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1.
Phytomedicine ; 109: 154552, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36610157

RESUMO

BACKGROUND: Mitochondrial dynamics plays a crucial role in tubular injury in diabetic kidney disease (DKD). Asiatic acid (AA) has demonstrated renal protective effects in DKD; however, its therapeutic effect on tubular injury in DKD remains unclear. PURPOSE: This study aimed to verify the effects of AA on tubular injury in DKD and underlying mechanisms. STUDY DESIGN: In the present study, the effects of AA on tubular injury were assessed in rats with streptozotocin-induced diabetes and advanced glycation end products (AGEs)-stimulated HK-2 cells models. METHODS: After oral administration with or without AA for ten weeks, body weight and levels of fast blood glucose, serum creatinine (sCr), blood urea nitrogen (BUN), urinary albumin, and kidney injury molecule-1 (KIM-1) were detected. Histological analysis was performed to evaluate the renal function of rats. Moreover, the expression of proteins associated with the Nrf-2 pathway and mitochondrial dynamics was analyzed. AGEs-stimulated HK-2 cells were examined to evaluate the tubular protection and the mechanism of AA in vitro. RESULTS: AA remarkably decreased albumin levels, KIM-1 levels in urine, and serum Cr, and BUN levels. In addition, AA prevented tubular injury and mitochondrial injury by regulating the Nrf-2 pathway and mitochondrial dynamics. Furthermore, the effects of AA on mitochondrial dynamics and tubular protection were eliminated after treatment with ML385 (Nrf2 inhibitor). CONCLUSION: These findings suggested that AA might be developed as a potential candidate for the treatment of tubular injury in DKD, and its effects are potentially mediated via the regulation of the Nrf-2 pathway and mitochondrial dynamics.


Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Ratos , Animais , Nefropatias Diabéticas/metabolismo , Túbulos Renais , Dinâmica Mitocondrial , Rim/patologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Albuminas/metabolismo , Produtos Finais de Glicação Avançada/metabolismo
2.
Int J Mol Sci ; 24(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36675044

RESUMO

Free drug concentration in the blood sera is crucial for its appropriate activity. Serum albumin, the universal blood carrier protein, is responsible for transporting drugs and releasing them into the bloodstream. Therefore, a drug's binding to SA is especially important for its bioavailability and it is a key problem in the drug design process. In this paper, we present crystal structures of three animal serum albumin complexes: ovine, caprine, and leporine, with diclofenac, a popular non-steroidal anti-inflammatory drug that is used in therapy of chronic and acute pain. Details of diclofenac binding mode by the presented serum albumins are compared with analogous complexes of human and equine serum albumins. The analysis of the occupied binding pockets in crystal structures of the investigated serum albumins from different mammals shows that they have two common and a number of unique diclofenac binding sites. The most intriguing is the fact that the albumins from the described species are able to bind different numbers of molecules of this popular anti-inflammatory drug, but none of the binding sites overlap with ones in the human serum albumin.


Assuntos
Diclofenaco , Albumina Sérica , Animais , Ovinos , Cavalos , Humanos , Diclofenaco/química , Albumina Sérica/metabolismo , Cabras/metabolismo , Anti-Inflamatórios não Esteroides/química , Sítios de Ligação , Albuminas/metabolismo , Ligação Proteica
3.
Cells ; 11(23)2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36496994

RESUMO

Availability of oxygen plays an important role in tissue organization and cell-type specific metabolism. It is, however, difficult to analyze hypoxia-related adaptations in vitro because of inherent limitations of experimental model systems. In this study, we establish a microfluidic tissue culture protocol to generate hypoxic gradients in vitro, mimicking the conditions found in the liver acinus. To accomplish this, four microfluidic chips, each containing two chambers, were serially connected to obtain eight interconnected chambers. HepG2 hepatocytes were uniformly seeded in each chamber and cultivated under a constant media flow of 50 µL/h for 72 h. HepG2 oxygen consumption under flowing media conditions established a normoxia to hypoxia gradient within the chambers, which was confirmed by oxygen sensors located at the inlet and outlet of the connected microfluidic chips. Expression of Hif1α mRNA and protein was used to indicate hypoxic conditions in the cells and albumin mRNA and protein expression served as a marker for liver acinus-like zonation. Oxygen measurements performed over 72 h showed a change from 17.5% to 15.9% of atmospheric oxygen, which corresponded with a 9.2% oxygen reduction in the medium between chamber1 (inlet) and 8 (outlet) in the connected microfluidic chips after 72 h. Analysis of Hif1α expression and nuclear translocation in HepG2 cells additionally confirmed the hypoxic gradient from chamber1 to chamber8. Moreover, albumin mRNA and protein levels were significantly reduced from chamber1 to chamber8, indicating liver acinus zonation along the oxygen gradient. Taken together, microfluidic cultivation in interconnected chambers provides a new model for analyzing cells in a normoxic to hypoxic gradient in vitro. By using a well-characterized cancer cell line as a homogenous hepatocyte population, we also demonstrate that an approximate 10% reduction in oxygen triggers translocation of Hif1α to the nucleus and reduces albumin production.


