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The role of plasma-derived exosomal miRNA in premature ovarian failure (POF) remains unclear. This study aimed to investigate the epigenetic pathogenesis of POF through exosomal miRNA sequencing. Exosomes were isolated and characterized from six POF patients and four healthy individuals using nanoparticle tracking analysis, transmission electron microscopy and western blot analysis. Exosomal miRNA sequencing was performed to identify differentially expressed miRNAs with |fold change| greater than 1.5 and p value less than 0.05. Bioinformatics analysis in GSE39501 dataset and our sequencing data was conducted to investigate underlying mechanisms of POF. The functional role of hsa-miR-19b-3p was assessed using CCK8, western blot, flow cytometry and fluorescence staining. The regulatory effect of hsa-miR-19b-3p on BMPR2 was investigated through miRNA transfection, qPCR analysis, and luciferase reporter assay. Statistical significance was determined using t-tests and one-way ANOVA (p < 0.05). Exosomal miRNA sequencing revealed 18 dysregulated miRNAs in POF patients compared to healthy controls. Functional enrichment analysis demonstrated their involvement in cell growth, oocyte meiosis and PI3K-Akt signaling pathways. Moreover, the constructed miRNA-mRNA network unveiled potential regulatory mechanisms underlying POF, particularly implicating hsa-miR-19b-3p in the regulation of BMPR2. In vitro assays conducted on KGN cells confirmed that hsa-miR-19b-3p promoted apoptosis, as evidenced by reduced cell viability, decayed mitochondrial membrane potential and increased apoptotic rate, thereby supporting its role in POF. Notably, hsa-miR-19b-3p was found to significantly downregulate BMPR2 expression via targeting its 3'UTR, while co-expression analysis revealed strong associations between BMPR2 and POF-related processes. This study sheds light on the epigenetic pathogenesis of POF by investigating exosomal miRNA profiles. Particularly, hsa-miR-19b-3p emerged as a potential regulator of BMPR2 and demonstrated its functional significance in POF through modulation of apoptosis.
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Haemoglobin H (Hb H) disease (intermediate status of α-thalassemia) shows marked phenotypic variability from asymptomatic to severe anaemia. Apart from the combined ß-thalassemia allele ameliorating clinical severity, reports of genetic modifier genes affecting the phenotype of Hb H disease are scarce which bring inconvenience to precise diagnosis and genetic counselling of the patients. Here, we present a novel mutation (c.948C>A, p.S316R) in the PIP4K2A gene in a female Hb H disease patient who displayed moderate anaemia and a relatively high Hb H level. Haematological analysis in her family members revealed that individuals carrying this mutation have upregulated ß-globin expression, leading to a more imbalanced ß/α-globin ratio and more Hb H inclusion bodies in peripheral red blood cells. According to functional experiments, the mutant PIP4K2A protein exhibits enhanced protein stability, increased kinase activity and a stronger regulatory effect on downstream proteins, suggesting a gain-of-function mutation. Moreover, introduction of the S316R mutation into HUDEP-2 cells increased expression of ß-globin, further inhibiting erythroid differentiation and terminal enucleation. Thus, the S316R mutation is a novel genetic factor associated with ß-globin expression, and the PIP4K2A gene is a new potential modifier gene affecting the α-thalassemia phenotype.
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Talassemia alfa , Talassemia beta , Feminino , Humanos , Talassemia alfa/genética , Mutação com Ganho de Função , Globinas beta/genética , Mutação , Talassemia beta/genética , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
Re-activation of fetal haemoglobin (HbF) has been proved to be an effective strategy for the treatment of ß-haemoglobinopathies. In this study, we identified TEA domain transcription factor 4 (TEAD4) as a new potential regulator of HbF by integrating public data sets with quantitative polymerase chain reaction analysis in ß-thalassaemia patients. Significant negative correlation was observed between the expression of TEAD4 and HbF levels in ß-thalassaemia patients. Functional validations of TEAD4 inhibition in both ß-thalassaemia CD34+ cells and HUDEP-2 cells indicated that depletion of TEAD4 led to a significant increase of HbF. Finally, we identified a binding motif of TEAD4 on γ-globin gene promoters; its disruption consistently led to de-repression of HbF. Taken together, these results demonstrate that TEAD4 could act as a transcriptional inhibitor of the γ-globin gene through direct binding on its promoter. Our findings demonstrate a novel role of TEAD4 on the regulation of HbF, which may benefit patients with ß-haemoglobinopathies.
