In the last two decades, astrocytes have gained significant interest as key regulators of physiological CNS function. This suggests that any alterations in astrocyte function may have significant repercussions on neuron survival and functioning. This evidence was key in promoting a new way of studying neurodegenerative diseases, wherein the focus was shifted from neuronal loss to astrocyte alteration, further opening new possibilities regarding the identification of new cellular and molecular targets for effective therapies. Consistent evidence suggests that the transcription factor STAT3 is a key component in the regulation of CNS inflammation, mostly contributing to astrocytes’ activation and modulating the expression of enzymes, such as COX2; transcription factors, such as AP1; and cytokines, such as IL-1β. Additionally, STAT3 signaling has been reported to interact with the transcription factor NF-κB, widely regarded as one of the key controllers of CNS inflammation via the expression of several inflammatory molecules and enzymes. Therefore, the dysregulation of the STAT3 signaling pathway may have a significant impact on neuronal survival and disease outcome progression in the case of CNS pathological conditions characterized by an inflammatory state, such as Amyotrophic Lateral Sclerosis (ALS). In the present study, we performed a pharmacological screening in vitro using the immortalized P0-17D astrocytic cell line in order to identify new molecules capable of preventing astrocytic activation of STAT3 and NF-κB. Among the small molecules we tested, Stattic proved unable to inhibit STAT3, while WP1066 and SH4-54 were both able to reduce its transcriptional activity. Moreover, the plant derivative Curcumin was shown to have a similar inhibitory effect, and we further confirmed its ability to inhibit the transcription factor NF-κB. On the other hand, Cucurbitacin I proved toxic at all concentrations tested. We then proceeded to test the anti-inflammatory effect of WP1066, SH4-54 and Curcumin in P0-17D cells, confirming their ability to curb the expression of the genes COX-2, iNOS and MMP-9, all involved in the inflammatory response of reactive astrocytes. Given the results obtained, we concluded that WP1066, SH4-54 and Curcumin may be relevant in case of treatment of neuroinflammatory conditions, as well as treatment of neurodegenerative disorders.
Nell'ultimo ventennio numerosi studi hanno confermato l'importanza degli astrociti quali regolatori della funzione fisiologica del sistema nervoso centrale, suggerendo che qualsiasi alterazione della loro funzione possa avere ripercussioni significative su sopravvivenza e funzionalità delle cellule neuronali. Questo ha promosso un nuovo modo di studiare le malattie neurodegenerative, laddove il focus è spostato dalla morte dei neuroni all'alterazione della funzione astrocitaria, aprendo nuove possibilità per l'identificazione di target cellulari e molecolari per lo sviluppo di terapie efficaci di malattie neurodegenerative. Il fattore di trascrizione STAT3 si è rivelato un fattore chiave nella regolazione dell'infiammazione del sistema nervoso centrale, contribuendo principalmente all'attivazione delle cellule astrocitarie e all'espressione di enzimi, fattori di trascrizione e citochine implicati nei processi infiammatori. Inoltre, è stato confermato che il pathway di STAT3 interagisca con quello del fattore di trascrizione NF-κB, il quale è considerato uno dei principali regolatori della neuroinfiammazione. Dunque, l'alterazione del pathway di STAT3 potrebbe avere un impatto significativo sulla sopravvivenza neuronale e sulla progressione di malattie caratterizzate da uno stato infiammatorio, come la Sclerosi Laterale Amiotrofica (SLA). Nel presente studio abbiamo portato a termine uno screening farmacologico in vitro sulla linea astrocitaria immortalizzata P0-17D, al fine di identificare nuove molecole capaci di prevenire l'attivazione astrocitaria dei fattori di trascrizione STAT3 e NF-κB. Tra le piccole molecole che abbiamo testato la molecola Stattic si è rivelata incapace di inibire l'attività trascrizionale di STAT3, mentre sia WP1066 che SH4-54 hanno avuto un effetto inibitorio significativo. Allo stesso modo, il derivato naturale Curcumina è stata capace di prevenire l'attivazione di STAT3, oltre che di NF-κB, mentre la Cucurbitacina I si è rivelata tossica a tutte le concentrazioni utilizzate. Abbiamo poi confermato l'effetto anti-infiammatorio delle molecole WP1066, SH4-54 e Curcumina, verificando la loro capacità di ridurre in maniera significativa l'espressione dei geni COX-2, iNOS e MMP-9, tutti implicati nella risposta infiammatoria astrocitaria. Abbiamo dunque concluso che le molecole WP1066, SH4-54 e la Curcumina possano essere rilevanti nel caso di terapie volte a trattare condizioni neuroinfiammatorie e malattie nerudegenerative.
Evaluation of the anti-inflammatory activity of small molecules and plant derivatives in an in vitro model of neuroinflammatory astrocytes. (Valutazione dell'attività anti-infiammatoria di piccole molecole e derivati naturali in un modello astrocitario di neuroinfiammazione in vitro)
RESCIGNO, ANTONIO
2018/2019
Abstract
In the last two decades, astrocytes have gained significant interest as key regulators of physiological CNS function. This suggests that any alterations in astrocyte function may have significant repercussions on neuron survival and functioning. This evidence was key in promoting a new way of studying neurodegenerative diseases, wherein the focus was shifted from neuronal loss to astrocyte alteration, further opening new possibilities regarding the identification of new cellular and molecular targets for effective therapies. Consistent evidence suggests that the transcription factor STAT3 is a key component in the regulation of CNS inflammation, mostly contributing to astrocytes’ activation and modulating the expression of enzymes, such as COX2; transcription factors, such as AP1; and cytokines, such as IL-1β. Additionally, STAT3 signaling has been reported to interact with the transcription factor NF-κB, widely regarded as one of the key controllers of CNS inflammation via the expression of several inflammatory molecules and enzymes. Therefore, the dysregulation of the STAT3 signaling pathway may have a significant impact on neuronal survival and disease outcome progression in the case of CNS pathological conditions characterized by an inflammatory state, such as Amyotrophic Lateral Sclerosis (ALS). In the present study, we performed a pharmacological screening in vitro using the immortalized P0-17D astrocytic cell line in order to identify new molecules capable of preventing astrocytic activation of STAT3 and NF-κB. Among the small molecules we tested, Stattic proved unable to inhibit STAT3, while WP1066 and SH4-54 were both able to reduce its transcriptional activity. Moreover, the plant derivative Curcumin was shown to have a similar inhibitory effect, and we further confirmed its ability to inhibit the transcription factor NF-κB. On the other hand, Cucurbitacin I proved toxic at all concentrations tested. We then proceeded to test the anti-inflammatory effect of WP1066, SH4-54 and Curcumin in P0-17D cells, confirming their ability to curb the expression of the genes COX-2, iNOS and MMP-9, all involved in the inflammatory response of reactive astrocytes. Given the results obtained, we concluded that WP1066, SH4-54 and Curcumin may be relevant in case of treatment of neuroinflammatory conditions, as well as treatment of neurodegenerative disorders.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/22334