RESUMO
The etiology of preeclampsia (PE), a complex and multifactorial condition, remains incompletely understood. DNA methylation, which is primarily regulated by three DNA methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B, plays a vital role in early embryonic development and trophectoderm differentiation. Yet, how DNMTs modulate trophoblast fusion and PE development remains unclear. In this study, we found that the DNMTs expression was downregulated during trophoblast cells fusion. Downregulation of DNMTs was observed during the reconstruction of the denuded syncytiotrophoblast (STB) layer of placental explants. Additionally, overexpression of DNMTs inhibited trophoblast fusion. Conversely, treatment with the DNA methylation inhibitor 5-aza-CdR decreased the expression of DNMTs and promoted trophoblast fusion. A combined analysis of DNA methylation data and gene transcriptome data obtained from the primary cytotrophoblasts (CTBs) fusion process identified 104 potential methylation-regulated differentially expressed genes (MeDEGs) with upregulated expression due to DNA demethylation, including CD59, TNFAIP3, SDC1, and CDK6. The transcription regulation region (TRR) of TNFAIP3 showed a hypomethylation with induction of 5-aza-CdR, which facilitated CREB recruitment and thereby participated in regulating trophoblast fusion. More importantly, clinical correlation analysis of PE showed that the abnormal increase in DNMTs may be involved in the development of PE. This study identified placental DNA methylation-regulated genes that may contribute to PE, offering a novel perspective on the role of epigenetics in trophoblast fusion and its implication in PE development.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Pré-Eclâmpsia , Trofoblastos , Trofoblastos/metabolismo , Feminino , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Humanos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Fusão Celular , Placenta/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genéticaRESUMO
Based on the association network of "drug pair-disease", the effect characteristics of Astragali Radix-Chuanxiong Rhizoma drug pair in the treatment of ischemic stroke(IS) with Qi deficiency and blood stasis and the matching mechanism of the two were explored. Through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction Database, the effective chemical components of the drug pair were screened, and the candidate targets were predicted. Databa-ses such as GeneCards, DrugBank, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD) were searched to obtain gene targets related to IS. Through STRING and Cytoscape 3.9.1 software, the protein-protein interaction(PPI) network was constructed by using the interaction information of disease syndrome-related genes and candidate targets of drug pairs, and the core targets were screened according to the network topological feature values. Based on the Metascape platform and DAVID database, the biomolecular interaction information was integrated to analyze the Kyoto Encyclopedia of Genes and Genomes(KEGG) and mine biological functions, so as to further explore the mechanism of action and compatibility characteristics of Astragali Radix-Chuan-xiong Rhizoma. The results showed that the candidate biological process was mainly involved in the regulation of functional modules such as immune, blood circulation, neurotransmitter, and oxidative stress, and it was enriched in lipid and atherosclerosis, calcium signaling pathway, and platelet activation. Astragali Radix and Chuanxiong Rhizoma have their own characteristics. Astragali Radix has a regulatory response to growth factors while maintaining the body's immune balance, while Chuanxiong Rhizoma mainly improves the circulatory system and participates in hormone metabolism, so as to indicate the compatibility mechanism of Astragali Radix-Chuanxiong Rhizoma drug pair for multi-target and multi-pathway synergistic treatment of IS. Through further experimental verification, it was found that the Astragali Radix-Chuanxiong Rhizoma drug pair could significantly down-regulate the expression of key targets including TLR4, NF-κB, IL-1ß, F2R, PLCß1, and MYLK. This study preliminarily reveals that the Astragali Radix-Chuanxiong Rhizoma drug pair may play the three replenishing effects of promoting blood circulation, benefiting Qi, and clearing collaterals by correcting immune di-sorders, blood circulation disorders, and inflammation, which provide support for the clinical research on the subsequent improvement of Qi deficiency and blood stasis in the treatment of IS and provide a new idea for the analysis of modern biological connotation of the compatibility of seven emotions of traditional Chinese medicine.
