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Adipogenesis, a pivotal cellular process involving the differentiation of mesenchymal stem cells (MSCs) to mature adipocytes, plays a significant role in various physiological functions. Dysregulation of adipogenesis is implicated in conditions such as obesity. However, the complete molecular understanding of adipogenesis remains elusive. This study aimed to uncover the novel role of lamina-associated polypeptide 2 alpha (LAP2α) in human adipose-derived stem cells (hASCs) adipogenesis and its impact on high-fat diet (HFD)-induced obesity and associated metabolic disturbances. LAP2α expression was assessed during the adipogenic differentiation of hASCs using RT-qPCR and western blotting. The functional role of LAP2α in adipogenesis was explored both in vitro and in vivo through loss- and gain-of-function studies. Moreover, mice with HFD-induced obesity received lentivirus injection to assess the effect of LAP2α knockdown on fat accumulation. Molecular mechanisms underlying LAP2α in adipogenic differentiation were investigated using RT-qPCR, Western blotting, immunofluorescence staining, and Oil Red O staining. LAP2α expression was upregulated during hASCs adipogenic differentiation. LAP2α knockdown hindered adipogenesis, while LAP2α overexpression promoted adipogenic differentiation. Notably, LAP2α deficiency resisted HFD-induced obesity, improved glucose intolerance, mitigated insulin resistance, and prevented fatty liver development. Mechanistically, LAP2α knockdown attenuated signal transducer and activator of transcription 3 (STAT3) activation by reducing the protein level of phosphorylated STAT3. A STAT3 activator (Colivelin) counteracted the negative impact of LAP2α deficiency on hASCs adipogenic differentiation. Taken together, our current study established LAP2α as a crucial regulator of hASCs adipogenic differentiation, unveiling a new therapeutic target for obesity prevention.
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Adipogenia , Dieta Hiperlipídica , Células-Tronco Mesenquimais , Obesidade , Humanos , Dieta Hiperlipídica/efeitos adversos , Obesidade/metabolismo , Obesidade/genética , Obesidade/etiologia , Animais , Camundongos , Células-Tronco Mesenquimais/metabolismo , Masculino , Diferenciação Celular , Camundongos Endogâmicos C57BL , Tecido Adiposo/metabolismo , Tecido Adiposo/citologia , Adipócitos/metabolismo , Células Cultivadas , Técnicas de Silenciamento de Genes , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Proteínas de Ligação a DNA , Proteínas de MembranaRESUMO
Setting 7 subsection in abstract Objectives: Necroptosis, a form of programmed cell death, can occur in the placenta of patients with preeclampsia (PE). Hydrogen sulfide (H2S) can inhibit necroptosis of human umbilical vein endothelial cells under the high-glucose-induced injury. Whether H2S can protect trophoblasts against necroptosis underlying PE has not been elucidated. This study was aimed to explore the protective role of H2S in trophoblast cells against necroptosis underlying PE. DESIGN: This is an in vitro experimental study. PARTICIPANTS: A total of 10 pregnant women with severe preeclampsia (PE) and 10 matched control normotensive pregnant women were included. The placenta tissues were extracted from participators. The human JEG-3 trophoblasts were commercially available. METHODS: The expression and localization of necrotic proteins were assayed in human placenta samples and the effect of necrotic cell death on the proliferation and apoptosis of human JEG-3 trophoblasts was evaluated. The component expressions of inflammatory cytokine and p38MAPK signaling pathway were measured in samples pretreated with or without NaHS (H2S donor) and SB203580 (p38 inhibitor). RESULTS: RIPA1, RIPA3, and p-p38 levels were significantly higher in PE placental tissue, whereas cystathionine-ß-synthase expression was decreased. In JEG-3 trophoblasts, necroptosis increased apoptotic cell numbers, suppressed cell proliferation, increased inflammatory cytokine expression, and increased p38MAPK activation, which can be prevented by NaHS. LIMITATIONS: In the present study, we did not provide sufficient evidence that necroptosis was a part of the pathogenesis of preeclampsia. CONCLUSIONS: we proposed the putative role of necroptosis in early-onset PE, reflected by the blockage of caspase-8/3 and increased expression of RIPA1, and RIPA3 in PE placenta tissues. Furthermore, we demonstrated that exogenous H2S protected cytotrophoblasts against CER-induced necroptosis via the p38MAPK pathway.
