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[This corrects the article DOI: 10.3389/fphar.2021.680351.].
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Curcumin is identified that it has the potential to treat Parkinson's disease (PD), but its instability limits its further application in clinic. The mono-carbonyl analogs of curcumin (MACs) with diketene structure can effectively improve its stability, but it is highly toxic. In the present study, a less cytotoxic and more stable monoketene MACs skeleton S2 was obtained, and a series of monoketene MACs were synthesized by combining 4-hydroxy-3methoxy groups of curcumin. In the 6-OHDA-induced PD's model in-vitro, some compounds exhibited significant neurotherapeutic effect. The quantitative structure-activity relationship (QSAR) model established by the random forest algorithm (RF) for the cell viability rate of above compounds showed that the statistical results are good (R2 = 0.883507), with strong reliability. Among all compounds, the most active compound A4 played an important role in neuroprotection in the PD models both in vitro and in vivo by activating AKT pathway, and then inhibiting the apoptosis of cells caused by endoplasmic reticulum (ER) stress. In the PD model in-vivo, compound A4 significantly improved survival of dopaminergic neurons and the contents of neurotransmitters. It also enhanced the retention of nigrostriatal function which was better than the effect in the mice treated by Madopar, a classical clinical drug for PD. In summary, we screened out the compound A4 with high stability, less cytotoxic monoketene compounds. And these founding provide evidence that the compound A4 can protect dopaminergic neurons via activating AKT and subsequently suppressing ER stress in PD.
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Curcumina , Fármacos Neuroprotetores , Doença de Parkinson , Animais , Camundongos , Apoptose , Curcumina/farmacologia , Curcumina/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reprodutibilidade dos TestesRESUMO
Ultraviolet irradiation, especially ultraviolet B (UVB) irradiation, increases the risks of various skin diseases, such as sunburn, photo-aging and cancer. However, few drugs are available to treat skin lesions. Therefore, the discovery of drugs to improve the health of irradiated skin is urgently needed. Fibroblast growth factor 21 (FGF21) is a metabolic factor that plays an important role in the protection and repair of various types of pathological damage. The effects of FGF21 on skin injury caused by UVB-irradiation were the focus of this study. We found that UVB irradiation promoted the expression of FGF21 protein in mouse epidermal cells, and exogenous recombinant human FGF21 (rhFGF21) protected mouse skin tissue against UVB-induced injury. RhFGF21 inhibited the inflammatory responses and epidermal cell apoptosis as well as promotion of autophagy in UVB-irradiated mice. Moreover, we found that rhFGF21 protected HaCaT cells against UVB-induced apoptosis, and the protective effect was enhanced by treatment with an autophagy activator (rapamycin) but was inhibited by treatment with an autophagy inhibitor (3-methyladenine, 3MA). AMP-activated protein kinase (AMPK), as a cellular energy sensor, regulates autophagy. RhFGF21 increased the expression of p-AMPK protein in epidermal cells irradiated with UVB in vivo and in vitro. Moreover, rhFGF21 increased autophagy levels and the viability were diminished by treatment with an AMPK inhibitor (compound C). RhFGF21 protects epidermal cells against UVB-induced apoptosis by inducing AMPK-mediated autophagy.
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Proteínas Quinases Ativadas por AMP , Autofagia , Humanos , Camundongos , Animais , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose , Raios Ultravioleta/efeitos adversos , Células Epidérmicas/metabolismo , Sirolimo/farmacologiaRESUMO
Microglia are immune cells in the central nervous system (CNS) that participate in response to pathological process after ischemic injury. Non-mitogenic fibroblast growth factor 1 (nmFGF1) is an effective neuroprotective factor that is also known as a metabolic regulator. The present study aimed to investigate the effects and mechanism of the neuroprotective ability of nmFGF1 on microglia in mice after photothrombosis (PT) stroke model, to determine whether it could ameliorate ischemic injury in stroke experiment. We discovered that the intranasal administration of nmFGF1 reduced infarct size and ameliorated neurological deficits in behavioral assessment by regulating the secretion of proinflammatory and anti-inflammatory cytokines. Furthermore, in the in vitro experiments, we found that nmFGF1 regulated the expression levels of proinflammatory and anti-inflammatory cytokines in oxygen-glucose deprivation (OGD) and lipopolysaccharide (LPS) stimulation. Evidence have shown that when nuclear factor erythroid 2-related factor 2 (Nfr2) is activated, it inhibits nuclear factor-kappa B (NF-κB) activation to alleviate inflammation. Interestingly, nmFGF1 treatment in vivo remarkably inhibited NF-κB pathway activation and activated Nrf2 pathway. In addition, nmFGF1 and NF-κB inhibitor (BAY11-7082) inhibited NF-κB pathway in LPS-stimulated BV2 microglia. Moreover, in LPS-stimulated BV2 microglia, the anti-inflammatory effect produced by nmFGF1 was knocked down by Nrf2 siRNA. These results indicate that nmFGF1 promoted functional recovery in experimental stroke by modulating microglia/macrophage-mediated neuroinflammation via Nrf2 and NF-κB signaling pathways, making nmFGF1 a potential agent against ischemic stroke.
