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Oxidative stress caused by excess ROS often leads to cellular macromolecule damage and eventually causes various biological catastrophes. Sirt6, a member of the mammalian homolog family of yeast Sir2 NAD+-dependent histone deacetylases, regulates multiple biological processes. Sirt6 exerts antioxidative functions by enhancing DNA repair and DNA end resection. In our study, we found that Sirt6 expression was induced by H2O2 and paraquat (PQ) in cells. When exposed to PQ, the Sirt6+/- C57BL/6 mice showed more serious liver damage and lower survival rate than the Sirt6+/+ mice. The Nrf2 protein levels and the mRNA levels of its target genes in mouse tissues were decreased by Sirt6 deficiency, and Sirt6 overexpression increased the Nrf2 protein content. Moreover, the endogenous H2O2 levels were increased in the tissues of Sirt6-deficient mice and were decreased in Sirt6 overexpression cells. Then, we found that Nrf2 was degraded faster in the Sirt6-deficient mouse embryonic fibroblasts (MEFs) than in the wild type MEFs and that Sirt6 enhanced the protein accumulation of Nrf2 in the nucleus. Lastly, we found that Sirt6 interacted with Nrf2 in co-IP and GST pull-down assays and that Sirt6 overexpression decreased the binding of Nrf2 to Keap1. Taken together, the results of the present study suggest that Sirt6 exerts antioxidative functions by increasing the Nrf2 protein level via Keap1-mediated regulation.
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Factor 2 Relacionado con NF-E2 , Sirtuinas , Animales , Ratones , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Antioxidantes/metabolismo , Peróxido de Hidrógeno/metabolismo , Sirtuinas/genética , Sirtuinas/metabolismo , Ratones Endogámicos C57BL , Fibroblastos/metabolismo , Estrés Oxidativo , Paraquat , Mamíferos/metabolismoRESUMEN
AIMS: The mechanisms underlying ageing-induced vascular remodelling remain unclear. This study investigates the role and underlying mechanisms of the cytoplasmic deacetylase sirtuin 2 (SIRT2) in ageing-induced vascular remodelling. METHODS AND RESULTS: Transcriptome and quantitative real-time PCR data were used to analyse sirtuin expression. Young and old wild-type and Sirt2 knockout mice were used to explore vascular function and pathological remodelling. RNA-seq, histochemical staining, and biochemical assays were used to evaluate the effects of Sirt2 knockout on the vascular transcriptome and pathological remodelling and explore the underlying biochemical mechanisms. Among the sirtuins, SIRT2 had the highest levels in human and mouse aortas. Sirtuin 2 activity was reduced in aged aortas, and loss of SIRT2 accelerated vascular ageing. In old mice, SIRT2 deficiency aggravated ageing-induced arterial stiffness and constriction-relaxation dysfunction, accompanied by aortic remodelling (thickened vascular medial layers, breakage of elastin fibres, collagen deposition, and inflammation). Transcriptome and biochemical analyses revealed that the ageing-controlling protein p66Shc and metabolism of mitochondrial reactive oxygen species (mROS) contributed to SIRT2 function in vascular ageing. Sirtuin 2 repressed p66Shc activation and mROS production by deacetylating p66Shc at lysine 81. Elimination of reactive oxygen species by MnTBAP repressed the SIRT2 deficiency-mediated aggravation of vascular remodelling and dysfunction in angiotensin II-challenged and aged mice. The SIRT2 coexpression module in aortas was reduced with ageing across species and was a significant predictor of age-related aortic diseases in humans. CONCLUSION: The deacetylase SIRT2 is a response to ageing that delays vascular ageing, and the cytoplasm-mitochondria axis (SIRT2-p66Shc-mROS) is important for vascular ageing. Therefore, SIRT2 may serve as a potential therapeutic target for vascular rejuvenation.