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OBJECTIVE: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro. METHODS: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo. RESULTS: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type â (COLâ ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLâ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLâ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group. CONCLUSION: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo, and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.
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Subunidade alfa 1 de Fator de Ligação ao Core , Osteogênese , Tioléster Hidrolases , Animais , Humanos , Camundongos , Tecido Adiposo/citologia , Diferenciação Celular/genética , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Camundongos Nus , Osteogênese/genética , RNA Mensageiro/metabolismo , Células-Tronco/metabolismo , Proteases Específicas de Ubiquitina/genética , Tioléster Hidrolases/metabolismoRESUMO
OBJECTIVE: We aimed to investigate the correlation between blood homocysteine (Hcy) levels and pre-eclampsia (PE) risk in pregnant women. METHODS: Related articles were searched using PubMed, Embase, and Web of Science databases. Methodological quality of included studies was evaluated using the Newcastle-Ottawa Quality Assessment Scale (NOS). Cochran's Q and I2 tests were used to evaluate heterogeneity. Egger's test was used to evaluate publication bias. A sensitivity analysis was performed to test stability of the results using a one-by-one elimination method. Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) was used to assess certainty of evidence. RESULTS: Nine studies (4384 PE and 26021 non-PE patients) were included in the meta-analysis. The methodology of them was of good quality, with NOS scores of 5-8. However, there was a significant heterogeneity among included studies. Therefore, the random effect model was generated and combined results suggested a significant association between increased level of Hcy in pregnant women and PE risk. Although a significant publication bias was found in the current study with a P value of 0.006 in the Egger test, sensitivity analysis showed that the combined results were stable and did not vary significantly from any single study. However, the GRADE evidence quality was very low, which may lower the recommendation of pooled results. CONCLUSIONS: Increased levels of Hcy in maternal blood were significantly associated with the risk of PE, but low certainty of evidence need to be improved by more high-quality studies.
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Pré-Eclâmpsia , Feminino , Homocisteína , Humanos , Pré-Eclâmpsia/epidemiologia , Gravidez , GestantesRESUMO
Urocortins (UCNs), belonging to corticotropin-releasing hormone (CRH) family, exert their function via CRH receptor type 1 (CRHR1) and 2 (CRHR2). Our previous studies have demonstrated that CRH acts on CRHR1 to potentiate prostaglandins (PGs) output induced by inflammatory stimuli in myometrial cells. In the present study, we sought to investigate the effects of UCNs on prostaglandin (PG) output via CRHR2 in cultured human uterine smooth muscle cells (HUSMCs) from pregnant women at term. We found that UCN and UCN 3 treatment promoted PGE2 and PGF2α secretion in a dose-dependent manner. In contrast, UCN2 dose-dependently inhibited PGE2 and PGF2α secretion. Their effects were reversed by CRHR2 antagonist and CRHR2 siRNA. Mechanically, we showed that UCN and UCN3 suppressed cAMP production and led to Gi activation while UCN2 stimulated cAMP production and activated Gs signaling. Further, UCN and UCN3 but not UCN2 activated NF-κB and MAPK signaling pathways through Gi signaling. UCN and UCN3 stimulation of PGs secretion were dependent on Gi/adenylyl cyclase (AC)/cAMP, NF-κB and MAPK signaling pathways. UCN2 suppression of PGs output was through Gs/AC/cAMP signaling pathways. Our data suggest that UCN, UCN2 and UCN3 can finely regulate PGs secretion via CRHR2, which facilitates the functional status of the uterus during pregnancy.
