RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Rosmarinic acid (RA), a natural polyphenol abundant in numerous herbal remedies, has been attracting growing interest owing to its exceptional ability to protect the liver. Toosendanin (TSN), a prominent bioactive compound derived from Melia toosendan Siebold & Zucc., boasts diverse pharmacological properties. Nevertheless, TSN possesses remarkable hepatotoxicity. Intriguingly, the potential of RA to counteract TSN-induced liver damage and its probable mechanisms remain unexplored. AIM OF THE STUDY: This study is aimed at exploring whether RA can alleviate TSN-induced liver injury and the potential mechanisms involved autophagy. MATERIALS AND METHODS: CCK-8 and LDH leakage rate assay were used to evaluate cytotoxicity. Balb/c mice were intraperitoneally administered TSN (20 mg/kg) for 24 h after pretreatment with RA (0, 40, 80 mg/kg) by gavage for 5 days. The autophagic proteins P62 and LC3B expressions were detected using western blot and immunohistochemistry. RFP-GFP-LC3B and transmission electron microscopy were applied to observe the accumulation levels of autophagosomes and autolysosomes. LysoTracker Red and DQ-BSA staining were used to evaluate the lysosomal acidity and degradation ability respectively. Western blot, immunohistochemistry and immunofluorescence staining were employed to measure the expressions of JAK2/STAT3/CTSC pathway proteins. Dual-luciferase reporter gene was used to measure the transcriptional activity of CTSC and RT-PCR was used to detect its mRNA level. H&E staining and serum biochemical assay were employed to determine the degree of damage to the liver. RESULTS: TSN-induced damage to hepatocytes and livers was significantly alleviated by RA. RA markedly diminished the autophagic flux blockade and lysosomal dysfunction caused by TSN. Mechanically, RA alleviated TSN-induced down-regulation of CTSC by activating JAK2/STAT3 signaling pathway. CONCLUSION: RA could protect against TSN-induced liver injury by activating the JAK2/STAT3/CTSC pathway-mediated autophagy and lysosomal function.
Assuntos
Autofagia , Doença Hepática Induzida por Substâncias e Drogas , Cinamatos , Depsídeos , Janus Quinase 2 , Lisossomos , Ácido Rosmarínico , Fator de Transcrição STAT3 , Transdução de Sinais , Animais , Humanos , Masculino , Camundongos , Autofagia/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Cinamatos/farmacologia , Depsídeos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Janus Quinase 2/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos Endogâmicos BALB C , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismoRESUMO
AIMS: Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by ß-hexosaminidase A (HexA) leads to the accumulation of GM2 ganglioside in neuronal lysosomes. Despite the storage phenotype, the role of autophagy and its regulation by mTOR has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. RESULTS: Pathological autophagosomes with impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities. INNOVATION: Our data provide a novel molecular mechanism underlying GM2 gangliosidosis. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for these diseases. CONCLUSIONS: We contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy.
Assuntos
Arginina/farmacologia , Autofagia , Gangliosidoses GM2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Catepsinas/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Hexosaminidase A/química , Hexosaminidase A/metabolismo , Hexosaminidase B/química , Hexosaminidase B/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Mutação/genética , Permeabilidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Doença de Sandhoff/patologia , Transdução de Sinais/efeitos dos fármacos , Doença de Tay-Sachs/patologia , Transcriptoma/genéticaRESUMO
Type 1 diabetes mellitus is an autoimmune disease caused by the destruction of pancreatic beta cells. Many patients with type 1 diabetes experience skeletal muscle wasting. Although the link between type 1 diabetes and muscle wasting is not clearly known, insulin insufficiency and hyperglycemia may contribute to decreased muscle mass. In this study, we investigated the therapeutic effect of the ethanolic extract of Schisandrae chinensis Fructus (SFe) on muscle wasting in streptozotocin (STZ)-induced diabetic mice. STZ-diabetic C57BL/6 mice (blood glucose level ≥300 mg/dL) were orally administered SFe (250 or 500 mg/kg/day) for 6 weeks. We observed that SFe administration did not change blood glucose levels but increased gastrocnemius muscle weight, cross-sectional area, and grip strength in STZ-induced diabetic mice. Administration of SFe (500 mg/kg) decreased the expression of atrophic factors, such as MuRF1 and atrogin-1, but did not alter the expression of muscle synthetic factors. Further studies showed that SFe administration decreased the expression of KLF15 and p-CREB, which are upstream molecules of atrophic factors. Examination of the expression of molecules involved in autophagy-lysosomal pathways (e.g., p62/SQSTM1, Atg7, Beclin-1, ULK-1, LC3-I, and LC3-II) revealed that SFe administration significantly decreased the expression of p62/SQSTM1, LC3-I, and LC3-II; however, no changes were observed in the expression of Atg7, Beclin-1, or ULK-1. Our results suggest that SFe ameliorated muscle wasting in STZ-induced diabetic mice by decreasing protein degradation via downregulation of the CREB-KLF15-mediated UPS system and the p62/SQSTM1-mediated autophagy-lysosomal pathway.
