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1.
Zhongguo Zhong Yao Za Zhi ; 46(15): 3949-3959, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34472272

RESUMO

Qishen Yiqi Dripping Pills(QSYQ) are used clinically to treat various myocardial ischemic diseases, such as angina pectoris, myocardial infarction, and heart failure; however, the molecular mechanism of QSYQ remains unclear, and the scientific connotation of traditional Chinese medicine(TCM) compatibility has not been systematically explained. The present study attempted to screen the critical pathway of QSYQ in the treatment of myocardial ischemia by network pharmacology and verify the therapeutic efficacy with the oxygen-glucose deprivation(OGD) model, in order to reveal the molecular mechanism of QSYQ based on the critical pathway. The key targets of QSYQ were determined by active ingredient identification and target prediction, and underwent pathway enrichment analysis and functional annotation with David database to reveal the biological role and the critical pathway of QSYQ. Cell counting Kit-8(CCK-8), lactate dehydrogenase(LDH), and Western blot tests were launched on high-content active ingredients with OGD cell model to reveal the molecular mechanism of QSYQ based on the critical pathway. The results of network pharmacology indicated that QSYQ, containing 18 active ingredients and 82 key targets, could protect cardiomyocytes by regulating biological functions, such as nitric oxide biosynthesis, apoptosis, inflammation, and angiogenesis, through TNF signaling pathway, HIF-1 signaling pathway, PI3 K-Akt signaling pathway, etc. HIF-1 signaling pathway was the critical pathway. As revealed by CCK-8 and LDH tests, astragaloside Ⅳ, salvianic acid A, and ginsenoside Rg_1 in QSYQ could enhance cell viability and reduce LDH in the cell supernatant in a concentration-dependent manner(P<0.05). As demonstrated by the Western blot test, astragaloside Ⅳ significantly down-regulated the protein expression of serine/threonine-protein kinase(Akt1) and hypoxia-inducible factor 1α(HIF-1α) in the HIF-1 signaling pathway, and up-regulated the protein expression of vascular endothelial growth factor A(VEGFA). Salvianic acid A significantly down-regulated the protein expression of upstream phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3 CA) and downstream HIF-1α of Akt1. Ginsenoside Rg_1 significantly down-regulated the expression of HIF-1α protein and up-regulated the expression of VEGFA. The therapeutic efficacy of QSYQ on myocardial ischemia was achieved by multiple targets and multiple pathways, with the HIF-1 signaling pathway serving as the critical one. The active ingredients of QSYQ could protect cardiomyocytes synergistically by regulating the targets in the HIF-1 signaling pathway to inhibit its expression.


Assuntos
Medicamentos de Ervas Chinesas , Isquemia Miocárdica , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/genética , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular
2.
Braz J Biol ; 83: e245330, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495146

RESUMO

BACKGROUND: The brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. AIM: The study examined the expression of Neuroglobin (Ngb) and Hypoxia-inducible factor-1α (Hif-1α) in adult and young yak brain tissues, and provided researchers with meaningful insight into the anatomy, physiology, and biochemistry of this mammal. METHOD: The study employed immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR), and Western blot (WB) to obtain the results. RESULTS: Ngb and Hif-1α were significantly (P<0.05) expressed in the cerebellar cortex, piriform lobe, medulla, and corpus callosum of the adult yak while in the young yak brain tissues, the protein expressions were significantly found in the white matter of the cerebellum, pineal gland, corpus callosum, and cerebellar cortex. The Ngb and Hif-1α expression showed similarities and differences. This may have resulted from similar animal species, source of nutrition, age factors, brain size, emotional activities, and communication. The findings documented that Ngb and Hif-1α are commonly expressed in various adult and young yak brain tissues. Multiple roles in the brain tissues of the adult and young yaks are involved in the expression and distribution and are proposed to play a significant role in the adaptation of the yak to the high altitude environment. CONCLUSION: This study provides meaningful data to understand the adaptive mechanism to hypoxia and recommended researchers to expand on the adaptive mechanism and brain tissues that are not recorded.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Hipóxia , Animais , Encéfalo , Bovinos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neuroglobina , RNA Mensageiro
3.
Braz J Biol ; 83: e248911, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495167

