RESUMO
Hair loss is a common condition that can have a negative impact on an individual's quality of life. The severe side effects and the low efficacy of current hair loss medications create unmet needs in the field of hair loss treatment. Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1), one of the components of the extracellular matrix, has been shown to play a role in maintaining its integrity. HAPLN1 was examined for its ability to impact hair growth with less side effects than existing hair loss treatments. HAPLN1 was predominantly expressed in the anagen phase in three stages of the hair growth cycle in mice and promotes the proliferation of human hair matrix cells. Also, recombinant human HAPLN1 (rhHAPLN1) was shown to selectively increase the levels of transforming growth factor-ß receptor II in human hair matrix cells. Furthermore, we observed concomitant activation of the ERK1/2 signaling pathway following treatment with rhHAPLN1. Our results indicate that rhHAPLN1 elicits its cell proliferation effect via the TGF-ß2-induced ERK1/2 pathway. The prompt entering of the hair follicles into the anagen phase was observed in the rhHAPLN1-treated group, compared to the vehicle-treated group. Insights into the mechanism underlying such hair growth effects of HAPLN1 will provide a novel potential strategy for treating hair loss with much lower side effects than the current treatments.
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Cardiovascular diseases (CVDs) are the most common cardiovascular system disorders. Cellular senescence is a key mechanism associated with dysfunction of aged vascular endothelium. Hyaluronic acid and proteoglycan link protein 1 (HAPLN1) has been known to non-covalently link hyaluronic acid (HA) and proteoglycans (PGs), and forms and stabilizes HAPLN1-containing aggregates as a major component of extracellular matrix. Our previous study showed that serum levels of HAPLN1 decrease with aging. Here, we found that the HAPLN1 gene expression was reduced in senescent human umbilical vein endothelial cells (HUVECs). Moreover, a recombinant human HAPLN1 (rhHAPLN1) decreased the activity of senescence-associated ß-gal and inhibited the production of senescence-associated secretory phenotypes, including IL-1ß, CCL2, and IL-6. rhHAPLN1 also down-regulated IL-17A levels, which is known to play a key role in vascular endothelial senescence. In addition, rhHAPLN1 protected senescent HUVECs from oxidative stress by reducing cellular reactive oxygen species levels, thus promoting the function and survival of HUVECs and leading to cellular proliferation, migration, and angiogenesis. We also found that rhHAPLN1 not only increases the sirtuin 1 (SIRT1) levels, but also reduces the cellular senescence markers levels, such as p53, p21, and p16. Taken together, our data indicate that rhHAPLN1 delays or inhibits the endothelial senescence induced by various aging factors, such as replicative, IL-17A, and oxidative stress-induced senescence, thus suggesting that rhHAPLN1 may be a promising therapeutic for CVD and atherosclerosis.
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The development of atherosclerotic cardiovascular disease is associated with the phenotypic switching of vascular smooth muscle cells (SMCs) from a contractile to a synthetic state, leading to cell migration and proliferation. Plateletderived growth factorBB (PDGFBB) modulates this de-differentiation by initiating a number of biological processes. In this study, we show that gene expression of hyaluronic acid (HA) and proteoglycan link protein 1 (HAPLN1) was upregulated during differentiation of human aortic SMCs (HASMCs) into a contractile state, but downregulated upon during PDGF-BB-induced dedifferentiation. This is the first study showing that the treatment of HASMCs with full-length recombinant human HAPLN1 (rhHAPLN1) significantly reversed PDGF-BB-induced decrease in the protein levels of contractile markers (SM22α, α-SMA, calponin, and SM-MHC), and inhibited the proliferation and migration of HASMCs induced by PDGF-BB. Furthermore, our results show that rhHAPLN1 significantly inhibited the phosphorylation of FAK, AKT, STAT3, p38 MAPK and Raf mediated by the binding of PDGF-BB to PDGFRß. Together, these results indicated that rhHAPLN1 can suppress the PDGF-BB-stimulated phenotypic switching and subsequent de-differentiation of HASMCs, highlighting its potential as a novel therapeutic target for atherosclerosis and other vascular diseases. [BMB Reports 2023; 56(8): 445-450].