Assuntos
Autofagia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Diabetes Mellitus Experimental/complicações , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Lisossomos/metabolismo , Atrofia Muscular/tratamento farmacológico , Extratos Vegetais/farmacologia , Schisandra/química , Animais , Frutas/química , Lisossomos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologiaRESUMO
The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a blocker of an emerging class of human intracellular channels, namely the two-pore channel (TPC) family, whose role has been established in several diseases. Indeed, Nar was shown to be effective against neoangiogenesis, a process essential for solid tumor progression, by specifically impairing TPC activity. The goal of the present review is to illustrate the rationale that links TPC channels to the mechanism of coronavirus infection, and how their inhibition by Nar could be an efficient pharmacological strategy to fight the current pandemic plague COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Flavanonas/farmacologia , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Arabidopsis/metabolismo , COVID-19/epidemiologia , COVID-19/patologia , COVID-19/virologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/virologia , Flavanonas/uso terapêutico , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/virologia , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Pandemias/prevenção & controle , SARS-CoV-2/patogenicidade , Vacúolos/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
Bisphenol A (BPA) is a widely distributed environmental endocrine disruptor. The accumulation of BPA has been proved that produce various toxic effects both on human and animals. However, the strategies to reduce the damage of BPA on the body and related mechanisms remain to be studied. Coenzyme Q10 (CoQ10), as a powerful antioxidant, is ubiquitous in many eukaryotic cells, which can improve the integrity of lysosomal membrane, lysosomal degradation function and promote autophagy. Here, we examined the ability of CoQ10 to alleviate oxidative stress and apoptosis in BPA-induced damages in C2C12 cells, and how to alleviate it. Our results showed that BPA treatment significantly reduced cell viability, increased the number of cell apoptosis and ROS production, decreased mitochondrial membrane potential, and inhibited the gene expression of mitochondria biogenesis. Moreover, we demonstrated that exposure to BPA increased expression levels of autophagy protein (LC3-II, p62), inhibited autophagy flux, and disrupted the acidic pH environment of lysosomes. Importantly, CoQ10 supplementation effectively restored these abnormalities caused by BPA. CoQ10 significantly decreased the apoptotic incidence and ROS levels, improved mitochondrial membrane potential. Moreover, CoQ10 improved lysosome function and enhanced autophagy flux. Taken together, our results indicate that CoQ10 supplementation is a feasible and effective way to promote the level of autophagy by improving lysosomal function, thereby reducing the apoptosis caused by BPA accumulation. This study aims to provide evidence for the role of CoQ10 in repairing BPA-induced cell damage in clinical practice.
Assuntos
Antioxidantes/toxicidade , Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Fenóis/toxicidade , Ubiquinona/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular , Lisossomos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Ubiquinona/farmacologiaRESUMO
SCOPE: The muscle loss during aging results from the blunt of protein synthesis and poses threat to the elderly health. This study aims to investigate whether betaine affects muscle loss by improving protein synthesis. METHODS AND RESULTS: Male C57BL/6J mice are raised from age 12 or 15 months. Mice are fed with AIN-93M diet without or with 2% w/v betaine in distilled water as control group or betaine intervention group (Bet), respectively. Betaine supplementation to mice demonstrates better body composition, grip strength, and motor function. Muscle morphology upregulates expression of myogenic regulate factors, and elevates myosin heavy chain and also improves in Bet group. Betaine promotes muscle protein synthesis via tethering mammalian target of rapamycin complex1 protein kinase (mTORC1) on the lysosomal membrane thereby activating mTORC1 signaling. All these effects aforementioned are time-dependent (p < 0.05). Ultrahigh-performance liquid chromatography results show that betaine increases S-adenosyl-l-methionine (SAM) via methionine cycle. SAM sensor-Samtor-overexpression in C2C12 cells could displace mTORC1 from lysosome thereby inhibiting the mTORC1 signaling. Addition of betaine attenuates this inhibition by increasing SAM level and then disrupting interaction of Samtor complex. CONCLUSIONS: These observations indicate that betaine could promisingly promote protein synthesis to delay age-related muscle loss.