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Fator 1 de Crescimento de Fibroblastos , AVC Isquêmico , Macrófagos , Microglia , Fator 2 Relacionado a NF-E2 , NF-kappa B , Acidente Vascular Cerebral , Animais , Anti-Inflamatórios/farmacologia , Polaridade Celular/efeitos dos fármacos , Citocinas/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 1 de Crescimento de Fibroblastos/farmacologia , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Microglia/metabolismo , Microglia/patologia , Mitógenos/metabolismo , Mitógenos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
Diabetes increases the risk of stroke, exacerbates neurological deficits, and increases mortality. Non-mitogenic fibroblast growth factor 1 (nmFGF1) is a powerful neuroprotective factor that is also regarded as a metabolic regulator. The present study aimed to investigate the effect of nmFGF1 on the improvement of functional recovery in a mouse model of type 2 diabetic (T2D) stroke. We established a mouse model of T2D stroke by photothrombosis in mice that were fed a high-fat diet and injected with streptozotocin (STZ). We found that nmFGF1 reduced the size of the infarct and attenuated neurobehavioral deficits in our mouse model of T2D stroke. Angiogenesis plays an important role in neuronal survival and functional recovery post-stroke. NmFGF1 promoted angiogenesis in the mouse model of T2D stroke. Furthermore, nmFGF1 reversed the reduction of tube formation and migration in human brain microvascular endothelial cells (HBMECs) cultured in high glucose conditions and treated with oxygen glucose deprivation/re-oxygenation (OGD). Amp-activated protein kinase (AMPK) plays a critical role in the regulation of angiogenesis. Interestingly, we found that nmFGF1 increased the protein expression of phosphorylated AMPK (p-AMPK) both in vivo and in vitro. We found that nmFGF1 promoted tube formation and migration and that this effect was further enhanced by an AMPK agonist (A-769662). In contrast, these processes were inhibited by the application of an AMPK inhibitor (compound C) or siRNA targeting AMPK. Furthermore, nmFGF1 ameliorated neuronal loss in diabetic stroke mice via AMPK-mediated angiogenesis. In addition, nmFGF1 ameliorated glucose and lipid metabolic disorders in our mouse model of T2D stroke without causing significant changes in body weight. These results revealed that nmFGF1-regulated glucolipid metabolism and angiogenesis play a key role in the improvement of functional recovery in a mouse model of T2D stroke and that these effects are mediated by the AMPK signaling pathway.
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BACKGROUND: Bee pollen (BP) has been used as a traditional medicine and food diet additive due to its nutritional and biological properties. The potential biological properties of bee pollen vary greatly with the botanical and geographical origin of the pollen grains. This study was conducted to characterize the botanical origin and assess the antioxidant effects of ethanol extracts of 18 different bee pollen (EBP) samples from 16 locations in South Korea and their inhibitory activities on human ß-amyloid precursor cleavage enzyme (BACE1), acetylcholinesterase (AChE), human intestinal bacteria, and 5 cancer cell lines. METHODS: The botanical origin and classification of each BP sample was evaluated using palynological analysis by observing microscope slides. We measured the biological properties, including antioxidant capacity, inhibitory activities against human BACE1, and AChE, and antiproliferative activities toward five cancer cell lines, of the 18 EBPs. In addition, the growth inhibitory activities on four harmful intestinal bacteria, six lactic acid-producing bacteria, two nonpathogenic bacteria, and an acidulating bacterium were also assessed. RESULTS: Four samples (BP3, BP4, BP13 and BP15) were found to be monofloral and presented four dominant pollen types: Quercus palustris, Actinidia arguta, Robinia pseudoacacia, and Amygdalus persica. One sample (BP12) was found to be bifloral, and the remaining samples were considered to be heterofloral. Sixteen samples showed potent antioxidant activities with EC50 from 292.0 to 673.9 µg mL- 1. Fourteen samples presented potent inhibitory activity against human BACE1 with EC50 from 236.0 to 881.1 µg mL- 1. All samples showed antiproliferative activity toward the cancer cell lines PC-3, MCF-7, A549, NCI-H727 and AGS with IC50 from 2.7 to 14.4 mg mL- 1, 0.9 to 12.7 mg mL- 1, 5.0 to > 25 mg mL- 1, 2.7 to 17.7 mg mL- 1, and 2.4 to 8.7 mg mL- 1, respectively. In addition, total phenol and flavonoid contents had no direct correlation with antioxidant, anti-human BACE1, or antiproliferative activities. CONCLUSION: Fundamentally, Korean bee pollen-derived preparations could be considered a nutritional addition to food to prevent various diseases related to free radicals, neurodegenerative problems, and cancers. The botanical and geographical origins of pollen grains could help to establish quality control standards for bee pollen consumption and industrial production.