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Sirtuina 2 , Remodelación Vascular , Ratones , Humanos , Animales , Anciano , Sirtuina 2/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Especies Reactivas de Oxígeno/metabolismo , Envejecimiento , Ratones NoqueadosRESUMEN
OBJECTIVES: Chronic kidney disease (CKD) is a long-term condition characterized by poor prognosis and a high mortality rate. Panax notoginseng saponins (PNS) are the main active ingredient of the traditional Chinese herb Panaxnotoginseng(Burk.)F.H.Chen, which has been widely reported to have various pharmacological effects. Here, we examined the effect of PNS on renal function and the modulation of intestinal flora and intestinal barrier in a rat model of adenine-induced CKD. METHODS: Adenine was used to establish a rat model of CKD, biochemical testing, histopathologic examination, ELISA, immunohistochemical assay, western blot assay, and fecal microbiota 16s rRNA analysis was used to test the effect of PNS on CKD rats. RESULTS: Adenine induced a significant decrease in glomerular filtration rate, an increase in urinary protein excretion rate, and pathological damage to renal tissue in CKD rats. TNF-α, MCP-1, IL-1ß, IL-18, TMAO, and endotoxin levels were increased in the blood of the model rats. Application of PNS countered the effects of adenine, restoring the above parameters to the level observed in healthy rats. In addition, activation of the inflammatory proteins NF-κB (p65) and NLRP3 and the fibrosis-associated proteins α-SMA and smad3 were inhibited in the kidneys of CKD rats. Furthermore, PNS promoted the expression of the tight junction proteins Occludin and ZO-1, increased SIgA levels, strengthened intestinal immunity, reduced mechanical damage to the intestine, was reduced levels of DAO and D-LA. Our data suggest PNS may delay CKD by restoring gut microbiota, and through the subsequent generation of a microbial barrier and modulation of microbiota metabolites. CONCLUSIONS: In conclusion, PNS may inhibit the development of inflammation and fibrosis in the kidney tissue through regulation of intestinal microorganisms and inhibition of the activation of pro-inflammatory and pro-fibrotic proteins in the kidney.
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Panax notoginseng , Insuficiencia Renal Crónica , Saponinas , Ratas , Animales , Panax notoginseng/química , Saponinas/farmacología , Saponinas/uso terapéutico , ARN Ribosómico 16S , Intestinos , Insuficiencia Renal Crónica/tratamiento farmacológico , Fibrosis , AdeninaRESUMEN
Irinotecan is a kind of alkaloid with antitumour activity, but its low solubility and high toxicity limit its application. Epigallocatechin-3-gallate (EGCG) is one of the main bioactive components in tea. The epidemiological investigation and animal and cell experiments show that EGCG has a preventive and therapeutic effect on many kinds of tumours. Here, colorectal cancer cells RKO and HCT116 were employed, and the CCK8 proliferation test was used to screen the appropriate concentration of EGCG and irinotecan, and the effects of single and/or combined drugs on migration, invasion, DNA damage, cell cycle and autophagy of tumour cells were investigated. The results showed that EGCG combined with irinotecan (0.5 µmol L- ) not only had a stronger inhibitory effect on tumour cells than EGCG or irinotecan alone but also prevented tumour cell migration and invasion. EGCG alone did not cause DNA damage in colorectal cancer cells, but its combination with irinotecan could induce S or G2 phase arrest by inhibiting topoisomerase I to cause more extensive DNA damage. EGCG also induced apoptosis by promoting autophagy with irinotecan synergistically. These results indicated that EGCG in combination with irinotecan could be a promising strategy for colorectal cancer.
