RESUMEN
BACKGROUND: Patients with hip fracture often experience severe pain, particularly during movement or slight positional change, prior to the occurrence of surgery. It is essential to explore the appropriate analgesic methods before surgery in patients with hip fracture, especially those capable of alleviating dynamic pain. Pericapsular nerve group (PENG) block was introduced as a useful technique for hip analgesia. In this study, we aimed to compare the reduction in dynamic pain between the PENG block and supra-inguinal fascia iliaca compartment block (SIFICB). METHODS: This prospective trial included 80 hip fracture patients aged ≥19 years, with an American Society of Anesthesiologists Physical Status of 1-4 and a baseline dynamic pain score ≥4 on the numerical rating scale. The patients were randomly allocated into the PENG block (n=40) and SIFICB group (n=40). For the PENG block and SIFICB, 20 mL and 30 mL of 0.3% ropivacaine was used, respectively. The primary outcome was reduction in dynamic pain scores at 30 min following the peripheral nerve block. Dynamic pain score was evaluated when the leg was passively raised. RESULTS: A total of 79 patients were included in the final analysis, and the reductions in pain score during hip flexion were 3.1±2.4 and 2.9±2.5 in the PENG block and SIFICB groups, respectively, which was statistically insignificant (p=0.75). Moreover, no significant differences were observed in any of the outcomes. CONCLUSIONS: PENG block and SIFICB could effectively provide analgesia for dynamic pain in patients with hip fractures, with no significant difference between the two groups. TRIAL REGISTRATION NUMBER: NCT04677348.
RESUMEN
Atopic dermatitis is a chronic inflammatory skin disease characterized by intense itching and frequent skin barrier dysfunctions. EGR-1 is a transcription factor that aggravates the pathogenesis of atopic dermatitis by promoting the production of various inflammatory cytokines. Three 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides (IT21, IT23, and IT25) were identified as novel inhibitors of EGR-1 DNA-binding activity. In silico docking experiments were performed to elucidate the binding conditions of the EGR-1 zinc-finger (ZnF) DNA-binding domain. Electrophoretic mobility shift assays confirmed the targeted binding effect on the EGR-1 ZnF DNA-binding domain, leading to dose-dependent dissociation of the EGR-1-DNA complex. At the functional cellular level, IT21, IT23, and IT25 effectively reduced mRNA expression of TNFα-induced EGR-1-regulated inflammatory genes, particularly in HaCaT keratinocytes inflamed by TNFα. In the in vivo efficacy study, IT21, IT23, and IT25 demonstrated the potential to alleviate atopic dermatitis-like skin lesions in the ear skin of BALB/c mice. These findings suggest that targeting the EGR-1 ZnF DNA-binding domain with 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamide derivatives (IT21, IT23, and IT25) could serve as lead compounds for the development of potential therapeutic agents against inflammatory skin disorders, including atopic dermatitis.
