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1.
Oxid Med Cell Longev ; 2022: 9004738, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36092160

RESUMO

Accumulating evidence has suggested the significant role of long noncoding RNAs (lncRNA) in regulating ferroptosis, while its regulatory mechanism in diabetic retinopathy (DR) remains unelucidated. In this work, we first demonstrated that lncRNA zinc finger antisense 1 (ZFAS1) is upregulated in high glucose-cultured human retinal endothelial cells (hRECs) and ZFAS1 inhibition attenuated high glucose- (HG-) induced ferroptosis, which was evidenced by cell viability, total iron and ferrous iron levels, reactive oxygen species (ROS) level, and Glutathione Peroxidase 4 (GPX4) expression detection. Mechanistically, we validated that ZFAS1 may act as a competing endogenous RNA by competitively binding with microRNA-7-5p (miR-7-5p) and modulating the expression of its downstream molecule acyl-CoA synthetase long-chain family member 4 (ACSL4), which is now identified as a classic driver gene of ferroptosis process. In conclusion, our results demonstrate that HG-induced ZFAS1 elevation activates ferroptosis in hRECs and the ZFAS1/miR-7-5p/ACSL4 axis may serve as a therapeutic target for endothelial dysfunction in DR.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Ferroptose , MicroRNAs , RNA Longo não Codificante , Proliferação de Células/fisiologia , Retinopatia Diabética/genética , Células Endoteliais/metabolismo , Glucose/farmacologia , Humanos , Ferro , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Zinco
2.
PLoS One ; 17(9): e0274420, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36107941

RESUMO

UDP-glucose dehydrogenase (UGDH) generates essential precursors of hyaluronic acid (HA) synthesis, however mechanisms regulating its activity are unclear. We used enzyme histostaining and quantitative image analysis to test whether cytokines that stimulate HA synthesis upregulate UGDH activity. Fibroblast-like synoviocytes (FLS, from N = 6 human donors with knee pain) were cultured, freeze-thawed, and incubated for 1 hour with UDP-glucose, NAD+ and nitroblue tetrazolium (NBT) which allows UGDH to generate NADH, and NADH to reduce NBT to a blue stain. Compared to serum-free medium, FLS treated with PDGF showed 3-fold higher UGDH activity and 6-fold higher HA release, but IL-1beta/TGF-beta1 induced 27-fold higher HA release without enhancing UGDH activity. In selected proliferating cells, UGDH activity was lost in the cytosol, but preserved in the nucleus. Cell-free assays led us to discover that diaphorase, a cytosolic enzyme, or glutathione reductase, a nuclear enzyme, was necessary and sufficient for NADH to reduce NBT to a blue formazan dye in a 1-hour timeframe. Primary synovial fibroblasts and transformed A549 fibroblasts showed constitutive diaphorase/GR staining activity that varied according to supplied NADH levels, with relatively stronger UGDH and diaphorase activity in A549 cells. Unilateral knee injury in New Zealand White rabbits (N = 3) stimulated a coordinated increase in synovial membrane UGDH and diaphorase activity, but higher synovial fluid HA in only 2 out of 3 injured joints. UGDH activity (but not diaphorase) was abolished by N-ethyl maleimide, and inhibited by peroxide or UDP-xylose. Our results do not support the hypothesis that UGDH is a rate-liming enzyme for HA synthesis under catabolic inflammatory conditions that can oxidize and inactivate the UGDH active site cysteine. Our novel data suggest a model where UGDH activity is controlled by a redox switch, where intracellular peroxide inactivates, and high glutathione and diaphorase promote UGDH activity by maintaining the active site cysteine in a reduced state, and by recycling NAD+ from NADH.


Assuntos
Sinoviócitos , Animais , Cisteína/metabolismo , Fibroblastos/metabolismo , Formazans , Glucose/farmacologia , Glucose Desidrogenase/metabolismo , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Maleimidas , NAD/metabolismo , Nitroazul de Tetrazólio , Oxirredução , Peróxidos , Coelhos , Sinoviócitos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Difosfato de Uridina/metabolismo , Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/metabolismo , Xilose
3.
Eur Rev Med Pharmacol Sci ; 26(17): 6199-6207, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36111920

RESUMO

OBJECTIVE: Previous studies show the downregulation of miR-181d-5p in the vitreous humor of patients with diabetic retinopathy (DR); however, it is not known whether miR-181d-5p is implicated in the development of DR. The present study aimed at evaluating the beneficial effects of miR-181d-5p on high glucose (HG)­induced human retinal microvascular endothelial cells (hRMECs), as well as the underlying mechanism. MATERIALS AND METHODS: hRMECs treated with HG were used to induce an in vitro cell model of DR. The analysis of quantitative real-time PCR (qRT-PCR) and western blot were used to detect the expression of miR-181d-5p and vascular endothelial growth factor A (VEGFA). The cell viability was measured using CCK-8 assay. The migration was assessed by performing Transwell assay. The angiogenesis of hRMECs was evaluated using the tube formation assay. The binding between target genes was explored using bioinformatic prediction and the luciferase reporter assay. RESULTS: HG treatment decreased miR-181d-5p but upregulated VEGFA expression in hRMECs. MiR-181d-5p inhibition augmented cell proliferation, migration and angiogenesis of hRMECs caused by HG. Upregulation of miR-181d-5p led to opposing effects, and miR-181d-5p directly targeted and negatively regulated VEGFA. The overexpression of VEGFA reversed the anti-proliferative, anti-migratory and antiangiogenic effects of miR-181d-5p in HG-treated hRMECs. CONCLUSIONS: These findings indicate that miR-181d-5p ameliorates HG-stimulated proliferation, migration and angiogenesis of hRMECs through inhibition of VEGFA, which might provide a new target for the treatment of DR.