Assuntos
Ácido Hialurônico , Músculo Liso Vascular , Humanos , Becaplermina/farmacologia , Ácido Hialurônico/farmacologia , Proteínas Proto-Oncogênicas c-sis/farmacologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , Músculo Liso Vascular/metabolismo , Células Cultivadas , Proteoglicanas/metabolismo , Movimento Celular , Proliferação de Células , Miócitos de Músculo Liso/metabolismoRESUMO
Rheumatoid arthritis (RA) is a multifactorial immune-mediated disease, the pathogenesis of which involves different cell types. T-cell activation plays an important role in RA. Therefore, inhibiting T-cell activation is one of the current therapeutic strategies. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), also known as abatacept, reduces cytokine secretion by inhibiting T-cell activation. To achieve a homeostatic therapeutic effect, CTLA4-Ig has to be administered repeatedly over several weeks, which limits its applicability in RA treatment. To overcome this limitation, we increased the number of sialic acid-capped antennas by genetically engineering the CTLA4 region to increase the therapeutic effect of CTLA4-Ig. N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST) were co-overexpressed in Chinese hamster ovary (CHO) cells to generate a highly sialylated CTLA4-Ig fusion protein, named ST6. The therapeutic and immunogenic effects of ST6 and CTLA4-Ig were compared. ST6 dose-dependently decreased paw edema in a mouse model of collagen-induced arthritis and reduced cytokine levels in a co-culture cell assay in a similar manner to CTLA4-Ig. ST6- and CTLA4-Ig-induced T cell-derived cytokines were examined in CD4 T cells isolated from peripheral blood mononuclear cells after cell killing through irradiation followed by flow- and magnetic-bead-assisted separation. Interestingly, compared to CTLA4-Ig, ST6 was substantially less immunogenic and more stable and durable. Our data suggest that ST6 can serve as a novel, less immunogenic therapeutic strategy for patients with RA.
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Histone deacetylase inhibitors (HDACis) are well-known epigenetic regulators with therapeutic potential in various diseases. Recent studies have shown that HDACis are involved in immune-mediated anti-cancer effects and may modulate the activity of immunotherapy agents. CG-745, a histone deacetylase inhibitor, has shown anti-cancer effects in pancreatic cancer, colorectal cancer, and non-small cell lung cancer. However, the exact role of CG-745 within the immune system is largely unknown. In this study, we have shown that CG-745 induces microenvironment changes promoting anti-cancer effect of anti-PD-1 antibody in syngeneic mouse models. Specifically, CG-745 induces or extends IL-2 and IFN-γ expression with or without additional stimulation, and increases proliferation of cytotoxic T cells and NK cells, while inhibiting proliferation of regulatory T cells. The analysis of immune cell distribution in the tumor microenvironment and spleen reveals that CG-745 suppresses M2 macrophage polarization and decreases the myeloid-derived suppressor cells. Recent advances in immunotherapy highlight the anti-cancer effects of immune checkpoint inhibitor despite a relatively limited clinical benefit in the subset of patients. Our results indicate that CG-745 enables the synergistic effects of the immune checkpoint inhibitor combination therapy in various cancers by suppressing tumor microenvironment.
