CHAPTER 1: ANTI-INFLAMMATORY ACTION OF ARACHIDONOYL AMIDE DERIVATIVES IN A HUMAN KERATINOCYTES CELL LINE MODEL OF ACUTE INFLAMMATION. CHAPTER 2: THE POLYPHENILPROPANOID VERBASCOSIDE INDUCES AUTOPHAGY IN HUMAN HEPATOCYTES CELL LINE BY AKT MODULATION.

ABSTRACT 1 Arachidonic acid (AA) is a poly unsaturated fatty acid present in the membrane phospholipids and it is released in the cytosol by phospholipase A2 after stimulation. AA is the substrate of cyclooxygenase-1 and 2 producing prostaglandins and prostacyclins that are well known inflammatory mediators. However, the effect of AA on inflammation is rather contradictory: in fact, some papers report its specific anti-inflammatory effect. Since AA is not easy to be used for its instability and light sensitivity, we synthesized seven new arachidonoyl amide derivatives (AA-Ds). To mimic cell inflammation, human keratinocyte cells (HaCaT) were induced with TNFα and IFNγ and the cells were pre-treated with AA-Ds compounds. Gene expression levels were measured by real time PCR. Western blot experiments were performed to measure total or phosphorylated protein levels, and transcription factors activity were tested by EMSA. We have demonstrated the anti-inflammatory action of AA-Ds since AA-Ds pre-treated cells showed a strong decrease in iNOS mRNA levels with respect to induced ones. In addition, Tumor Necrosis Factor α (TNFα), NF-kB Inhibitor α (IkBα), Class II major histocompatibility complex Transactivator (CIITA), Chemokine C-X-C motif ligand 9 (CXCL9) and C-X-C motif ligand 10 (CXCL10) gene expressions were down-regulated. The possible shortening effect of AA-Ds on iNOS mRNA half-life was also excluded by using the transcriptional inhibitor Actinomycin D. Western Blot results show a significant inhibition of kBp65-phosphorylated protein levels in time course assay in pre-treatment with AA-Ds. These data suggest a possible involvement of NF-kB transcriptional activity in the inhibition of iNOS induction. EMSA results show the inhibition of NF-kB-DNA binding starting from 3 hrs after cells induction, while, they show little effects on STAT1-DNA binding. Others potential mechanisms of AA-Ds gene expression inhibition involve others different transcriptional factor. EMSA data shows the increase in DNA binding of transcription factor AP1 at the different times. We also show the gene expression level of some components of AP-1 heterodimer, in particular, AA-Ds pre-treated samples show high increase in Fra-1, c-Jun and c-Fos mRNA levels. These results could lead up to development of new molecules against inflammatory processes which should be able to break off cytokines-induced signal transduction. ABSTRACT 2 Supplements of plant origin are increasingly used in food and feed. Recently it has been dispensed with food a polyphenyl propanoid glycoside, verbascoside as a supplement for animal feed. Presutti T. shows in his thesis on lambs, significant increases in growth, reduction in serum triglycerides, total cholesterol, LDL cholesterol and increase HDL cholesterol and bilirubin, as well as significant increases in the levels of vitamins A and E. These data have interested the Institute of Biotechnology Research (IRB) in Altavilla Vicentina, that extracted and purified verbascoside. In this project our aim is to investigate, in an in vitro model, the possible metabolic changes induced by verbascoside that would lead to a modulation of the energy balance of the cell. We used human hepatocytes cell line (HepG2) as an in vitro cellular model, cultured with or without increasing doses of verbascoside, though, we did not find any significant differences in gene expression of several genes involved in the biosynthesis and oxidation of fatty acids. Only the transcript for LDL receptor increased with verbascoside treatment. However, by morphological analysis, we showed an increase in the number and size of vacuoles in the cells treated with verbascoside compared to untreated cells. Using the Monodansylcadaverine, a specific probe which is incorporated in autophagic vacuoles, we demonstrated the activation of the autophagic process in cells treated with verbascoside. The autophagic process is useful to recycle damage proteins, mitochondria organelles and obtain energy in stressful conditions, and it is essential for cell homeostasis. The AMP-activated protein kinase (AMPK) is a protein kinase that can lead to the activation of autophagy. We verified, in our cell system, the phosphorylation levels of AMPK. Western blot analysis showed no significant differences in the state of phosphorylation of this kinase in the samples treated with verbascoside in comparison to the control ones. Autophagy, however, can also be activated by the blockade of inhibitory signals of mammalian target of rapamycin (mTOR) kinase. In this pathway we observed a significant inhibition of phosphorylation of serine 473 of protein kinase AKT. AKT phosphorylation activates mTOR cascade that is known antagonist of autophagy. An inhibition of the activation of AKT may lead to blockage of mTOR and it can trigger autophagy. These preliminary data may direct us to understand the molecular mechanism of verbascoside in our cell system.