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Autofagia , Catequina/análogos & derivados , Neoplasias Colorrectales/tratamiento farmacológico , Daño del ADN , Sinergismo Farmacológico , Irinotecán/farmacología , Antioxidantes/farmacología , Catequina/farmacología , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Quimioterapia Combinada , Humanos , Inhibidores de Topoisomerasa I/farmacologíaRESUMEN
For the basic research on the traditional Chinese medicine(TCM), objective syndrome of traditional Chinese medicine and evaluation criteria of traditional Chinese medicine compounds are hardly to break though. While, the modern immunology points out that the body is a counterbalance state and immune imbalance is the root of sickness. The thinking mode of treating diseases in traditional Chinese medicine is also "balance", considering disease is the result of bias which present the imbalance of "Yin counters Yang", "exterior counters interior", "cold counters heat" and "weak counters strong". The Chinese herbal compound formula preparation was applied on disease therapy based on theory of Chinese medicine, which was confirmed by long period clinical application. It is composed of multi-compounds and has the characteristic of multi-targeting. Integrative medicine has spawned pan-immunomics, and the evaluation of immune function (immune balance) has become an important basis for diagnosis and treatment models of integrative medicine. In addition, balance is the core idea of whole-systemic conception of traditional Chinese medicine. Therefore, we speculate that immune balance under pan-immunomic can bridge the traditional Chinese medicine and modern integrative medicine and is the important basis for objective syndrome of traditional Chinese medicine and evaluation criteria of traditional Chinese medicine compounds. According to the bridging theory, we attempt to utilize informatics and statistical methods to construct an evaluation system for pharmacodynamics of traditional Chinese medicine based on its moderate regulation and the balanced adjustment of immunity under pan-immunomic, which further reveal the scientific essence of the whole-systemic view of traditional Chinese medicine. This research brings out a new valuable strategy and provides a theoretical basis for accelerating the transformation of traditional Chinese medicine, especially the exploitation of Chinese herbal compound formula, and constructing the new drug innovation and review system for traditional Chinese medicine. Besides as a reference for traditional Chinese medicine objective syndrome and pharmacodynamics of traditional Chinese medicine compounds, the evaluation system can screen the immunity of sub-health population also. With the continuous accumulation of clinical sample and data, the evaluation system will be more accurate and intelligent.
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Medicamentos Herbarios Chinos/farmacología , Sistema Inmunológico/efectos de los fármacos , Medicina Tradicional China , Humanos , Síndrome , Yin-YangRESUMEN
Based on the systematic summary of the results of the fourth general survey of traditional Chinese medicine resources, the cultivation of large varieties of Chinese material medica and the latest research on health industrial development, the novel concepts and scientific connotations of generalized science of Chinese material medica are put forward, and the basic ideas and methods of a new Chinese medicine academic system, the cultivation system of large varieties of Chinese medicinal materials and the application system of the large health industry are constructed. This kind of generalized science of Chinese material medica, rooted in the traditional Chinese culture and the theory of "preventive treatment of disease", can avoid the narrow prospect induced by the increasing specialization and refinement of knowledge of science of Chinese material medica. It will play an important role in the modernization, industrialization, internationalization of traditional Chinese medicine.
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Materia Medica/uso terapéutico , Medicina Tradicional China , Industria Farmacéutica , Humanos , InvestigaciónRESUMEN
Chronic kidney disease (CKD) is a progressive disorder characterized by structural and functional changes in the kidneys, providing a global health challenge with significant impacts on mortality rates. Extracellular vesicles (EVs), are vital in the physiological and pathological processes associated with CKD. They have been shown to modulate key pathways involved in renal injury, including inflammation, fibrosis, apoptosis, and oxidative stress. Currently, the application research of EVs in the diagnosis and treatment of CKD is highly prevalent. However, there is currently a lack of standardized guidelines for their application, and various methodologies have advantages and limitations. Consequently, we present an comprehensive summary elucidating the multifaceted involvement of EVs in both physiological and pathological aspects in CKD. Furthermore, we explore their potential as biomarkers and diverse therapeutic roles in CKD. This review provides an overview of the current state of research on application of EVs in the diagnosis and therapeutic management of CKD.