Assuntos
Astragalus propinquus , Medicamentos de Ervas Chinesas , AVC Isquêmico , Mapas de Interação de Proteínas , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/química , Humanos , Astragalus propinquus/química , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/genética , AVC Isquêmico/metabolismo , Rizoma/química , Ligusticum/químicaRESUMO
This study aims to decipher the mechanism of tetramethylpyrazine(TMP) in regulating the migration of neural stem cells(NSCs) in the rat model of middle cerebral artery occlusion(MCAO) via the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase 1(HO-1)/C-X-C motif chemokine receptor 4(CXCR4) pathway. SD rats were randomized into sham, MCAO(model), and tetramethylpyrazine(TMP, 20 mg·kg~(-1) and 40 mg·kg~(-1)) groups. The neurological impairment was assessed by the modified neurological severity score(mNSS). The immunofluorescence assay was employed to detect the cells stained with both 5-bromodeoxyuridine(BrdU) and doublecortin(DCX) in the brain tissue. The effect of TMP on the migration of C17.2 cells was observed. Western blot was employed to determine the protein levels of Nrf2, HO-1, p62, NAD(P)H quinone oxidoreductase 1(NQO1), stromal cell-derived factor 1(SDF-1), and CXCR4 in the brain tissue and C17.2 cells. The results showed that after 7 days and 21 days of mode-ling, the mNSS and BrdU~+/DCX~+ cells were increased, and the expression of Nrf2 and CXCR4 in the brain tissue was up-regulated. Compared with the model group, TMP(40 mg·kg~(-1)) reduced the mNSS, increased the number of BrdU~+/DCX~+ cells, and up-regulated the expression of Nrf2, CXCR4, and SDF-1. In addition, TMP promoted the migration of C17.2 cells and up-regulated the expression of p62, Nrf2, HO-1, and NQO1 in a time-and dose-dependent manner. The expression was the highest at the time point of 12 h in the TMP(50 µg·mL~(-1)) group(P<0.01). In conclusion, TMP activates the Nrf2/HO-1/CXCR4 pathway to promote the migration of NSCs to the ischemic area, thus exerting the therapeutic effect on the ischemia-reperfusion injury. This study provides experimental support for the application of TMP in ischemic stroke.
Assuntos
Movimento Celular , Heme Oxigenase-1 , Fator 2 Relacionado a NF-E2 , Células-Tronco Neurais , Pirazinas , Ratos Sprague-Dawley , Receptores CXCR4 , Animais , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Pirazinas/farmacologia , Ratos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Movimento Celular/efeitos dos fármacos , Masculino , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/genética , Proteína Duplacortina , Transdução de Sinais/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , HumanosRESUMO
This study aimed to investigate the intervention effect of tetramethylpyrazine(TMP) combined with transplantation of neural stem cells(NSCs) on middle cerebral artery occlusion(MCAO) rat model and to explore the mechanism of TMP combined with NSCs transplantation on ischemic stroke based on the regulation of stem cell biological behavior. MCAO rats were randomly divided into a model group, a TMP group, an NSCs transplantation group, and a TMP combined with NSCs transplantation group according to neurological function scores. A sham group was set up at the same time. The neurological function score was used to evaluate the improvement of neurological function in MCAO rats after TMP combined with NSCs transplantation. The proliferation, migration, and differentiation of NSCs were evaluated by BrdU, BrdU/DCX, BrdU/NeuN, and BrdU/GFAP immunofluorescence labeling. The protein expression of stromal cell-derived factor 1(SDF-1), C-X-C motif chemokine receptor 4(CXCR4), as well as oxidative stress pathway proteins nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(KEAP1), heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1) was detected by Western blot to study the migration mechanism of TMP combined with NSCs. The results showed that TMP combined with NSCs transplantation significantly improved the neurological function score in MCAO rats. Immunofluorescence staining showed a significant increase in the number of BrdU~+, BrdU~+/DCX~+, BrdU~+/NeuN~+, and BrdU~+/GFAP~+ cells in the TMP, NSCs transplantation, and combined treatment groups, with the combined treatment group showing the most significant increase. Further Western blot analysis revealed significantly elevated expression of CXCR4 protein in the TMP, NSCs transplantation, and combined treatment groups, along with up-regulated protein expression of Nrf2, HO-1, and NQO1, and decreased KEAP1 protein expression. This study showed that both TMP and NSCs transplantation can promote the recovery of neurological function by promoting the proliferation, migration, and differentiation of NSCs, and the effect of TMP combined with NSCs transplantation is superior. The mechanism of action may be related to the activation of the Nrf2/HO-1/CXCR4 pathway.