Assuntos
Retinopatia Diabética , MicroRNAs , Indutores da Angiogênese , Retinopatia Diabética/genética , Células Endoteliais/metabolismo , Glucose/farmacologia , Humanos , MicroRNAs/metabolismo , Neovascularização Patológica/genética , Sincalida , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Hum Exp Toxicol ; 41: 9603271221125928, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36113040

RESUMO

To elucidate the effect of tricin in cerebral ischemia/reperfusion (I/R) injury and examine its possible underlying mechanisms. Rats were randomly divided into Sham (exposed the right internal carotid arteries), I/R, and tricin (administered at various doses) groups. After the cerebral I/R injury model was established, a Morris water maze test and a tetrazolium chloride assay were performed. Apoptosis and autophagy were assessed in the nerve cells of hippocampus tissue, and the levels of inflammatory markers within animal serum were detected. Proteins related to apoptosis and the PI3K/Akt pathway were evaluated. To further investigate the mechanisms by which tricin affects brain damage, mouse neuroblastoma cells N2a were divided into control, oxygen-glucose deprivation and reoxygenation (OGD/R), tricin, PI3K/Akt activator, and tricin + PI3K/Akt inhibitor groups. The cell viability, apoptosis, inflammatory factors, and PI3K/Akt pathway related proteins in N2a cells were also detected. The results revealed that I/R-induced learning and memory dysfunction was improved by tricin treatment. The area of cerebral infarction, the levels of apoptosis and autophagy in nerve cells, and the serum inflammatory marker content were all decreased following tricin treatment. Additionally, the expression of Beclin-1 protein was downregulated, while the expression of Bcl-2 protein, p-PI3K/PI3K and p-Akt/Akt was upregulated after tricin treatment. Mechanistically, tricin or PI3K/Akt activator ameliorated OGD/R-induced apoptosis, autophagy, and inflammation. However, these effects were reversed following PI3K/Akt inhibitor treatment in OGD/R-induced N2a cells. In summary, this study suggested that tricin can against I/R-induced brain injury by inhibiting autophagy, apoptosis and inflammation, and activating the PI3K/Akt signaling pathway.


Assuntos
Isquemia Encefálica , Traumatismo por Reperfusão , Animais , Apoptose , Autofagia , Proteína Beclina-1 , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Cloretos/farmacologia , Flavonoides , Glucose/farmacologia , Inflamação/tratamento farmacológico , Camundongos , Neurônios/metabolismo , Oxigênio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo
5.
Drug Des Devel Ther ; 16: 3071-3085, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36118165

RESUMO

Background: Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury. Methods: We generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting. Results: DHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR). Conclusion: This research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment.


Assuntos
Ferroptose , Traumatismo por Reperfusão , Animais , Coenzima A/metabolismo , Coenzima A/farmacologia , Coenzima A/uso terapêutico , Flavonóis , Glucose/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Ferro , Ligases/metabolismo , Ligases/farmacologia , Ligases/uso terapêutico , Lipídeos/farmacologia , Mamíferos/metabolismo , Oxigênio/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/farmacologia , Proteína de Ligação a Fosfatidiletanolamina/uso terapêutico , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Fosfotransferases (Aceptor do Grupo Álcool) , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais , Sincalida/metabolismo , Sincalida/farmacologia , Sincalida/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Água
6.
PLoS One ; 17(9): e0272986, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36054206

RESUMO

Acyl-CoA synthetase 1 (ACSL1) is an enzyme that converts fatty acids to acyl-CoA-derivatives for lipid catabolism and lipid synthesis in general and can provide substrates for the production of mediators of inflammation in monocytes and macrophages. Acsl1 expression is increased by hyperglycemia and inflammatory stimuli in monocytes and macrophages, and promotes the pro-atherosclerotic effects of diabetes in mice. Yet, surprisingly little is known about the mechanisms underlying Acsl1 transcriptional regulation. Here we demonstrate that the glucose-sensing transcription factor, Carbohydrate Response Element Binding Protein (CHREBP), is a regulator of the expression of Acsl1 mRNA by high glucose in mouse bone marrow-derived macrophages (BMDMs). In addition, we show that inflammatory stimulation of BMDMs with lipopolysaccharide (LPS) increases Acsl1 mRNA via the transcription factor, NF-kappa B. LPS treatment also increases ACSL1 protein abundance and localization to membranes where it can exert its activity. Using an Acsl1 reporter gene containing the promoter and an upstream regulatory region, which has multiple conserved CHREBP and NF-kappa B (p65/RELA) binding sites, we found increased Acsl1 promoter activity upon CHREBP and p65/RELA expression. We also show that CHREBP and p65/RELA occupy the Acsl1 promoter in BMDMs. In primary human monocytes cultured in high glucose versus normal glucose, ACSL1 mRNA expression was elevated by high glucose and further enhanced by LPS treatment. Our findings demonstrate that CHREBP and NF-kappa B control Acsl1 expression under hyperglycemic and inflammatory conditions.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Coenzima A Ligases/genética , Hiperglicemia , Inflamação/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , NF-kappa B , Animais , Coenzima A/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Hiperglicemia/genética , Hiperglicemia/metabolismo , Inflamação/genética , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , RNA Mensageiro/genética
7.
Front Endocrinol (Lausanne) ; 13: 926129, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36082077