Assuntos
Fígado , Oxigênio , Humanos , Oxigênio/metabolismo , Fígado/metabolismo , Hipóxia/metabolismo , RNA Mensageiro/metabolismo , Albuminas/metabolismo
4.
BMC Neurosci ; 23(1): 66, 2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36384553

RESUMO

AIMS: Esketamine upregulates Zn2+-dependent matrix metalloproteinase 9 (MMP9) and increases the neuronal apoptosis in retinal ganglion cell layer during the early development. We aimed to test whether albumin can alleviate esketamine-induced apoptosis through downregulating Zn2+-dependent MMP9. METHODS: We investigate the role of Zn2+ in esketamine-induced neuronal apoptosis by immunofluorescence. MMP9 protein expression and enzyme activity were investigated by zymography in situ., western blot and immunofluorescence. Whole-mount retinas from P7 Sprague-Dawley rats were used. RESULTS: We demonstrated that esketamine exposure increased Zn2+ in the retinal GCL during the early development. Zn2+-dependent MMP9 expression and enzyme activity up-regulated, which eventually aggravated apoptosis. Albumin effectively down-regulated MMP9 expression and activity via binding of free zinc, ultimately protected neurons from apoptosis. Meanwhile albumin treatment promoted activated microglia into multi-nucleated macrophagocytes and decreased the inflammation. CONCLUSION: Albumin alleviates esketamine-induced neuronal apoptosis through decreasing Zn2+ accumulation in GCL and downregulating Zn2+-dependent MMP9.


Assuntos
Metaloproteinase 9 da Matriz , Retina , Ratos , Animais , Metaloproteinase 9 da Matriz/metabolismo , Regulação para Baixo , Ratos Sprague-Dawley , Retina/metabolismo , Apoptose , Albuminas/metabolismo , Albuminas/farmacologia , Zinco/farmacologia
5.
J Control Release ; 352: 1009-1023, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36375619

RESUMO

Kidney diseases are a serious health issue worldwide, and novel therapeutics are urgently needed. Extracellular vesicles (EVs) have emerged as potent drug delivery systems (DDSs), but their therapeutic potential is limited by short circulation times and insufficient renal retention. Here, we report that endogenous ligand (albumin, ALB) binding is an efficient modification strategy to improve the therapeutic potency of EV-based DDSs for kidney diseases. Surface albumin-binding peptide (ABP)-displayed EVs (ABP-EVs) were produced by transfecting parent cells with the ABP-Lamp2b fusion plasmid. Compared with unmodified EVs (NC-EVs), ABP-EVs showed increased binding to ALB in vitro and elevated circulation time and multiple organ retention in vivo after systemic (iv) injection. Moreover, ABP-EVs had higher renal retention than NC-EVs in mice with acute kidney injury through a complex mechanism involving microvascular injury and megalin-mediated endocytosis. As a result, delivery of small molecule drugs (e.g., curcumin) or proteins (e.g., hepatocyte growth factor) by ABP-EVs had superior therapeutic (e.g., anti-apoptotic, antioxidant, anti-inflammatory) effects in vitro and in vivo. This study highlights that ABP-EVs are versatile DDSs for kidney diseases and provides insights into the new strategies of engineering EVs for drug delivery.


Assuntos
Vesículas Extracelulares , Nefropatias , Camundongos , Animais , Ligantes , Vesículas Extracelulares/metabolismo , Rim , Nefropatias/tratamento farmacológico , Nefropatias/metabolismo , Peptídeos/metabolismo , Albuminas/metabolismo
6.
J Insect Physiol ; 143: 104453, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36341969