Asunto(s)
Dermatitis Atópica , Diseño de Fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/patología , Humanos , Animales , Ratones , Relación Estructura-Actividad , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Estructura Molecular , Relación Dosis-Respuesta a Droga , Simulación del Acoplamiento Molecular , Ratones Endogámicos BALB C , Indoles/química , Indoles/farmacología , Indoles/síntesis química , Hidrazinas/farmacología , Hidrazinas/química , Hidrazinas/síntesis químicaRESUMEN
Epidermal hyperinnervation is a critical feature of pruritus during skin inflammation. However, the mechanisms underlying epidermal hyperinnervation are unclear. This study investigates the role of the transcription factor EGR1 in epidermal innervation by utilizing wild-type (Egr1+/+) and Egr1-null (Egr1â/â) mice topically applied Dermatophagoides farinae extract from dust mite. Our findings revealed that Egr1â/â mice exhibited reduced scratching behaviors and decreased density of epidermal innervation compared with Egr1+/+ mice. Furthermore, we identified artemin, a neurotrophic factor, as an EGR1 target responsible for Dermatophagoides farinae extract-induced hyperinnervation. It has been demonstrated that Dermatophagoides farinae extract stimulates toll-like receptors in keratinocytes. To elucidate the cellular mechanism, we stimulated keratinocytes with Pam3CSK4, a toll-like receptor 1/2 ligand. Pam3CSK4 triggered a toll-like receptor 1/2-mediated signaling cascade involving IRAK4, IκB kinase, MAPKs, ELK1, EGR1, and artemin, leading to increased neurite outgrowth and neuronal migration. In addition, increased expression of EGR1 and artemin was observed in the skin tissues of patients with atopic dermatitis. These findings highlight the significance of the EGR1-artemin axis in keratinocytes, promoting the process of epidermal innervation and suggesting it as a potential therapeutic target for alleviating itch and pain associated with house dust mite-induced skin inflammation.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz , Epidermis , Queratinocitos , Proteínas del Tejido Nervioso , Células Receptoras Sensoriales , Animales , Queratinocitos/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Epidermis/inervación , Epidermis/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Células Receptoras Sensoriales/metabolismo , Dermatophagoides farinae/inmunología , Prurito/inmunología , Prurito/etiología , Prurito/metabolismo , Modelos Animales de Enfermedad , Humanos , Antígenos Dermatofagoides/inmunología , Transducción de Señal , Ratones Noqueados , Masculino , Dermatitis Atópica/inmunología , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patologíaRESUMEN
Mesenchymal stem cells (MSCs) can differentiate into endothelial cells; however, the mechanisms underlying this process in the tumor microenvironment (TME) remain elusive. This study shows that tumor necrosis factor alpha (TNF-α), a key cytokine present in the TME, promotes the endothelial differentiation of MSCs by inducing vascular endothelial growth factor receptor 2 (VEGFR2) gene expression. EGR1 is a member of the zinc-finger transcription factor family induced by TNF-α. Our findings indicate that EGR1 directly binds to the VEGFR2 promoter and transactivates VEGFR2 expression. We also demonstrate that EGR1 forms a complex with c-JUN activated by c-JUN N-terminal kinase (JNK) to promote VEGFR2 transcription and endothelial differentiation in MSCs in response to TNF-α stimulation. The shRNA-mediated silencing of EGR1 or c-JUN abrogates TNF-α-induced VEGFR2 transcription and the endothelial differentiation of MSCs. To further evaluated the role of EGR1 in the endothelial differentiation of BM-MSCs, we used a syngenic tumor implantation model. 4T1 mouse mammary tumor cells were injected subcutaneously into BALB/c mice with primary mBM-MSCs isolated from wild-type (Egr1+/+) or Egr1-null (Egr1-/-) mice. CD31-positive cells were predominantly observed at the border of the tumor in the 4T1 plus wild-type MSC group, while staining less in the 4T1 alone or 4T1 plus Egr1-null MSC group. Collectively, these findings demonstrate that the JNK-EGR1 signaling axis plays a crucial role in the TNF-α-induced endothelial differentiation of MSCs in the TME, which could be a potential therapeutic target for solid tumors vasculatures.
Asunto(s)
Células Madre Mesenquimatosas , Factor de Necrosis Tumoral alfa , Humanos , Ratones , Animales , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Células Endoteliales/metabolismo , Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Células Cultivadas , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismoRESUMEN
Atopic dermatitis (AD) is one of the most common inflammatory skin diseases accompanied by severe itching. ß-caryophyllene (BCP), which displays anti-inflammatory activity, is a natural agonist of cannabinoid receptor 2. However, the therapeutic effects of BCP on atopic dermatitis (AD) remain poorly understood. The current study aimed to evaluate the topical therapeutic efficacy of BCP in an AD-like mouse model. Thymic Stromal Lymphopoietin (TSLP) is a keratinocyte-derived cytokine that drives AD pathogenesis. This study also investigated the effect of BCP on the interleukin 4 (IL-4)-induced expression of TSLP in HaCaT keratinocytes. We found that the topical application of BCP alleviated AD-like skin inflammation and inhibited the infiltration of proinflammatory cells into skin lesions. Moreover, the topical application of BCP reduced EGR1 (Early Growth Response 1) and TSLP expression in AD-like skin lesions. We also found that BCP inhibited IL-4-induced TSLP expression by downregulating mitogen-activated protein kinase (MAPK)-mediated EGR1 expression in HaCaT keratinocytes. These findings demonstrate that BCP ameliorates DNCB-induced AD-like skin lesions through the downregulation of the MAPK/EGR1/TSLP signaling axis. BCP may be applicable for developing topical therapeutic agents for chronic skin inflammatory diseases, such as AD.