RESUMO

The telencephalon refers to the most highly developed and anterior part of the forebrain, consisting mainly of the cerebral hemispheres. The study determined Neuroglobin (Ngb) and Hypoxia-inducible factor (HIF-1α) expression in the telencephalon of yak and cattle, and compare the expression and distribution pattern of Ngb and HIF-1α in the two animals. Immunohistochemistry (IHC), quantitative real-time Polymerase Chain Reaction (qRT-PCR), and Western blot (WB) were employed to investigate Ngb and Hif-1α expression in the telencephalon of yak and cattle. mRNA and protein expressions of Ngb and HIF-1α showed positive in different tissues of the yak and cattle telencephalon. Ngb expression in tissues of the yak recorded higher as compare to cattle while HIF-1α expression was found higher in cattle than yak. The HIF-1α expression in some tissues of yak telencephalon was consistent with the cattle. The results documented that HIF-1α may have a direct or indirect synergistic effect on Ngb expression in the yak telencephalon to improve hypoxia adaptation. It is suggested that yak may need more Ngb expression for adaptation, but the expression of HIF-1α seems to be down-regulated during long-term adaptation, and the specific causes of this phenomenon needs to be further verified.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Telencéfalo , Adaptação Fisiológica , Animais , Bovinos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neuroglobina , RNA Mensageiro/genética
4.
Stem Cell Res Ther ; 12(1): 432, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344474

RESUMO

BACKGROUND: Bone tissue engineering is a new concept bringing hope for the repair of large bone defects, which remains a major clinical challenge. The formation of vascularized bone is key for bone tissue engineering. Growth of specialized blood vessels termed type H is associated with bone formation. In vivo and in vitro studies have shown that low level laser therapy (LLLT) promotes angiogenesis, fracture healing, and osteogenic differentiation of stem cells by increasing reactive oxygen species (ROS). However, whether LLLT can couple angiogenesis and osteogenesis, and the underlying mechanisms during bone formation, remains largely unknown. METHODS: Mouse bone marrow mesenchymal stem cells (BMSCs) combined with biphasic calcium phosphate (BCP) grafts were implanted into C57BL/6 mice to evaluate the effects of LLLT on the specialized vessel subtypes and bone regeneration in vivo. Furthermore, human BMSCs and human umbilical vein endothelial cells (HUVECs) were co-cultured in vitro. The effects of LLLT on cell proliferation, angiogenesis, and osteogenesis were assessed. RESULTS: LLLT promoted the formation of blood vessels, collagen fibers, and bone tissue and also increased CD31hiEMCNhi-expressing type H vessels in mBMSC/BCP grafts implanted in mice. LLLT significantly increased both osteogenesis and angiogenesis, as well as related gene expression (HIF-1α, VEGF, TGF-ß) of grafts in vivo and of co-cultured BMSCs/HUVECs in vitro. An increase or decrease of ROS induced by H2O2 or Vitamin C, respectively, resulted in an increase or decrease of HIF-1α, and a subsequent increase and decrease of VEGF and TGF-ß in the co-culture system. The ROS accumulation induced by LLLT in the co-culture system was significantly decreased when HIF-1α was inhibited with DMBPA and was followed by decreased expression of VEGF and TGF-ß. CONCLUSIONS: LLLT enhanced vascularized bone regeneration by coupling angiogenesis and osteogenesis. ROS/HIF-1α was necessary for these effects of LLLT. LLLT triggered a ROS-dependent increase of HIF-1α, VEGF, and TGF-ß and resulted in subsequent formation of type H vessels and osteogenic differentiation of mesenchymal stem cells. As ROS also was a target of HIF-1α, there may be a positive feedback loop between ROS and HIF-1α, which further amplified HIF-1α induction via the LLLT-mediated ROS increase. This study provided new insight into the effects of LLLT on vascularization and bone regeneration in bone tissue engineering.