RESUMO

Aims/Objectives: Wound healing in people with diabetes is delayed secondary to impaired nitric oxide generation, advanced glycation end products (AGE), and poor migration of epithelial cells. We developed a novel topical esmolol hydrochloride (Galnobax) and assessed its efficacy for wound healing in streptozocin-induced diabetic hairless rat. Methods: All experiments were performed at an animal laboratory and tertiary-care research facility. Ex vivo aldose reductase inhibition was assessed from enzymes obtained from a bacterial culture (spectrophotometer), sorbitol content in homogenized red blood cells, and AGE in glucose and bovine serum by fluorometry following the addition of esmolol in varying concentrations. A scratch assay of human fibroblasts, endothelial cells, and keratinocytes was assessed under a high-glucose environment and after esmolol by phase-contrast microscopy. The efficacy evaluation of the topical application of Galnobax (14 and 20%) or vehicle was conducted in streptozotocin-induced diabetic hairless rats, and endogenous nitrite and hydroxyproline from homogenized wound tissue were measured along with pharmacokinetic and dermal toxicity in Hanford miniature swine. Results: Esmolol inhibited the formation of sorbitol by 59% in erythrocytes in comparison to glucose-induced sorbitol levels. AGE generation in bovine serum albumin was reduced at 1 mM esmolol concentrations (2.6 ± 1.7) compared with control (p < 0.05) and similar to that of diclofenac (2.5 ± 1.3). Esmolol at 1 and 10 µM enhanced the migration of fibroblasts, epithelial cells, and keratinocytes compared with control. The nitric oxide levels (day 7) were 44 and 112% higher with Galnobax (14%) than those of the diabetic group (p < 0.05) and the vehicle control group (p < 0.05), respectively. The days 7 and 14 hydroxyproline in the wound was higher by 22 and 44% following Galnobax (14%) compared with the diabetic and vehicle control groups. The wound area exhibited better reduction with Galnobax at 14% up to day 10 follow-up compared with the controls. The pharmacokinetic and dermal toxicity in miniature swine suggested no significant adverse event with Galnobax. Conclusions: Topical esmolol hydrochloride is a novel, safe, and effective treatment modality that acts through pleotropic mechanisms to hasten wound healing in diabetes.


Assuntos
Diabetes Mellitus , Produtos Finais de Glicação Avançada , Aldeído Redutase/farmacologia , Animais , Células Endoteliais , Fibroblastos , Glucose/farmacologia , Humanos , Hidroxiprolina/farmacologia , Óxido Nítrico/farmacologia , Propanolaminas , Ratos , Sorbitol/farmacologia , Estreptozocina , Suínos , Porco Miniatura , Cicatrização
8.
Oxid Med Cell Longev ; 2022: 5142381, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36062192

RESUMO

Background: It is well-known that dysfunctions of vascular smooth muscle cells (VSMCs) act an essential part in vascular complications of diabetes. Studies have shown that circular RNAs (circRNAs) and microRNAs (miRNAs) play a crucial role in regulating cell functions. However, their influence on the proliferation, calcification, and autophagy of VSMCs remains to be further explored. Therefore, this study elucidates the role and mechanism of hsa_circRNA_0008028 in high glucose- (HG-, 30 mM) treated VSMCs in vitro. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was chosen to detect the levels of hsa_circRNA_0008028, miR-182-5p, and tribble 3 (TRIB3). Then, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to predict and verify the binding relationship between miR-182-5p and hsa_circRNA_0008028 or TRIB3. Cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, corresponding commercial kits, and western blotting were used to measure indexes reflecting cell viability, proliferation, calcification, and autophagy of VSMCs, respectively. Results: In HG-induced VSMCs, hsa_circRNA_0008028 and TRIB3 were highly expressed, whereas miR-182-5p decreased. Meanwhile, cell proliferation, calcification, and autophagy could be repressed by silencing of hsa_circRNA_0008028. However, these effects can be eliminated by miR-182-5p inhibition. Furthermore, it was demonstrated that hsa_circRNA_0008028 could promote the expression of TRIB3, a target of miR-182-5p, by directly sponging miR-182-5p. The expression of TRIB3 was suppressed by hsa_circRNA_0008028 knockout, which was rescued by miR-182-5p inhibition. Conclusion: This study reveals that hsa_circRNA_0008028 can act as a sponge of miR-182-5p and promote HG-induced proliferation, calcification, and autophagy of VSMCs partly by regulating TRIB3.