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Dinoprosta/metabolismo , Dinoprostona/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Miométrio/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Urocortinas/farmacologia , Útero/metabolismo , Dinoprosta/genética , Feminino , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miométrio/efeitos dos fármacos , Receptores de Hormônio Liberador da Corticotropina/genética , Útero/efeitos dos fármacosRESUMO
The nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays a critical role in various inflammatory diseases. We sought to investigate the role of NLRP3 inflammasome in uterine activation for labor at term and preterm. We found that NLRP3 inflammasome was activated in the myometrium tissues obtained from the pregnant women undergoing labor at term (TL) compared with those not undergoing labor (TNL) at term. NLRP3 inflammasome was also activated in amnion and chorion-deciduas in TL and preterm labor (PTL) groups. In the mouse model, uterine NLRP3 inflammasome and nuclear factor kappaB (NF-κB) were activated toward term and during labor. Treatment of pregnant mice with lipopolysaccharide (LPS) and RU38486 induced preterm birth (PTB) and also promoted uterine NLRP3 inflammasome and NF-κB activation. Treatment of pregnant mice with NLRP3 inflammasome inhibitor BAY11-7082 and MCC950 delayed the onset of labor and suppressed NLRP3 inflammasome and NF-κB activation in uterus. MCC950 postponed labor onset of the mice with LPS and RU38486 treatment and inhibited NLRP3 inflammasome activation in uterus. Our data provide the evidence that NLRP3 inflammasome is involved in uterine activation for labor onset in term and PTB in humans and mouse model.
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Inflamassomos , Nascimento Prematuro , Animais , Feminino , Humanos , Recém-Nascido , Camundongos , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Gravidez , Útero/metabolismoRESUMO
Polycystic ovary syndrome (PCOS) is a complicated reproductive endocrine disease characterized by hyperandrogenism, polycystic ovaries, and anovulation. Previous studies have revealed that androgen receptors (ARs) are strongly associated with hyperandrogenism and abnormalities in folliculogenesis in patients with PCOS. However, the kinases responsible for androgen receptor activity, especially in granulosa cells, and the role of casein kinase 2α (CK2α) specifically in the pathogenesis of PCOS, remain unknown. Here, we show that both CK2α protein and mRNA levels were higher in luteinized granulosa cells of patients with PCOS compared with non-PCOS, as well as in the ovarian tissues of mice with a dehydroepiandrosterone-induced PCOS-like phenotype, compared with controls. In addition, CK2α not only interacted with AR in vivo and in vitro, but it also phosphorylated and stabilized AR, triggering AR and ovulation related genes excessive expression. CK2α also promoted cell proliferation in the KGN cell line and inhibited apoptosis. Collectively, the finding highlighted that the CK2α-AR axis probably caused the etiology of the PCOS. Thus, CK2α might be a promising clinical therapeutic target for PCOS treatment.
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High circulating androgen in women with polycystic ovary syndrome (PCOS) may increase the risk of cardiovascular disease in offspring. The aim of the present study is to investigate whether maternal androgen excess in the rat PCOS model would lead to cardiac hypertrophy in offspring. Maternal testosterone propionate (maternal-TP)-treated adult female offspring displayed cardiac hypertrophy associated with local high cardiac dihydrotestosterone (DHT). The molecular markers of cardiac hypertrophy along with androgen receptor (AR) and PKCδ, were increased in the Maternal-TP group. Treatment of primary neonatal rat ventricular cardiomyocytes (NRCMs) and H9c2 cells with DHT significantly increased cell size and upregulated PKCδ expression, which could be attenuated by AR antagonist. Treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly increased cell size and upregulated myh7 level. Rottlerin, that may inhibit PKCδ, significantly reduced the hypertrophic effect of DHT and PMA on NRCMs and H9c2 cells. Chromatin immunoprecipitation revealed that AR could bind to Pkcδ promoter. Our results indicate that prenatal exposure to testosterone may induce cardiac hypertrophy in adult female rats through enhanced Pkcδ expression in cardiac myocytes.