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Intestinal epithelial cells are adapted in mucosal hypoxia and hypoxia-inducible factors in these cells can fortify barrier integrity to support mucosal tissue healing. Here we investigated whether hypoxia-related pathways could be proposed as potential therapeutic targets for inflammatory bowel disease. We developed a novel hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor, CG-598 which stabilized HIF-1α in the gut tissue. Treatment of CG-598 did not affect extra-intestinal organs or cause any significant adverse effects such as erythropoiesis. In the experimental murine colitis model, CG-598 ameliorated intestinal inflammation with reduction of inflammatory lesions and pro-inflammatory cytokines. CG-598 treatment fortified barrier function by increasing the expression of intestinal trefoil factor, CD73, E-cadherin and mucin. Also, IL-10 and IL-22 were induced from lamina propria CD4+ T-cells. The effectiveness of CG-598 was comparable to other immunosuppressive therapeutics such as TNF-blockers or JAK inhibitors. These results suggest that CG-598 could be a promising therapeutic candidate to treat inflammatory bowel disease.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Animais , Células CACO-2 , Linhagem Celular Tumoral , Colite/tratamento farmacológico , Colite/imunologia , Colite/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Células HCT116 , Células HeLa , Humanos , Hipóxia/tratamento farmacológico , Hipóxia/imunologia , Hipóxia/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Inibidores de Prolil-Hidrolase/farmacologia , Fator Trefoil-3/imunologia , Fator Trefoil-3/metabolismoRESUMO
Neurotransmitter release is mediated by ceramide, which is generated by sphingomyelin hydrolysis. In the present study, we examined whether synaptosomal-associated protein 25 (SNAP-25) is involved in ceramide production and exocytosis. Neutral sphingomyelinase 2 (nSMase2) was partially purified from bovine brain and we found that SNAP-25 was enriched in the nSMase2-containing fractions. In rat synaptosomes and PC12 cells, the immunoprecipitation pellet of anti-SNAP-25 antibody showed higher nSMase activity than the immunoprecipitation pellet of anti-nSMase2 antibody. In PC12 cells, SNAP-25 was colocalized with nSMase2. Transfection of SNAP-25 small interfering RNA (siRNA) significantly inhibited nSMase2 translocation to the plasma membrane. A23187-induced ceramide production was concomitantly reduced in SNAP-25 siRNA-transfected PC12 cells compared with that in scrambled siRNA-transfected cells. Moreover, transfection of SNAP-25 siRNA inhibited dopamine release, whereas addition of C6-ceramide to the siRNA-treated cells moderately reversed this inhibition. Additionally, nSMase2 inhibition reduced dopamine release. Collectively, our results indicate that SNAP-25 interacts with nSMase2 during ceramide production, which mediates exocytosis and neurotransmitter release.
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Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Transporte Biológico , Bovinos , Membrana Celular/metabolismo , Ceramidas/biossíntese , Dopamina/metabolismo , Células PC12 , Ratos , Esfingomielina Fosfodiesterase/química , SinaptossomosRESUMO
BACKGROUND/AIM: Interleukin (IL)-1ß is a pro-inflammatory cytokine that has recently been established as a stimulator of angiogenesis via regulation of proangiogenic factor expression in the tumor microenvironment. This study aimed to demonstrate the inhibitory effects of Robinia pseudoacacia leaf extract (RP) on IL-1ß-mediated tumor angiogenesis. MATERIALS AND METHODS: Secreted embryonic alkaline phosphatase (SEAP) reporter gene assay, ex vivo and in vitro tube formation assay, western blot, and quantitative PCR were used to analyze the inhibitory effect of RP on IL-1ß-mediated angiogenesis. RESULTS: RP inhibited secretion of SEAP, blocked IL-1ß signaling, and inhibited IL-1ß-mediated angiogenesis in ex vivo and in vitro assays. RP inhibited nuclear translocation of NF-ĸB by suppressing phosphorylation of IL-1ß signaling protein kinases and inhibited mRNA expression of IL-1ß-induced pro-angiogenic factors including VEGFA, FGF2, ICAM1, CXCL8, and IL6. CONCLUSION: RP suppressed IL-1ß-mediated angiogenesis and, thus, could be a promising agent in anticancer therapy.
Assuntos
Interleucina-1beta/metabolismo , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Extratos Vegetais/farmacologia , Folhas de Planta/química , Robinia/química , Animais , Citocinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LCMS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 â 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.
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Drug repositioning is a strategy that explores new pharmaceutical applications of previously launched or failed drugs, and is advantageous since it saves capital and time. In this study, we examined the inhibition of TLR2 signaling by drug candidates. HEK-Blue™-hTLR2 cells were pretreated with drugs and stimulated using the TLR2 ligand, Pam3CSK4. Among the drugs that inhibited TLR2 signaling, we selected TRIAC, which is yet to be patented. Pretreatment with TRIAC decreased the TLR2 level and the phosphorylation of Akt and MAPKs in HEK-Blue™-hTLR2 cells. Since TLR2 is overexpressed in patients with acute hepatitis, we confirmed that TRIAC alleviates necrosis in a mouse model of Con A-induced acute hepatitis. The serum AST and ALT levels are indicators of liver damage, and are increased in Con A-induced hepatitis. Additionally, TLR2 and inflammatory cytokine levels are increased following administration of Con A and lead to liver damage. TRIAC decreased the serum levels of AST and ALT, and reduced liver tissue necrosis in mice with Con A-induced acute fulminant liver damage, by reducing the levels of inflammatory cytokines. In conclusion, TRIAC alleviates inflammation in mouse models of Con A-induced hepatitis by inhibiting the phosphorylation of Akt and MAPKs, the sub-mechanisms underlying TLR2 signaling.