Assuntos
Terapia com Luz de Baixa Intensidade , Osteogênese , Animais , Regeneração Óssea , Células Endoteliais da Veia Umbilical Humana , Humanos , Peróxido de Hidrogênio , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica
5.
Nat Commun ; 12(1): 4700, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349124

RESUMO

During skin injury, immune response and repair mechanisms have to be coordinated for rapid skin regeneration and the prevention of microbial infections. Natural Killer (NK) cells infiltrate hypoxic skin lesions and Hypoxia-inducible transcription factors (HIFs) mediate adaptation to low oxygen. We demonstrate that mice lacking the Hypoxia-inducible factor (HIF)-1α isoform in NK cells show impaired release of the cytokines Interferon (IFN)-γ and Granulocyte Macrophage - Colony Stimulating Factor (GM-CSF) as part of a blunted immune response. This accelerates skin angiogenesis and wound healing. Despite rapid wound closure, bactericidal activity and the ability to restrict systemic bacterial infection are impaired. Conversely, forced activation of the HIF pathway supports cytokine release and NK cell-mediated antibacterial defence including direct killing of bacteria by NK cells despite delayed wound closure. Our results identify, HIF-1α in NK cells as a nexus that balances antimicrobial defence versus global repair in the skin.


Assuntos
Células Matadoras Naturais/imunologia , Pele/imunologia , Pele/microbiologia , Cicatrização , Animais , Hipóxia Celular , Citocinas/metabolismo , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Camundongos , Neovascularização Fisiológica , Pele/irrigação sanguínea , Dermatopatias Bacterianas/prevenção & controle
6.
J Pak Med Assoc ; 71(7): 1832-1837, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34410257

RESUMO

OBJECTIVE: To find the association of single nucleotide polymorphism of hypoxia-inducible factor-1 alpha, rs11549465 (1772 Cytosine > Thymine) with metabolic syndrome, and to compare the anthropometric and biochemical variables in different genotypes of hypoxia-inducible factor-1 alpha. METHODS: The cross-sectional comparative study was conducted at the University of Health Sciences, Lahore, Pakistan, from July 2016 to April 2019, and comprised patients of metabolic syndrome selected from the Sheikh Zayed Hospital, Lahore. Healthy controls were also enrolled. Fasting venous sample was taken for the determination of study parameters. The genetic variant of hypoxia-inducible factor-1 alpha was analysed by restriction fragment length polymorphism polymerase chain reaction. Data was analysed using SPSS 22. RESULTS: Out of 400 subjects, 200(50%) each were patients and controls. The frequency of CC genotype of hypoxia-inducible factor-1 alpha Cytosine > Thymine in patients was 166(83%) and in controls 147(73.5%); CT genotype was 34(17%) and 53(26.5%) respectively, while TT genotype was not observed. There was a significant association of the C allele and CC genotype (p=0.03) with the increased risk of metabolic syndrome (p=0.02). On comparison of study variables in the two genotypes, systolic blood pressure, anthropometric and lipid parameters were significantly higher in the wild CC genotype compared to CT in the control group (p<0.05), but there was no significant difference in the patients (p>0.05). CONCLUSIONS: Major allele C of hypoxia-inducible factor-1 alpha 1772 Cytosine > Thymine was found to be associated with increased risk of metabolic syndrome.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Síndrome Metabólica , Estudos de Casos e Controles , Estudos Transversais , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Hipóxia , Síndrome Metabólica/epidemiologia , Síndrome Metabólica/genética , Polimorfismo de Nucleotídeo Único
7.
Gene ; 803: 145893, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34384864