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Cardiomegalia/genética , Síndrome do Ovário Policístico/genética , Proteína Quinase C-delta/genética , Receptores Androgênicos/genética , Acetofenonas/farmacologia , Androgênios/genética , Androgênios/metabolismo , Animais , Animais Recém-Nascidos , Benzopiranos/farmacologia , Cardiomegalia/etiologia , Cardiomegalia/patologia , Di-Hidrotestosterona/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Síndrome do Ovário Policístico/complicações , Síndrome do Ovário Policístico/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/patologia , Proteína Quinase C-delta/antagonistas & inibidores , Ratos , Transdução de Sinais/efeitos dos fármacos , Testosterona/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Chorionic NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) plays a pivotal role in controlling the amount of prostaglandins in the uterus and has been implicated in the process of labor. Prior studies identified hydrogen sulfide-generating enzymes cystathionine-ß-synthetase (CBS) and cystathionine-γ-lyase (CSE) in fetal membranes. We investigated whether hydrogen sulfide is involved in the regulation of PGDH expression in the chorion during labor. The chorionic tissues were obtained from pregnant women at preterm in labor and at term in labor or not in labor at term. Levels of CSE and CBS and hydrogen sulfide production rate were down-regulated in term in labor and preterm in labor groups compared with not in labor at term group. The CBS level correlated to PGDH expression in the chorion. Hydrogen sulfide donor NaHS and precursor l-cysteine dose-dependently stimulated PGDH expression and activity in cultured chorionic trophoblasts. The effect of l-cysteine was blocked by CBS inhibitor and CBS siRNA but not by CSE inhibitor and CSE siRNA. Hydrogen sulfide treatment suppressed miR-26b and miR-199a expression in chorionic trophoblasts. miR-26b and miR-199a mimics blocked hydrogen sulfide upregulation of PGDH expression. Our results indicate that hydrogen sulfide plays pivotal roles in maintenance of PGDH expression in the chorion during human pregnancy. Reduced expression of hydrogen sulfide-generating enzymes contributes to an increased amount of prostaglandins in the uterus during labor.
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Córion/enzimologia , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Hidroxiprostaglandina Desidrogenases/metabolismo , Trabalho de Parto Prematuro/metabolismo , Nascimento a Termo/metabolismo , Cistationina gama-Liase/genética , Regulação para Baixo , Feminino , Humanos , Sulfeto de Hidrogênio/metabolismo , Hidroxiprostaglandina Desidrogenases/genética , Trabalho de Parto Prematuro/genética , Gravidez , Nascimento a Termo/genéticaRESUMO
A test strip has been developed for the rapid detection of the illegal additive ethyl anthranilate (EA) in wine. The detection scheme is based on a combination of photonic crystal based detection and molecular imprinting based recognition. The resulting molecularly imprinted photonic crystal (MIPC) undergoes a gradual color change from green to yellow to red upon binding of EA. A semi-quantitative colorimetric card can be used to estimate the content of EA, either visually or by making use of an optical fiber spectrometer. A linear relationship was found between the Bragg diffraction peak shift and the concentration of EA in the range from 0.1 mM to 10 mM. The detection limit is 10 µM. The test has been successfully used to screening for the presence of EA in grape wine. The test strip is selective, and can be re-used after re-activation. Graphical abstract Schematic representation of the fabrication and application of the molecularly imprinted photonic crystal (MIPC) based test trip. The resulting MIPC undergoes a gradual color change from green to yellow to red upon binding of the illegal wine additive ethyl anthranilate (EA).
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Prostaglandin (PG) E2 plays critical roles during pregnancy and parturition. Emerging evidence indicates that human labour is an inflammatory event. We sought to investigate the effect of PGE2 on the output of proinflammatory cytokines in cultured human uterine smooth muscle cells (HUSMCs) from term pregnant women and elucidate the role of subtypes of PGE2 receptors (EP1, EP2, EP3 and EP4). After drug treatment and/or transfection of each receptor siRNA, the concentrations of inflammatory secreting factors in HUSMCs culture medium were detected by the corresponding ELISA kits. The results showed that, PGE2 increased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) output, decreased chemokine (c-x-c motif) ligand 8 (CXCL8) output in a dose-dependent manner, but had no effect on IL-1ß and chemokine (c-c motif) ligand 2 (CCL-2) secretion of HUSMCs. EP1/EP3 agonist 17-phenyl-trinor-PGE2 stimulated IL-6 and TNFα whilst suppressing IL-1ß and CXCL8 output. The effects of 17-phenyl-trinor-PGE2 on IL-1ß and CXCL8 secretion were remained whereas its effect on IL-6 and TNFα output did not occur in the cells with EP3 knockdown. The stimulatory effects of 17-phenyl-trinor-PGE2 on IL-6 and TNFα were remained whereas the inhibitory effects of 17-phenyl-trinor-PGE2 on IL-1ß secretion was blocked in the cells with EP1 knockdown. Either of EP2 and EP4 agonists stimulated IL-1ß and TNFα output, which was reversed by EP2 and EP4 siRNA, respectively. The inhibitors of phospholipase C (PLC) and protein kinase C (PKC) blocked EP1/EP3 modulation of TNFα and CXCL8 output. PI3K inhibitor LY294002 and P38 inhibitor SB202190 blocked 17-phenyl-trinor-PGE2-induced IL-1ß and IL-6 output, respectively. The inhibitors of adenylyl cyclase and PKA prevented EP2 and EP4 stimulation of IL-1ß and TNFα output, whereas PLC and PKC inhibitors blocked EP2- and EP4-induced TNFα output but not IL-1ß output. Our data suggest that PGE2 receptors exhibit different effects on the output of various cytokines in myometrium, which can subtly modulate the inflammatory microenvironment in myometrium during pregnancy.