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Background: Sorafenib is a classic molecular targeted drug approved for hepatocellular carcinoma (HCC) therapy. However, a poor response rate and increasing resistance to sorafenib make its therapeutic efficacy suboptimal. Combination treatment with an agent capable of potentiating sorafenib sensitivity may be a promising solution. Aim: The aim of this study was to determine the synergistic effect of ellagic acid (EA), a natural polyphenol, and sorafenib on HCC. Methods: CCK-8, EdU incorporation and colony formation assays were used to study the effect of EA on HCC cell proliferation. Apoptosis was detected by flow cytometry in HCC cells and TUNEL assay in xenograft tumors. Transcriptome analysis was utilized to investigate alterations in signaling pathways with EA treatment. A xenograft mouse model was used to confirm the synergistic effect of sorafenib and EA on HCC tumors in vivo. Results: We found that EA inhibited growth and induced apoptosis in both HCC cells and xenograft tumors. Mechanistically, EA treatment reduced the activation of the MAPK and Akt/mTOR signaling pathways in HCC cells. Furthermore, combined EA and sorafenib treatment further inhibited the MAPK and Akt/mTOR signaling pathways compared to EA or sorafenib alone. EA synergistically potentiated the anticancer activity of sorafenib against HCC both in vitro and in vivo. Conclusion: EA inhibits HCC growth by inducing apoptosis through attenuation of the MAPK and Akt/mTOR signaling pathways. EA potentiates the response of HCC tumors to sorafenib both in vitro and in vivo, an effect that may be attributed to further inhibition of the MAPK and Akt/mTOR signaling pathways. These results suggest that EA is an effective adjuvant option for sorafenib therapy.
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BACKGROUND: Renal fibrosis is related to impaired kidney function and can eventually lead to end-stage renal disease, for which no effective treatment is available. Panax notoginseng saponins (PNS), as a commonly used traditional Chinese medicine, is considered a possible alternative for the treatment of fibrosis. OBJECTIVE: The purpose of the present study was to investigate the effects and possible mechanisms of PNS on renal fibrosis. METHODS: HK-2 cells were used to induce renal fibrosis cell model by lipopolysaccharide (LPS), and the cytotoxicity of PNS on HK-2 cells was investigated. Cell damage, pyroptosis, and fibrosis were analyzed to investigate the effects of PNS on LPS-induced HK-2 cells. NLRP3 agonist Nigericin was used further to explore the inhibitory effect of PNS on LPS-induced pyroptosis so as to clarify the possible mechanism of PNS on renal fibrosis. RESULTS: PNS had no cytotoxicity on HK-2 cells, and could reduce the apoptosis and the release of lactate dehydrogenase (LDH) and inflammatory cytokines of LPS-induced HK-2 cells, showing an alleviating effect on cell damage. PNS also reduced the expression of pyroptosis proteins NLRP3, IL-1ß, IL-18, and Caspase-1, as well as fibrosis proteins α-SMA, collagen â and p-Smad3/Smad3, which showed an inhibitory effect on LPS-induced pyroptosis and fibrosis. In addition, LPS-induced cell damage, pyroptosis, and fibrosis were aggravated after Nigericin treatment, while PNS alleviated the aggravation caused by Nigericin. CONCLUSION: PNS inhibits pyroptosis by inhibiting the activation of NLRP3 inflammasome in LPS-induced HK-2 cells, which ultimately alleviates renal fibrosis and plays a good role in the treatment of kidney diseases.
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Endovascular thrombectomy (EVT) is the recommended option for acute ischemic stroke (AIS) patients with large vessel occlusion (LVO) that within 6 h onset of stroke. The EVT treatment time window has been extended to 24 h in carefully selected patients by DAWN trial. Recent evidences indicated that some patients presented beyond 24 h still potentially benefit from EVT treatment. Herein, we describe one case of successful delayed EVT in a 50-year-old male AIS patient with an 8-day history of left middle cerebral artery occlusion. Before surgery, CT perfusion demonstrated a marked left hypoperfusion with penumbra volume of 127 mL and ischemic core volume of 10 mL. EVT was performed with complete recanalization and significant improvement in his neurological deficits at 90-days post-surgery follow-up. In future, more randomized clinical trials are warranted to further confirm the safety, efficacy, as well as the applicable population of delayed EVT.
