Unconjugated bilirubin (UCB) is a metabolic end-product of heme catabolism. Bilirubin's behavior in a human body has two faces similar to Janus Bifrons. Elevated serum/plasma UCB concentration exposes babies to the risk of neurotoxicity. Conversely, mildly elevated systemic bilirubin concentrations such as in Gilbert syndrome protect against various oxidative stress-mediated and metabolic diseases including cardiovascular diseases and diabetic nephropathy (DN). The project was developed in three main parts: Task 1 is an in vitro comparative study focused on the evaluation of the effects of increasing concentration of bilirubin on redox balance. UCB effect on cellular redox state among four in vitro systems coming from different organs was evaluated. Hepatic (HepG2), hearth endothelial (H5V), kidney tubular (HK2) and neuronal (SH-SY5Y) cell lines were exposed to the same UCB treatments and intracellular bilirubin concentration (iUCB), cytotoxicity, intracellular ROS concentrations, and antioxidant capacity (EC50)were determined. We observed that iUCB around 7 ng/mg acts as an antioxidant, while at iUCB higher than 25 ng/mg is associated with a prooxidant and cytotoxic effects. Task 2 investigates the effect of life-long hyperbilirubinemia and bilirubin-priming on the main pathways activated in atherosclerosis and DN at the cellular level. Fibrosis in tubular epithelial cells, apoptosis in podocytes, ER-stress and inflammation in endothelial cells were analyzed. Primary aortic endothelial cells and podocytes from hyperbilirubinemic jj and normobilirubinemic Nj Gunn rats were exposed to Palmitic acid (PA) and Angiotensin II (AngII), respectively. We observed that the viability of jj cells was significantly higher than the viability of Nj. Reduction in CHOP expression and IL-6 release was detected in jj primary aortic endothelial cells exposed to PA compared to Nj. The same occurred on H5V pretreated with UCB. Upon AngII treatment, jj podocytes showed lower DNA fragmentation, cleaved caspase-3, and cleaved PARP induction. By investigating the possible mechanism responsible for UCB anti-fibrotic and anti-apoptotic effect, we observed that jj podocytes showed a higher basal expression of LC3 II compared to Nj. In HK2 cells, the induction by AngII of HIF-1α and LOXl2 was significantly reduced by UCB pre-treatment. Task 3 The association between total bilirubin levels, age, pubertal stage, anthropometric, clinical, and laboratory data were evaluated in a retrospective cross-sectional study performed on 1672 consecutive extremely obese children and adolescents. Total bilirubin level correlates positively with age and increased according to the pubertal stage. High triglycerides, waist /height ratio, insulin, homeostatic model assessment (HOMA), and C-reactive protein showed a negative weak correlation with bilirubin. However, any significative relationship between serum total bilirubin Mets and NAFLD was not detected. We believe that the present thesis work have contributed to a better understanding of the molecular mechanism involved in the protective impact of bilirubin through 3 statements: 1)The definition of the intracellular UCB thresholds that set the switch between anti- and prooxidant effects of bilirubin. iUCB around 7 ng/mg had antioxidant activities; iUCB >25 ng/mg resulted in prooxidant and cytotoxic effects; 2) the demonstration that life-long hyperbilirubinemia exposure or bilirubin-priming significantly contribute to the activation of protection against metabolic syndrome, by enhancing responses to damage and increasing cell viability in in vitro models of atherosclerosis and DN; 3) the observation that serum bilirubin levels, even in their normal ranges, might have graded associations with the prevalence of the MetS and NAFLD in young ages, when the metabolic dysregulation is in its early stage, but not in extremely obese population.