Assuntos
Isquemia Encefálica , Proteína Duplacortina , Fator 2 Relacionado a NF-E2 , Células-Tronco Neurais , Pirazinas , Ratos Sprague-Dawley , Receptores CXCR4 , Animais , Pirazinas/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/transplante , Células-Tronco Neurais/metabolismo , Ratos , Masculino , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Isquemia Encefálica/terapia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Transplante de Células-Tronco/métodos , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Humanos , Traumatismo por Reperfusão/terapia , Traumatismo por Reperfusão/metabolismo , Infarto da Artéria Cerebral Média/terapia , NAD(P)H Desidrogenase (Quinona)/metabolismo , NAD(P)H Desidrogenase (Quinona)/genéticaRESUMO
This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.
Assuntos
Armadilhas Extracelulares , Neutrófilos , Peroxidase , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Animais , Neutrófilos/efeitos dos fármacos , Neutrófilos/citologia , Camundongos , Ratos , Peroxidase/metabolismo , Peroxidase/genética , Acetato de Tetradecanoilforbol/farmacologia , Masculino , Lipopolissacarídeos/farmacologia , Ratos Sprague-Dawley , Histonas/metabolismo , Histonas/genética , HumanosRESUMO
Based on the sarcoma receptor coactivator(Src)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway, the mechanism of action of bulleyaconitine A in the treatment of bone destruction of experimental rheumatoid arthritis(RA) was explored. Firstly, key targets of RA bone destruction were collected through GeneCards, PharmGKB, and OMIM databa-ses. Potential targets of bulleyaconitine A were collected using SwissTargetPrediction and PharmMapper databases. Next, intersection targets were obtained by the Venny 2.1.0 platform. Protein-protein interaction(PPI) network and topology analysis were managed by utilizing the STRING database and Cytoscape 3.8.0. Then, Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were conducted in the DAVID database. AutoDock Vina was applied to predict the molecular docking and binding ability of bulleyaconitine A with key targets. Finally, a receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model was established in vitro. Quantitative real-time polymerase chain reaction(qRT-PCR) was used to detect the mRNA expression levels of related targets, and immunofluorescence and Western blot were adopted to detect the protein expression level of key targets. It displayed that there was a total of 29 drug-disease targets, and Src was the core target of bulleyaconitine A in anti-RA bone destruction. Furthermore, KEGG enrichment analysis revealed that bulleyaconitine A may exert an anti-RA bone destruction effect by regulating the Src/PI3K/Akt signaling pathway. The molecular docking results showed that bulleyaconitine A had better bin-ding ability with Src, phosphatidylinositol-4,5-diphosphate 3-kinase(PIK3CA), and Akt1. The result of the experiment indicated that bulleyaconitine A not only dose-dependently inhibited the mRNA expression levels of osteoclast differentiation-related genes cathepsin K(CTSK) and matrix metalloproteinase-9(MMP-9)(P<0.01), but also significantly reduced the expression of p-c-Src, PI3K, as well as p-Akt in vitro osteoclasts(P<0.01). In summary, bulleyaconitine A may inhibit RA bone destruction by regulating the Src/PI3K/Akt signaling pathway. This study provides experimental support for the treatment of RA bone destruction with bulleyaconitine A and lays a foundation for the clinical application of bulleyaconitine A.