Assuntos
Calcinose , Proteínas de Ciclo Celular , MicroRNAs , Proteínas Serina-Treonina Quinases , RNA Circular , Proteínas Repressoras , Autofagia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Glucose/farmacologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Circular/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
9.
Eur Rev Med Pharmacol Sci ; 26(16): 5683-5688, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36066140

RESUMO

OBJECTIVE: High glucose can promote the apoptosis of glomerular mesangial cells and cause diabetic nephropathy (DN). However, the mechanism remains unclear. In the present study, we investigated the effects of high glucose on the survival of human renal mesangial cells (HRMCs). MATERIALS AND METHODS: Cells were treated with high glucose (30 mM) or normal glucose (5 mM) for 48 hours. Cell proliferation was determined by trypan blue assay. The relative expression of metalloproteinase-3 (TIMP3) and inflammatory factors detected by real-time polymerase chain reaction (PCR). Protein expression of Smad2/3, p-Smad2/3 and Smad7 in HRMCs were analyzed by Western blot. RESULTS: Compared with normal glucose, we found that high glucose significantly inhibited cell survival, accompanied by the decrease of tissue metalloproteinase-3 (TIMP3) mRNA expression. Western blot results showed that the expression of p-Smad2/3 was significantly up-regulated, the expression of Smad7 was significantly downregulated, and inflammatory factors IL-6/IL-8 mRNA expression were increased in the HRMCs cultured with the high glucose. We also found that, compared with the normal glucose, the level of MDA was significantly increased (p<0.01), and the level of SOD was significantly lower (p<0.05) in the HRMCs cultured with the high glucose. CONCLUSIONS: These findings suggested that high glucose inhibited the survival of HRMCs and may be associated with the downregulation of TIMP3 expression, Smad signaling pathway, inflammation and oxidative stress.


Assuntos
Nefropatias Diabéticas , Células Mesangiais , Nefropatias Diabéticas/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , RNA Mensageiro/metabolismo , Transdução de Sinais
10.
Pestic Biochem Physiol ; 187: 105199, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36127070

RESUMO

Biocontrol microbes are environment-friendly and safe for humans and animals. To seek biocontrol microbes effective in suppressing tomato gray mold is important for tomato production. Therefore, serial experiments were conducted to characterize the antagonism of Bacillus velezensis HY19, a novel self-isolated biocontrol bacterium, against Botrytis cinerea in vitro and the control on tomato gray mold in greenhouse. This bacterium produced extracellular phosphatase, protease, cellulose and siderophores, and considerably inhibited the growth of B. cinerea. A liquid chromatography-mass spectrometry (LC-MS) detected salicylic acid and numerous antifungal substances present in B. velezensis HY19 fermentation liquid (BVFL). When B. cinerea was grown on potato glucose agar, BVFL crude extract remarkably suppressed the fungal growth and reduced protein content and the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD). Transcriptome studies showed that BVFL crude extract significantly induced different expression of numerous genes in B. cinerea, most of which were down-regulated. Theses differently expressed genes were involved in the biological process, cell compartment, molecular functions, and metabolisms of glycine, serine, threonine, and sulfur in pathogen hyphae. Thus, this biocontrol bacterium antagonized B. cinerea in multiple ways due to the production of numerous antifungal substances that acted on multiple targets in the cells. BVFL significantly increased antioxidant enzyme activities in tomato leaves and decreased the incidence of tomato gray mold, with the control efficacies of 73.12-76.51%. Taken together, B. velezensis HY19 showed a promising use potential as a powerful bioagent against tomato gray mold.


Assuntos
Lycopersicon esculentum , Ágar/farmacologia , Animais , Antifúngicos/farmacologia , Antioxidantes/farmacologia , Bacillus , Catalase , Celulose/farmacologia , Misturas Complexas/farmacologia , Glucose/farmacologia , Glicina/farmacologia , Humanos , Lycopersicon esculentum/microbiologia , Peptídeo Hidrolases/farmacologia , Monoéster Fosfórico Hidrolases/farmacologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Ácido Salicílico/farmacologia , Serina/farmacologia , Sideróforos/farmacologia , Enxofre/farmacologia , Superóxido Dismutase , Treonina/farmacologia
11.
PLoS One ; 17(9): e0274663, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36112590

RESUMO

OBJECTIVES: The aim of this study is to compare acute effects of consuming extra virgin coconut oil (EVCO) as a source of medium chain fatty acids and extra virgin olive oil (EVOO) as a source of long chain fatty acids in normal weight and obese subjects. DESIGN: Randomised, crossover design. PARTICIPANTS: Metabolically healthy twenty male subjects (10 normal weight; 10 obese) aged 19-40 years. INTERVENTION: Subjects consumed breakfast meals containing skimmed milk, fat-free white cheese, bread and EVCO (25 g) or EVOO (25 g). OUTCOME MEASURES: Visual analog scale evaluations, resting metabolic rate measurements and selected blood parameters analysis (glucose, triglyceride, insulin and plasma peptide YY) were performed before and after the test breakfast meals. In addition, energy intakes were evaluated by ad libitum lunch meal at 180 min. RESULTS: Visual analogue scale values of hunger and desire to eat decreased significantly after EVCO consumption than EVOO consumption in normal weight subjects at 180 min. There was an increase trend in plasma PYY at 30 and 180 min after EVCO breakfast compared to EVOO breakfast. Ad libitum energy intakes after EVCO and EVOO consumption in normal weight subjects were 924 ± 302; 845 ± 158 kcal (p = 0.272), respectively whereas in obese subjects were 859 ± 238; 994 ± 265 kcal (p = 0.069) respectively. CONCLUSION: The results of this study shows that consumption of EVCO compared to EVOO may have suppressive effect on hunger and desire to eat, may affect postprandial PYY levels differently and have no effect on postprandial energy expenditure. TRIAL REGISTRATION: Clinical Trials NCT04738929.