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Hemoglobina Fetal/genética , Regiões Promotoras Genéticas , Fatores de Transcrição de Domínio TEA/metabolismo , gama-Globinas/genética , Linhagem Celular , Células Eritroides/metabolismo , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Talassemia beta/genética , Talassemia beta/metabolismoRESUMO
Acetaminophen (APAP) is a widely used over-the-counter analgesic and antipyretic. It can cause hepatotoxicity. Recent studies demonstrated that hydrogen sulfide (H2 S) exhibits cell protection in several cell types. This study was designed to investigate whether H 2 S ameliorated APAP-induced acute liver injury and to elucidate its mechanisms. In this study, we analyzed the detailed biological and molecular processes of APAP-induced hepatotoxicity using a bioinformatics analysis, which showed that apoptosis and the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase pathway were confirmed to play critical roles in these processes. We further investigated the protective effects of H 2 S on APAP-induced hepatotoxicity. In vivo, we observed that the exogenous supplement of H 2 S ameliorated APAP-induced liver injury. Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) systems were the endogenous pathway of H 2 S. The expression of CBS/CSE was decreased in APAP-treated mice, while H 2 S could significantly restore it. In addition, APAP-induced JNK activation was inhibited by H 2 S in vivo. In vitro, H 2 S abolished the active effects of APAP on caspase3, Bax, and Bcl-2 expressions as well as JNK phosphorylation in hepatocytes. It was found through flow cytometry that the amount of APAP-induced apoptotic hepatocytes was decreased in the presence of H 2 S. In conclusion, our results suggested that H 2 S attenuated APAP-induced apoptosis in hepatocytes through JNK/MAPK siganaling pathway.
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Acetaminofen/efeitos adversos , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Acetaminofen/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatócitos/patologia , Humanos , Sulfeto de Hidrogênio , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Contrast-induced acute kidney injury (CIAKI) is a common cause of hospital-acquired acute kidney injury (AKI). S100A8/A9-TLR4-NLRP3 inflammasome pathway triggers inflammation, apoptosis and tissue injury in several AKI models. Nevertheless, the underlying mechanism of S100A8/A9-TLR4-NLRP3 inflammasome pathway in CIKAI is not clear. We aimed to investigate the possible role of S100A8/A9-TLR4-NLRP3 inflammasome in the pathophysiology of CIAKI. METHODS: We treated male rats and NRK-52E cells by iopromide to establish in vivo and in vitro models of CIAKI. We collected serum and urine samples to detect renal function. We obtained kidney tissue for histological analysis and detection of protein concentration. We used inhibitor of TLR4 and NLRP3-siRNA to further testify their role in CIAKI in NRK-52E cells. RESULTS: Iopromide caused elevation of SCr, BUN and NGAL level, decrease of endogenous creatinine clearance, morphological injury and tubular apoptosis, enhanced IL-1ß and IL-18 expression, and increased expression of S100A8/A9, TLR4 and NLRP3 inflammsome. In NRK-52E cells, iopromide caused enhanced apoptotic rates and ROS generation, which could be ameliorated by inhibitor of TLR4 and NLRP3-siRNA. Moreover, inhibition of TLR4 dampened NLRP3 expression. CONCLUSION: S100A8/A9-TLR4-NLRP3 inflammasome pathway represented a key mechanism of CI-AKI, which provided a potential therapeutic target.