RESUMO

Insect nephrocytes are ultrafiltration cells that remove circulating proteins and exogenous toxins from the haemolymph. Experimental disruption of nephrocyte development or function leads to systemic impairment of insect physiology as evidenced by cardiomyopathy, chronic activation of immune signalling and shortening of lifespan. The genetic and structural basis of the nephrocyte's ultrafiltration mechanism is conserved between arthropods and mammals, making them an attractive model for studying human renal function and systemic clearance mechanisms in general. Although dynamic changes to intracellular calcium are fundamental to the function of many cell types, there are currently no studies of intracellular calcium signalling in nephrocytes. In this work we aimed to characterise calcium signalling in the pericardial nephrocytes of Drosophila melanogaster. To achieve this, a genetically encoded calcium reporter (GCaMP6) was expressed in nephrocytes to monitor intracellular calcium both in vivo within larvae and in vitro within dissected adults. Larval nephrocytes exhibited stochastically timed calcium waves. A calcium signal could be initiated in preparations of adult nephrocytes and abolished by EGTA, or the store operated calcium entry (SOCE) blocker 2-APB, as well as RNAi mediated knockdown of the SOCE genes Stim and Orai. Neither the presence of calcium-free buffer nor EGTA affected the binding of the endocytic cargo albumin to nephrocytes but they did impair the subsequent accumulation of albumin within nephrocytes. Pre-treatment with EGTA, calcium-free buffer or 2-APB led to significantly reduced albumin binding. Knock-down of Stim and Orai was non-lethal, caused an increase to nephrocyte size and reduced albumin binding, reduced the abundance of the endocytic cargo receptor Amnionless and disrupted the localisation of Dumbfounded at the filtration slit diaphragm. These data indicate that pericardial nephrocytes exhibit stochastically timed calcium waves in vivo and that SOCE mediates the localisation of the endocytic co-receptor Amnionless. Identifying the signals both up and downstream of SOCE may highlight mechanisms relevant to the renal and excretory functions of a broad range of species, including humans.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Humanos , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Ácido Egtázico/metabolismo , Endocitose , Sinalização do Cálcio , Larva/metabolismo , Albuminas/metabolismo , Mamíferos/metabolismo
7.
Lipids Health Dis ; 21(1): 115, 2022 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-36335399

RESUMO

BACKGROUND: Compared with typical visceral fat deposits in obesity and metabolic syndrome, perirenal adipose tissue (PRAT) dysfunction is more closely linked to obesity-related chronic kidney disease (OB-CKD). The myokine irisin reportedly promotes positive outcomes in metabolic disease. This study investigated whether irisin could reduce urinary albumin excretion and demonstrate renoprotective effects through the regulation of PRAT function in obese mice. METHODS: C57BL/6 J mice received a high-fat diet (HFD) with or without concurrent administration of irisin. Glucose tolerance, plasma levels of free fatty acids, and urinary albumin excretion were assessed, along with renal morphology. The vascular endothelial growth factor and nitric oxide in glomeruli were also analyzed, in addition to PRAT function-associated proteins. RESULTS: Irisin administration significantly reduced the final body weight, fat mass, and free fatty acids, without reducing PRAT mass, in HFD mice. Furthermore, irisin decreased urinary albumin excretion and attenuated both renal fibrosis and lipid accumulation. Irisin administration led to increases in PRAT function-associated proteins, including sirtuin1, uncoupling protein-1, and heme-oxygenase-1. Ex vivo treatment of PRAT and glomeruli with irisin also restored PRAT function. Finally, irisin treatment restored the vascular endothelial growth factor-nitric oxide axis. CONCLUSIONS: Irisin attenuated metabolic disorders and protected against OB-CKD by normalizing the PRAT-kidney axis. These results suggest that agents targeting PRAT activation might be useful for treatment of OB-CKD.


Assuntos
Fibronectinas , Insuficiência Renal Crônica , Camundongos , Animais , Camundongos Obesos , Óxido Nítrico/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Tecido Adiposo/metabolismo , Dieta Hiperlipídica/efeitos adversos , Rim/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/prevenção & controle , Insuficiência Renal Crônica/metabolismo , Albuminas/metabolismo
8.
Free Radic Biol Med ; 193(Pt 2): 551-566, 2022 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-36336230

RESUMO

Protein modification occurs in biological milieus that are characterized by high concentrations of (macro)molecules (i.e. heterogeneous and packed environments). Recent data indicate that crowding can modulate the extent and rate of protein oxidation, however its effect on other post-translational modifications remains to be explored. In this work we hypothesized that crowding would affect the glycation of plasma proteins. Physiologically-relevant concentrations of albumin (35 mg mL-1) and transferrin (2 mg mL-1) were incubated with methylglyoxal and glyoxal (5 µM-5 mM), two α-oxoaldehyde metabolites that are elevated in the plasma of people with diabetes. Crowding was induced by adding dextran or ficoll polymers. Electrophoresis, electron microscopy, fluorescence spectroscopy and mass spectrometry were employed to investigate the structural consequences of glycation under crowded conditions. Our data demonstrate that crowding modulates the extent of formation of transferrin cross-links, and also the modification pathways in both albumin and transferrin. Arginine was the most susceptible residue to modification, with lysine and cysteine also affected. Loss of 0.48 and 7.28 arginine residues per protein molecule were determined on incubation with 500 µM methylglyoxal for albumin and transferrin, respectively. Crowding did not influence the extent of loss of arginine and lysine for either protein, but the sites of modification, detected by LC-MS, were different between dilute and crowded conditions. These data confirm the relevance of studying modification processes under conditions that closely mimic biological milieus. These data unveil additional factors that influence the pattern and extent of protein modification, and their structural consequences, in biological systems.