Asunto(s)
Dermatitis Atópica , Ratones , Animales , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/metabolismo , Dinitroclorobenceno , Interleucina-4/metabolismo , Linfopoyetina del Estroma Tímico , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Citocinas/metabolismo , Queratinocitos/metabolismo , Piel/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismoRESUMEN
Filaggrin (FLG), an essential structural protein for skin barrier function, is down-regulated under chronic inflammatory conditions, leading to disruption of the skin barrier. However, the detailed molecular mechanisms of how FLG changes in the context of chronic inflammation are poorly understood. Here, we identified the molecular mechanisms by which inflammatory cytokines inhibit FLG expression in the skin. We found that the AP1 response element within the -343/+25 of the FLG promoter was necessary for TNFα + IFNγ-induced down-regulation of FLG promoter activity. Using DNA affinity precipitation assay, we observed that AP1 subunit composition binding to the FLG promoter was altered from c-FOS:c-JUN (at the early time) to FRA1:c-JUN (at the late time) in response to TNFα + IFNγ stimulation. Knockdown of FRA1 or c-JUN abrogated TNFα + IFNγ-induced FLG suppression. Histone deacetylase (HDAC) 1 interacted with FRA1:c-JUN under TNFα + IFNγ stimulation. Knockdown of HDAC1 abrogated the inhibitory effect of TNFα + IFNγ on FLG expression. The altered expression of FLG, FRA1, c-JUN, and HDAC1 was confirmed in mouse models of 2,4-dinitrochlorobenzene-induced atopic dermatitis and imiquimod-induced psoriasis. Thus, the current study demonstrates that TNFα + IFNγ stimulation suppresses FLG expression by promoting the FRA1:c-JUN:HDAC1 complex. This study provides insight into future therapeutic strategies targeting the FRA1:c-JUN:HDAC1 complex to restore impaired FLG expression in chronic skin inflammation.
Asunto(s)
Proteínas Filagrina , Histona Desacetilasa 1 , Queratinocitos , Proteínas Proto-Oncogénicas c-fos , Proteínas Proto-Oncogénicas c-jun , Animales , Enfermedad Crónica , Dermatitis/genética , Dermatitis/metabolismo , Regulación hacia Abajo , Proteínas Filagrina/genética , Proteínas Filagrina/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Interferón gamma/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The circadian clock system is closely associated with inflammatory responses. Dysregulation of the circadian clock genes in the skin impairs the skin barrier function and affects the pathophysiology of atopic dermatitis. Interleukin 4 (IL-4) is a proinflammatory cytokine derived from T-helper type 2 cells; it plays a critical role in the pathogenesis of atopic dermatitis. Agerarin (6,7-dimethoxy-2,2-dimethyl-2H-chromene) is a natural JAK1/2/3 inhibitor isolated from Ageratum houstonianum that has a protective effect on the epidermal skin barrier. However, it remains unclear whether agerarin affects the circadian clock system. The aim of this study is to investigate the effect of agerarin on IL-4-induced PER2 gene expression in human keratinocytes through reverse transcription (RT)-PCR, quantitative real-time PCR (qPCR), immunoblotting, immunofluorescence microscopic analysis, and real-time bioluminescence analysis. We found that agerarin reduced IL-4-induced PER2 mRNA expression by suppressing the JAK-STAT3 pathway. In addition, real-time bioluminescence analysis in PER2:luc2p promoter-reporter cells revealed that agerarin restored the oscillatory rhythmicity of PER2 promoter activity altered by IL-4. These findings suggest that agerarin may be useful as a cosmeceutical agent against inflammatory skin conditions associated with disrupted circadian rhythms, such as atopic dermatitis.