Assuntos
Betaína/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metiltransferases/antagonistas & inibidores , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , S-Adenosilmetionina/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Metionina/metabolismo , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann-Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Saponinas/farmacologia , Serina Endopeptidases/metabolismo , Triterpenos/farmacologia , Internalização do Vírus/efeitos dos fármacos , Antivirais/química , COVID-19/metabolismo , Linhagem Celular , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Modelos Moleculares , Platycodon/química , SARS-CoV-2/fisiologia , Saponinas/química , Triterpenos/químicaRESUMO
BACKGROUND: The survival rate and therapeutic options for patients with bladder cancer have improved little in recent decades. Guggulsterone (GS), a phytoestrogen, has been investigated as an anticancer drug in various malignancies. PURPOSE: The present study aimed to evaluate the anticancer effects of E-isomer and Z-isomer GS in the human bladder cancer cell lines TSGH8301 (low-grade) and T24 (high-grade) and their underlying mechanisms. METHODS: The cell survival effect of GS was investigated by the MTT and colony formation assays in bladder cancer cell lines. Flow cytometry was used to analyze the cell cycle and cell death. Migration ability was measured by wound healing and transwell assays. Protein expression was determined by Western blot after GS treatment. The potency of GS on subcutaneous TSGH8301 bladder tumors was evaluated using an in vivo imaging system. RESULTS: E-isomer GS reduced the survival rate of both low- and high-grade human bladder cancer cells. GS caused cell cycle arrest, accompanied by the decrease and increase in cyclin A and p21 levels, respectively. Additionally, caspase-dependent apoptosis was observed following GS treatment. Furthermore, GS treatment downregulated mTOR-Akt signaling and induced autophagy with p62 and LC3ß-II expression. Moreover, the farnesoid X receptor was involved in GS-inhibited cell growth. In addition, GS reduced the migration ability with a decrease in integrin-focal adhesion kinase and myosin light chain. Interestingly, the suppression of GS-mediated migration was prevented by the lysosomal inhibitor ammonium chloride (NH4Cl). GS also reduced TSGH8301 bladder cancer cell progression by increasing the level of p21, cleaved caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and LC3ß-II in vivo. CONCLUSIONS: The current findings suggest that GS treatment may serve as a potential anticancer therapy for different grades of urothelial carcinoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Lisossomos/efeitos dos fármacos , Pregnenodionas/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos Endogâmicos BALB C , Serina-Treonina Quinases TOR/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The clinical use of doxorubicin (DOX) is limited by its cardiotoxicity, which is closely associated with oxidative stress. Xinmailong (XML) is a bioactive peptide extracted from American cockroaches, which has been mainly applied to treat chronic heart failure in China. Our previous study showed that XML attenuates DOX-induced oxidative stress. However, the mechanism of XML in DOX-induced cardiotoxicity remains unclear. Heme oxygenase-1 (HO-1), an enzyme that is ubiquitously expressed in all cell types, has been found to take antioxidant effects in many cardiovascular diseases, and its expression is protectively upregulated under DOX treatment. Lysosome and autophagy are closely involved in oxidative stress as well. It is still unknown whether XML could attenuate doxorubicin-induced lysosomal dysfunction and oxidative stress in H9c2 cells via HO-1. Thus, this study was aimed at investigating the involvement of HO-1-mediated lysosomal function and autophagy flux in DOX-induced oxidative stress and cardiotoxicity in H9c2 cells. Our results showed that XML treatment markedly increased cell proliferation and SOD activity, improved lysosomal function, and ameliorated autophagy flux block in DOX-treated H9c2 cells. Furthermore, XML significantly increased HO-1 expression following DOX treatment. Importantly, HO-1-specific inhibitor (Znpp) or HO-1 siRNA could significantly attenuate the protective effects of XML against DOX-induced cell injury, oxidative stress, lysosomal dysfunction, and autophagy flux block. These results suggest that XML protects against DOX-induced cardiotoxicity through HO-1-mediated recovery of lysosomal function and autophagy flux and decreases oxidative stress, providing a novel mechanism responsible for the protection of XML against DOX-induced cardiomyopathy.