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Citocinas/metabolismo , Miócitos de Músculo Liso/citologia , Miométrio/citologia , Receptores de Prostaglandina E/fisiologia , Células Cultivadas , Cromonas/farmacologia , Feminino , Humanos , Imidazóis/farmacologia , Inflamação , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases , Gravidez , Piridinas/farmacologiaRESUMO
Recent evidence suggests that uterine activation for labor is associated with inflammation within uterine tissues. Hydrogen sulfide (H2S) plays a critical role in inflammatory responses in various tissues. Our previous study has shown that human myometrium produces H2S via its generating enzymes cystathionine-γ-lyase (CSE) and cystathionine-ß-synthetase (CBS) during pregnancy. We therefore explored whether H2S plays a role in the maintenance of uterine quiescence during pregnancy. Human myometrial biopsies were obtained from pregnant women at term. Uterine smooth muscle cells (UMSCs) isolated from myometrial tissues were treated with various reagents including H2S. The protein expression of CSE, CBS and contraction-associated proteins (CAPs) including connexin 43, oxytocin receptor and prostaglandin F2α receptor determined by Western blot. The levels of cytokines were measured by ELISA. The results showed that CSE and CBS expression inversely correlated to the levels of CAPs and activated NF-κB in pregnant myometrial tissues. H2S inhibited the expression of CAPs, NF-κB activation and the production of interleukin (IL)-1ß, IL-6 and tumor necrosis factor α (TNFα) in cultured USMCs. IL-1ß treatment reversed H2S inhibition of CAPs. Knockdown of CSE and CBS prevented H2S suppression of inflammation. H2S modulation of inflammation is through KATP channels and phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways. H2S activation of PI3K and ERK signaling is dependent on KATP channels. Our data suggest that H2S suppresses the expression of CAPs via inhibition of inflammation in myometrium. Endogenous H2S is one of the key factors in maintenance of uterine quiescence during pregnancy.
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Poluentes Atmosféricos/farmacologia , Sulfeto de Hidrogênio/farmacologia , Inflamação/tratamento farmacológico , Trabalho de Parto/efeitos dos fármacos , Contração Uterina/efeitos dos fármacos , Útero/efeitos dos fármacos , Células Cultivadas , Cistationina gama-Liase/metabolismo , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Trabalho de Parto/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miométrio/citologia , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Transdução de Sinais/efeitos dos fármacos , Contração Uterina/metabolismo , Útero/metabolismoRESUMO
Preeclampsia, characterized by high blood pressure, albuminuria and other systemic disorders, is a serious complication during pregnancy. It has been reported that GNA12 is overexpressed during preeclampsia. In this study, we investigated the potential association between the methylation of the GNA12 promoter and preeclampsia. The methylation level at eight CpG sites of the GNA12 promoter was analyzed by MassARRAY in placenta and peripheral blood DNA samples from 50 preeclampsia patients and 50 normal pregnant women. In the placenta DNA samples, the methylation level at three CpG sites of the GNA12 promoter was significantly lower in the preeclampsia patients than in the controls. The difference was also significant at two of the three CpG sites in the peripheral blood DNA samples. The mRNA expression level of GNA12 in placenta was analyzed by real-time quantitative PCR in 20 cases and 20 controls. Consistent with the decreased methylation level, the mRNA expression level of GNA12 was higher in preeclampsia patients than in controls. Our results showed that preeclampsia is associated with decreased methylation of the GNA12 promoter, which can be detected in both the placenta and the peripheral blood of the pregnant women.