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Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/fisiopatologia , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Meios de Contraste , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose/efeitos dos fármacos , Calgranulina A/genética , Calgranulina B/genética , Linhagem Celular , Creatinina/sangue , Modelos Animais de Doenças , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genéticaRESUMO
Background: Diabetic kidney disease (DKD) is an intricate complication of diabetes with limited treatment options. Anoikis, a programmed cell death activated by loss of cell anchorage to the extracellular matrix, participated in various physiological and pathological processes. Our study aimed to elucidate the role of anoikis-related genes in DKD pathogenesis. Methods: Differentially expressed genes (DEGs) associated with anoikis in DKD were identified. Weighted gene co-expression network analysis (WGCNA) was conducted to identify DKD-correlated modules and assess their functional implications. A diagnostic model for DKD was developed using LASSO regression and Gene set variation analysis (GSVA) was performed for enrichment analysis. Experimental validation was employed to validate the significance of selected genes in the progression of DKD. Results: We identified 10 anoikis-related DEGs involved in key signaling pathways impacting DKD progression. WGCNA highlighted the yellow module's significant enrichment in immune response and regulatory pathways. Correlation analysis further revealed the association between immune infiltration and anoikis-related DEGs. Our LASSO regression-based diagnostic model demonstrated a well-predictive efficacy with seven identified genes. GSVA indicated that gene function in the high-risk group was primarily associated with immune regulation. Further experimental validation using diabetic mouse models and data analysis in the single-cell dataset confirmed the significance of PYCARD and SFN in DKD progression. High glucose stimulation in RAW264.7 and TCMK-1 cells showed significantly increased expression levels of both Pycard and Sfn. Co-expression analysis demonstrated distinct functions of PYCARD and SFN, with KEGG pathway analysis showing significant enrichment in immune regulation and cell proliferation pathway. Conclusion: In conclusion, our study provides valuable insights into the molecular mechanisms involved in DKD pathogenesis, specifically highlighting the role of anoikis-related genes in modulating immune infiltration. These findings suggest that targeting these genes may hold promise for future diagnostic and therapeutic approaches in DKD management.
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Background: Diabetic nephropathy (DN) is one of the primary causes of end-stage renal disease, yet effective therapeutic targets remain elusive. This study aims to identify novel diagnostic biomarkers and potential therapeutic candidates for DN. Methods: Differentially expressed genes (DEGs) in GSE96804 and GSE142025 were identified and functional enrichment analysis was performed. Diagnostic biomarkers were selected using machine learning algorithms and evaluated by Receiver Operating Characteristic analysis. c-Fos expression was validated in an established DN mouse model. Immune infiltration levels were assessed with Single-Sample Gene Set Enrichment Analysis. Co-expression analysis revealed regulatory relationships involving FOS. cMAP predicted potential therapeutic candidates. Transcriptome sequencing and experiments in RAW264.7 cells was performed to investigate molecular mechanisms of emetine. Results: In both datasets, we identified 44 upregulated and 74 downregulated DEGs involved in focal adhesion, ECM-receptor interaction, and the PI3K-Akt signaling pathway. FOS emerged as a robust diagnostic marker with decreased expression in DN patients and DN mouse. Co-expression analysis revealed potential regulatory mechanisms of FOS, implicating the MAPK signaling pathway, regulation of cell proliferation and apoptotic signaling pathways. Immune dysregulation was observed in DN patients. Notably, emetine was identified as a potential therapeutic candidate. Transcriptome sequencing and experimental validation demonstrated emetine suppressed M1 macrophage polarization by inhibiting the activation of NF-κB signaling pathway, as well as reducing the expression of Il-18 and Ccl5. Conclusion: In conclusion, our study identified FOS as a promising diagnostic biomarker and emetine as a potential therapeutic candidate for DN. These findings enhance our understanding of DN pathogenesis and present novel prospects for therapeutic strategies.
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Background: Kidney stones or nephrolithiasis is a chronic metabolic disease characterized by renal colic and hematuria. Currently, a pathogenetic mechanism resulting in kidney stone formation remains elusive. We performed a multi-omic study investigating urinary microbial compositions and metabolic alterations during nephrolithiasis. Method: Urine samples from healthy and individuals with nephrolithiasis were collected for 16S rRNA gene sequencing and liquid chromatography-mass spectroscopy. Microbiome and metabolome profiles were analyzed individually and combined to construct interactome networks by bioinformatic analysis. Results: Distinct urinary microbiome profiles were determined in nephrolithiasis patients compared with controls. Thirty-nine differentially abundant taxa between controls and nephrolithiasis patients were identified, and Streptococcus showed the most significant enrichment in nephrolithiasis patients. We also observed significantly different microbial compositions between female and male nephrolithiasis patients. The metabolomic analysis identified 112 metabolites that were differentially expressed. Two significantly enriched metabolic pathways, including biosynthesis of unsaturated fatty acids and tryptophan metabolism, were also identified in nephrolithiasis patients. Four potentially diagnostic metabolites were also identified, including trans-3-hydroxycotinine, pyroglutamic acid, O-desmethylnaproxen, and FAHFA (16:0/18:2), and could function as biomarkers for the early diagnosis of nephrolithiasis. We also identified three metabolites that contributed to kidney stone size. Finally, our integrative analysis of the urinary tract microbiome and metabolome identified distinctly different network characteristics between the two groups. Conclusions: Our study has characterized important profiles and correlations among urinary tract microbiomes and metabolomes in nephrolithiasis patients for the first time. These results shed new light on the pathogenesis of nephrolithiasis and could provide early clinical biomarkers for diagnosing the disease.