Assuntos
Doxorrubicina/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Heme Oxigenase (Desciclizante)/metabolismo , Lisossomos/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/efeitos adversos , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular , Doxorrubicina/efeitos adversos , Interações Medicamentosas , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Lisossomos/enzimologia , Miócitos Cardíacos/enzimologia , RatosRESUMO
The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson's Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.
Assuntos
Endossomos/efeitos dos fármacos , Indazóis/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Lisossomos/efeitos dos fármacos , Doença de Parkinson/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Avaliação Pré-Clínica de Medicamentos , Endossomos/fisiologia , Mutação com Ganho de Função , Técnicas de Introdução de Genes , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Lisossomos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Mutação Puntual , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Distribuição Aleatória , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Doenças Priônicas/tratamento farmacológico , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Dobramento de Proteína , Animais , Sítios de Ligação , Simulação por Computador , Retículo Endoplasmático/metabolismo , Fibroblastos , Células HEK293 , Humanos , Ligantes , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos TestesRESUMO
A major pathological feature of Parkinson's disease (PD) is the aberrant accumulation of misfolded assemblies of alpha-synuclein (α-Syn). Protein clearance appears as a regulator of the 'α-Syn burden' underlying PD pathogenesis. The picture emerging is that a combination of pathways with complementary roles, including the Proteasome System and the Autophagy-Lysosome Pathway, contributes to the intracellular degradation of α-Syn. This study addresses the mechanisms governing the degradation of α-Syn species seeded by exogenous fibrils in neuronally differentiated SH-SY5Y neuroblastoma cells with inducible expression of α-Syn. Using human α-Syn recombinant fibrils (pre-formed fibrils, PFFs), seeding and aggregation of endogenous Proteinase K (PK)-resistant α-Syn species occurs within a time frame of 6 days, and is still prominent after 12 days of PFF addition. Clearance of α-Syn assemblies in this inducible model was enhanced after switching off α-Syn expression with doxycycline. Lysosomal inhibition led to accumulation of SDS-soluble α-Syn aggregates 6 days after PFF-addition or when switching off α-Syn expression. Additionally, the autophagic enhancer, rapamycin, induced the clearance of α-Syn aggregates 13 days post-PFF addition, indicating that autophagy is the major pathway for aggregated α-Syn clearance. SDS-soluble phosphorylated α-Syn at S129 was only apparent at 7 days of incubation with a higher amount of PFFs. Proteasomal inhibition resulted in further accumulation of SDS-soluble phosphorylated α-Syn at S129, with limited PK resistance. Our data suggest that in this inducible model autophagy is mainly responsible for the degradation of fibrillar α-Syn, whereas the proteasome system is responsible, at least in part, for the selective clearance of phosphorylated α-Syn oligomers.
Assuntos
Lisossomos/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Autofagia , Linhagem Celular , Sobrevivência Celular , Doxiciclina/farmacologia , Humanos , Imuno-Histoquímica , Lisossomos/efeitos dos fármacos , Fosforilação , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , alfa-Sinucleína/antagonistas & inibidoresRESUMO
Recently, we identified a novel mechanism of lipotoxicity in the kidney proximal tubular cells (PTECs); lipid overload stimulates macroautophagy/autophagy for the renovation of plasma and organelle membranes to maintain the integrity of the PTECs. However, this autophagic activation places a burden on the lysosomal system, leading to a downstream suppression of autophagy, which manifests as phospholipid accumulation and inadequate acidification in lysosomes. Here, we investigated whether pharmacological correction by eicosapentaenoic acid (EPA) supplementation could restore autophagic flux and alleviate renal lipotoxicity. EPA supplementation to high-fat diet (HFD)-fed mice reduced several hallmarks of lipotoxicity in the PTECs, such as phospholipid accumulation in the lysosome, mitochondrial