 sebocyte treatment with PLA2 fractions lead to the
production of fatty acids and eicosanoids which activate PPAR. Interestingly, Botmo
GF 11-101 was not able to activate any PPAR and Botmo GF 11-117 significantly
activated PPARa (p < 0.001) in PPARa, d or g2 transiently transfected SZ95
sebocytes.
Phospholipase activates the arachidonic acid (AA) metabolism. AA metabolised with
cyclooxygenase (COX) and lipoxygenase (LOX) to prostaglandins as well as
leukotrines. To get more knowledge about the lipogenesis pathway, we prestimulated
SZ95 sebocytes with arachidonic acid and treated sebocytes with
cyclooxygenase-2 inhibitor (NS398), LOX inhibitor (NDGA), 5-LOX inhibitor (MK886)
and PLA2 inhibitor (AACOCF3). Interestingly, most of the inhibitors stimulated neutral
lipid synthesis in SZ95 sebocytes. Only, PLA2 inhibitor showed no neutral lipid
stimulation.
Additionally, SZ95 sebocytes transiently expressing PPAR were pre-stimulated with
arachidonic acid. Treatment with NS398 reduced the level of PPAR isotype activation
which, however, remained higher than that of untreated control cells. Since NS398 is
a known prostaglandin E2 (PGE2) inhibitor, this effect is assumed to be caused by a
reduction of PGE2. The LOX inhibitor NDGA activated all transiently expressed PPAR
in SZ95 sebocytes. The epidermal LOX products such as 15-HETE and HPETE may
act on PPAR in a non specific manner. In conclusion, the enzymatically active PLA2 may activate a pathway of arachidonic
acid and mediators which activate lipid synthesis. Botmo GF 11-117 activated
PPARa. PLA2 inactive Botmo GF 11-101 still significantly activated lipid synthesis,
while no PPAR activation was measurable. Thus, it is suspected that both Bothrops
moojeni fractions act via different mechanisms on lipid stimulation in SZ95 sebocytes.
However, the exact pathway is still not identified. Botmo GF 11-101 and 11-117
might be interesting tools for the investigation of sebocyte lipogenesis and may be
helpful to the development of therapeutic concepts for the treatment of age-related
skin dryness.