Asunto(s)
Dermatitis Atópica , Inhibidores de las Cinasas Janus , Benzopiranos , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/genética , Dermatitis Atópica/metabolismo , Humanos , Interleucina-4/metabolismo , Inhibidores de las Cinasas Janus/farmacología , Queratinocitos , Proteínas Circadianas Period/genéticaRESUMEN
Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases worldwide, characterized by intense pruritus and eczematous lesions. Aberrant expression of thymic stromal lymphopoietin (TSLP) in keratinocytes is associated with the pathogenesis of AD and is considered a therapeutic target for the treatment of this disease. Saikosaponin A (SSA) and saikosaponin C (SSC), identified from Radix Bupleuri, exert anti-inflammatory effects. However, the topical effects of SSA and SSC on chronic inflammatory skin diseases are unclear. In this study, we investigated the effects of SSA and SSC on TSLP suppression in an AD-like inflammatory environment. We observed that SSA and SSC suppressed tumor necrosis factor-α-induced TSLP expression by downregulating the expression of the transcription factor early growth response 1 (EGR1) via inhibition of the extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and p38 mitogen-activated protein kinase pathways. We also confirmed that topical application of SSA or SSC reduced AD-like skin lesions in BALB/c mice challenged with 2,4-dinitrochlorobenzene. Our findings suggest that suppression of EGR1-regulated TSLP expression in keratinocytes might be attributable to the anti-inflammatory effects of SSA and SSC in AD-like skin lesions.
Asunto(s)
Dermatitis Atópica , Enfermedades de la Piel , Animales , Antiinflamatorios/farmacología , Citocinas/metabolismo , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Células HaCaT , Humanos , Queratinocitos/metabolismo , Ratones , Ácido Oleanólico/análogos & derivados , Saponinas , Enfermedades de la Piel/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Linfopoyetina del Estroma TímicoRESUMEN
PER2 is a core circadian clock gene that regulates circadian rhythms. IL-4 plays a critical role in the pathogenesis of skin inflammation, including atopic dermatitis. IL-4 enhances PER2 expression, suggesting a relationship between inflammation and the circadian clock. However, little is known about the molecular and cellular mechanisms regulating PER2 expression by inflammatory cytokines. This study showed that transcription factor EGR1 interacted with the PER2 promoter and promoted IL-4âinduced transcriptional activation of the PER2, as revealed by promoterâreporter assay, electrophoretic mobility shift assay, DNA affinity precipitation assay, and chromatin immunoprecipitation analysis. We also found that IL-4 can use both MAPK and Jak signaling pathways to induce EGR1-mediated PER2 expression, and c-Jun N-terminal kinase 1/2 can augment IL-4âinduced activation of the Jakâsignal transducer and activator of transcription 3 pathway. Consistently, Per2 expression was reduced in dinitrochlorobenzene-induced atopic dermatitisâlike skin lesions in Egr1â/â mice compared with that in Egr1+/+ mice. In addition, using a real-time bioluminescence assay, we observed that EGR1 is required for rhythmic oscillation of PER2 expression under IL-4 exposure. These findings provide further insight into the role of EGR1 in regulating PER2 expression in impaired circadian rhythm in skin inflammation.