dysfunction, inflammation, and fibrosis. In addition to improving the metabolic syndrome, EPA alleviated renal lipotoxicity via several mechanisms. EPA supplementation to HFD-fed mice or the isolated PTECs cultured in palmitic acid (PA) restored lysosomal function with significant improvements in the autophagic flux. The PA-induced redistribution of phospholipids from cellular membranes into lysosomes and the HFD-induced accumulation of SQSTM1/p62 (sequestosome 1), an autophagy substrate, during the temporal and genetic ablation of autophagy were significantly reduced by EPA, indicating that EPA attenuated the HFD-mediated increases in autophagy demand. Moreover, a fatty acid pulse-chase assay revealed that EPA promoted lipid droplet (LD) formation and transfer from LDs to the mitochondria for beta-oxidation. Noteworthy, the efficacy of EPA on lipotoxicity is autophagy-dependent and cell-intrinsic. In conclusion, EPA counteracts lipotoxicity in the proximal tubule by alleviating autophagic numbness, making it potentially suitable as a novel treatment for obesity-related kidney diseases.Abbreviations: 4-HNE: 4-hydroxy-2-nonenal; ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; ATG: autophagy-related; ATP: adenosine triphosphate; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; cKO: conditional knockout; CML: N-carboxymethyllysine; COL1A1: collagen type I alpha 1 chain; COX: cytochrome c oxidase; CTRL: control; DGAT: diacylglycerol O-acyltransferase; EPA: eicosapentaenoic acid; FA: fatty acid; FFA: free fatty acid; GFP: green fluorescent protein; HFD: high-fat diet; iKO: inducible knockout; IRI: ischemia-reperfusion injury; LAMP1: lysosomal-associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor-related protein 2; MAP1LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; OA: oleic acid; PAS: periodic-acid Schiff; PPAR: peroxisome proliferator activated receptor; PPARGC1/PGC1: peroxisome proliferator activated receptor, gamma, coactivator 1; PTEC: proximal tubular epithelial cell; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SDH: succinate dehydrogenase complex; SFC/MS/MS: supercritical fluid chromatography triple quadrupole mass spectrometry; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TG: triglyceride; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Autofagia/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Ácido Eicosapentaenoico/farmacologia , Ácido Eicosapentaenoico/uso terapêutico , Injúria Renal Aguda/induzido quimicamente , Animais , Rim/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Fosfolipídeos/metabolismoRESUMO
Measurement of autophagic flux in vivo is critical to understand how autophagy can be used to combat disease. Neurodegenerative diseases have a special relationship with autophagy, which makes measurement of autophagy in the brain a significant research priority. Currently, measurement of autophagic flux is possible through use of transgenic constructs, or application of a lysosomal inhibitor such as chloroquine. Unfortunately, chloroquine is not useful for measuring autophagic flux in the brain and the use of transgenic animals necessitates cross-breeding of transgenic strains and maintenance of lines, which is costly. To find a drug that could block lysosomal function in the brain for the measurement of autophagic flux, we selected compounds from the literature that appeared to have similar properties to chloroquine and tested their ability to inhibit autophagic flux in cell culture and in mice. These chemicals included chloroquine, quinacrine, mefloquine, promazine and trifluoperazine. As expected, chloroquine blocked lysosomal degradation of the autophagic protein LC3B-II in cell culture. Quinacrine also inhibited autophagic flux in cell culture. Other compounds tested were not effective. When injected into mice, chloroquine caused accumulation of LC3B-II in heart tissue, and quinacrine was effective at blocking LC3B-II degradation in male, but not female skeletal muscle. None of the compounds tested were useful for measuring autophagic flux in the brain. During this study we also noted that the vehicle DMSO powerfully up-regulated LC3B-II abundance in tissues. This study shows that chloroquine and quinacrine can both be used to measure autophagic flux in cells, and in some peripheral tissues. However, measurement of flux in the brain using lysosomal inhibitors remains an unresolved research challenge.