La bilirubina è il prodotto finale del catabolismo dell'eme. Livelli molto elevati di bilirubina sono neurotossici, al contrario, concentrazioni leggermente più elevate del normale, proteggono da varie malattie metaboliche comprese quelle cardiovascolari e la nefropatia diabetica (DN). Il progetto è stato sviluppato in tre parti: Nella prima parte è stato effettuato uno studio comparativo in vitro, utilizzando quattro linee cellulari provenienti da organi differenti. Sono stati valutati gli effetti di diverse concentrazioni di bilirubina sull'equilibrio redox delle cellule. HepG2, H5V, HK2 e SH-SY5Y sono state esposte agli stessi trattamenti di bilirubina e sono statedeterminate le corrispettive concentrazione di bilirubina intracellulare (iUCB), valutata la citotossicità, la produzione di ROS e la capacità antiossidante. Si è visto che iUCB intorno a 7 ng / mg agisce come antiossidante, mentre iUCB > 25 ng / mg hanno effetti proossidanti e citotossici. La seconda parte è uno studio degli effetti della lieve iperbilirubinemia (condizione pre-esistente e duratura nel tempo, che caratterizza la colonia di ratti Gunn così come i soggetti affetti dalla sindrome di Gilbert) e dal “priming” della bilirubina (priming inteso come trattamento con la bilirubina prima dell’esposizione delle linee cellulari al danno metabolico) in modelli in vitro di aterosclerosi e DN. Sono state analizzate la fibrosi nelle cellule epiteliali tubulari, l'apoptosi nei podociti, lo ER-stress e l'infiammazione nelle cellule endoteliali. Cellule endoteliali aortiche e podociti primari sono stati isolati da ratti Gunn iperbilirubinemici jj e normobilirubinemici Nj ed esposti rispettivamente ad acido palmitico (PA) e angiotensina II (AngII). In entrambi i modelli di cellule primarie, la vitalità delle cellule jj è superiore alla vitalità di quelle Nj. La bilirubina attenua il rilascio di IL-6 e l’induzione di CHOP sia nelle cellule endoteliali aortiche primarie che nelle endoteliali immortalizzate H5V. I podociti jj, dopo il trattamento con AngII, hanno mostrato una minore frammentazione del DNA,minor induzione della forma attiva della caspasi-3 e della forma attiva di PARP. Studiando il possibile meccanismo responsabile dell'effetto antifibrotico e anti-apoptotico della bilirubina, abbiamo osservato che i podociti jj mostravano una maggiore espressione basale di LC3 II rispetto a quelli Nj.In più si è visto che nelle cellule HK2, l'induzione da Ang II di HIF-1α e LOXl2 è stata ridotta dal pretrattamento con bilirubina. Infine, nella terza parte, ci siamo focalizzati sull'associazione tra i livelli di bilirubina sierica, la prevalenza di NAFLD e MetS. Il livello di bilirubina totale è correlato positivamente con l'età e aumenta in base alla fase puberale. Trigliceridi alti, rapporto vita / altezza, insulina, valutazione del modello omeostatico (HOMA) e proteina C-reattiva hanno mostrato una debole correlazione negativa con la bilirubina. Tuttavia, non è stata rilevata alcuna relazione significativa tra la bilirubina totale sierica Mets e NAFLD. Riteniamo che il presente lavoro abbia contribuito a una migliorare la comprensione del meccanismo molecolare coinvolto nell'impatto protettivo della bilirubina attraverso: 1) La definizione delle soglie di iUCB che impostano il passaggio tra gli effetti antiossidanti e proossidanti della bilirubina; 2) la dimostrazione che life-long hyperbilirubinemia o bilirubin-priming contribuiscono alla protezione contro la sindrome metabolica, migliorando le risposte al danno e aumentando la vitalità cellulare nei modelli in vitro analizzati; 3) l'osservazione che i livelli di bilirubina sierica, anche nei loro range normali, potrebbero avere associazioni graduali con la prevalenza di MetS e NAFLD in giovane età, quando la disregolazione metabolica è nella sua fase iniziale, ma non nella popolazione estremamente obesa.
Meccanismi di protezione della bilirubina contro il danno metabolico a livello cellulare / Bianco, Annalisa. - (2021 Apr 23).