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Cálculos Renais , Ácido Pirrolidonocarboxílico , Biomarcadores/urina , Feminino , Humanos , Rim , Masculino , RNA Ribossômico 16S/genética , TriptofanoRESUMO
Background: Icariin presents protective effect in several kidney diseases. However, the role of icariin in contrast-induced acute kidney injury (CIAKI) is still unclear. This study aimed to investigate the effect of icariin in CIAKI, as well as exploring the underlying mechanism from the aspect of interaction between protein-coding genes and non-coding RNAs. Methods: The effect of icariin was evaluated in both in vivo and in vitro CIAKI models. Rat kidneys were collected for genome-wide sequencing. The differentially expressed genes (DEGs) were screened and visualized by R software. The function annotation of DEGs was analyzed by Metascape. By Cytoscape software, the competing endogenous RNA (ceRNA) network was constructed, and hub genes were selected. Expressions of hub genes were validated by PCR. Association of hub genes in the ceRNA network and renal function was also examined. Results: Icariin protected against CIAKI in both in vivo and in vitro models. Based on DEGs in icariin pretreated CIAKI rats, lncRNA- and circRNA-associated ceRNA networks were constructed, respectively. Function annotation showed the ceRNA networks were enriched in ERK1 and ERK2 cascade, MAPK signaling and NF-κB signaling. Further, two circRNAs, six lncRNAs, four miRNAs and nine mRNAs were selected as hub genes of the ceRNA network. Among them, eight mRNAs (Acot1, Cbwd1, Ly6i, Map3k14, Mettl2b, Nyap1, Set and Utp20) were negatively correlated with renal function, while one mRNA (Tmem44) was positively correlated with renal function. Conclusion: Icariin presented a protective effect against CIAKI. The ceRNA network, involving Acot1, Cbwd1, Ly6i, Map3k14, Mettl2, Nyap1, Set, Tmem44 and Utp20, might partially contribute to the underlying mechanism of icariin protection by regulation of ERK1 and ERK2 cascade, MAPK signaling and NF-κB signaling.
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Injúria Renal Aguda , MicroRNAs , RNA Longo não Codificante , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/prevenção & controle , Animais , Flavonoides , Redes Reguladoras de Genes , MicroRNAs/metabolismo , NF-kappa B/genética , RNA Circular , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RatosRESUMO
In recent years hydrogen sulfide (H2S) has demonstrated vasculoprotective effects against cell death, which suggests its promising therapeutic potential for numerous types of disease. Additionally, a protective effect of exogenous H2S in HGinduced injuries in HUVECs was demonstrated, suggesting a potential protective effect for diabetic vascular complications. The present study aimed to investigate the mechanism accounting for the cytoprotective role of exogenous H2S against high glucose [HG (40 mM glucose)]induced injury and inflammation in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to HG for 24 h to establish an in vitro model of HGinduced cytotoxicity. The cells were pretreated with sodium hydrosulfide (NaHS), a donor of H2S, or inhibitors of necroptosis and p38 MAPK prior to the exposure to HG. Cell viability, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), IL1ß, IL6, IL8, TNFα, phosphorylated(p)38 and receptorinteracting protein 3 (RIP3) expression levels were detected using the indicated methods, including Cell Counting Kit 8, fluorescence detection, western blotting, immunofluorescence assay and ELISAs. The results demonstrated that necroptosis and the p38 MAPK signaling pathway mediated HGinduced injury and inflammation. Notably, NaHS was discovered to significantly ameliorate p38 MAPK/necroptosismediated injury and inflammation in response to HG, as evidenced by an increase in cell viability, a decrease in ROS generation and loss of MMP, as well as the reduction in the secretion of proinflammatory cytokines. In addition, the upregulated expression of RIP3 induced by HG was repressed by treatment with SB203580, while the HGinduced upregulation of pp38 expression levels were significantly downregulated following the treatment of Nec1 and RIP3siRNA. In conclusion, the findings of the present study indicated that NaHS may protect HUVECs against HGinduced injury and inflammation by inhibiting necroptosis via the p38 MAPK signaling pathway, which may represent a promising drug for the therapy of diabetic vascular complications.