Assuntos
Autofagia/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cloroquina/farmacologia , Lisossomos/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Células HeLa , Humanos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Masculino , Mefloquina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Promazina/farmacologia , Quinacrina/farmacologia , Trifluoperazina/farmacologiaRESUMO
Arsenic is an environmental toxicant. Its overdose can cause liver damage. Autophagy has been reported to be involved in arsenite (iAs3+ ) cytotoxicity and plays a dual role in cell proliferation and cell death. However, the effect and molecular regulative mechanisms of iAs3+ on autophagy in hepatocytes remains largely unknown. Here, we found that iAs3+ exposure lead to hepatotoxicity by inducing autophagosome and autolysosome accumulation. On the one hand, iAs3+ promoted autophagosome synthesis by inhibiting E2F1/mTOR pathway in L-02 human hepatocytes. On the other, iAs3+ blocked autophagosome degradation partially via suppressing the expression of INPP5E and Rab7 as well as impairing lysosomal activity. More importantly, autophagosome and autolysosome accumulation induced by iAs3+ increased the protein level of E2F7a, which could further inhibit cell viability and induce apoptosis of L-02 cells. The treatment of Ginkgo biloba extract (GBE) effectively reduced autophagosome and autolysosome accumulation and thus alleviated iAs3+ -induced hepatotoxicity. Moreover, GBE could also protect lysosomal activity, promote the phosphorylation level of E2F1 (Ser364 and Thr433) and Rb (Ser780) as well as suppress the protein level of E2F7a in iAs3+ -treated L-02 cells. Taken together, our data suggested that autophagosome and autophagolysosome accumulation play a critical role for iAs3+ -induced hepatotoxicity, and GBE is a promising candidate for intervening iAs3+ induced liver damage by regulating E2F1-autophagy-E2F7a pathway and restoring lysosomal activity.
Assuntos
Arsenitos/toxicidade , Autofagia , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F7/metabolismo , Fígado/patologia , Lisossomos/metabolismo , Extratos Vegetais/farmacologia , Transdução de Sinais , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ginkgo biloba , Humanos , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacosRESUMO
The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.
Assuntos
Endocitose , Ferro/farmacologia , Receptores da Transferrina/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Apoferritinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Células HeLa , Células Hep G2 , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Transporte Proteico/efeitos dos fármacos , Transferrina/metabolismoRESUMO
BACKGROUND: Gypenoside (GP) is the major bioactive constituent of G. pentaphyllum, a traditional Chinese medicine. It has been reported that GP can affect autophagy and lipid metabolism in cultured cells. We hypothesize that GP can inhibit foam cell formation in cultured macrophages through autophagy modulation. METHODS: THP1 cells were cultured and treated with oxidized low-density lipoprotein (ox-LDL), followed by GP treatment at different concentrations. The autophagy flux was evaluated using western blot and confocal microscope analyses. The ox-LDL uptake and foam cell formation abilities were measured. RESULTS: We found that ox-LDL impaired the autophagy flux in the cultured macrophages, indicated by a significant reduction of LC3-II and autophagosome puncta quantification, as well as an accumulation of p62 proteins. GP treatment, however, dose-dependently restored the autophagy flux impaired by ox-LDL and reduced the ox-LDL uptake and foam cell transformation from THP1 cells, which can be alleviated, or exacerbated, by modulation of autophagy status using autophagy enhancer or inhibitor. Coimmunoprecipitation assays showed that GP up-regulated Srit1 and FOXO1 expression and enhanced their direct interaction, and thus contributed to the regulation of autophagy. CONCLUSION: GP inhibits ox-LDL uptake and foam cell formation through enhancing Sirt1-FOXO1 mediated autophagy flux restoration, suggesting this compound has therapeutic potential for atherosclerosis.
Assuntos
Autofagia , Células Espumosas/metabolismo , Proteína Forkhead Box O1/metabolismo , Lipoproteínas LDL/metabolismo , Sirtuína 1/metabolismo , Autofagia/efeitos dos fármacos , Células Espumosas/efeitos dos fármacos , Proteína Forkhead Box O1/genética , Gynostemma , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Extratos Vegetais/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirtuína 1/genética , Células THP-1 , Regulação para Cima/efeitos dos fármacosRESUMO
Autophagy is an important tightly controlled cellular process that regulates cellular homeostasis and is involved in deciding cell fate such as cell survival and death. The role of autophagy in many intracellular signaling pathways explains its interaction with other different types of cell death, including apoptosis and immunogenic cell death (ICD). The reports showed the complex and intriguing relationship existing between autophagy and immune system signaling pathways. However, the role of autophagy in ICD remains to be clearly elucidated. In this study, we demonstrated that Brucine, a clinically-used small molecule in traditional Chinese medicine, elicited autophagy inhibition. Brucine also triggered cell stress and induced features of ICD, including calreticulin (CRT) exposure and high-mobility group box 1 (HMGB1) release in MDA-MB-231 and CT26 cancer cells. Brucine impaired autolysosomal degradation and exerted a feedback regulation of ERK1/2-mTOR-p70S6K signaling cascade. Brucine-elicited ICD was confirmed by the rejection of CT26 tumor cells, implanted in the mice after vaccination with Brucine-treated CT26 cells. The impaired autophagy contributed to Brucine-induced ICD, as knock-down of Atg5 significantly reduced Brucine-elicited CRT exposure and HMGB1 release. Our results revealed Brucine as a novel autophagy regulator, ICD inducer and hitherto undocumented role of autophagy in ICD. Thus, these results imply the importance of Brucine in cancer immunotherapy. Therefore, Brucine may be used as an ICD inducer and improve its application in cancer treatment with minimized toxicity.
