Atherosclerosis is a chronic inflammatory disease affecting medium-to-large caliber arteries. It is characterized by the accumulation of lipids, inflammatory cells, and fibrotic components within the arterial wall, leading to the formation of atherosclerotic plaques. Such plaques can obstruct blood flow, thereby causing ischemia, or to rupture, which may result in thrombus formation. Atherosclerotic cardiovascular disease (ASCVD) represents a significant cause of mortality worldwide, with risk factors such as hypertension, hypercholesterolemia, smoking, and an unbalanced diet. Prevention is based on the reduction LDL cholesterol and inflammation through targeted therapies, early diagnosis, and the management of modifiable risk factors. In the treatment of dyslipidemias, LDL receptors (LDLR) play a crucial role in reducing plasma LDL cholesterol levels. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein that degrades LDLR, can be inhibited to prolong receptor activity and thus enhance the clearance of LDL. Current guidelines recommend the use of statins, which reduce endogenous cholesterol synthesis and increase LDLR expression. However, natural nutraceuticals are emerging as a valid alternative due to their higher tolerability, fewer side effects, and better safety profile, especially for patients with statin intolerance or low cardiovascular risk. Recent research has focused on identifying new natural molecules and phytocomplexes with cholesterol-lowering properties and potential benefits in the treatment of metabolic diseases. This experimental study investigates the cholesterol-lowering potential of four isoquinoline alkaloids (chaeronepaline-A, -B, -C, -D) derived from the Corydalis chaerophylla plant, tested in vitro on HuH7 hepatocarcinoma cell lines. Firstly, the cytotoxicity of the compounds was evaluated, and the optimal concentrations were selected for further experiments. The concentrations used were 5 and 50 μM for chaeronepaline-A and -C, 10 and 100 μM for chaeronepaline-D, and 2.5 and 25 μM for chaeronepaline-B. Subsequently, the alkaloids were tested for their capacity to modulate LDLR and PCSK9 expression, influence cholesterol biosynthesis, and regulate PCSK9 secretion through the utilization of assays such as Western Blot, Real-time qPCR, and ELISA. The results demonstrate that chaeronepaline-B and chaeronepaline-C upregulate LDLR protein expression and stabilize PCSK9 levels. Notably, chaeronepaline-B showed a significant reduction in cholesterol biosynthesis (25%) and PCSK9 secretion (62%), demonstrating considerable potential. In addition, chaeronepaline-B was observed to effectively reduce intracellular lipid accumulation, as evidenced by Oil Red-O staining, suggesting a positive impact on lipid metabolism. This observation is supported by a significant reduction in the expression of the genes FASN and SCD-1, which are involved in the process of lipogenesis. Overall, the data suggest that chaeronepaline-B may act through a mechanism similar to that of berberine, exhibiting a superior cholesterol-lowering effect compared to the other alkaloids tested. This compound represents a promising therapeutic alternative for the treatment of hypercholesterolemia and metabolic diseases, warranting further in vivo studies to validate these preliminary findings.
L'aterosclerosi è una patologia cronica infiammatoria che colpisce le arterie di medio-grande calibro. È caratterizzata dall'accumulo di lipidi, cellule infiammatorie e componenti fibrotici nella parete arteriosa, che porta alla formazione di placche aterosclerotiche. Queste placche possono ostruire il flusso sanguigno, causando ischemia, o rompersi, con conseguente formazione di trombi. La malattia cardiovascolare aterosclerotica (ASCVD) rappresenta una delle principali cause di mortalità a livello globale, con fattori di rischio quali ipertensione, ipercolesterolemia, tabagismo e dieta non equilibrata. La prevenzione si basa sulla riduzione del colesterolo LDL e dell'infiammazione attraverso terapie mirate, diagnosi precoce e gestione dei fattori di rischio modificabili. Nel trattamento delle dislipidemie, i recettori delle LDL (LDLR) svolgono un ruolo cruciale nel ridurre i livelli plasmatici di colesterolo LDL. L'inibizione di PCSK9 (proproteina convertasi subtilisina/Kexin tipo 9), una proteina che degrada LDLR, prolunga l'attività del recettore, migliorando la clearance delle LDL. Le attuali linee guida raccomandano l'uso di statine, farmaci che riducono la sintesi endogena di colesterolo e aumentano l'espressione dei recettori LDLR. Tuttavia, stanno emergendo sempre più i nutraceutici naturali come valida alternativa, in quanto presentano una maggiore tollerabilità, minori effetti collaterali e sono considerati più sicuri, soprattutto per i pazienti con intolleranze o basso rischio cardiovascolare. Negli ultimi anni, la ricerca si è focalizzata sull’identificazione di nuove molecole e fitocomplessi naturali con proprietà ipocolesterolemizzanti e potenziali benefici nel trattamento delle malattie metaboliche. In questo studio sperimentale, si è indagato il potenziale effetto ipocolesterolemizzante di quattro alcaloidi isochinolinici (chaeronepaline-A, -B, -C, -D) derivati dalla pianta Corydalis chaerophylla, testati in vitro utilizzando una linea cellulare