Assuntos
Lisina , Aldeído Pirúvico , Humanos , Aldeído Pirúvico/metabolismo , Lisina/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Transferrina , Arginina/metabolismo , Albuminas/metabolismo , Proteínas Sanguíneas/metabolismo
9.
J Diabetes Res ; 2022: 3411123, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36330072

RESUMO

Aim: Patients with diabetic ketosis often exhibit albuminuria. We previously found that acute hyperglycaemia can cause nephrotoxic injury. Here, we explored whether an excessive ketone body level causes kidney injury and the potential underlying mechanism. Methods: Fifty-six type 2 diabetes without ketosis (NDK group), 81 type 2 diabetes with ketosis (DK group), and 38 healthy controls (NC group) were enrolled. Clinical data were collected before and after controlling diabetic ketosis. Beta-hydroxybutyric acid (BOHB), an AKT activator, an AKT inhibitor, or plasmids encoding DAB2 were transformed into human renal proximal tubule epithelial cells (HK-2 cells). Results: The urinary albumin-to-creatinine ratio (ACR), transferrin (TF), immunoglobulin G (IgG), Beta2-microglobulin (ß2-MG), retinol-binding protein (RBP), N-acetyl-beta-glucosaminidase (NAG), and Beta-galactosidase (GAL) were higher in the DK than NC and NDK groups. The proportion of patients with an increased urinary level of TF, IgG, ß2-MG, RBP, NAG, or GAL was higher in the DK group too. After controlling ketosis, urinary microalbumin, TF, IgG, ß2-MG, and RBP decreased significantly. In HK-2 cells, albumin endocytosis and megalin expression decreased with increasing BOHB concentration. Compared with BOHB treatment, BOHB with AKT activator significantly increased the DAB2, megalin levels and albumin endocytosis; the AKT inhibitor treatment exhibited the opposite effects. Compared with BOHB treatment, megalin expression and albumin endocytosis were significantly increased after BOHB with DAB2 overexpression treatment. Conclusions: Patients with diabetic ketosis may suffer from glomerular and tubular injuries that recover after ketosis control. High concentrations of BOHB downregulate megalin expression by inhibiting the AKT/DAB2/megalin signalling pathway and albumin endocytosis in proximal renal tubules.


Assuntos
Diabetes Mellitus Tipo 2 , Cetoacidose Diabética , Humanos , Ácido 3-Hidroxibutírico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Albuminas/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Imunoglobulina G/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
10.
Nutrients ; 14(19)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36235547

RESUMO

INTRODUCTION: Observational studies reported inverse associations between serum total 25-hydroxyvitamin D (25(OH)D) concentrations and mortality. Evolving evidence indicated, however, that bioavailable or free 25(OH)D may be even better predictors of mortality. We conducted a systematic review and meta-analysis to summarize the epidemiological evidence on associations of vitamin D-binding protein (VDBP), albumin-bound, bioavailable, and free 25(OH)D, with mortality. METHODS: We systematically searched PubMed and Web of Science, up to 27 May 2022. Predictors of interest included serum or plasma concentrations of VDBP, albumin-bound, bioavailable, and free 25(OH)D. Assessed health outcomes were all-cause and cause-specific mortality. We included studies reporting associations between these biomarkers and mortality outcomes. We applied random-effects models for meta-analyses to summarize results from studies assessing the same vitamin D biomarkers and mortality outcomes. RESULTS: We identified twelve eligible studies, including ten on VDBP, eight on bioavailable 25(OH)D, and eight on free 25(OH)D. No study reported on albumin-bound 25(OH)D and mortality. In meta-analyses, the highest levels of bioavailable and free 25(OH)D were associated with 37% (hazard ratio (HR): 0.63, 95% confidence interval (CI): 0.46, 0.87), and 29% (HR: 0.71, 95% CI: 0.53, 0.97) decrease in all-cause mortality, respectively, compared with the lowest levels. These estimates were similar to those for total 25(OH)D (HR: 0.67, 95% CI: 0.56, 0.80) observed in the same studies. Higher VDBP levels were associated with lower all-cause mortality in cancer patient cohorts. However, no such association was observed in general population cohorts. CONCLUSIONS: Similar inverse associations of total, bioavailable, and free 25(OH)D with mortality suggest that bioavailable and free 25(OH)D do not provide incremental value in predicting mortality.