Assuntos
Apetite , Peptídeo YY , Adulto , Óleo de Coco/farmacologia , Ingestão de Alimentos , Ácidos Graxos/farmacologia , Glucose/farmacologia , Humanos , Insulina , Masculino , Obesidade , Azeite de Oliva/farmacologia , Triglicerídeos/farmacologia , Adulto Jovem
12.
Mol Med ; 28(1): 116, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104669

RESUMO

BACKGROUND: Cataracts are the leading cause of blindness and a common ocular complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) and altered autophagic activity occur during the development of diabetic cataracts. The disturbed interaction of autophagy with EMT in LECs stimulated by high glucose levels may participate in cataract formation. METHODS: A rat diabetic cataract model induced by streptozotocin (STZ) and human lens epithelial cells (HLE-B3) stimulated with a high glucose concentration were employed in the study. These models were treated with rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)), and N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT, an inhibitor of γ-secretase) alone or in combination. Lens opacity was observed and photographed under a slit-lamp microscope. Histological changes in paraffin sections of lenses were detected under a light microscope after hematoxylin and eosin staining. Alterations of autophagosomes in LECs were counted and evaluated under a transmission electron microscope. The expression levels of proteins involved in the EMT, autophagy, and the signaling pathways in LECs were measured using Western blotting and immunofluorescence staining. Cell migration was determined by performing transwell and scratch wound assays. Coimmunoprecipitation (Co-IP) was performed to verify protein-protein interactions. Proteins were overexpressed in transfected cells to confirm their roles in the signaling pathways of interest. RESULTS: In LECs, a high glucose concentration induces the EMT by activating Jagged1/Notch1/Notch intracellular domain (NICD)/Snail signaling and inhibits autophagy through the AKT/mTOR/unc 51-like kinase 1 (ULK1) signaling pathway in vivo and in vitro, resulting in diabetic cataracts. Enhanced autophagic activity induced by rapamycin suppressed the EMT by inducing Notch1 degradation by SQSTM1/p62 and microtubule-associated protein light chain 3 (LC3) in LECs, while inhibition of the Notch signaling pathway with DAPT not only prevented the EMT but also activated autophagy by decreasing the levels of NICD, which bound to ULK1, phosphorylated it, and then inhibited the initiation of autophagy. CONCLUSIONS: We describe a new interaction of autophagy and the EMT involving NICD/ULK1 signaling, which mediates crosstalk between these two important events in the formation of diabetic cataracts. Activating autophagy and suppressing the EMT mutually promote each other, revealing a potential target and strategy for the prevention of diabetic cataracts.


Assuntos
Catarata , Diabetes Mellitus , Animais , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Catarata/etiologia , Transição Epitelial-Mesenquimal , Glucose/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mamíferos/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo
13.
Neuroreport ; 33(13): 583-589, 2022 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-36049163

RESUMO

Brain-derived neurotrophic factor (BDNF) is expressed in both hypothalamic neurons and microglia, and plays a critical role in the regulation of metabolism. Although hypothalamic expression of BDNF is regulated by metabolic signals such as nutrients and hormones, it remains unknown whether these signals differentially regulate BDNF expression in different cell types. The present study aimed to determine whether glucose and fructose regulate BDNF expression in microglia via the specific glucose transporter. To determine the effect of glucose and fructose on Bdnf mRNA and protein expression, murine microglial cell line SIM-A9 cells were exposed to the maintenance concentration of glucose (17.5 mmol/l), high glucose (25 mmol/l), or fructose (7.5 mmol/l) for 40 min to 24 h. To determine whether the blockade of glucose transporter 5 (GLUT5) negates the effect of glucose on Bdnf mRNA expression, cells were exposed to 25 mmol/l glucose in the presence or absence of the GLUT5 inhibitor for 4 h. Levels of Bdnf mRNA and protein were measured by real-time PCR and ELISA, respectively. High glucose caused a significant increase in both pan-Bdnf and long-form Bdnf (L-Bdnf) mRNA as well as protein levels when compared with the maintenance of concentration of glucose in a time-dependent manner. Fructose treatment also increased L-Bdnf mRNA expression. Pharmacological blockade of GLUT5 did not affect glucose-induced Bdnf mRNA expression. These findings suggest that glucose and fructose directly stimulate Bdnf mRNA expression in microglia and these responses may mediate the metabolic actions of glucose and fructose.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Frutose , Glucose , Microglia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Frutose/metabolismo , Frutose/farmacologia , Expressão Gênica , Glucose/metabolismo , Glucose/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/farmacologia , Camundongos , Microglia/metabolismo , RNA Mensageiro/metabolismo
14.
Crit Rev Eukaryot Gene Expr ; 32(7): 35-45, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36004694