RESUMO

Glycolysis and heat shock proteins (HSPs) play an important role in mediating the physiological response to hypoxia. The changes of glycolysis and HSPs with altitude would provide important information regarding ways to prevent hypoxia-related sickness in both animals and humans. In this study, the expression pattern of HIF1A, PDK4, HSP27 and HSP60, indexes activity and content of glucose metabolism were detected in heart, lung, brain, and quadriceps femoris taken from Tibetan sheep (Ovis aries) that were raised at different altitudes (2,500 m, 3,500 m and 4,500 m). The expression of HIF1A and PDK4 was increased with increasing altitude in all of the tissues. The lactate dehydrogenase (LDH) activities and adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NADH (redox state), NAD+), lactic acid (LA), pyruvic acid (PA) contents were all increased with increasing altitude in all of the tissues. The ratio of NADH/NAD+ and LA/PA were higher in sheep at an altitude of 4,500 m than of 3,500 m and 2,500 m in all tissues, except for the NADH/NAD+ ratio in lung and quadriceps femoris. An increase in the protein and mRNA expression of ATP-independent HSP27 during hypoxia condition was detected. The expression of ATP-dependent HSP60 mRNA and protein was increased in all of the tissues at an altitude of 3,500 m than of 2,500 m, but was decreased at an altitude of 4,500 m. These results suggest that glycolysis and HSPs are upregulated to ensure energy supply and proteostasis during hypoxia, but energy conservation may be prioritized over cytoprotective protein chaperoning in Tibetan sheep tissues during extreme hypoxia.


Assuntos
Aclimatação , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Altitude , Animais , Hipóxia Celular , Regulação da Expressão Gênica , Glicólise , Ovinos , Tibet , Regulação para Cima
8.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445365

RESUMO

In this study, we aimed to investigate the influence of N-acetylcysteine (NAC) on the gene expression profile, neoangiogenesis, neutrophils and macrophages in a rat model of incisional wounds. Before creating wounds on the backs of 24 Sprague-Dawley rats, intradermal injections were made. Lidocaine-epinephrin solutions were supplemented with 0.015%, 0.03% or 0.045% solutions of NAC, or nothing (control group). Scars were harvested on the 3rd, 7th, 14th and 60th day post-surgery. We performed immunohistochemical staining in order to visualize macrophages (anti-CD68), neutrophils (anti-MPO) and newly formed blood vessels (anti-CD31). Additionally, RT-qPCR was used to measure the relative expression of 88 genes involved in the wound healing process. On the 14th day, the number of cells stained with anti-CD68 and anti-CD31 antibodies was significantly larger in the tissues treated with 0.03% NAC compared with the control. Among the selected genes, 52 were upregulated and six were downregulated at different time points. Interestingly, NAC exerted a significant effect on the expression of 45 genes 60 days after its administration. In summation, a 0.03% NAC addition to the pre-incisional anesthetic solution improves neovasculature and increases the macrophages' concentration at the wound site on the 14th day, as well as altering the expression of numerous genes that are responsible for the regenerative processes.


Assuntos
Acetilcisteína/administração & dosagem , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Fator de Crescimento Transformador beta1/genética , Cicatrização/efeitos dos fármacos , Acetilcisteína/farmacologia , Anestesia Local , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ratos , Ratos Sprague-Dawley
9.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360981