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Purpose: Chemokine ligand 5 (CCL5), a vital member of the CC chemokine family, plays diverse roles in tumorigenesis, metastasis, and prognosis in various human tumors. However, no pan-cancer analysis has been conducted to illustrate its distinctive effects on clinical prognosis via underlying mechanisms and biological characteristics. Methods: Herein, we exploited the existed public bioinformatics database, primarily TCGA database and GTEx data, to comprehensively analyze the value of CCL5 involved in patient prognosis. Results: This study found that CCL5 was excessively expressed in most tumors and significantly associated with clinical prognosis in 10 out of 33 types of tumors. Notably, CCL5 might be an independent predictive biomarker of clinical outcome in SKCM patients, confirmed by univariate and multivariate Cox regression analysis. Furthermore, we acquired the genetic alteration status of CCL5 in multiple types of tumor tissues from TCGA cohorts. We revealed a potential correlation between the expression level of CCL5 and tumor mutational burden in 33 types of tumors. In addition, data showed that DNA methylation was associated with CCL5 gene expression in THCA, PRAD, LUSC, and BRCA cancers. Immune infiltration and immune checkpoints are fine indexes for evaluating immunotherapy. We uncovered that CCL5 was negatively correlated with the immune infiltration of CD8+ T cell, CD4+ T cell, macrophages, and gamma delta T cells in BRCA-basal and CESC tumors, while a significant positive correlation was observed in BLCA, COAD and other 7 types of tumors. Besides, CCL5 was closely associated with the immune checkpoint molecules in 8 types of tumors. The TIDE score was less in the CCL5 high-expressed group than in the CCL5 low-expressed group in SKCM patients, which indicated that CCL5 might be a fine monitor of immune response for immunotherapy. GO enrichment analysis data uncovered that cytokine-cytokine receptor interaction and chemokine signaling might be involved in the role of CCL5 in regulating tumor pathogenesis and prognosis. Conclusion: In conclusion, CCL5 was preliminarly identified as a biomarker of immune response and prognosis for tumors patients via our first comprehensive pan-cancer analysis.
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ABSTRACT: Cardiovascular diseases, including heart failure, coronary artery disease, atherosclerosis, aneurysm, thrombosis, and hypertension, are a great economic burden and threat to human health and are the major cause of death worldwide. Recently, researchers have begun to appreciate the role of microbial ecosystems within the human body in contributing to metabolic and cardiovascular disorders. Accumulating evidence has demonstrated that the gut microbiota is closely associated with the occurrence and development of cardiovascular diseases. The gut microbiota functions as an endocrine organ that secretes bioactive metabolites that participate in the maintenance of cardiovascular homeostasis, and their dysfunction can directly influence the progression of cardiovascular disease. This review summarizes the current literature demonstrating the role of the gut microbiota in the development of cardiovascular diseases. We also highlight the mechanism by which well-documented gut microbiota-derived metabolites, especially trimethylamine N-oxide, short-chain fatty acids, and phenylacetylglutamine, promote or inhibit the pathogenesis of cardiovascular diseases. We also discuss the therapeutic potential of altering the gut microbiota and microbiota-derived metabolites to improve or prevent cardiovascular diseases.
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Peritoneal macrophages (PMs), which resided in peritoneal cavity, are crucial to maintain tissue homeostasis and immunity. Macrophage self-renewal and polarization states are critical for PM population homeostasis and function. However, the underlying molecular mechanism that regulates self-renewal and polarization of PMs is still unclear and needs to be explored. Here, we demonstrated that PMs self-renewal was stimulated by granulocyte macrophage colony-stimulating factor (GM-CSF), but not by macrophage colony-stimulating factor (M-CSF). Pharmacological inhibition of Bromodomain & Extraterminal (BET) Proteins by either JQ1 or ARV-825 significantly reduced GM-CSF-dependent peritoneal macrophage self-renewal by abrogating cell proliferation and decreasing self-renewal-related gene expression, such as MYC and Klf4, at transcriptional and protein levels. In addition, transcriptomic analysis showed that JQ1 blocked alternative PMs polarization by downregulating key transcriptional factor IRF4 expression, but not the activation of AKT or STAT6 in PMs. These findings illustrated that the significance of BET family proteins in GM-CSF-induced PMs self-renewal and IL-4-induced alternative polarization.