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Metilação de DNA , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pré-Eclâmpsia/genética , Regiões Promotoras Genéticas , Adulto , Ilhas de CpG , Feminino , Humanos , Placenta/metabolismo , Gravidez , Proteínas RepressorasRESUMO
Chorionic NAD-dependent 15-hydroxy prostaglandin dehydrogenase (PGDH) plays a pivotal role in controlling the amount of prostaglandins in the uterus. Peroxisome proliferator-activated receptors (PPARs) are implicated to be involved in parturition. In this study, we investigated whether PPARs are involved in control of PGDH expression in chorion. The chorionic tissues were collected from the following groups of the women with singleton pregnancy: term no labor (TNL), term labor (TL) and preterm labor (PTL). Chorionic trophoblasts were isolated and cultured in vitro. Immunocytochemistry analysis showed that PPARα, PPARß, and PPARγ were localized to trophoblasts in chorion. The protein levels of PGDH, PPARß, and PPARγ were localized to trophoblasts in chorion. The protein levels of PPARα, PPARß, and PPARγ were reduced in TL tissues compared to that of TNL group. PPARα, PPARß, and PPARγ expression correlated to PGDH in TNL tissues, whereas only PPARγ expression correlated to PGDH in TL chorion tissues. PGDH expression was decreased in PTL tissues compared with TL group, whereas the expression of PPARs was not significantly different between TL and PTL groups. The agonists of three PPARs dose-dependently stimulated PGDH activity, mRNA, and protein expression in cultured chorionic cells. PPARs did not affect the stability of PGDH mRNA but stimulated the transcriptional activity of HPGD gene. Our results suggest that PPARs play pivotal roles in maintenance of PGDH expression in chorion during human pregnancy.
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Córion/metabolismo , Hidroxiprostaglandina Desidrogenases/metabolismo , PPAR alfa/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Adulto , Células Cultivadas , Córion/enzimologia , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Recém-Nascido , Masculino , PPAR alfa/genética , Receptores Ativados por Proliferador de Peroxissomo/genética , Gravidez , Nascimento Prematuro , Prostaglandinas/metabolismo , Isoformas de Proteínas , RNA Mensageiro/genética , Nascimento a Termo , Trofoblastos/metabolismoRESUMO
Prostaglandin F2α (PGF2α) plays a critical role in the initiation and process of parturition. Since human labor has been described as an inflammatory event, we investigated the role of PGF2α in the inflammatory process using cultured human uterine smooth muscle cells (HUSMCs) isolated from term pregnant women as a model. Using a multiplex assay, HUSMCs treated with PGF2α changed their output of a number of cytokines and chemokines, with a distinct response pattern that differed between HUSMCs isolated from the upper and lower segment region of the uterus. Confirmatory enzyme-linked immunosorbent assays (ELISAs) showed that PGF2α stimulated increased output of interleukin (IL) 1ß, IL6, IL8 (CXCL8) and monocyte chemotactic protein-1 (MCP1, also known as chemokine (c-c motif) ligand 2, CCL2) by HUSMCs isolated from both upper and lower uterine segments. In contrast, PGF2α inhibited tumor necrosis factor α (TNFα) release by HUMSCs from the lower uterine segment while the output of TNFα was undetectable in the upper segment. Small interfering (si) RNA mediated knockdown of the PGF2α receptor prevented the changes in cytokine and chemokine output by the HUSMCs. Since the PGF2α receptor (PTGFR) couples via the Gq protein and subsequently activates the phospholipase C (PLC) and protein kinase C (PKC) signaling pathways, we examined the role of these pathways in PGF2α modulation of the cytokines. Inhibition of PLC and PKC reversed the effects of PGF2α. PGF2α activated multiple signaling pathways including extracellular signal-regulated kinases (ERK) 1/2, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), P38, calcineurin/nuclear factor of activated T-cells (NFAT) and NF-κB signaling. Inhibition of ERK reversed PGF2α-induced IL1ß, IL6 and CCL2 output, while inhibition of PI3K blocked the effect of PGF2α on IL6, CXCL8 and CCL2 output and inhibition of NF-κB reversed PGF2α-induced IL1ß and CCL2 output. NFAT was involved in PGF2α modulation of CCL2 and TNFα output. In conclusion, our results support a role of PGF2α in creating an inflammatory environment during the late stage of human pregnancy.