di epatocarcinoma umano, HuH7. In primo luogo, è stata valutata la citotossicità dei composti, per poi selezionare le concentrazioni ottimali per gli esperimenti successivi. Le concentrazioni selezionate sono state 5 e 50 M per i composti chaeronepaline-A e chaeronepaline-C, 10 e 100 M per il composto chaeronepaline-D e 2,5 e 25 M per il composto chaeronepaline-B. Gli alcaloidi sono stati quindi testati per valutare la loro capacità di modulare l’espressione di LDLR e PCSK9, influenzare la biosintesi del colesterolo e la secrezione di PCSK9, tramite saggi quali Western Blot, Real-time qPCR ed ELISA. I risultati indicano che i composti chaeronepaline-B e chaeronepaline-C sovraregolano l’espressione proteica di LDLR e stabilizzano quella di PCSK9. In particolare, chaeronepaline-B ha mostrato ridurre la biosintesi del colesterolo (25%) e della secrezione di PCSK9 (62%), risultando così l’alcaloide di maggior interesse. Anche per quanto riguarda l’accumulo lipidico intracellulare, valutato attraverso una colorazione con Oil Red-O, chaeronepaline-B ha dimostrato un’efficace riduzione, suggerendo un’azione positiva sul metabolismo lipidico. Questi dati sono supportati da una riduzione significativa dell’espressione genica di FASN e SCD-1, geni coinvolti nella de novo lipogenesi. Nel complesso, i dati suggeriscono che chaeronepaline-B possa agire con un meccanismo simile alla berberina, con un effetto ipocolesterolemizzante migliore rispetto agli altri alcaloidi testati. Questo composto rappresenta una promettente alternativa terapeutica per il trattamento dell’ipercolesterolemia e delle malattie metaboliche, con la necessità di ulteriori studi in vivo per confermare questi risultati preliminari.
Identificazione di nuovi alcaloidi derivati da Corydalis chaerophylla D.C. e caratterizzazione in vitro della loro potenziale attività farmacologica sul metabolismo del colesterolo
BENETAZZO, VERONICA
2023/2024
Abstract
Atherosclerosis is a chronic inflammatory disease affecting medium-to-large caliber arteries. It is characterized by the accumulation of lipids, inflammatory cells, and fibrotic components within the arterial wall, leading to the formation of atherosclerotic plaques. Such plaques can obstruct blood flow, thereby causing ischemia, or to rupture, which may result in thrombus formation. Atherosclerotic cardiovascular disease (ASCVD) represents a significant cause of mortality worldwide, with risk factors such as hypertension, hypercholesterolemia, smoking, and an unbalanced diet. Prevention is based on the reduction LDL cholesterol and inflammation through targeted therapies, early diagnosis, and the management of modifiable risk factors. In the treatment of dyslipidemias, LDL receptors (LDLR) play a crucial role in reducing plasma LDL cholesterol levels. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein that degrades LDLR, can be inhibited to prolong receptor activity and thus enhance the clearance of LDL. Current guidelines recommend the use of statins, which reduce endogenous cholesterol synthesis and increase LDLR expression. However, natural nutraceuticals are emerging as a valid alternative due to their higher tolerability, fewer side effects, and better safety profile, especially for patients with statin intolerance or low cardiovascular risk. Recent research has focused on identifying new natural molecules and phytocomplexes with cholesterol-lowering properties and potential benefits in the treatment of metabolic diseases. This experimental study investigates the cholesterol-lowering potential of four isoquinoline alkaloids (chaeronepaline-A, -B, -C, -D) derived from the Corydalis chaerophylla plant, tested in vitro on HuH7 hepatocarcinoma cell lines. Firstly, the cytotoxicity of the compounds was evaluated, and the optimal concentrations were selected for further experiments. The concentrations used were 5 and 50 μM for chaeronepaline-A and -C, 10 and 100 μM for chaeronepaline-D, and 2.5 and 25 μM for chaeronepaline-B. Subsequently, the alkaloids were tested for their capacity to modulate LDLR and PCSK9 expression, influence cholesterol biosynthesis, and regulate PCSK9 secretion through the utilization of assays such as Western Blot, Real-time qPCR, and ELISA. The results demonstrate that chaeronepaline-B and chaeronepaline-C upregulate LDLR protein expression and stabilize PCSK9 levels. Notably, chaeronepaline-B showed a significant reduction in cholesterol biosynthesis (25%) and PCSK9 secretion (62%), demonstrating considerable potential. In addition, chaeronepaline-B was observed to effectively reduce intracellular lipid accumulation, as evidenced by Oil Red-O staining, suggesting a positive impact on lipid metabolism. This observation is supported by a significant reduction in the expression of the genes FASN and SCD-1, which are involved in the process of lipogenesis. Overall, the data suggest that chaeronepaline-B may act through a mechanism similar to that of berberine, exhibiting a superior cholesterol-lowering effect compared to the other alkaloids tested. This compound represents a promising therapeutic alternative for the treatment of hypercholesterolemia and metabolic diseases, warranting further in vivo studies to validate these preliminary findings.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/73568