RESUMO

Granulosa-lutein cells (GLCs) from PCOS women display reduced HIF-1α and EDN2 levels, suggesting their role in PCOS etiology. Here, we investigated the mechanisms involved in aberrant EDN2 expression in PCOS, and its association with HIF-1α. Various HIF-1α-dependent factors were studied in GLCs from PCOS and compared to normally ovulating women. MicroRNA-210 (miR-210), its target genes (SDHD and GPD1L), and HIF-1α-responsive genes (EDN2 and VEGFA) differed in GLCs from PCOS, compared with those of healthy women. Levels of miR-210-designated hypoxiamiR-and EDN2 were reduced in the PCOS GLCs; concomitantly, GPD1L and SDHD levels were elevated. Cultured GLCs retained low EDN2 expression and had low HIF-1α levels, providing evidence for a disrupted hypoxic response in the PCOS GLCs. However, VEGFA expression was elevated in these cells. Next, miR-210 levels were manipulated. miR-210-mimic stimulated EDN2 twice as much as the miR-NC-transfected cells, whereas miR-210-inhibitor diminished EDN2, emphasizing the importance of hypoxiamiR for EDN2 induction. Intriguingly, VEGFA transcripts were reduced by both miR-210-mimic and -inhibitor, demonstrating that EDN2 and VEGFA are distinctly regulated. Disrupted hypoxic response in the GLCs of periovulatory follicles in PCOS women may play a role in ovulation failure, and in the reduced fertility prevalent in this syndrome.


Assuntos
Endotelina-2/metabolismo , Células da Granulosa/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Lúteas/metabolismo , Síndrome do Ovário Policístico/metabolismo , Transdução de Sinais , Adulto , Células Cultivadas , Endotelina-2/genética , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 50(3): 352-360, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34402252

RESUMO

To investigate the effects of salt-inducible kinase 2 (SIK2) on energy metabolism in rats with cerebral ischemia-reperfusion. Adult SD male rats were divided into 5 groups: sham group, ischemia group, reperfusion group, adenovirus no-load group, and SIK2 overexpression group with 5 animals in each group. The middle cerebral artery occlusion (MCAO) was induced with the modified Zea-Longa line thrombus method to establish the cerebral ischemia reperfusion model. Eight days before the MCAO, SIK2 overexpression was induced by injecting 7 µL adenovirus in the right ventricle, then MCAO was performed for followed by reperfusion HE staining was used to observe the pathological changes of cerebral tissue in rats; TTC staining was used to observe the volume of cerebral infarct. The levels of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) in rat brain tissue were detected by ELISA; the levels of SIK2 and hypoxia-inducible factor 1α (HIF-1α) in the rat brain tissues were detected by RT-qPCR and Western blotting. Compared with the sham group, SIK2 level was decreased in the ischemia group, and it was further declined in the reperfusion group (<0.05). Compared with the sham group and ischemic group, the pathological injury in reperfusion group were more severe, and the infarct size was larger; compared with the reperfusion group and adenovirus no-load group, the pathological injury of the SIK2 overexpression group was milder, and the infarct size is less. Compared with the sharn group, HIF-1α was increased in both ischemia group and reperfusion group, especially in ischemia group (all <0.05); HIF-1α level in the SIK2 overexpression group was higher than that in the reperfusion group and adenovirus no-load group (all <0.05). ATP level in ischemia group and reperfusion group was lower than that in the sham group, and the reperfusion group decreased more significantly than the ischemia group (<0.05); ADP content was increased in the ischemia and reperfusion group, and the ADP content in reperfusion group was significantly higher than that in the ischemia group (<0.05). ATP level in the SIK2 overexpression group was higher than that in the reperfusion group and adenovirus no-load group (all <0.05), and ADP was decreased in the SIK2 overexpression group (all <0.05). SIK2 can up-regulate the ATP level and down-regulate the ADP level in rat brain tissue and alleviate cerebral ischemia-reperfusion injury by increase the level of HIF-1α.