Assuntos
Deficiência de Vitamina D , Proteína de Ligação a Vitamina D , Albuminas/metabolismo , Biomarcadores , Humanos , Vitamina D/análogos & derivados , Deficiência de Vitamina D/complicações
11.
Sci Rep ; 12(1): 17961, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289430

RESUMO

Nanoparticles in medicine can integrate actively targeted imaging agents and drug delivery vehicles, and combining multiple types of therapeutics in a single particle has numerous advantages, especially in multiple myeloma. MM is an incurable hematological disorder characterized by clonal proliferation of plasma cells in the bone marrow. In this study, we evaluated the anti-myeloma activity of 3 nanocomposites (3NPs): As4S4/ZnS/Fe3O4 (1:4:1), As4S4/ZnS/Fe3O4 with folic acid (FA), and As4S4/ZnS/Fe3O4 with FA and albumin with reduced survival MM cell lines and primary MM samples by each of 3NP. Cytotoxic effects of 3NPs were associated with caspase- and mitochondria-dependent apoptosis induction and reduced c-Myc expression. Modulation of cell cycle regulators, such as p-ATM/ATM and p-ATR/ATR, and increases in p-Chk2, cyclin B1, and histones were accompanied by G2/M arrest triggered by 3NPs. In addition, 3NPs activated several myeloma-related signaling, including JNK1/2/3, ERK1/2 and mTOR. To overcome BM microenvironment-mediated drug resistance, nanocomposites retained its anti-MM activity in the presence of stroma. 3NPs significantly decreased the stem cell-like side population in MM cells, even in the context of stroma. We observed strong synergistic effects of 3NPs combined with lenalidomide, pomalidomide, or melphalan, suggesting the potential of these combinations for future clinical studies.


Assuntos
Mieloma Múltiplo , Nanocompostos , Humanos , Albuminas/metabolismo , Apoptose , Caspases/metabolismo , Linhagem Celular Tumoral , Ciclina B1/metabolismo , Ácido Fólico/farmacologia , Histonas/farmacologia , Lenalidomida/farmacologia , Melfalan/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Microambiente Tumoral
12.
BMC Vet Res ; 18(1): 371, 2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253770

RESUMO

BACKGROUND: Abnormal or stereotyped behaviours in dairy cows are common in large-scale indoor farms and are usually accompanied by high physiological stress levels. Feed tossing is an abnormal behaviour commonly seen in cows while being fed, making farm management difficult. However, the reasons behind this behaviour have not been sufficiently reported. The objective of this study was to explore the changes in rumen fermentation, serum indicators, inflammatory conditions and the performance of cows with feed tossing behaviour. Holstein cows with similar lactation stages in the same barn were subjected to behaviour observations two times per day for 21 consecutive days. Ten cows with feed tossing behaviour (FT) and ten cows without abnormal behaviours (CON) were selected for further sampling. Plasma samples, rumen fluid, milk yield data of cows, and an indoor environment temperature-humidity index (THI) were collected. RESULTS: There was no significant difference in average daily milk yield during the observation period between feed-tossing cows (n = 68) and the other cows (n = 112). The number of cows showing FT behaviour had a moderately strong negative linear correlation with the THI of the environment. Compared to the CON cows, the FT cows had higher cortisol, norepinephrine and urea nitrogen levels in plasma, as well as higher plasma levels of inflammatory indicators, including total protein, lactate dehydrogenase, albumin, aspartate aminotransferase levels, and the ratio of aspartate aminotransferase to alanine aminotransferase. The FT cows had no significant variations from the CON cows regarding their rumen fermentation indicators, such as pH, ammonia nitrogen, and volatile fatty acids. In addition, 16S rRNA analysis revealed that there might be no clear association between the diversity and abundance of rumen bacteria and feed tossing behaviour. CONCLUSIONS: Our findings suggested that cows might have suffered from high levels of physiological stress and immune state for a long period when they exhibited FT behaviour. The environmental THI could affect the FT behaviour of cows; as the THI increases, the willingness of cows to throw decreases. This work provided the first evidence that feed tossing might be a response associated with high levels of physiological stress and immune. It also explored our insights into a commonly observed behavioural response to cow welfare traits.


Assuntos
Amônia , Rúmen , Alanina Transaminase , Albuminas/metabolismo , Amônia/análise , Ração Animal/análise , Animais , Aspartato Aminotransferases , Bovinos , Dieta/veterinária , Ácidos Graxos Voláteis/metabolismo , Feminino , Fermentação , Hidrocortisona/metabolismo , Lactato Desidrogenases/metabolismo , Lactação , Leite/química , Nitrogênio/metabolismo , Norepinefrina , RNA Ribossômico 16S , Rúmen/metabolismo , Estresse Fisiológico , Ureia/análise
13.
Int J Mol Sci ; 23(19)2022 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-36232524

RESUMO

Methamphetamine (METH) is a highly addictive drug abused by millions of users worldwide, thus becoming a global health concern with limited management options. The inefficiency of existing treatment methods has driven research into understanding the mechanisms underlying METH-induced disorders and finding effective treatments. This study aims to understand the complex interactions of the gastrointestinal-immune-nervous systems following an acute METH dose administration as one of the potential underlying molecular mechanisms concentrating on the impact of METH abuse on gut permeability. Findings showed a decreased expression of tight junction proteins ZO-1 and EpCAm in intestinal tissue and the presence of FABP-1 in sera of METH treated mice suggests intestinal wall disruption. The increased presence of CD45+ immune cells in the intestinal wall further confirms gut wall inflammation/disruption. In the brain, the expression of inflammatory markers Ccl2, Cxcl1, IL-1ß, TMEM119, and the presence of albumin were higher in METH mice compared to shams, suggesting METH-induced blood-brain barrier disruption. In the spleen, cellular and gene changes are also noted. In addition, mice treated with an acute dose of METH showed anxious behavior in dark and light, open field, and elevated maze tests compared to sham controls. The findings on METH-induced inflammation and anxiety may provide opportunities to develop effective treatments for METH addiction in the future.