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Quimiocinas/metabolismo , Citocinas/metabolismo , Dinoprosta/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miométrio/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Feminino , Humanos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Miométrio/citologia , Miométrio/metabolismo , Gravidez , Transdução de Sinais/fisiologiaRESUMO
The chorion laeve controls the levels of active prostaglandins within the uterus by NAD-dependent 15-hydroxy prostaglandin dehydrogenase (PGDH). The expression of PGDH in chorion is modulated by glucocorticoids and progesterone. In this study, we investigated glucocorticoid receptor (GR) and progesterone receptor A and B (PRA and PRB) in the regulation of PGDH expression in chorion, and we determined whether reduced PGDH expression in chorion during labor is associated with the changes in GR and PR expression by real-time RT-PCR and Western blot analysis. Dexamethasone (DEX) inhibited PGDH expression whereas progesterone stimulated PGDH expression in chorionic trophoblasts. DEX suppressed PGDH expression in GR overexpression and PR knockdown cells. The inhibitory effect of DEX did not occur in GR knockdown cells. Progesterone inhibited PGDH in GR overexpression and PR knockdown cells and it stimulated PGDH in PRB overexpression cells whereas it suppressed PGDH in PRA overexpression cells. Knockdown of c-Jun resulted in a loss of progesterone- and DEX-induced effects. PGDH was down-regulated in chorion tissues during labor. PRB was decreased whereas PRA and GR were increased in chorion during labor. Glucocorticoids inhibit PGDH expression via GR in chorionic trophoblasts. Progesterone enhances PGDH expression through PRB, whereas it inhibits PGDH expression via GR and PRA. Decreased PGDH expression is associated with increased GR and PRA, although decreased PRB, in chorion during labor.
Assuntos
Córion/enzimologia , Hidroxiprostaglandina Desidrogenases/metabolismo , Trabalho de Parto/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Células Cultivadas , Córion/citologia , Córion/efeitos dos fármacos , Dexametasona/farmacologia , Di-Hidrotestosterona/análogos & derivados , Di-Hidrotestosterona/farmacologia , Membranas Extraembrionárias/efeitos dos fármacos , Membranas Extraembrionárias/metabolismo , Feminino , Imunofluorescência , Glucocorticoides/farmacologia , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Trabalho de Parto/efeitos dos fármacos , Gravidez , Progesterona/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transcrição Gênica/efeitos dos fármacos , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Mangrove aquatic ecosystems receive substantial nitrogen (N) inputs from both land and sea, playing critical roles in modulating coastal N fluxes. The microbially-mediated competition between denitrification and dissimilatory nitrate reduction to ammonium (DNRA) in mangrove sediments significantly impacts the N fate and transformation processes. Despite their recognized role in N loss or retention in surface sediments, how these two processes vary with sediment depths and their influential factors remain elusive. Here, we employed a comprehensive approach combining 15N isotope tracer, quantitative PCR (qPCR) and metagenomics to verify the vertical dynamics of denitrification and DNRA across five 100-cm mangrove sediment cores. Our results revealed a clear vertical partitioning, with denitrification dominated in 0-30 cm sediments, while DNRA played a greater role with increasing depths. Quantification of denitrification and DNRA functional genes further explained this phenomenon. Taxonomic analysis identified Pseudomonadota as the primary denitrification group, while Planctomycetota and Pseudomonadota exhibited high proportion in DNRA group. Furthermore, genome-resolved metagenomics revealed multiple salt-tolerance strategies and aromatic compound utilization potential in denitrification assemblages. This allowed denitrification to dominate in oxygen-fluctuating and higher-salinity surface sediments. However, the elevated C/N in anaerobic deep sediments favored DNRA, tending to generate biologically available NH4+. Together, our results uncover the depth-related variations in the microbially-mediated competition between denitrification and DNRA, regulating N dynamics in mangrove ecosystems.