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Secretases da Proteína Precursora do Amiloide/metabolismo , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Pólen , Acetilcolinesterase/metabolismo , Animais , Apiterapia , Abelhas , Linhagem Celular Tumoral , Humanos , República da CoreiaRESUMO
Ischemic strokes account for about 80% of all strokes and are associated with a high risk of mortality. Angiogenesis of brain microvascular endothelial cells may contribute to functional restoration following ischemia. Fibroblast growth factor 1 (FGF1), a member of FGF superfamily, involved in embryonic development, angiogenesis, wound healing, and neuron survival. However, the mitogenic activity of FGF1 is known to contribute to several human pathologies, thereby questioning the safety of its clinical applications. Here, we explored the effects and mechanism of action of non-mitogenic FGF1 (nmFGF1) on angiogenesis in mice after ischemia stroke and an oxygen-glucose deprivation (OGD)-induced human brain microvascular endothelial cells (HBMECs) injury model. We found that intranasal administration nmFGF1 significantly promoted angiogenesis in mice after stroke, and significantly increased the formation of matrigel tube and promoted scratch migration in a dose-dependent manner in OGD-induced HBMECs in vitro. However, the co-administration of an FGF receptor 1 (FGFR1)-specific inhibitor PD173074 significantly reversed the effects of nmFGF1 in vitro, suggesting that nmFGF1 functions via FGFR1 activation. Moreover, nmFGF1 activated sphingosine-1-phosphate receptor 1 (S1PR1, S1P1) in mice after stroke in vivo. S1P1 protein antagonist VPC23019 and agonist FTY720 were used to confirm that nmFGF1 promotes angiogenesis in vitro partially through the S1P1 pathway. OGD induced downregulation of S1P1 expression. The S1P1 antagonist VPC23019 blocked the stimulatory effects of nmFGF1, whereas the S1P1 agonist FTY720 exerted effects comparable with those of nmFGF1. Furthermore, PD173074 reversed the effect of nmFGF1 on upregulating S1P1 signaling. In conclusion, nmFGF1 enhanced angiogenesis in mice following stroke and OGD-induced HBMECs through S1P1 pathway regulation mediated via FGFR1 activation. This new discovery suggests the potential therapeutic role of nmFGF1 for the treatment of ischemic strokes.
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Major depressive disorder is a serious neuropsychiatric disorder with high rates of recurrence and mortality. Many studies have supported that inflammatory processes play a central role in the etiology of depression. Fibroblast growth factor 21 (FGF21), a member of the fibroblast growth factors (FGFs) family, regulates a variety of pharmacological activities, including energy metabolism, glucose and lipid metabolism, and insulin sensitivity. In addition, recent studies showed that the administration of FGF21, a regulator of metabolic function, had therapeutic effects on mood stabilizers, indicating that FGF21 could be a common regulator of the mood response. However, few studies have highlighted the antidepressant effects of FGF21 on lipopolysaccharide (LPS)-induced mice, and the anti-inflammatory mechanism of FGF21 in depression has not yet been elucidated. The purpose of the current study was to determine the antidepressant effects of recombinant human FGF21 (rhFGF21). The effects of rhFGF21 on depression-like behaviors and the inflammatory signaling pathway were investigated in both an LPS-induced mouse model and primary microglia in vitro. The current study demonstrated that LPS induced depressive-like behaviors, upregulated proinflammatory cytokines, and activated microglia in the mouse hippocampus and activated the inflammatory response in primary microglia, while pretreatment with rhFGF21 markedly improved depression-like behavior deficits, as shown by an increase in the total distance traveled and number of standing numbers in the open field test (OFT) and a decrease in the duration of immobility in the tail suspension test (TST) and forced swimming test (FST). Furthermore, rhFGF21 obviously suppressed expression levels of the proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) and inhibited microglial activation and the nuclear factor-κB (NF-κB) signing pathway. Moreover, coadministration of rhFGF21 with the fibroblast growth factor receptor 1 (FGFR1) inhibitor PD173074 significantly reversed these protective effects, indicating that the antidepressant effects of rhFGF21 occur through FGFR1 activation. Taken together, the results of the current study demonstrated for the first time that exogenous rhFGF21 ameliorated LPS-induced depressive-like behavior by inhibiting microglial expression of proinflammatory cytokines through NF-κB suppression. This new discovery suggests rhFGF21 as a new therapeutic candidate for depression treatment.