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Anti-Inflamatórios/farmacologia , Receptor 2 Toll-Like/metabolismo , Tri-Iodotironina/análogos & derivados , Animais , Células Cultivadas , Humanos , Inflamação/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais/efeitos dos fármacos , Tri-Iodotironina/farmacologiaRESUMO
Autophagy is essential for optimal cell function and survival, and the entire process accompanies membrane dynamics. Ceramides are produced by different enzymes at different cellular membrane sites and mediate differential signaling. However, it remains unclear which ceramide-producing pathways/enzymes participate in autophagy regulation under physiological conditions such as nutrient starvation, and what the underlying mechanisms are. In this study, we demonstrate that among ceramide-producing enzymes, neutral sphingomyelinase 2 (nSMase2) plays a key role in autophagy during nutrient starvation. nSMase2 was rapidly and stably activated upon starvation, and the enzymatic reaction in the Golgi apparatus facilitated autophagy through the activation of p38 MAPK and inhibition of mTOR. Moreover, nSMase2 played a protective role against cellular damage depending on autophagy. These findings suggest that nSMase2 is a novel regulator of autophagy and provide evidence that Golgi-localized ceramides participate in cytoprotective autophagy against starvation.
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Autofagia , Ceramidas/metabolismo , Complexo de Golgi/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Ativação Enzimática , Masculino , Camundongos Endogâmicos C57BL , Células PC12 , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Inanição , Estresse Fisiológico , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Dopamine (DA) reuptake is the primary mechanism to terminate dopaminergic transmission in the synaptic cleft. The dopamine transporter (DAT) has an important role in the regulation of DA reuptake. This study provides anatomical and physiological evidence that DAT recycling is regulated by ceramide kinase via the sphingomyelin pathway. First, the results show that DAT and neutral sphingomyelinase 2 (nSMase2) were successfully co-precipitated from striatal samples and were colocalized in the mouse striatum or PC12 cells. We also identified a protein-protein interaction between nSMase2 and DAT through in situ proximity ligation assay experiments in the mouse striatum. Second, dopamine (DA) stimulated the formation of ceramide and increased nSMase activity in PC12 cells, while treatment with a cell-permeable ceramide-1-phosphate (C1P) increased DA uptake. Third, we used inhibitors and siRNA to inhibit nSMase2 and ceramide kinase and observed the effects on DAT recycling in PC12 cells. Treatment with ceramide kinase inhibitor K1, or nSMase inhibitor GW4869, decreased DA uptake in PC12 cells, although the application of FB1, a ceramide synthase inhibitor, did not affect DA uptake. Transfection of nSMase2 and CERK siRNA decreased DAT surface level in PC12 cells. These results suggested that SM-derived C1P affects cell surface levels of DAT.