Assuntos
Isquemia Encefálica , Traumatismo por Reperfusão , Animais , Metabolismo Energético , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Infarto da Artéria Cerebral Média , Masculino , Proteínas Serina-Treonina Quinases , Ratos , Ratos Sprague-Dawley , Reperfusão
11.
World J Surg Oncol ; 19(1): 210, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34256803

RESUMO

OBJECTIVE: This meta-analysis was implemented to evaluate the association between hypoxia-inducible factor-1α (HIF-1α) C1772T/G1790A polymorphisms and susceptibility to head and neck cancer (HNC). MATERIAL AND METHODS: This meta-analysis has been registered on PROSPERO platform ( CRD42021257309 ). The PubMed, Embase and Web of Science databases were searched to retrieve eligible published papers. STATA software was used to calculate the pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) to assess the correlation strength. RESULTS: Our results demonstrated that the HIF-1α C1772T polymorphism was significantly related to an increased HNC risk (OR = 2.27, 95% CI = 1.17-4.42 for the homozygous model; OR = 11.53, 95% CI = 1.11-120.4 for the recessive model), especially in Caucasians (OR = 2.16, 95% CI = 1.09-4.27 for the homozygous model; OR = 2.28, 95% CI = 1.15-5.51 for the recessive model). Similarly, a remarkable correlation was discovered between the G1790A polymorphism and HNC risk (OR = 72.11, 95% CI = 2.08-2502.4 for the homozygous model; OR = 58.05, 95% CI = 1.70-1985.77 for the recessive model). Moreover, in the subgroup analysis by source of controls, a statistically significant correlation was discovered in the population-based (PB) subgroup (OR = 9.43, 95% CI = 1.20-73.9 for allelic model; OR = 72.11, 95% CI = 2.08-2502.4 for the homozygous model; OR = 3.22, 95% CI = 1.28-8.08 for the heterozygous model; OR = 7.83, 95% CI = 1.48-41.37 for the dominant model; OR = 58.05, 95% CI = 1.70-1985.8 for the recessive model) but not in the hospital-based (HB) subgroup. CONCLUSION: Our study found that both HIF-1α C1772T and G1790A polymorphisms might be a higher risk of HNC, especially in the Caucasian group with the C1772T polymorphism.


Assuntos
Neoplasias de Cabeça e Pescoço , Subunidade alfa do Fator 1 Induzível por Hipóxia , Predisposição Genética para Doença , Neoplasias de Cabeça e Pescoço/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Polimorfismo de Nucleotídeo Único , Prognóstico
12.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199089

RESUMO

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape "fibro-chondrocytic" cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.


Assuntos
Diferenciação Celular , Hipóxia/metabolismo , Menisco/citologia , Menisco/metabolismo , Animais , Biomarcadores , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Suínos , Engenharia Tecidual/métodos
13.
Analyst ; 146(17): 5255-5263, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34324622

RESUMO

In this article, we present a novel nitrocellulose-based microfluidic chip with 3-dimensional (3D) printing technology to study the effect of oxygen gradient on cells. Compared with conventional polydimethylsiloxane (PDMS) chips of oxygen gradient for cell cultures that can only rely on fluorescence microscope analysis, this hybrid nitrocellulose-based microfluidic platform can provide a variety of analysis methods for cells, including flow cytometry, western blot and RT-PCR, because the nitrocellulose-based chips with cells can be taken out from the growth chambers of 3D printed microfluidic chip and then used for cell collection or lysis. These advantages allow researchers to acquire more information and data on the basic biochemical and physiological processes of cell life. The effect of oxygen gradient on the zebrafish cells (ZF4) was used as a model to show the performance and application of our platform. Hypoxia caused the increase of intercellular reactive oxygen species (ROS) and accumulation of hypoxia-inducible factor 1α (HIF-1α). Hypoxia stimulated the transcription of hypoxia-responsive genes vascular endothelial growth factor (VEGF) and induced cell cycle arrest of ZF4 cells. The established platform is able to obtain more information from cells in response to different oxygen concentration, which has potential for analyzing the cells under a variety of pathological conditions.