RESUMO

Background - Diabetic nephropathy (DN) is a principal reason for kidney disease worldwide. High glucose (HG) is a major factor for DN. Kruppel like factor 5 (KLF5) participates in DN development. In the present study, we aim to elaborate the role of KLF5 in HG-induced renal tubular epithelial cell (RTEC) transdifferentiation in DN. Methods - RTECs (HK-2 cells) were treated with HG and were transfected with si-KLF5 or oe-HMGB1. Afterwards, expression of KLF5 and HMGB1 was detected, HK cell viability was determined, and levels of alpha-smooth muscle actin (α-SMA), E-cadherin, vimentin, and transforming growth factor beta 1 (TGF-ß1) were assessed. Additionally, the binding relation between KLF5 and HMGB1 was analyzed. Results - In HK-2 cells with HG treatment, expression of KLF5 and HMGB1 was upregulated; levels of α-SMA, vimentin, and TGF-ß1 were increased; and E-cadherin level was decreased. Moreover, KLF5 silencing resulted in down-regulated levels of α-SMA, vimentin, and TGF-ß1 but upregulated level of E-cadherin. On the other hand, KLF5 could bind to the HMGB1 promoter and activate HMGB1 transcription. HMGB1 overexpression partially counteracted the inhibitive effect of KLF5 silencing on HG-induced HK-2 transdifferentiation. Conclusion - HG induced overexpressed KLF5 in RTECs, and as a transcription factor, KLF5 could bind to the HMGB1 promoter, thereby promoting HMGB1 transcription and RTEC transdifferentiation.


Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Proteína HMGB1 , Caderinas/genética , Caderinas/metabolismo , Transdiferenciação Celular/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Células Epiteliais/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Proteína HMGB1/metabolismo , Proteína HMGB1/farmacologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Vimentina/genética , Vimentina/metabolismo , Vimentina/farmacologia
15.
Neurotoxicology ; 92: 166-179, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35985417

RESUMO

Diabetes mellitus can result in severe complications, such as neurodegenerative diseases including cognitive impairment and dementia. The glucagon-like peptide-1 (GLP-1) receptor agonist, liraglutide, is a novel antidiabetic drug with neuroprotective effects against neurodegenerative diseases. In this study, we explored the protective effect of liraglutide on SH-SY5Y cells exposed to methylglyoxal (MG), a byproduct of glucose metabolism that plays a key role in the development of diabetic encephalopathy. We found that liraglutide reduced the MG-induced oxidative stress, increased the activity of superoxide dismutase (SOD) and expression levels of P22phox, Gp91phox, and Xdh genes, and reduced reactive oxygen species (ROS) content. Metabolomics analysis based on 1H nuclear magnetic resonance showed that liraglutide induced alterations in metabolites involved in energy metabolism,including promotion of gluconeogenesis. Moreover, we found that liraglutide promoted oxidative phosphorylation and inhibited glycolysis in SH-SY5Y cells. This study revealed that liraglutide improved diabetes-related neuropathy damage by reducing the level of oxidative stress and maintaining the balance of energy metabolism, thus offering new insights into the potential mechanism of liraglutide in neuronal protection.


Assuntos
Neuroblastoma , Doenças Neurodegenerativas , Fármacos Neuroprotetores , Metabolismo Energético , Glucose/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Liraglutida/farmacologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo , Aldeído Pirúvico/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
16.
Biochim Biophys Acta Mol Basis Dis ; 1868(11): 166509, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-35914653

RESUMO

Type 2 diabetes is associated with an inflammatory phenotype in the pancreatic islets. We previously demonstrated that proinflammatory cytokines potently activate the tryptophan/kynurenine pathway (TKP) in INS-1 cells and in normal rat islets. Here we examined: (1) the TKP enzymes expression in the diabetic GK islets; (2) the TKP enzymes expression profiles in the GK islets before and after the onset of diabetes; (3) The glucose-stimulated insulin secretion (GSIS) in vitro in GK islets after KMO knockdown using specific morpholino-oligonucleotides against KMO or KMO blockade using the specific inhibitor Ro618048; (4) The glucose tolerance and GSIS after acute in vivo exposure to Ro618048 in GK rats. We report a remarkable induction of the kmo gene in GK islets and in human islets exposed to proinflammatory conditions. It occurred prominently in beta cells. The increased expression and activity of KMO reflected an acquired adaptation. Both KMO knockdown and specific inhibitor Ro618048 enhanced GSIS in vitro in GK islets. Moreover, acute administration of Ro618048 in vivo improved glucose tolerance, GSIS and basal blood glucose levels in GK rats. These results demonstrate that targeting islet TKP is able to correct defective GSIS. KMO inhibition could represent a potential therapeutic strategy for type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animais , Glicemia/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Cinurenina/metabolismo , Quinurenina 3-Mono-Oxigenase/metabolismo , Morfolinos , Ratos , Ratos Wistar , Triptofano/metabolismo
17.
Am J Physiol Gastrointest Liver Physiol ; 323(3): G283-G293, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35916424