Assuntos
Aconitina/análogos & derivados , Artrite Experimental , Artrite Reumatoide , Medicamentos de Ervas Chinesas , Animais , Proteínas Proto-Oncogênicas c-akt/genética , Fosfatidilinositol 3-Quinases/genética , Simulação de Acoplamento Molecular , Transdução de Sinais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , RNA Mensageiro , Medicamentos de Ervas Chinesas/farmacologiaRESUMO
OBJECTIVE: The purpose of this study is to investigate the role of high mobility group protein B1 (HMGB1) in placental development and fetal growth. METHODS: We employed the Cre-loxP recombination system to establish a placenta-specific HMGB1 knockout mouse model. Breeding HMGB1flox/flox mice with Elf5-Cre mice facilitated the knockout, leveraging Elf5 expression in extra-embryonic ectoderm, ectoplacental cone, and trophoblast giant cells at 12.5 days of embryonic development. The primary goal of this model was to elucidate the molecular mechanism of HMGB1 in placental development, assessing parameters such as placental weight, fetal weight, and bone development. Additionally, we utilized lentiviral interference and overexpression of HMGB1 in human trophoblast cells to further investigate HMGB1's functional role. RESULTS: Our findings indicate that the HMGB1flox/floxElf5cre/+ mouse displays fetal growth restriction, characterized by decreased placental and fetal weight and impaired bone development. The absence of HMGB1 inhibits autophagosome formation, impairs lysosomal degradation, and disrupts autophagic flux. Depletion of HMGB1 in human trophoblast cells also suppresses cell viability, proliferation, migration, and invasion by inhibiting the ERK signaling pathway. Overexpression of HMGB1 observed the opposite phenotypes. CONCLUSIONS: HMGB1 participates in the regulation of autophagy through the ERK signaling pathway and affects placental development.
Assuntos
Autofagia , Proteína HMGB1 , Sistema de Sinalização das MAP Quinases , Placenta , Trofoblastos , Animais , Feminino , Humanos , Camundongos , Gravidez , Autofagia/fisiologia , Proteína HMGB1/metabolismo , Proteína HMGB1/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos Knockout , Placenta/metabolismo , Placentação/fisiologia , Trofoblastos/metabolismo , Trofoblastos/fisiologia , MasculinoRESUMO
BACKGROUND AND AIMS: Intrahepatic cholestasis of pregnancy (ICP) is a special liver disease during pregnancy, characterized by abnormal bile acid metabolism. However, there is no consensus on how to group women with ICP based on the time of diagnosis worldwide. This study aimed to adopt a new grouping model of women with ICP, and the time from diagnosis to delivery was defined as the monitoring period. METHODS: This retrospective real-world data study was conducted across multiple centers and included 3172 women with ICP. The study first evaluated the significant difference in medication and nonmedication during different monitoring times. The least absolute shrinkage and selection operator (LASSO) model was then used to screen nine risk factors based on the predictors. The model's discrimination, clinical usefulness, and calibration were assessed using the area under the receiver operating characteristic (ROC) curve, decision curve, and calibration analysis. RESULTS: The incidence of preeclampsia risk in ICP patients without drug intervention increased with the extension of the monitoring period. However, the risk of preeclampsia decreased in ICP patients treated with ursodeoxycholic acid. A predictive nomogram and risk score model was developed based on nine risk factors. The area under the ROC curve of the nomogram was 0.765 [95% confidence interval (CI): 0.724-0.807] and 0.812 (95% CI: 0.736-0.889) for the validation cohort. CONCLUSIONS: This study found that a longer ICP monitoring period could lead to adverse pregnancy outcomes in the absence of drug intervention, especially preeclampsia. A predictive nomogram and risk score model was developed to better manage ICP patients, maintain pregnancy to term delivery, and minimize the risk of severe adverse maternal and fetal outcomes.