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BACKGROUND: According to Chinese constitutional theory, people can be divided into nine constitutions, which represent distinctive vulnerability to different diseases such as metabolic syndrome, atherosclerosis, and immunity-related disease, and so forth in modern medicine, phlegm-dampness constitution (PDC) is one of the nine constitutions, which is susceptible to metabolic syndrome (MS) and atherosclerosis that associate with lipid metabolism and immunity dysregulation closely. OBJECTIVES: In this study, we aimed to investigate the metabolic and immunity profiles of phlegm-damp constitution (PDC), including metabolites, lymphocytes distribution, and inflammatory cytokines. METHODS: A total of 74 patients with PDC and 66 individuals with gentle constitution (GC) were enrolled in this study. We utilized biochemical methods to detect metabolic parameters, flow cytometry to survey T/B/NK/NKT lymphocyte subgroups distribution, and ELISA to assay inflammatory cytokines. RESULTS: The subjects with PDC had higher GLU, AI TC, TG, and LDL-C and lower HDL-C levels. The immunity profile indicated that PDC subjects had higher percentage of WBCs, neutrophils, lymphocytes, B cells, and natural killer T cells compared with subjects with GC (P < 0.05). Serum levels of IL-10 decreased significantly in the subjects with phlegm-damp constitution, whereas IL-12 levels increased dramatically in the PDC group compared with the GC group (both P < 0.05). Additionally, logistic regression identified four independent variables (GLU, TG, LDL-C, and lymphocytes) that were highly correlated with PDC (P < 0.05). The area under the curve of the receiver operating characteristic curve was 0.878, which indicated the data were reliable to distinguish the subjects with PDC from the ones with GC. CONCLUSION: Phlegm-damp constitution was prone to hyperglycemia and hyperlipidemia syndrome, promoting the occurrence and progression of metabolic-related diseases. Interestingly, proinflammatory cells and cytokines were activated in the PDC group as well. Our findings could offer a profile of early screening indicators to identify high-risk patients of metabolic- and immunity-related diseases from Chinese constitution.
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Considerable calcification and stenosis frequently occur in the radial artery (RA) in diabetic nephropathy. PTA was performed successfully using a balloon to expand and restrictively tear the longitudinal axis of the RA. This approach seems to be a useful measure of promoting the maturation of AVF in diabetic nephropathy.
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Chronic kidney disease is a devastating condition resulting from irreversible loss of nephron numbers and function and leading to end-stage renal disease and mineral disorders. Vascular calcification, an ectopic deposition of calcium-phosphate salts in blood vessel walls and heart valves, is an independent risk factor of cardiovascular morbidity and mortality in chronic kidney disease. Moreover, aging and related metabolic disorders are essential risk factors for chronic kidney disease and vascular calcification. Marked progress has been recently made in understanding and treating vascular calcification in chronic kidney disease. However, there is a paucity of systematic reviews summarizing this progress, and investigating unresolved issues is warranted. In this systematic review, we aimed to overview the underlying mechanisms of vascular calcification in chronic kidney diseases and discuss the impact of chronic kidney disease on the pathophysiology of vascular calcification. Additionally, we summarized potential clinical diagnostic biomarkers and therapeutic applications for vascular calcification with chronic kidney disease. This review may offer new insights into the pathogenesis, diagnosis, and therapeutic intervention of vascular calcification.