Assuntos
Transtornos Relacionados ao Uso de Anfetaminas , Estimulantes do Sistema Nervoso Central , Metanfetamina , Albuminas/metabolismo , Transtornos Relacionados ao Uso de Anfetaminas/metabolismo , Animais , Ansiedade , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Molécula de Adesão da Célula Epitelial/metabolismo , Inflamação/metabolismo , Metanfetamina/metabolismo , Metanfetamina/toxicidade , Camundongos
14.
Int J Mol Sci ; 23(19)2022 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-36232707

RESUMO

Hepatic fibrosis is a form of irregular wound-healing response with acute and chronic injury triggered by the deposition of excessive extracellular matrix. Epithelial-mesenchymal transition (EMT) is a dynamic process that plays a crucial role in the fibrogenic response and pathogenesis of liver fibrosis. In the present study, we postulated a protective role of 3,3'-diindolylmethane (DIM) against TGF-ß1 mediated epithelial-mesenchymal transition (EMT) in vitro and carbon tetrachloride (CCl4)-induced liver fibrosis in mice. TGF-ß1-induced AML-12 hepatocyte injury was evaluated by monitoring cell morphology, measuring reactive oxygen species (ROS) and mitochondrial membrane potential, and quantifying apoptosis, inflammatory, and EMT-related proteins. Furthermore, CCl4-induced liver fibrosis in mice was evaluated by performing liver function tests, including serum ALT and AST, total bilirubin, and albumin to assess liver injury and by performing H&E and Sirius red staining to determine the degree of liver fibrosis. Immunoblotting was performed to determine the expression levels of inflammation, apoptosis, and Nrf2/HO-1 signaling-related proteins. DIM treatment significantly restored TGF-ß1-induced morphological changes, inhibited the expression of mesenchymal markers by activating E-cadherin, decreased mitochondrial membrane potential, reduced ROS intensity, and upregulated levels of Nrf2-responsive antioxidant genes. In the mouse model of CCl4-induced liver fibrosis, DIM remarkably attenuated liver injury and liver fibrosis, as reflected by the reduced ALT and AST parameters with increased serum Alb activity and fewer lesions in H&E staining. It also mitigated the fibrosis area in Sirius red and Masson staining. Taken together, our results suggest a possible molecular mechanism of DIM by suppressing TGF-ß1-induced EMT in mouse hepatocytes and CCl4-induced liver fibrosis in mice.


Assuntos
Tetracloreto de Carbono , Fator de Crescimento Transformador beta1 , Albuminas/metabolismo , Animais , Antioxidantes/farmacologia , Bilirrubina/metabolismo , Caderinas/metabolismo , Tetracloreto de Carbono/toxicidade , Transição Epitelial-Mesenquimal , Fator de Transcrição de Proteínas de Ligação GA , Hepatócitos/metabolismo , Indóis , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
15.
J Control Release ; 351: 581-596, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36181916

RESUMO

Nanotechnology-enabled ferroptosis therapy is an emerging paradigm for tumor treatment, but amplifying ferroptotic damage in tumor cells in a safe and selective manner is still challenging, which severely hinders its clinical translation. In this study, we constructed a bio-inspired protein nanocomplex based on natural-occurring bovine serum albumin (BSA) and ferritin for efficient tumor elimination via cooperatively enhanced ferroptosis therapy. The long-circulating BSA molecules provided multiple anchoring points for the efficient loading of the GPX4-inhibiting ferroptosis inducer (1S, 3R) RAS-selective lethal 3 (RSL3), which was further complexed with ferritin via acidity-responsive glutaraldehyde linkers. The ferritin moieties may not only bind to transferrin receptor 1 overexpressed on tumor cell membrane for targeted endocytic uptake but also be degraded in lysosomes to induce iron overload, which could substantially promote the lipid peroxidation in tumor cells and cooperate with the glutathione peroxidase 4 (GPX4)-inhibiting capability of RSL3 to induce pronounced ferroptosis. The in vitro and in vivo results collectively demonstrated that the albumin-ferritin-based nanocomplex could present superior antitumor effects with no obvious adverse effects, which may open new avenues for the clinical translation of ferroptosis-dependent therapeutic modalities.


