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

Abstract

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.