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Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Transporte Biológico , Ceramidas/metabolismo , Camundongos Endogâmicos C57BL , Oxirredutases/antagonistas & inibidores , Células PC12 , Monoéster Fosfórico Hidrolases/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Ligação Proteica , RatosRESUMO
Acute lung injury (ALI) is a respiratory failure disease and the major source of mortality in the critically ill patients. The main pathological changes involved in ALI include the excessive recruitment and activation of neutrophils by increased pro-inflammatory mediators. However, any specific therapy for ALI has not been developed. The objective of this study was to investigate protective effects of parthenolide, a sesquiterpene lactone produced in feverfew, on LPS-induced lung injury. In the present study, parthenolide treatment reduced infiltration of inflammatory cells, airway permeability and production of pro-inflammatory cytokines in LPS-induced ALI mouse model. Further, LPS-stimulated phosphorylation of NF-κB, the key regulatory transcription factor in ALI, was inhibited by parthenolide treatment in lung epithelial BEAS-2B cells and alveolar macrophage MH-S cells. These results suggest that parthenolide may provide a beneficial therapeutic strategy for ALI.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Anti-Inflamatórios não Esteroides/uso terapêutico , Lactonas/uso terapêutico , Lipopolissacarídeos/toxicidade , Substâncias Protetoras/uso terapêutico , Sesquiterpenos/uso terapêutico , Células A549 , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Humanos , Lactonas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Substâncias Protetoras/farmacologia , Sesquiterpenos/farmacologiaRESUMO
Paraquat dichloride (N,N-dimethyl-4-4'-bipiridinium, PQ) is an extremely toxic chemical that is widely used in herbicides. PQ generates reactive oxygen species (ROS) and causes multiple organ failure. In particular, PQ has been reported to be an immunotoxic agrochemical compound. PQ was shown to decrease the number of macrophages in rats and suppress monocyte phagocytic activity in mice. However, the effect of PQ on macrophage cell viability remains unclear. In this study, we evaluated the cytotoxic effect of PQ on the mouse macrophage cell line, RAW264.7 and its possible mechanism of action. RAW264.7 cells were treated with PQ (0, 75, and 150 µM), and cellular apoptosis, mitochondrial membrane potential (MMP), and intracellular ROS levels were determined. Morphological changes to the cell nucleus and cellular apoptosis were also evaluated by DAPI and Annexin V staining, respectively. In this study, PQ induced apoptotic cell death by dose-dependently decreasing MMP. Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker. In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway. Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.
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Paraquat (PQ), one of the most widely used herbicides, has been used for several decades in agriculture. Some studies suggest that PQ has effects on the immune system. Moreover, previous studies have shown that PQ imparted some immunosuppressive effects. In the present study, cytotoxicity assays using splenic NK cells from mice treated for 28 days with PQ (at 0.2, 1, and 5 mg/kg) were performed to determine whether PQ altered the function of NK cells. Given that PQ was expected to induce an immunosuppressive effect, it was hypothesized that a gene involved in cellular metal ion homeostasis, metallothionein-1 (MT-1), could play an important role in this outcome. This belief was based on the fact that MT1 encodes a protein responsible for zinc ion homeostasis, and that a reduction in free zinc ion levels impairs NK cell function. The results showed that PQ treatments led to increased MT expression in several organs (liver, kidneys, testes) and in splenocytes, caused a reduction of both free zinc ions in sera and in free intracellular zinc, and reduced the expression of GATA-3, a zinc-finger transcription factor important for maturation and activity of T-cells and NK cells. These results provide a basis for a new molecular mechanism to describe potential immunosuppressive effects of PQ in vivo.
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Imunossupressores/farmacologia , Células Matadoras Naturais/imunologia , Metalotioneína/imunologia , Paraquat/farmacologia , Baço/imunologia , Animais , Citotoxinas/farmacologia , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Células Matadoras Naturais/patologia , Masculino , Camundongos , Baço/patologia , Zinco/imunologiaRESUMO
In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.
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Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Preparações de Plantas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , Proteínas de Neurofilamentos/metabolismo , Células PC12 , Proteínas Proto-Oncogênicas c-raf , RatosRESUMO
Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH-optimum and Mg(2+)-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.
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Chaperonina 60/fisiologia , Dopamina/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Bovinos , Células HEK293 , Humanos , Células PC12 , Ratos , Ratos Sprague-DawleyRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator of the maturation and function of cells in the granulocyte and macrophage lineages, and also plays a significant role in wound healing. In a previous study, we expressed human GM-CSF in rice cells (rice cell-derived human GM-CSF; rhGM-CSF). The purpose of the present study was to evaluate its effect on wound healing in oral mucositis. Oral mucositis was induced in Syrian hamster cheek pouches by 5-fluorouracil treatment and mechanical scratching. Ulcerated areas were treated from days 3 to 14 with an application of 200 µL saline, or of the same volume of a solution containing 0.04, 0.2, or 1 µg/mL rhGM-CSF. Treatment of hamsters with rhGM-CSF reduced the ulcerated areas of the oral mucosa, compared with the control. Early in the healing process, the mucositis tissue layer of the rhGM-CSF-treated group showed significantly decreased myeloperoxidase activity and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells. Treatment with rhGM-CSF also affected expression of inflammatory cytokines in the ulcerative mucosal tissue. These results demonstrate the efficacy of plant-produced rhGM-CSF in wound healing and have significant implications for the development of rhGM-CSF as a therapeutic agent for ulcerative oral mucositis.
