CHAPTER 1 L'acido arachidonico (AA) è un acido grasso poli-insaturo presente nei fosfolipidi di membrana. Esso viene rilasciato nel citosol delle cellule ad opera della fosfolipasi A2. L’AA è il substrato per le cicloossigenasi-1 e 2 che portano alla produzione di prostaglandine e prostacicline, noti mediatori dell’infiammazione. Tuttavia in letteratura la somministrazione di AA non ha sempre attività pro-infiammatoria, infatti, alcuni articoli riportano uno suo specifico effetto anti-infiammatorio (Adam Olaf., 2002). Una linea cellulare di cheratinociti umani in coltura (HaCaT), è stata attivata con citochine: TNFα e IFNγ per simulare e indurre l'infiammazione. Dato che l’uso diretto dell’AA nel terreno di coltura cellulare non è raccomandato per la sua instabilità e sensibilità alla luce, abbiamo fatto sintetizzare sette nuovi derivati ammidici dell’acido arachidonico (AA-Ds) e questi sono stati addizionati al terreno di coltura di cellule HaCaT prima della loro induzione con citochine. I livelli di espressione di geni pro-infiammatori sono stati misurati mediante Real Time-PCR, e tramite Western Blot sono state analizzate le variazioni dei livelli di specifiche proteine totali o fosforilate. L'attività dei fattori di trascrizionali attivati dagli stimoli pro-infiammatori è stata misurata tramite EMSA. I nostri risultati dimostrano l'azione anti-infiammatoria di AA-Ds poiché le cellule pre-trattate con AA-Ds hanno mostrato una forte diminuzione dei livelli di espressione del gene Ossido Nitrico Sintasi inducibile (iNOS) rispetto alle cellule di controllo indotte con le sole citochine. Inoltre, altri geni pro-infiammatori quali: Tumor Necrosis Factor α (TNFα), l’inibitore di NF-kB (IkBα), Transattivatore di Classe II (CIITA), chemochine CXC motivo legante 9 (CXCL9) e 10 (CXCL10), sono risultati down-regolati dal pretrattamento. Utilizzando l'inibitore trascrizionale Actinomicina D abbiamo potuto dimostrare che l’emivita del trascritto di iNOS non è modificata con i pretrattamenti, ciò ci ha indirizzato allo studio della regolazione trascrizionale del gene. In esperimenti di time-course in campioni pre-trattamenti con AA-Ds gli immunoblot mostrano una significativa inibizione dei livelli di fosforilazione della sub-unità p65 di NF-kB. Questi dati suggeriscono un possibile coinvolgimento dell’attività trascrizionale di NF-kB nel meccanismo di inibizione dell'induzione del gene iNOS. Risultati di EMSA mostrano, nei campioni trattati, l’inibizione del legame di NF-kB al DNA a partire da 3 ore dopo l'induzione con citochine, mentre, mostrano solo piccoli effetti inibitori sul legame STAT1-DNA. Data la complessa regolazione del promotore del gene iNOS si è supposto che possano essere coinvolti anche altri fattori di trascrizione. Dati di EMSA mostrano, a diversi tempi, un aumento del legame al DNA del fattore trascrizionale AP1. Inoltre, nei campioni pre-trattati con AA-Ds alcuni componenti dell’eterodimero AP-1 mostrano aumentati livelli di trascritto come: Fra-1, c-Jun e c-Fos. Noi ipotizziamo un effetto inibitorio del complesso trascrizionale AP1 sul gene iNOS in accordo con dati di letteratura che indicano come il complesso AP-1 eserciti una regolazione negativa sull’espressione di questo gene. Questi risultati potrebbero portare in futuro allo sviluppo di nuove molecole ad attività anti-infiammatoria capaci di interrompere la trasduzione del segnale indotta dalle citochine TNFα e IFNγ. CHAPTER 2 Gli integratori di origine vegetale sono sempre più usati nell’alimentazione umana e animale. Recentemente è stato somministrato ad agnelli assieme al cibo come integratore un polifenilpropanoide glicoside, il verbascoside. Presutti T. mostra, nella sua tesi sperimentale su agnelli, aumenti significativi di crescita, riduzione nei livelli serici di trigliceridi, colesterolo totale, colesterolo LDL ed aumento di colesterolo HDL e bilirubina, oltre a significativi aumenti dei livelli di vitamine A ed E (Presutti T., 2009). Questi dati hanno suscitato l’interesse dell’Istituto di Ricerche Biotecnologiche (IRB) di Altavilla Vicentina, che estrae e purifica il verbascoside da colture di cellule vegetali e con il quale collaboriamo. Il nostro obiettivo era di investigare, in un sistema modello in vitro, i possibili cambiamenti metabolici indotti dal verbascoside capaci di portare ad una modulazione del bilancio energetico della cellula. È stata utilizzata una linea di epatociti umani (HepG2) come modello cellulare in vitro. Dai primi test effettuati, addizionando il terreno di coltura con dosi crescenti di verbascoside, non abbiamo riscontrato differenze significative nell’espressione genica di alcuni geni implicati nella biosintesi e ossidazione degli acidi grassi eccetto un aumento del recettore per LDL. Tuttavia, da una analisi morfologica, le cellule trattate con verbascoside presentavano un aumento del numero e grandezza dei vacuoli rispetto alle cellule non trattate. Utilizzando la Monodansylcadaverina (MDC), una sonda specifica che viene incorporata nei vacuoli autofagici, abbiamo dimostrato l’attivazione del processo autofagico in cellule trattate con verbascoside. Il processo autofagico è utile per la cellula per riciclare proteine, mitocondri, organuli cellulari e ricavarne energia in condizioni di stress, ed è fondamentale per l’omeostasi della cellula. L’AMP-activated protein kinase (AMPK) è una proteina chinasi che può portare all’attivazione dell’autofagia. Abbiamo verificato, nel nostro sistema di epatociti in coltura, i livelli di fosforilazione di AMPK. I risultati di Western blotting non hanno mostrato differenze significative nello stato di fosforilazione di questa proteina tra i campioni trattati con verbascoside e quelli di controllo. L’autofagia però può essere attivata anche dal blocco di segnali inibitori tramite la via dipendente da mammalian target of rapamycin (mTOR) chinasi. Abbiamo osservato una inibizione significativa dei livelli di fosforilazione della proteina chinasi AKT fosforilata in Serina 473. AKT fosforilata attiva a cascata mTOR che è un noto antagonista dell’autofagia. Una inibizione dell’attivazione di AKT può portare ad un blocco di mTOR e all’innesco dell’autofagia. Questi dati preliminari possono indirizzarci a comprendere il meccanismo d’azione del verbascoside come induttore di autofagia nel modello cellulare da noi usato.
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.
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.
Gregorelli, Alex
2015-01-01
Abstract
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.File | Dimensione | Formato | |
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