Asunto(s)
Dermatitis Atópica , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Interleucina-4/metabolismo , Proteínas Circadianas Period , Animales , Ritmo Circadiano/fisiología , ADN/genética , Dermatitis Atópica/genética , Dinitroclorobenceno , Humanos , Inflamación , Queratinocitos/metabolismo , Ratones , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Novel (Z)-3-((4,6-diphenylpyrimidin-2-ylamino)methylene)-2,3-dihydrochromen-4-one derivatives were designed and synthesized to find chemotherapeutic agents. Derivative 9 was selected based on its clonogenicity against cancer cells and synthetic yield for further biological experiments. It showed decreases in aurora kinase A, B, and C phosphorylation from western blot analysis. Derivative 9 upregulated the expression of G1 cell cycle inhibitory proteins including p21 and p27, and G1 progressive cyclin D1, and downregulated G1-to-S progressive cyclins, resulting in cell cycle arrest at the G1/S boundary. It stimulated the cleavage of caspase-9, -3, -7, and poly (ADP-ribose) polymerase, resulting in triggering apoptosis through a caspase-dependent pathway. In addition, derivative 9 inhibited in vivo tumor growth in a syngeneic tumor implantation mouse model. The findings of this study suggest that derivative 9 can be considered as a lead compound for chemotherapeutic agents.
Asunto(s)
Antineoplásicos , Caspasas , Animales , Antineoplásicos/farmacología , Apoptosis , Caspasas/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/farmacología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/farmacología , Ratones , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that acts as a critical mediator in the pathogenesis of atopic dermatitis (AD). Various therapeutic agents that prevent TSLP function can efficiently relieve the clinical symptoms of AD. However, the downregulation of TSLP expression by therapeutic agents remains poorly understood. In this study, we investigated the mode of action of chrysin in TSLP suppression in an AD-like inflammatory environment. We observed that the transcription factor early growth response (EGR1) contributed to the tumor necrosis factor alpha (TNFα)-induced transcription of TSLP. Chrysin attenuated TNFα-induced TSLP expression by downregulating EGR1 expression in HaCaT keratinocytes. We also showed that the oral administration of chrysin improved AD-like skin lesions in the ear and neck of BALB/c mice challenged with 2,4-dinitrochlorobenzene. We also showed that chrysin suppressed the expression of EGR1 and TSLP by inhibiting the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 mitogen-activated protein kinase pathways. Collectively, the findings of this study suggest that chrysin improves AD-like skin lesions, at least in part, through the downregulation of the ERK1/2 or JNK1/2-EGR1-TSLP signaling axis in keratinocytes.
Asunto(s)
Citocinas/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Enfermedades de la Piel/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/farmacología , Animales , Células Cultivadas , Citocinas/genética , Dinitroclorobenceno/toxicidad , Humanos , Queratinocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Enfermedades de la Piel/inducido químicamente , Enfermedades de la Piel/metabolismo , Enfermedades de la Piel/patología , Linfopoyetina del Estroma TímicoAsunto(s)
Antiinflamatorios/farmacología , ADN/metabolismo , Dermatitis Atópica/tratamiento farmacológico , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Animales , Antiinflamatorios/uso terapéutico , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Dinitroclorobenceno/administración & dosificación , Dinitroclorobenceno/toxicidad , Modelos Animales de Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Células HaCaT , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Noqueados , Simulación del Acoplamiento Molecular , Unión Proteica/efectos de los fármacos , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Kallikrein-related peptidase 7 (KLK7) is a chymotrypsin-like serine peptidase that plays a crucial role in regulating skin desquamation. KLK7 expression is highly upregulated in atopic dermatitis (AD) skin lesions in both humans and mice. Th2-lymphocyte-derived cytokines, including interleukin (IL)-4 and IL-13, have been shown to promote KLK7 expression in keratinocytes in patients with AD. However, the molecular mechanism underlying KLK7 expression remains poorly understood. Here, we