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Aging is a key risk factor for angiogenic dysfunction and cardiovascular diseases, including heart failure, hypertension, atherosclerosis, diabetes, and stroke. Members of the NAD+-dependent class III histone deacetylase family, sirtuins, are conserved regulators of aging and cardiovascular and cerebrovascular diseases. The sirtuin SIRT6 is predominantly located in the nucleus and shows deacetylase activity for acetylated histone 3 lysine 56 and lysine 9 as well as for some non-histone proteins. Over the past decade, experimental analyses in rodents and non-human primates have demonstrated the critical role of SIRT6 in extending lifespan. Recent studies highlighted the pleiotropic protective actions of SIRT6 in angiogenesis and cardiovascular diseases, including atherosclerosis, hypertension, heart failure, and stroke. Mechanistically, SIRT6 participates in vascular diseases via epigenetic regulation of endothelial cells, vascular smooth muscle cells, and immune cells. Importantly, SIRT6 activators (e.g., MDL-800/MDL-811) have provided therapeutic value for treating age-related vascular disorders. Here, we summarized the roles of sirtuins in cardiovascular diseases; reviewed recent advances in the understanding of SIRT6 in vascular biology, cardiovascular aging, and diseases; highlighted its therapeutic potential; and discussed future perspectives.
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Transgene silencing is one of two major obstacles in both basic biomedical research for transgene and clinical practice of gene therapy. Based on the model of HT1080 cell clones, which transduced single copy of retroviral vector MGPN2, the mechanism of transgene silencing was explored in this investigation by a serial molecular techniques. In the HT1080 cell clone that absence of GFP protein synthesized, no significant aberration of epigenetic modification was detected, but the transcript size and the sequence changed that resulted in the reading frame shift. In addition to chromosomal position effect leading to transgene silencing, the transcript reading frame shift associated with retroviral vector rearrangements could induce complete silencing of transgene.
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Reordenamiento Génico , Silenciador del Gen/fisiología , Vectores Genéticos/genética , Retroviridae/genética , Transgenes/genética , Línea Celular Tumoral , Ingeniería Genética , Humanos , Retroviridae/metabolismoRESUMEN
This study was aimed to construct Sirt1 shRNA interfering vector and to analyze the effects of Sirtl on cell proliferation and apoptosis in HepG2, A549 and 293T cell lines. To design and synthisize Sirtl shRNA sequence then recombinate it to pGenesil-1.0 plasmid, the positive pGenesil-1.0-Sirtl vector clone was screened by effective detections and sequencing. The vectors were transfected into HepG2, A549, 293T cell lines, and Sirtl expression levels in these clones were detected by RT-PCR and Western-blot. These clone cell proliferation activities were detected by MTT, and these cells apoptosis incidences were detected by MTT experiment after treated with DOX. The results showed that Sirt1 shRNA interfering vectors were successfully screened. The levels of Sirtl expression in HepG2-sh, A549-sh and 293T-sh cells were significantly reduced compared with their control cells. It was indicated that the proliferation activities of these cells were impaired and anti-apoptosis capabilities of HepG2-sh, A549-sh and 293T-sh were also impaired notably. Sirt1 took an important role in maintaining cell proliferation and resisting cell apoptosis caused by DNA damage, and this result also provided theoretical information for the further research.
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Apoptosis/genética , Proliferación Celular , Vectores Genéticos , ARN Interferente Pequeño/genética , Sirtuina 1/genética , Línea Celular Tumoral , Humanos , Interferencia de ARN , TransfecciónRESUMEN
OBJECTIVE: To investigate the permeability of quercetin across blood-brain barrier (BBB) and its impact on the proliferation and apoptosis of U251 cells. METHODS: The BBB model was established through culture of primary brain microvessel endothelial cells (BMVEC) and primary astroglia cells (AC), which was confirmed by transmission electron microscopy (TEM) and trans epithelial electric resistance (TEER). High pressure liquid chromatography (HPLC) was performed to determine quercetin permeability across BBB. U251 cells were exposed to quercetin. MTT assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), single-cell gel electrophoresis (SCGE) and flow cytometry (FCM) were performed to analyze cell proliferation, apoptosis, DNA damage and cell cycle distribution. RESULTS: The BBB was constructed successfully. Up to 65.54% of quercetin permeated across the BBB. Quercetin attenuated the proliferation of U251 and induced apoptosis. The FCM revealed that the U251 cells were inhibited at the G2/M point. CONCLUSION: Quercetin can permeate across the BBB effectively, restraining the proliferation of U251 cells and inducing apoptosis.