Assuntos
Ferroptose , Ferritinas , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Morte Celular , Albuminas/metabolismo
16.
Nat Commun ; 13(1): 5943, 2022 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-36209212

RESUMO

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.


Assuntos
COVID-19 , Doenças Transmissíveis , Albuminas/metabolismo , Diferenciação Celular/fisiologia , Cisplatino/metabolismo , Cisplatino/farmacologia , Doenças Transmissíveis/metabolismo , Humanos , Rim , Néfrons/metabolismo , Organoides/metabolismo , SARS-CoV-2
17.
Front Endocrinol (Lausanne) ; 13: 960835, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36237185

RESUMO

Objective: To investigate the effects and mechanism of hyperinsulinemia on the metabolic switch to ß-hydroxybutyrate (BHB) absorption and utilization under a starvation or hypoxic environment in proximal tubular epithelial cells. Methods: A high-fat diet-induced hyperinsulinemia model in ZDF rats was used to test the expression of key enzymes/proteins of ketone body metabolism in the kidney. Notably, 12-week-old renal tubule SMCT1 specific knockout mice (SMCT1 flox/floxCre+) and control mice (SMCT1 flox/floxCre-) were used to confirm the roles of SMCT1 in kidney protection under starvation. The changes of key enzymes/proteins of energy metabolism, mitochondrial function, and albumin endocytosis in HK2 cells under low glucose/hypoxic environments with or without 50 ng/mL insulin were studied. Silent information regulation 2 homolog 3 (SIRT3) was overexpressed to evaluate the effect of hyperinsulinemia on the metabolic switch to BHB absorption and utilization through the SIRT3/SMCT1 pathway in HK2 cells. Results: In ZDF rats, the expression of HMGCS2 increased, the SMCT1 expression decreased, while SCOT remained unchanged. In renal tubule SMCT1 gene-specific knockout mice, starvation for 48 h induced an increase in the levels of urine retinol-binding protein, N-acetyl-ß-glucosaminidase, and transferrin, which reflected tubular damages. In HK2 cells under an environment of starvation and hypoxia, the levels of key enzymes related to fatty acid oxidation and ketone body metabolism were increased, whereas glucose glycolysis did not change. The addition of 2 mmol/l BHB improved ATP production, mitochondrial biosynthesis, and endocytic albumin function, while cell apoptosis was reduced in HK2 cells. The addition of 50 ng/ml insulin resulted in the decreased expression of SMCT1 along with an impaired mitochondrial function, decreased ATP production, and increased apoptosis. The overexpression of SIRT3 or SMCT1 reversed these alterations induced by a high level of insulin both in low-glucose and hypoxic environments. Conclusions: The increased absorption and utilization of BHB is part of the metabolic flexibility of renal tubular epithelial cells under starvation and hypoxic environments, which exhibits a protective effect on renal tubular epithelial cells by improving the mitochondrial function and cell survival. Moreover, hyperinsulinemia inhibits the absorption of BHB through the inhibition of the SIRT3/SMCT1 pathway.


Assuntos
Hiperinsulinismo , Sirtuína 3 , Inanição , Ácido 3-Hidroxibutírico , Trifosfato de Adenosina , Albuminas/metabolismo , Animais , Células Epiteliais/metabolismo , Glucose/metabolismo , Hexosaminidases/metabolismo , Insulina/metabolismo , Corpos Cetônicos , Camundongos , Camundongos Knockout , Ratos , Proteínas de Ligação ao Retinol , Sirtuína 3/metabolismo , Transferrinas
18.
Int J Nanomedicine ; 17: 4743-4756, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36238535

RESUMO

Introduction: Plasma albumins as protein nanoparticles (PNs) exert essential functions in the control of biological osmotic pressure (OP), being involved in regulating water metabolism, cell morphology and cell tension. Understanding how plasma albumins and different electrolytes co-determine biological OP effects is crucial for correct interpretation of hemodynamic disorders, and practical treatment of hypo/hyper-proteinemia. Methods: Optical measurement based on intermediate filament (IF) tension probe was used for real-time evaluation of transmembrane osmotic effects in live cells. Ion fluorescent probes were employed to evaluate intracellular ion levels, and a current clamp was used to measure membrane potential, thus exploring association of electrochemical and osmotic effects. Results: Albumins are involved in regulation of intracellular osmolarity by a quantitative relationship. Extracellular PNs can alter membrane potentials by adsorbing counterions, induce production of intracellular PNs and further control the opening of ion channels and ion flow, contributing to electrochemical and osmotic re-equilibrium. Furthermore, various ions interplay with extracellular PNs, showing different osmotic effects: increased levels of calcium ions result in a hypotonic effect, whereas potassium ions induce hyper-osmolarity. Conclusion: Extracellular PNs and Ca2+/K+ display antagonistic or synergetic effects in regulating biological OP. Live cells can spontaneously regulate osmotic effects through changing membrane potential and controlling intracellular ion content. Various plasma components need to be comprehensively analyzed, further developing a diagnostic index that considers the biological OP effects of various blood components and improves the evaluation of symptoms and diseases, such as calcium/potassium-hemodynamic disorders and edema.