demonstrated that the EGR-1-binding sequence (EBS) in the promoter region of KLK7 played a crucial role in IL-13-induced KLK7 transcription. Disruption of the EBS induced by a point mutation inhibited IL-13-induced KLK7 promoter activity. EGR-1 was shown to directly bind to the EBS, and EGR1 knockdown with shRNA abrogated IL-13-induced KLK7 expression. Using Egr1 knockout mice, we showed that Egr-1 was necessary for KLK7 expression in AD-like lesions induced by the repeated topical application of 2,4-dinitrobenzene on the dorsal skin of mice. We also demonstrated that the ERK1/2 mitogen-activated protein kinase (MAPK) pathway was responsible for EGR-1-dependent KLK7 transcription in response to IL-13 stimulation. Our findings delineate a signaling pathway that contributes to the regulation of KLK7 expression through the IL13-ERK MAPK-EGR1 signaling axis.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Interleucina-13/metabolismo , Calicreínas/genética , Animales , Dermatitis Atópica/genética , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Modelos Animales de Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 1 de la Respuesta de Crecimiento Precoz/deficiencia , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Técnicas de Silenciamiento del Gen , Células HaCaT , Humanos , Calicreínas/metabolismo , Queratinocitos/metabolismo , Queratinocitos/patología , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Chrysin (5,7-dihydroxyflavone) is a natural polyphenolic compound that induces an anti-inflammatory response. In this study, we investigated the molecular mechanism underlying the chrysin-induced suppression of C-C motif chemokine ligand 5 (CCL5) gene expression in atopic dermatitis (AD)-like inflammatory microenvironment. We showed that chrysin inhibited CCL5 expression at the transcriptional level through the suppression of nuclear factor kappa B (NF-κB) in the inflammatory environment. Chrysin could bind to the ATP-binding pocket of the inhibitor of κB (IκB) kinase (IKK) and, subsequently, prevent IκB degradation and NF-κB activation. The clinical efficacy of chrysin in targeting IKK was evaluated in 2,4-dinitrochlorobenzene-induced skin lesions in BALB/c mice. Our results suggested that chrysin prevented CCL5 expression by targeting IKK to reduce the infiltration of mast cells to the inflammatory sites and at least partially attenuate the inflammatory responses. These findings suggested that chrysin might be useful as a platform for the design and synthesis of small-molecule IKK-targeting drugs for the treatment of chronic inflammatory diseases, such as AD.
Asunto(s)
Quimiocina CCL5/genética , Dermatitis Atópica/genética , Flavonoides/farmacología , Quinasa I-kappa B/genética , Inflamación/tratamiento farmacológico , Animales , Microambiente Celular/efectos de los fármacos , Microambiente Celular/genética , Quimiocina CCL5/antagonistas & inhibidores , Dermatitis Atópica/patología , Flavonoides/química , Humanos , Inflamación/genética , Inflamación/patología , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , FN-kappa B/genética , Factor de Necrosis Tumoral alfaRESUMEN
WNT11 is a member of the non-canonical Wnt family and plays a crucial role in tumor progression. However, the regulatory mechanisms underlying WNT11 expression are unclear. Tumor necrosis factor-alpha (TNFα) is a major inflammatory cytokine produced in the tumor microenvironment and contributes to processes associated with tumor progression, such as tumor invasion and metastasis. By using site-directed mutagenesis and introducing a serial deletion in the 5'-regulatory region of WNT11, we observed that TNFα activates the early growth response 1 (EGR1)-binding sequence (EBS) in the proximal region of WNT11 and that the transcription factor EGR1 is necessary for the TNFα-induced transcription of WNT11. EGR1 bound directly to the EBSs within the proximal 5'-regulatory region of WNT11 and ectopic expression of EGR1 stimulated WNT11 promoter activity, whereas the knockdown of EGR1 expression by RNA interference reduced TNFα-induced WNT11 expression in T47D breast cancer cells. We also observed that mitogen-activated protein kinases (MAPK), extracellular signalregulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase mediated TNFα-induced transcription of WNT11 via EGR1. Our results suggest that EGR1 directly targets WNT11 in response to TNFα stimulation in breast cancer cells. [BMB Reports 2020; 53(12): 628-633].