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BACKGROUND: Extracellular matrix (ECM) comprising the environments of multicellular society has a dynamic network structure. Collagen is one of the ubiquitous components of ECM. Collagen affects the inflammatory response by regulating the release of pro-inflammatory cytokines from cells. Gelatin, denatured collagen found temporally in tissues, is supposed to be pathophysiologically involved in tissue remodeling, inflammation caused by tissue damage. Previous reports indicate that, phorbol myristate (PMA)-stimulated human U937 (lymphoma cell line) cells that are often used as macrophage-like cells, show cell aggregations when cultured on type I collagen (col I) or gelatin-coated dishes, accompanying the changes of production and release of proinflammatory factors. However, it still remains to be examined whether collagen and gelatin affects normal macrophages as well. AIM: This study aims to investigate the effect of col. I, the main component of collagenous protein and its denatured product, gelatin, on mouse peritoneal macrophages (MPMs). METHODS: MTT assay, flow cytometric analysis of ROS, biochemical detection of antioxidant levels, ELISA assay, and western blot were used. RESULTS: MPMs formed multicellular aggregates on col. I - and gelatin-coated dishes with a concentration- and time-dependent manner. Further studies showed that the culture on col. I and gelatin up-regulated the protein expression and secretion of pro-inflammatory molecules such as IL-1ß, TNFα and prostaglandin E2 (PGE2) in MPMs. The levels were higher in the cells on gelatin than those on col. I. ROS levels are significantly increased in the cells cultured on both col. I- and gelatin-coated dishes, accompanying decreased levels of antioxidant enzyme catalase (CAT) and anti-oxidant glutathione (GSH), and enhanced nuclear translocation of NF-κB. CONCLUSION: Col I - or gelatin-coated culture induced the formation of multicellular aggregates and increased production of NF-κB-associated pro-inflammatory molecules in MPMs through up-regulation of ROS levels.
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Colágeno Tipo I , Gelatina , Macrófagos Peritoneais/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Agregação Celular , Dinoprostona/metabolismo , Feminino , Interleucina-1beta/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Wound healing, especially for diabetic wounds, is a lengthy and complicated process involving interactions and responses at the protein, cell, and tissue levels. Loading of growth factors into a hydrogel to construct a sustained-release system is considered a promising approach to improve wound healing. The present study investigates the effect of thermosensitive heparin-poloxamer (HP) hydrogel-encapsulated recombinant human fibroblast growth factor 21 (rhFGF21) on wound healing in mice with streptozotocin-induced diabetes mellitus. First, we studied the in vitro release of rhFGF21 from the rhFGF21-HP coacervate. The results showed that HP might control the release of rhFGF21. Next, we examined the effect of rhFGF21-HP on skin wound healing in diabetic mice. Our data showed that rhFGF21-HP significantly improved wound closure; promoted granulation, collagen deposition, and re-epithelialization; and enhanced the expression of CD31. Moreover, rhFGF21-HP had obvious advantages in diabetic wound healing. Therefore, the results suggest that the rhFGF21-HP hydrogel polymer plays an important role in skin wound healing. This work provides a suitable sustained-release delivery system that can continuously release rhFGF21 and presents a promising therapeutic strategy for wound healing in patients with diabetes.-Liu, H., Zhao, Y., Zou, Y., Huang, W., Zhu, L., Liu, F., Wang, D., Guo, K., Hu, J., Chen, J., Ye, L., Li, X., Lin, L. Heparin-poloxamer hydrogel-encapsulated rhFGF21 enhances wound healing in diabetic mice.