Assuntos
Autofagia/efeitos dos fármacos , Morte Celular/genética , Morte Celular/imunologia , Medicamentos de Ervas Chinesas , Lisossomos/efeitos dos fármacos , Estricnina/análogos & derivados , Animais , Autofagia/fisiologia , Proteína 5 Relacionada à Autofagia/genética , Calreticulina , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Proteína HMGB1/metabolismo , Humanos , Imunoterapia , Lisossomos/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Neoplasias/tratamento farmacológico , Fitoterapia , Estricnina/farmacologia , Estricnina/uso terapêuticoRESUMO
The first phytochemical investigation of the seeds of Euphorbia peplus led to the isolation and characterization of five new (1-5), named euphopepluanones A-E, and five known diterpenoids (6-10). Their structures were established by extensive spectroscopic analysis and X-ray crystallographic experiments. Euphopepluanones A-E (1-3) feature a very rare 5/11/5-tricyclic skeleton, and euphopepluanones D-E (4-5) represent the first report of lathyrane type diterpenoids found in E. peplus. The new compounds 1-5 were assessed for their activities to induce lysosomal biogenesis through LysoTracker Red staining, in which compounds 1 and 3 could significantly induce lysosomal biogenesis. In addition, compounds 1 and 3 could promote the nuclear translocation of TFEB, a master transcriptional factor of lysosomal genes, indicating that compounds 1 and 3 induced lysosomal biogenesis through activation of TFEB.
Assuntos
Diterpenos/isolamento & purificação , Euphorbia/classificação , Lisossomos/efeitos dos fármacos , Compostos Macrocíclicos/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Sementes/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/química , Células HeLa , Humanos , Compostos Macrocíclicos/metabolismo , Estrutura Molecular , Biogênese de Organelas , Extratos Vegetais/metabolismoRESUMO
PURPOSE: The degradation of drugs within endolysosomes has been widely addressed as a cause of poor bioavailability. One of the strategies to allow molecules to escape from a destructive fate is to introduce a photosensitizing moiety into a drug carrier enabling the permeabilization of endosomes and endolysosomes upon irradiation. This paper presents an alternative delivery nanosystem composed of cost-effective soybean phosphatides mixed with IR-820, a near-infrared (NIR) sensitizer, to load various active compounds and trigger an endolysosomal escape with a low cytotoxic effect. METHODS: IR-820-incorporated phosphatides-based nanoparticles were formulated using a thin-film hydration method to encapsulate different molecular probes and a drug model. The nanoparticles were characterized in vitro using dynamic light scattering, transmission electron microscopy, as well as ultraviolet-visible and fluorescence spectroscopy techniques. The NIR-corresponding generation of the photochemical products, the content release, and the cytotoxicity toward the HaCaT keratinocyte cell line were evaluated. The cellular internalization and endolysosomal escape were monitored using a cytochemical marker and fluorescent probes with a colocalization analysis. RESULTS: The IR-820-combined nanoparticles revealed the NIR-triggered changes in the singlet oxygen presence, nanoparticle architecture, and release rate without being cytotoxic. Additionally, the nanoplatform appeared to enhance cellular uptake of the macromolecules. The localization of the cytochemical marker and the colocalization analysis on the fluorescence signals of the encapsulated fluorophore and the lysosome-labeling reporter implied the transient endolysosomal escape of the cargo within the HaCaT cells after NIR irradiation. CONCLUSION: The inclusion of IR-820 into a soybean-phosphatides base ingredient provides NIR responsiveness, particularly the endolysosomal escape of the payload, to the formulated nanoparticles, while preserving the beneficial properties as a drug carrier. This alternative delivery nanomedicine system has future potential to provide high bioavailability of cytosolic drugs utilizing time- and spatial-controllable NIR triggerability as well as the synergistic therapeutic effects with NIR-biomodulation.