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteínas Wnt/metabolismo , Sitios de Unión/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Transducción de Señal/genética , Transcripción Genética/genética , Microambiente Tumoral/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Wnt/genética , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Pro-opiomelanocortin (POMC) is a large precursor protein of and ß-endorphin. POMC expressed in keratinocytes regulates various pathophysiological responses, such as pruritus in atopic dermatitis. Interleukin (IL)-31 is a T helper 2 (Th2)-derived cytokine that functions as a pruritogen, stimulating the sensory neurons in the skin. However, the regulatory mechanism underlying IL-31-induced POMC expression in keratinocytes remains largely unknown. Herein, using a 5'-serial deletion and site-specific mutation constructs of the regulatory region of POMC, we demonstrated that a putative EGR1-binding sequence (EBS) motif in POMC is required for its upregulation by IL-31 in HaCaT keratinocytes. Notably, EGR-1 directly interacted with the EBS motif in POMC. The ectopic expression of EGR-1 stimulated the POMC promoter activity, whereas the knockdown of EGR-1 expression by RNA interference reduced IL-31-induced POMC expression. Furthermore, we observed that three major mitogen-activated protein kinases, ERK, JNK, and p38 kinase, mediated IL-31-induced EGR-1 expression. In summary, our results suggest that EGR-1 trans-activates POMC in response to IL-31 stimulation in HaCaT keratinocytes.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Interleucinas/farmacología , Queratinocitos/metabolismo , Proopiomelanocortina/genética , Transcripción Genética/efectos de los fármacos , Secuencias de Aminoácidos , Línea Celular Transformada , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Genes Reporteros , Genes Sintéticos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Mutación Puntual , Proopiomelanocortina/biosíntesis , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Activación Transcripcional , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Matrix metalloproteinase 1 (MMP-1), a calcium-dependent zinccontaining collagenase, is involved in the initial degradation of native fibrillar collagen. Tissue necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that is rapidly produced by dermal fibroblasts, monocytes/macrophages, and keratinocytes and regulates inflammation and damaged-tissue remodeling. MMP-1 is induced by TNFα and plays a critical role in tissue remodeling and skin aging processes. However, the regulation of the MMP1 gene by TNFα is not fully understood. We aimed to find additional cis-acting elements involved in the regulation of TNFα-induced MMP1 gene transcription in addition to the nuclear factor-kappa B (NF-κB) and activator protein 1 (AP1) sites. Assessments of the 5'-regulatory region of the MMP1 gene, using a series of deletion constructs, revealed the requirement of the early growth response protein 1 (EGR-1)-binding sequence (EBS) in the proximal region for proper transcription by TNFα. Ectopic expression of EGR-1, a zinc-finger transcription factor that binds to G-C rich sequences, stimulated MMP1 promoter activity. The silencing of EGR-1 by RNA interference reduced TNFα-induced MMP-1 expression. EGR-1 directly binds to the proximal region and transactivates the MMP1 gene promoter. Mutation of the EBS within the MMP1 promoter abolished EGR-1-mediated MMP-1 promoter activation. These data suggest that EGR-1 is required for TNFα-induced MMP1 transcriptional activation. In addition, we found that all three MAPKs, ERK1/2, JNK, and p38 kinase, mediate TNFα-induced MMP-1 expression via EGR-1 upregulation. These results suggest that EGR-1 may represent a good target for the development of pharmaceutical agents to reduce inflammation-induced MMP-1 expression. [BMB Reports 2020; 53(6): 323-328].