RESUMO
Mangrove sediments host a diverse array of microbial populations and are characterized by high heterogeneity along their vertical depths. However, the genetic diversity within these populations is largely unknown, hindering our understanding of their adaptive evolution across the sediment depths. To elucidate their genetic diversity, we utilized metagenome sequencing to identify 16 high-frequency microbial populations comprised of two archaea and 14 bacteria from mangrove sediment cores (0-100 cm, with 10 depths) in Qi'ao Island, China. Our analysis of the genome-wide genetic variation revealed extensive nucleotide diversity in the microbial populations. The genes involved in the transport and the energy metabolism displayed a high nucleotide diversity (HND; 0.0045-0.0195; an indicator of shared minor alleles with the microbial populations). By tracking the processes of homologous recombination, we found that each microbial population was subjected to different purification selection levels at different depths (44.12% genes). This selection resulted in significant differences in synonymous/non-synonymous mutation ratio between 0-20 and 20-100 cm layers, indicating the adaptive evolutionary process of microbial populations. Furthermore, our assessment of differentiation in the allele frequencies between these two layers showed that the functional genes involved in the metabolic processes of amino acids or cofactors were highly differential in more than half of them. Together, we showed that the nucleotide diversity of microbial populations was shaped by homologous recombination and gene-specific selection, finally resulting in the stratified differentiation occurring between 0-20 and 20-100 cm. These results enhance our cognition of the microbial adaptation mechanisms to vertical environmental changes during the sedimentation process of coastal blue carbon ecosystems.
RESUMO
Increasing nitrogen (N) input to coastal ecosystems poses a serious environmental threat. It is important to understand the responses and feedback of N removal microbial communities, particularly nitrifiers including the newly recognized complete ammonia-oxidizers (comammox), to improve aquaculture sustainability. In this study, we conducted a holistic evaluation of the functional communities responsible for nitrification by quantifying and sequencing the key functional genes of comammox Nitrospira-amoA, AOA-amoA, AOB-amoA and Nitrospira-nxrB in fish ponds with different fish feeding levels and evaluated the contribution of nitrifiers in the nitrification process through experiments of mixing pure cultures. We found that higher fish feeding dramatically increased N-related concentration, affecting the nitrifying communities. Compared to AOA and AOB, comammox Nitrospira and NOB were more sensitive to environmental changes. Unexpectedly, we detected an equivalent abundance of comammox Nitrospira and AOB and observed an increase in the proportion of clade A in comammox Nitrospira with the increase in fish feeding. Furthermore, a simplified network and shift of keystone species from NOB to comammox Nitrospira were observed in higher fish-feeding ponds. Random forest analysis suggested that the comammox Nitrospira community played a critical role in the nitrification of eutrophic aquaculture ponds (40-70 µM). Through the additional experiment of mixing nitrifying pure cultures, we found that comammox Nitrospira is the primary contributor to the nitrification process at 200 µM ammonium. These results advance our understanding of nitrifying communities and highlight the importance of comammox Nitrospira in driving nitrification in eutrophic aquaculture systems.
RESUMO
Drug local delivery system that directly supply anti-cancer drugs to the tumor microenvironment (TME) results in excellent tumor control and minimizes side effects associated with the anti-cancer drugs. Immune checkpoint inhibitors (ICIs) have been the mainstay of cancer immunotherapy. However, the systemic administration of ICIs is accompanied by considerable immunotherapy-related toxicity. To explore whether an anti-PD-L1 antibody administered locally via a sustained-release gel-forming carrier retains its effective anticancer function while causing fewer colitis-like side effects, CT, a previously reported depot system, was used to locally deliver an anti-PD-L1 antibody together with curcumin to the TME in bladder cancer-bearing ulcerative colitis model mice. We showed that CT-mediated intratumoral coinjection of an anti-PD-L1 antibody and curcumin enabled sustained release of both the loaded anti-PD-L1 antibody and curcumin, which contributed to substantial anticancer effects with negligible side effects on the colons of the UC model mice. However, although the anti-PD-L1 antibody administered systemically synergized with the CT-mediated intratumoral delivery of curcumin in inhibiting tumour growth, colitis was significantly worsened by intraperitoneal administration of anti-PD-L1 antibody. These findings suggested that CT is a promising agent for the local delivery of anticancer drugs, as it can allow effective anticancer functions to be retained while sharply reducing the adverse side effects associated with the systemic administration of these drugs.