Meccanismi di protezione della bilirubina contro il danno metabolico a livello cellulare
BIANCO, ANNALISA
2021-04-23
Abstract
Unconjugated bilirubin (UCB) is a metabolic end-product of heme catabolism. Bilirubin's behavior in a human body has two faces similar to Janus Bifrons. Elevated serum/plasma UCB concentration exposes babies to the risk of neurotoxicity. Conversely, mildly elevated systemic bilirubin concentrations such as in Gilbert syndrome protect against various oxidative stress-mediated and metabolic diseases including cardiovascular diseases and diabetic nephropathy (DN). The project was developed in three main parts: Task 1 is an in vitro comparative study focused on the evaluation of the effects of increasing concentration of bilirubin on redox balance. UCB effect on cellular redox state among four in vitro systems coming from different organs was evaluated. Hepatic (HepG2), hearth endothelial (H5V), kidney tubular (HK2) and neuronal (SH-SY5Y) cell lines were exposed to the same UCB treatments and intracellular bilirubin concentration (iUCB), cytotoxicity, intracellular ROS concentrations, and antioxidant capacity (EC50)were determined. We observed that iUCB around 7 ng/mg acts as an antioxidant, while at iUCB higher than 25 ng/mg is associated with a prooxidant and cytotoxic effects. Task 2 investigates the effect of life-long hyperbilirubinemia and bilirubin-priming on the main pathways activated in atherosclerosis and DN at the cellular level. Fibrosis in tubular epithelial cells, apoptosis in podocytes, ER-stress and inflammation in endothelial cells were analyzed. Primary aortic endothelial cells and podocytes from hyperbilirubinemic jj and normobilirubinemic Nj Gunn rats were exposed to Palmitic acid (PA) and Angiotensin II (AngII), respectively. We observed that the viability of jj cells was significantly higher than the viability of Nj. Reduction in CHOP expression and IL-6 release was detected in jj primary aortic endothelial cells exposed to PA compared to Nj. The same occurred on H5V pretreated with UCB. Upon AngII treatment, jj podocytes showed lower DNA fragmentation, cleaved caspase-3, and cleaved PARP induction. By investigating the possible mechanism responsible for UCB anti-fibrotic and anti-apoptotic effect, we observed that jj podocytes showed a higher basal expression of LC3 II compared to Nj. In HK2 cells, the induction by AngII of HIF-1α and LOXl2 was significantly reduced by UCB pre-treatment. Task 3 The association between total bilirubin levels, age, pubertal stage, anthropometric, clinical, and laboratory data were evaluated in a retrospective cross-sectional study performed on 1672 consecutive extremely obese children and adolescents. Total bilirubin level correlates positively with age and increased according to the pubertal stage. High triglycerides, waist /height ratio, insulin, homeostatic model assessment (HOMA), and C-reactive protein showed a negative weak correlation with bilirubin. However, any significative relationship between serum total bilirubin Mets and NAFLD was not detected. We believe that the present thesis work have contributed to a better understanding of the molecular mechanism involved in the protective impact of bilirubin through 3 statements: 1)The definition of the intracellular UCB thresholds that set the switch between anti- and prooxidant effects of bilirubin. iUCB around 7 ng/mg had antioxidant activities; iUCB >25 ng/mg resulted in prooxidant and cytotoxic effects; 2) the demonstration that life-long hyperbilirubinemia exposure or bilirubin-priming significantly contribute to the activation of protection against metabolic syndrome, by enhancing responses to damage and increasing cell viability in in vitro models of atherosclerosis and DN; 3) the observation that serum bilirubin levels, even in their normal ranges, might have graded associations with the prevalence of the MetS and NAFLD in young ages, when the metabolic dysregulation is in its early stage, but not in extremely obese population.File | Dimensione | Formato | |
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Thesis PhD_Bianco DEF.pdf
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