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Queratinocitos , Metaloproteinasa 1 de la Matriz/genética , Regiones Promotoras Genéticas/genética , Factor de Necrosis Tumoral alfa/metabolismo , Células Cultivadas , Humanos , Queratinocitos/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismoRESUMEN
Filaggrin (FLG) is a structural component of the stratum corneum that is essential for maintaining the barrier function of the skin and for the formation of natural moisturizing factors. 6,7-Dimethoxy-2,2-dimethyl-2H-chromene (Agerarin) is a bioactive compound derived from Ageratum houstonianum, a plant that is used as a traditional medicine to treat skin diseases. This study aimed to evaluate the effect of agerarin on skin inflammation in a dinitrochlorobenzene (DNCB)-induced atopic dermatitis mouse model. We found that the topical administration of agerarin ameliorates atopic dermatitis-like skin lesions. We also showed that agerarin restores the reduced filaggrin (FLG) expression in DNCB-applied skin sections. Moreover, agerarin decreased phosphorylation of JAK1 and JAK2 kinases to enhance FLG expression, which was reduced by TNFα+IFNγ and IL4+IL13 treatment, in HaCaT keratinocytes. These results demonstrate the feasibility of agerarin as a possible therapeutic against conditions of skin inflammation, such as atopic dermatitis, by improving the upregulation of FLG expression.
RESUMEN
AIM: This study was designed to compare the survival rates, follicular growth rates, and ovulation rates of vitrified preantral follicles (PF) from ovaries with those isolated from a vitrified ovarian cortical strip. METHODS: Mouse ovaries were divided into three groups: those not treated by vitrification of the PF (control), those treated by vitrification of the PF isolated from the ovaries (group I), and those treated by vitrification of ovarian tissue followed by PF isolation (group II). The group I samples were exposed to equilibration solution (EG-20) for 5.0 min plus vitrification solution (EFS-40) for 0.5 min, while the group II samples were exposed to EG-20 for 10.0 min plus EFS-40 for 2.0 min, before vitrification. They were subsequently placed on an electron microscope grid, and submerged immediately in liquid nitrogen. After thawing, the survival rate and the growth rate of the follicles were evaluated every 2 days. RESULTS: In the in vitro condition, the follicles grew and developed into antral follicles in groups I and II. The survival rate of the group I samples was higher than that of the group II samples during the in vitro culture (P<0.05). The growth rates of the follicles in group I were higher than those in group II after day 6 (P<0.05). The ovulation rate of the samples in group I was higher than that of group II (P<0.05). CONCLUSION: These results demonstrated that direct PF vitrification appeared to be better than vitrification of the PF isolated from ovarian tissue.
Asunto(s)
Criopreservación/métodos , Folículo Ovárico/fisiología , Ovulación/fisiología , Animales , Células Cultivadas , Femenino , Ratones , Ratones EndogámicosRESUMEN
Oocyte cryopreservation and intracytoplasmic sperm injection (ICSI) are advantageous to expand their usefulness in genetic engineering. Oocytes matured for 22 hr were vitrified in droplets of cryoprotectants (3.2 M ethylene glycol (EG), 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose) on copper electron microscope (EM) grids. After being warmed, the oocytes were cultured in IVM medium for an additional 2 hr. Sperm treated with dithiothreitol were utilized for ICSI. Oocytes injected with sperm were activated by combination of ionomycin with cycloheximide (CHX). The ICSI oocytes were compared for the rates of pronuclear formation, development, cell number, and the ratio of ICM to those of fresh ICSI and IVF control. The proportion of 2PN formation was significantly higher in IVF control (Group 1) than those in other treated groups. Among the treated groups a significant lower 2PN formation was observed in IVF-frozen-thawed than in ICSI-fresh and frozen-thawed groups. Cleavage rates in IVF-frozen-thawed and ICSI-frozen-thawed groups were significantly lower than those of IVF control and ICSI-fresh groups. In ICSI groups, the rates of cleavage and blastocyst in fresh oocytes were significantly higher than in frozen-thawed. Development rates into blastocysts in the ICSI-fresh and frozen-thawed groups were significantly lower than that of IVF control. Total cell number was significantly lower in both frozen-thawed IVF and ICSI groups than those in IVF-control and ICSI-fresh groups. However, the rates of the remaining cells that were found in the ICM were significantly higher in both frozen-thawed IVF and ICSI than in the IVF-control and ICSI-fresh groups. The results indicated that frozen-thawed bovine oocytes were suitable for ICSI procedure.