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Células Endoteliais da Veia Umbilical Humana , Inflamação , Sistema de Sinalização das MAP Quinases , Necroptose , Substâncias Protetoras , Sulfetos , Humanos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/prevenção & controle , Glucose/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfetos/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Immunotherapy offers a novel approach for the treatment of cutaneous melanoma, but the clinical efficiency varies for individual patients. In consideration of the high cost and adverse effects of immunotherapy, it is essential to explore the predictive biomarkers of outcomes. Recently, the tumor mutation burden (TMB) has been proposed as a predictive prognosticator of the immune response. METHOD: RNA-seq and somatic mutation datasets of 472 cutaneous melanoma patients were downloaded from The Cancer Genome Atlas (TCGA) database to analyze mutation type and TMB. Differently expressed genes (DEGs) were identified for functional analysis. TMB-related signatures were identified via LASSO and multivariate Cox regression analysis. The association between mutants of signatures and immune cells was evaluated from the TIMER database. Furthermore, the Wilcox test was applied to assess the difference in immune infiltration calculated by the CIBERSORT algorithm in risk groupings. RESULTS: C>T substitutions and TTN were the most common SNV and mutated gene, respectively. Patients with low TMB presented poor prognosis. DEGs were mainly implicated in skin development, cell cycle, DNA replication, and immune-associated crosstalk pathways. Furthermore, a prognostic model consisting of eight TMB-related genes was developed, which was found to be an independent risk factor for treatment outcome. The mutational status of eight TMB-related genes was associated with a low level of immune infiltration. In addition, the immune infiltrates of CD4+ and CD8+ T cells, NK cells, and M1 macrophages were higher in the low-risk group, while those of M0 and M2 macrophages were higher in the high-risk group. CONCLUSION: Our study demonstrated that a higher TMB was associated with favorable survival outcome in cutaneous melanoma. Moreover, a close association between prognostic model and immune infiltration was identified, providing a new potential target for immunotherapy.
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Biomarcadores Tumorais/genética , Melanoma/genética , Mutação , Neoplasias Cutâneas/genética , Biomarcadores Tumorais/imunologia , Biologia Computacional , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Melanoma/terapia , Modelos Genéticos , Modelos Imunológicos , Polimorfismo de Nucleotídeo Único , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Risco , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/terapiaRESUMO
Background: Papillary renal cell carcinoma (PRCC), although the second-most common type of renal cell carcinoma, still lacks specific biomarkers for diagnosis, treatment, and prognosis. TopBP1-interacting checkpoint and replication regulator (TICRR) is a DNA replication initiation regulator upregulated in various cancers. We aimed to evaluate the role of TICRR in PRCC tumorigenesis and prognosis. Methods: Based on the Kidney Renal Papillary cell carcinoma Project (KIRP) on The Cancer Genome Atlas (TCGA) database, we determined the expression of TICRR using the Wilcoxon rank sum test. The biological functions of TICRR were evaluated using the Metascape database and Gene Set Enrichment Analysis (GSEA). The association between TICRR and immune cell infiltration was investigated by single sample GSEA. Logistic analysis was applied to study the correlation between TICRR expression and clinicopathological characteristics. Finally, Cox regression analysis, Kaplan-Meier analysis, and nomograms were used to determine the predictive value of TICRR on clinical outcomes in PRCC patients. Results: TICRR expression was significantly elevated in PRCC tumors (P < 0.001). Functional annotation indicated enrichment with negative regulation of cell division, cell cycle, and corresponding pathways in the high TICRR expression phenotype. High TICRR expression in PRCC was associated with female sex, younger age, and worse clinical stages. Cox regression analysis revealed that TICRR was a risk factor for overall survival [hazard ratio (HR): 2.80, P = 0.002], progression-free interval (HR: 2.86, P < 0.001), and disease-specific survival (HR: 7.03, P < 0.001), especially in patients with male sex, age below 60 years, clinical stages II-IV and clinical T stage T1-T2. Conclusion: Increased TICRR expression in PRCC might play a role in tumorigenesis by regulating the cell cycle and has prognostic value for clinical outcomes.