RESUMO

Hepatic ischemia-reperfusion injury (HIRI) can lead to poor prognosis in patients undergoing liver transplantation or extensive liver resection. Maresin conjugate in tissue regeneration 1 (MCTR1) exerts a protective effect in several inflammatory disease models, but its role in HIRI remains unknown. In this study, we examined the effect of MCTR1 on HIRI and its underlying mechanism. HIRI mice and oxygen-glucose deprivation/reperfusion (OGD/R) AML12 cell models were used to evaluate the effects of MCTR1 at different doses on HIRI. Histological changes, inflammatory mediators, and ferroptosis-associated markers including iron content, oxidative stress and antioxidant activity, cell death marker (LDH), and the expression of Nuclear factor erythroid-derived 2-like 2 (NRF2) were analyzed. The results showed that MCTR1 treatment significantly ameliorated liver tissue damage and AST/ALT levels in HIRI mice. It also ameliorated ferroptosis in both HIRI mice and OGD/R AML12 cells, including a decrease in iron content, serum LDH release levels, reactive oxygen species (ROS), MDA, IL-1ß levels, and COX2 and transferrin receptor (TFRC) expression. In addition, it increased the levels of IL-10, the antioxidant stress markers SOD and GSH, and the expression of GPX4. With respect to the underlying mechanism, the expression of NRF2 in HIRI mice and OGD/R AML12 cells was significantly inhibited. MCTR1 treatment restored the inhibition of NRF2 expression caused by ischemia-reperfusion, and NRF2 inhibitors significantly inhibited nuclear aggregation of NRF2 promoted by MCTR1. In conclusion, the MCTR1 ameliorates ferroptosis-induced hepatic ischemia-reperfusion injury by promoting NRF2 expression and may represent a therapeutic strategy for treating HIRI.NEW & NOTEWORTHY MCTR1 exerts a protective effect in several inflammatory disease models, but its role in hepatic HIRI remains unknown. We confirm that the MCTR1 ameliorates ferroptosis-induced hepatic ischemia-reperfusion injury by promoting NRF2 expression. Our study illustrates the mechanism that MCTR1 protects from HIRI and identifies a therapeutic target for liver transplantation ischemia-reperfusion injury from the perspective of ferroptosis.


Assuntos
Ferroptose , Hepatopatias , Fator 2 Relacionado a NF-E2 , Traumatismo por Reperfusão , Animais , Antioxidantes/farmacologia , Glucose/farmacologia , Ferro , Hepatopatias/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Traumatismo por Reperfusão/metabolismo
18.
Mol Neurobiol ; 59(10): 6590-6607, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35972671

RESUMO

Glibenclamide (GLB) reduces brain edema and improves neurological outcome in animal experiments and preliminary clinical studies. Recent studies also suggested a strong anti-inflammatory effect of GLB, via inhibiting nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation. However, it remains unknown whether the anti-inflammatory effect of GLB is independent of its role in preventing brain edema, and how GLB inhibits the NLRP3 inflammasome is not fully understood. Sprague-Dawley male rats underwent 10-min asphyxial cardiac arrest and cardiopulmonary resuscitation or sham-operation. The Trpm4 siRNA and GLB were injected to block sulfonylurea receptor 1-transient receptor potential M4 (SUR1-TRPM4) channel in rats. Western blotting, quantitative real-time polymerase chain reaction, behavioral analysis, and histological examination were used to evaluate the role of GLB in preventing NLRP3-mediated neuroinflammation through inhibiting SUR1-TRPM4, and corresponding neuroprotective effect. To further explore the underlying mechanism, BV2 cells were subjected to lipopolysaccharides, or oxygen-glucose deprivation/reperfusion. Here, in rat model of cardiac arrest with brain edema combined with neuroinflammation, GLB significantly alleviated neurocognitive deficit and neuropathological damage, via the inhibition of microglial NLRP3 inflammasome activation by blocking SUR1-TRPM4. Of note, the above effects of GLB could be achieved by knockdown of Trpm4. In vitro under circumstance of eliminating distractions from brain edema, SUR1-TRPM4 and NLRP3 inflammasome were also activated in BV2 cells subjected to lipopolysaccharides, or oxygen-glucose deprivation/reperfusion, which could be blocked by GLB or 9-phenanthrol, a TRPM4 inhibitor. Importantly, activation of SUR1-TRPM4 in BV2 cells required the P2X7 receptor-mediated Ca2+ influx, which in turn magnified the K+ efflux via the Na+ influx-driven opening of K+ channels, leading to the NLRP3 inflammasome activation. These findings suggest that GLB has a direct anti-inflammatory neuroprotective effect independent of its role in preventing brain edema, through inhibition of SUR1-TRPM4 which amplifies K+ efflux and promotes NLRP3 inflammasome activation.


Assuntos
Edema Encefálico , Parada Cardíaca , Fármacos Neuroprotetores , Canais de Cátion TRPM , Animais , Anti-Inflamatórios/farmacologia , Edema Encefálico/complicações , Edema Encefálico/tratamento farmacológico , Glucose/farmacologia , Glibureto/farmacologia , Inflamassomos/metabolismo , Masculino , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Doenças Neuroinflamatórias , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Oxigênio/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Sulfonilureias
19.
J Ethnopharmacol ; 298: 115612, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-35987409