Assuntos
Albuminas , Nanopartículas , Albuminas/metabolismo , Água Corporal/metabolismo , Cálcio/metabolismo , Corantes Fluorescentes , Humanos , Canais Iônicos , Íons , Pressão Osmótica , Potássio/metabolismo
19.
J Immunol Res ; 2022: 3610935, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36249425

RESUMO

Objective: Diabetic nephropathy (DN), a diabetes-induced chronic complication, is the major trigger of end-stage renal disease. As the main active ingredient of Panax notoginseng (PNG), Panax notoginseng saponins (PNS) are crucial in treating renal diseases. This study is aimed at investigating the role played by PNS in renal protection and antioxidative stress (OS) in DN mice. Methods: A DN mouse model was constructed, and then low, medium, and high doses of PNS were used to intervene the model group mice. Eight weeks after intervention, the 24 h urine protein (UPro) and urinary albumin (UAlb) were quantitatively examined, and the related blood biochemical indices were measured. HE and PAS staining were performed for pathological changes of renal tissue. ELISA and western blotting were carried out to quantify the levels of OS indexes and inflammatory factors (IFs) in mouse kidney tissues and the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), respectively. Results: The weight of DN mice decreased first compared with control animals and then gradually increased after different doses of PNS treatment. Besides, DN mice presented elevated urine volume, UPro, and UAlb, all of which were reversed by PNS intervention. SOD activity and GSH content in renal tissues of the model group mice decreased markedly versus the control group, and MDA, CRP, IL-6, and TGF-ß1 contents elevated statistically, while different doses of PNS effectively reduced the OS injury and IFs in mice. Compared with the model group, PNS dose-dependently increased Nrf2 and HO-1 levels in DN mice. Conclusions: PNS is protective of HFF + STZ-induced DN mice against kidney tissue damage and can reduce the excretion of UPro and relieve the OS state of mice, possibly by activating Nrf2/HO-1 axis to play an antioxidant and anti-inflammatory role.


Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Panax notoginseng , Saponinas , Albuminas/metabolismo , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Diabetes Mellitus/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Heme Oxigenase-1/metabolismo , Interleucina-6/metabolismo , Rim/patologia , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Panax notoginseng/química , Saponinas/farmacologia , Saponinas/uso terapêutico , Superóxido Dismutase , Fator de Crescimento Transformador beta1/metabolismo
20.
Sci Rep ; 12(1): 17663, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36271135

RESUMO

Urine features an ideal source of non-invasive diagnostic markers. Some intrinsic and methodological issues still pose barriers to its full potential as liquid biopsy substrate. Unlike blood, urine concentration varies with nutrition, hydration and environmental factors. Urine is enriched with EVs from urinary-genital tract, while its conservation, purification and normalization can introduce bias in analysis of EV subsets in inter-and intra-individual comparisons. The present study evaluated the methods that decrease such biases such as appropriate and feasible urine storage, optimal single-step EV purification method for recovery of proteins and RNAs from small urine volumes and a normalization method for quantitative analysis of urine EV RNAs. Ultracentrifugation, chemical precipitation and immuno-affinity were used to isolate EVs from healthy donors' urine that was stored frozen or at room temperature for up to 6 months. Multiple urine biochemical and EV parameters, including particle count and protein content, were compared across urine samples. To this purpose nanoparticle tracking analysis (NTA) and protein assessment by BCA, ELISA and WB assays were performed. These measurements were correlated with relative abundances of selected EV mRNAs and miRNAs assessed by RT-PCR and ranked for the ability to reflect and correct for EV content variations in longitudinal urine samples. All purification methods enabled recovery and downstream analysis of EVs from as few as 1 ml of urine. Our findings highlight long term stability of EV RNAs upon urine storage at RT as well as excellent correlation of EV content in urine with some routinely measured biochemical features, such as total urine protein and albumin, but not creatinine most conventionally used for urine normalization. Comparative evaluation of mRNA and miRNAs in EV isolates revealed specific RNAs, in particular RNY4 and small miRNA panel, levels of which well reflected the inter-sample EV variation and therefore useful as possible post-analytical normalizers of EV RNA content. We describe some realistic urine processing and normalization solutions for unbiased readout of EV biomarker studies and routine clinical sampling and diagnostics providing the input for design of larger validation studies employing urine EVs as biomarkers for particular conditions and diseases.


Assuntos
Vesículas Extracelulares , MicroRNAs , Vesículas Extracelulares/metabolismo , MicroRNAs/genética , Biomarcadores/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Albuminas/metabolismo
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