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Hyperglycemia is a key factor in the development of diabetic complications, including the processes of atherosclerosis. Receptorinteracting protein 3 (RIP3), a mediator of necroptosis, is implicated in atherosclerosis development. Additionally, hydrogen sulfide (H2S) protects the vascular endothelium against hyperglycemiainduced injury and attenuates atherosclerosis. On the basis of these findings, the present study aimed to confirm the hypothesis that necroptosis mediates high glucose (HG)induced injury in human umbilical vein endothelial cells (HUVECs), and that the inhibition of necroptosis contributes to the protective effect of exogenous H2S against this injury. The results revealed that exposure of HUVECs to 40 mM HG markedly enhanced the expression level of RIP3, along with multiple injuries, including a decrease in cell viability, an increase in the number of apoptotic cells, an increase in the expression level of cleaved caspase3, generation of reactive oxygen species (ROS), as well as dissipation of the mitochondrial membrane potential (MMP). Treatment of the cells with sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to HG significantly attenuated the increased RIP3 expression and the aforementioned injuries by HG. Notably, treatment of cells with necrostatin1 (Nec1), an inhibitor of necroptosis, prior to exposure to HG ameliorated the HGinduced injuries, leading to a decrease in ROS generation and a loss of MMP. However, pretreatment of the cells with Nec1 enhanced the HGinduced increase in the expression levels of cleaved caspases3 and 9. By contrast, pretreatment with ZVADFMK, a pan caspase inhibitor, promoted the increased expression of RIP3 by HG. Taken together, the findings of the present study have demonstrated, to the best of our knowledge for the first time, that exogenous H2S protects HUVECs against HGinduced injury through inhibiting necroptosis. The present study has also provided novel evidence that there is a negative interaction between necroptosis and apoptosis in the HGtreated HUVECs.
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Apoptose/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Glucose/toxicidade , Células Endoteliais da Veia Umbilical Humana/patologia , Sulfeto de Hidrogênio/farmacologia , Substâncias Protetoras/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Caspase 3/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Necrose , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate whether exogenous hydrogen sulfide (H2S) inhibits the high-glucose (HG)-induced injury by modulating leptin/leptin receptor (LEPR) signal pathway in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were treated with 40 mmol/L glucose for 3-24 h, and the cell viability was examined by CCK-8 assay. The changes of cell morphology and the number of apoptotic cells were assessed by Hoechst 33258 nuclear staining followed by photofluorography. The intracellular levels of reactive oxygen species (ROS) was detected by DCFH-DA staining followed by photofluorography. Mitochondrial membrane potential (MMP) was determined by Rhodamine 123 (Rh123) staining and photofluorography. The expression levels of leptin and LEPR protein were measured by Western blotting. RESULTS: s The expression of leptin and LERP in HUVECs began to significantly increase at 3 h after HG exposure and reached the peak levels at 9 h (P<0.01). Pretreatment of HUVECs with 400 µmol/L sodium hydrosulfide (H2S donor) for 30 min inhibited HG-induced increase in leptin and leptin receptor expressions in HUVECs (P<0.01). Pretreatment of HUVECs with 400 µmol/L NaHS for 30 min or 50 ng/mL leptin antagonists (LA) for 1 h obviously alleviated HG-induced injury by increasing cell viability, decreasing cell apoptosis and lowering accumulation of intracellular ROS and MMP loss (P<0.01). CONCLUSION: Exogenous H2S protects against HG-induced injury by inhibiting leptin/LEPR pathway in HUVECs.