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Antimetabólitos Antineoplásicos/toxicidade , Fluoruracila/toxicidade , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Estomatite/tratamento farmacológico , Animais , Cricetinae , Interleucina-1beta/genética , Masculino , Mesocricetus , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/enzimologia , Oryza/genética , Peroxidase/metabolismo , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas Recombinantes/uso terapêutico , Estomatite/induzido quimicamente , Estomatite/patologia , Fator de Crescimento Transformador beta/genética , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: In the symptomatic discoid lateral meniscus, the effectiveness of preoperative magnetic resonance imaging (MRI) is not well documented. HYPOTHESIS: Magnetic resonance imaging classification will provide more information to the surgeon in choosing the appropriate treatment methods with the help of arthroscopic findings. STUDY DESIGN: Cohort study (diagnosis); Level of evidence, 2. METHODS: Sixty-seven patients (82 knees) were reviewed. The preoperative MRI was checked in 76 of 82 knees. The Lysholm and Ikeuchi grading scales were evaluated. Images were analyzed from MRI, and findings were classified into 4 categories: no shift, anterocentral shift, posterocentral shift, and central shift. Tear pattern classifications were based on arthroscopic findings: horizontal tear, peripheral tear, horizontal and peripheral tear, posterolateral corner loss, and others. The correlations between MRI classification tear patterns and surgical methods were analyzed using the chi-square test or the Fisher exact test. The sensitivity, specificity, and accuracy of shift in preoperative MRI-according to the existence of peripheral tear when corroborated with arthroscopy-were also analyzed with the chi-square test. Inter- and intraobserver reliability was statistically analyzed by producing the inter- and intraclass correlation coefficient. RESULTS: The mean preoperative Lysholm score was 77.3 (range, 43-97), and the last follow-up Lysholm score had increased to 96.8 (range, 84-100; P < .001). At last follow-up (100% follow-up), the Ikeuchi grading scale scored 48 knees as excellent, 30 as good, and 4 as fair. According to the MRI classification, 43 knees were no shift; 6, anterocentral shift; 15, posterocentral shift; and 12, central shift. Shift-type knees had a significantly larger number of peripheral tears, and repairs were performed in the shift-type knees (55%) more frequently than in the no-shift-type knees (28%). Among 82 knees, 31 were repaired simultaneously after a central partial meniscectomy. CONCLUSION: Magnetic resonance imaging classification provides more information to surgeons in choosing the appropriate treatment methods, although the final decision regarding procedure is made during arthroscopy after thorough analysis of the tear.
Assuntos
Imageamento por Ressonância Magnética/classificação , Meniscos Tibiais/cirurgia , Adolescente , Artroscopia , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Masculino , Auditoria Médica , Cuidados Pré-Operatórios , Estudos Retrospectivos , Ruptura/diagnóstico , Ruptura/patologiaRESUMO
During an arthroscopic examination for an anterior cruciate ligament (ACL) reconstruction, there is a relatively thick remnant ACL tibial stump attached to the posterior cruciate ligament (PCL) or rarely remained between the femur origin and the tibia insertion. We thought that preservation of the remnant ACL original bundle might promote graft healing or be helpful in preserving the proprioception and function to stabilize the knee. Therefore, we established a remnant preservation procedure without additional instruments during an ACL reconstruction using a bio-cross pin (RIGIDfix system: Mitek, Johnson & Johnson, USA) for the femoral tunnel fixation. The remnant ACL was sutured (usually three stitches) using a suture hook (Linvatec, Largo, FL), and both ends of the sutures were pulled to the far anteromedial (AM) portal. These sutures protected the remnant tissue during the ACL reconstruction because medial traction of these sutures can provide a wide view during the reconstruction. After the femoral and tibial tunnel formation, these sutures were pulled out to the inferior sleeve of the cross pin using a previously inserted wire loop via an inferior sleeve. After graft passage, a superior cross pin was first fixed and tibial fixation was then performed. Finally, inferior cross pin fixation was performed and ties were made at the entrance of the inferior cross pin.