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Diabetes Mellitus Experimental , Fatores de Crescimento de Fibroblastos/farmacologia , Heparina/química , Hidrogéis/química , Poloxâmero/química , Cicatrização/efeitos dos fármacos , Animais , Glicemia , Formas de Dosagem , Liberação Controlada de Fármacos , Fatores de Crescimento de Fibroblastos/administração & dosagem , Fatores de Crescimento de Fibroblastos/química , Teste de Tolerância a Glucose , Humanos , Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas RecombinantesRESUMO
Preadipocyte migration is a fundamental and important process for the development of tissue organization, especially in the development of primitive adipose tissue and adipocyte tissue wound healing. However, excessive migration may result in abnormal development and fibrosis-related diseases such as hypertrophic scar. We previously reported that type I collagen (collagen I) enhanced migration of 3T3-L1 preadipocytes via phosphorylation and/or acetylation of NF-κB p65, and the enhanced cell migration is repressed by silibinin treatment through sirt1. It is known that sirt1 has an ability to deacetylate acetylated NF-κB p65, but little is known about the effect of sirt1 on phosphorylated NF-κB p65. This study aims to examine the potential effect of sirt1 on the regulation of phosphorylated NF-κB p65 and the underlying mechanism. Autophagy is involved in many physiological and pathological processes, including regulation of cell migration as well as in cellular homeostasis. The present study demonstrates that silibinin induces autophagy in a dose-dependent manner in 3T3-L1 cells. Autophagy is under the regulation of sirt1/AMPK pathway, and inhibits collagen I-enhanced migration of 3T3-L1 cells through negative regulation of NF-κB p65 phosphorylation but not acetylation. The expression of peroxisome proliferator-activated receptor α (PPARα) is up-regulated with silibinin accompanying up-regulation of autophagy through activating sirt1 in 3T3-L1 cells. Taken together, these findings indicate that silibinin-induced autophagy is mediated by up-regulation of PPARα-sirt1-AMPK, contributing to repression of type I collagen-enhanced migration in murine 3T3-L1 preadipocytes through down-regulation of phosphorylated NF-κB p65.
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Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/metabolismo , Autofagia/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , PPAR alfa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Silibina/farmacologia , Sirtuína 1/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Camundongos , Fosforilação/efeitos dos fármacos , Fator de Transcrição RelA/metabolismoRESUMO
Purpose: Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultured on collagen I-coated dishes express lowered production of pro-inflammatory mediators in parallel through reduced reactive oxygen species (ROS) levels. By contrast, PMA-treated U937 cells on gelatin, the denatured collagen, show enhanced production of pro-inflammatory mediators, mediated by up-regulating autophagy levels. The present study is aimed to investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface. Material and methods: MTT assay, flow cytometric analysis of ROS and autophagy, biochemical detection of antioxidant levels, enzyme-linked immunosorbent assay, and western blot were used. Results: Gelatin-coating increased ROS levels in PMA-treated U937 cells. Increased ROS levels are involved in the regulation of cell aggregation and the release of pro-inflammatory mediators in gelatin-coated culture. These results lead to the query about the crosstalk between the two positive regulators, the autophagy and ROS. Autophagy induction is attenuated by N-acetyl-L-cysteine treatment, but the treatment with autophagy inhibitor, 3-methyladenine, does not affect ROS levels, suggesting ROS are upstream of autophagy in the regulation axis of differentiated U937 cells on gelatin-coated surface. Further study confirmed that upregulation of autophagy was responsible for ROS-induced cell aggregation and production of pro-inflammatory mediators. Conclusion: The results suggest that gelatin-coating promotes the aggregation of PMA-treated U937 cells and the production of pro-inflammatory mediators by ROS-autophagy signaling pathway.
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Autofagia/efeitos dos fármacos , Gelatina/química , Mediadores da Inflamação/metabolismo , Ésteres de Forbol/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Agregação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Dinoprostona/metabolismo , Humanos , Interleucina-1beta/metabolismo , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Suínos , Fator de Necrose Tumoral alfa/metabolismo , Células U937RESUMO
Reactive oxygen species (ROS) participate in various cell responses in association with cell proliferation, migration, differentiation, and death. Extracellular matrix (ECM) serves as cellular microenvironments for many kinds of cells, affecting cell activities. However, whether or not ECM influences cellular ROS levels has not been well studied. In this study, cells are cultured on collagen I molecule-coated (mol. coated) dishes and collagen I fibrous gel-covered (gel) dishes to explore their influence on cell behaviours. We found that the levels of ROS in murine 3T3-L1 preadipocytes increased both in cells on mol. coated and those on the gel. Much higher ROS levels were found in the cells cultured on the gel. Cell proliferation and migration were stimulated to opposite directions between the cells on mol. coated and the cells on gel. ROS in a moderate level were positive regulators in the proliferation and migration of cells on mol. coated; however, ROS in a high level served as negative regulators in the cells on gel. These opposite effects on cell proliferation and migration affected by different ROS levels are in parallel with opposite levels of NF-κB p65 activation.