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetic dermatopathy is one of the most serious and common complications of diabetes. It has been found that high glucose can lead to abnormal glycometabolism. The skin microenvironment pollution caused by the increase in glucose and the oxidative stress mediated by the deposition of advanced glycation end products can lead to invisible skin injury, and the interaction between them is the key factor that makes the skin wounds of diabetic rats difficult to heal. Therefore, the main task of promoting healing is to reduce blood glucose levels and relieve the deposition of advanced glycation end products. Polygonatum kingianum Collett & Hemsl (PK) of Asparagaceae is planted in Yunnan, China, and is used by the Bai, Hani and Wa nationalities as a traditional medicine for preventing and treating diabetes. AIM OF THE STUDY: To study the effects of PK extract on skin wound healing in diabetic rats and to explore the regulatory mechanism of PK on wound microenvironment pollution, the antioxidative stress signaling pathway and latent injury of wound skin tissue. METHODS: First, wounds were prepared after diabetic rats were given PK extract by gavage for 4 weeks, and then gavage was continued for 2 weeks to observe and calculate the wound healing rate. A scanning electron microscope was used to observe the pathomorphological changes in the skin tissue at the edge of the wound. Western blotting was used to detect protein expression. Immunohistochemistry was used to detect the expression of CD34, AGEs, bFGF and VEGF. The Nrf2/HO-1 signaling pathway in skin tissue was detected by fluorescence quantitative PCR. Serum biochemical indicators and inflammatory cytokine levels were detected by a kit. RESULTS: After PK treatment, the wound healing rate increased significantly (P < 0.001), the infiltration of inflammatory cells in skin tissue of DM lesion rats decreased, the number of new blood vessels increased, and the epidermis and dermis thickened. The content of glucose, AGEs, RAGE protein and RAGE mRNA in skin decreased significantly (P < 0.05, P < 0.01, P < 0.001), while the expression of Nrf2 mRNA, HO-1 mRNA, CD34, bFGF and VEGF increased significantly (P < 0.05, P < 0.01, P < 0.001). The levels of SOD, GSH, MMP-9 and MMP-2 in skin decreased (P < 0.05, P < 0.01, P < 0.001), but the level of TIMP-2 increased (P < 0.001). GSP, GHb and ICAM-1 in plasma decreased (P < 0.05, P < 0.01, P < 0.001), while T-AOC, SOD and FINS increased (P < 0.05, P < 0.01). The levels of MDA, TNF-, IL-6, IL-2 and IFN-γ in plasma and wound skin tissue decreased (P < 0.05, P < 0.01, P < 0.001). CONCLUSION: PK can reduce the infiltration of inflammatory cells and glucose content in the skin tissue at the edge of the wound, reduce inflammatory factors in skin and plasma, and increase angiogenesis, thus improving the wound healing rate. PK can alleviate the microenvironment pollution caused by AGEs and glucose metabolism disorder in diabetic rats and induce antioxidant activity through the Nrf 2/HO-1 signaling pathway, thus reducing oxidative damage and offsetting endogenous skin damage and hidden damage.


Assuntos
Diabetes Mellitus Experimental , Polygonatum , Animais , China , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Glucose/farmacologia , Produtos Finais de Glicação Avançada/metabolismo , Fator 2 Relacionado a NF-E2 , Polygonatum/metabolismo , RNA Mensageiro , Ratos , Rizoma/metabolismo , Superóxido Dismutase , Fator A de Crescimento do Endotélio Vascular/genética , Cicatrização
20.
PLoS One ; 17(8): e0273921, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36044512

RESUMO

Transplantation is lifesaving and the most effective treatment for end-stage organ failure. The transplantation success depends on the functional preservation of organs prior to transplantation. Currently, the University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) are the most commonly used preservation solutions. Despite intensive efforts, the functional preservation of solid organs prior to transplantation is limited to hours. In this study, we modified the UW solution containing components from both the UW and HTK solutions and analyzed their tissue-protective effect against ischemic injury. The composition of the UW solution was changed by reducing hydroxyethyl starch concentration and adding Histidine/Histidine-HCl which is the main component of HTK solution. Additionally, the preservation solutions were supplemented with melatonin and glucosamine. The protective effects of the preservation solutions were assessed by biochemical and microscopical analysis at 2, 10, 24, and 72 h after preserving the rat kidneys with static cold storage. Lactate dehydrogenase (LDH) activity in preservation solutions was measured at 2, 10, 24, and 72. It was not detectable at 2 h of preservation in all groups and 10 h of preservation in modified UW+melatonin (mUW-m) and modified UW+glucosamine (mUW-g) groups. At the 72nd hour, the lowest LDH activity (0.91 IU/g (0.63-1.17)) was measured in the mUW-m group. In comparison to the UW group, histopathological damage score was low in modified UW (mUW), mUW-m, and mUW-g groups at 10, 24, and 72 hours. The mUW-m solution at low temperature was an effective and suitable solution to protect renal tissue for up to 72 h.


Assuntos
Isquemia , Rim , Melatonina , Soluções para Preservação de Órgãos , Adenosina , Alopurinol/farmacologia , Animais , Glucosamina , Glucose/farmacologia , Glutationa/farmacologia , Histidina/farmacologia , Insulina/farmacologia , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Rim/patologia , Manitol/farmacologia , Melatonina/farmacologia , Preservação de Órgãos/métodos , Soluções para Preservação de Órgãos/química , Soluções para Preservação de Órgãos/farmacologia , Cloreto de Potássio/farmacologia , Rafinose/farmacologia , Ratos
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