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Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/química , Meios de Cultura/química , Células 3T3-L1 , Animais , Colágeno Tipo I/farmacologia , Camundongos , NF-kappa B/química , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/farmacologiaRESUMO
In organ fibrosis, mechanical stress and transforming growth factor beta-1 (TGF-ß1) promote differentiation into myofibroblast from mesenchymal cells, leading to extracellular matrix (ECM) remodeling or active synthesis, deposition or degradation of ECM components. A major component of ECM, type I collagen (col I) triple helical molecules assemble into fibrils or are denatured to gelatin without triple-helicity in remodeling. However, whether changes of ECM components in remodeling have influence on mesenchymal cell differentiation remains elusive. This study adopted three states of collagen I existing in ECM remodeling: molecular collagen, fibrillar collagen and gelatin to see what are characteristics in the effects on two cell lines of mesenchymal origin, murine 3T3-L1 embryonic fibroblast and murine C2C12 myoblasts. The results showed that all three forms of collagen I were capable of inducing these two cells to differentiate into myofibroblasts characterized by increased expression of alpha-smooth muscle actin (α-SMA) mRNA. The expression of α-SMA is positively regulated by TGF-ß1. Nuclear translocation of Yes-associated protein (YAP) is involved in this process. Focal adhesion kinase (FAK) is activated in the cells cultured on molecular collagen-coated plates, contributing to YAP activation. On the other hand, in the cells cultured on fibrillar collagen gel or gelatin-coated plates, oxidative stress but not FAK induce YAP activation. In conclusion, the three physicochemically distinct forms of col I induce the differentiation of mesenchymal cells into myofibroblasts through different pathways.
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Diferenciação Celular/fisiologia , Colágeno Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Proteínas de Ciclo Celular , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Proliferação de Células/fisiologia , Colágeno Tipo I/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Microscopia Confocal , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fator de Crescimento Transformador beta1/genética , Proteínas de Sinalização YAPRESUMO
Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway.
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Autofagia/efeitos dos fármacos , Citocinas/metabolismo , Endocitose/efeitos dos fármacos , Matriz Extracelular/metabolismo , Gelatina/metabolismo , Macrófagos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/análogos & derivados , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Carcinógenos/farmacologia , Agregação Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cloroquina/farmacologia , Gelatina/isolamento & purificação , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Lectinas de Ligação a Manose/antagonistas & inibidores , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/imunologia , Pseudópodes/metabolismo , Interferência de RNA , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Pele/química , Sus scrofa , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The present study is aimed to investigate the effect of collagen I on U937 cells, human monocyte-like histiocytic lymphoma cell line. Differentiation of U937 cells was induced by phorbol ester (PMA) treatment. The cells were cultured on the collagen I-coated plate. PMA-stimulated U937 cells formed multicellular aggregates on collagen I-coated surface, whereas PMA-unstimulated cells kept themselves away off each other. Moreover, the levels of reactive oxygen species (ROS) and productions of pro-inflammatory cytokines such as IL-1ß, TNFα and PGE2, pro-inflammatory mediator, were down-regulated in differentiated U937 cells cultured on collagen I-coated dishes. However, collagen I did not influence the capacity of E. coli phagocytosis. Cell aggregation as well as the down-regulation of IL-1ß, TNFα and PGE2 caused by the culture on collagen I-coated surface were suppressed by ROS donor, tert-butylhydroperoxide (tBHP). The sizes of cell aggregates became bigger in differentiated U937 cells by treatment with ROS scavengers such as N-acetylcysteine (NAC), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). In conclusion, collagen I-coated culture induces the differentiated U937 cells to form cell aggregates and decreases the production of pro-inflammatory cytokines through down-regulating ROS generation.
Assuntos
Colágeno Tipo I/metabolismo , Macrófagos/imunologia , Acetilcisteína/farmacologia , Agregação Celular , Diferenciação Celular , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Fagocitose , Ésteres de Forbol/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Células U937RESUMO
Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.