Assuntos
Microfluídica , Oxigênio , Animais , Hipóxia Celular , Colódio , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Impressão Tridimensional , Fator A de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/metabolismo
14.
Mol Med Rep ; 24(4)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34328190

RESUMO

Pulmonary microvascular endothelial cell (PMVEC) apoptosis is the initial stage of adult pulmonary hypertension (PH), which involves high pulmonary arterial pressure and pulmonary vascular remodeling. However, the mechanism regulating PMVEC apoptosis and its involvement in the early stages of neonatal hypoxic PH (HPH) pathogenesis are currently unclear. The present study aimed to investigate the effects of heat shock protein 70 (HSP70) on hypoxia­induced apoptosis in PMVECs. PMVECs isolated from neonatal Sprague­Dawley rats were transfected with lentivirus with or without HSP70, or treated with the synthetic HSP70 inhibitor N­formyl­3,4­methylenedioxy­benzylidene-g-butyrolactam under hypoxic conditions (5% O2) for 24, 48 or 72 h. PMVEC apoptosis was evaluated by performing flow cytometry and mitochondrial membrane potential (MMP) assays. The expression levels of HSP70, hypoxia­inducible factor­1α (HIF­1α) and apoptosis­associated proteins were determined by conducting reverse transcription­quantitative PCR and western blotting. Following 24, 48 or 72 h of hypoxia, the apoptotic rates of PMVECs were significantly elevated compared with cells under normoxic conditions. The MMP was significantly reduced, whereas the mRNA and protein expression levels of HIF­1α, cytochrome c (cyt C), caspase­3 and HSP70 were enhanced by hypoxia compared with those under normoxic conditions. Additionally, the mRNA and protein expression levels of B­cell lymphoma 2 (Bcl­2) were significantly downregulated in the hypoxia group compared with those in the normoxia group. In hypoxic PMVECs, HSP70 overexpression decreased the apoptotic rate and the expression levels of cyt C, downregulated the expression levels of caspase­3 and HIF­1α, and increased the MMP and the expression levels of Bcl­2. HSP70 inhibition resulted in the opposite outcomes compared with those of HSP70 overexpression. Therefore, the results of the present study suggested that HSP70 may inhibit mitochondrial pathway­mediated apoptosis in isolated neonatal rat PMVECs in early­stage hypoxia, which may be associated with HSP70­mediated HIF­1α downregulation. Overall, HSP70 may be protective against neonatal HPH through the HSP70/HIF­1α pathway.


Assuntos
Apoptose/genética , Células Endoteliais/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Hipertensão Pulmonar/metabolismo , Microvasos/metabolismo , Animais , Animais Recém-Nascidos , Caspase 3/genética , Caspase 3/metabolismo , Hipóxia Celular , Regulação para Baixo , Células Endoteliais/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metaloproteinases da Matriz/metabolismo , Microvasos/citologia , Mitocôndrias/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/genética , Regulação para Cima
15.
Nat Immunol ; 22(8): 1020-1029, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34312547

RESUMO

T cell exhaustion is an induced state of dysfunction that arises in response to chronic infection and cancer. Exhausted CD8+ T cells acquire a distinct epigenetic state, but it is not known whether that chromatin landscape is fixed or plastic following the resolution of a chronic infection. Here we show that the epigenetic state of exhaustion is largely irreversible, even after curative therapy. Analysis of chromatin accessibility in HCV- and HIV-specific responses identifies a core epigenetic program of exhaustion in CD8+ T cells, which undergoes only limited remodeling before and after resolution of infection. Moreover, canonical features of exhaustion, including super-enhancers near the genes TOX and HIF1A, remain 'epigenetically scarred.' T cell exhaustion is therefore a conserved epigenetic state that becomes fixed and persists independent of chronic antigen stimulation and inflammation. Therapeutic efforts to reverse T cell exhaustion may require new approaches that increase the epigenetic plasticity of exhausted T cells.


Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Memória Imunológica/imunologia , 2-Naftilamina/uso terapêutico , Anilidas/uso terapêutico , Antivirais/uso terapêutico , Cromatina/metabolismo , Ciclopropanos/uso terapêutico , Epigênese Genética/genética , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Lactamas Macrocíclicas/uso terapêutico , Prolina/análogos & derivados , Prolina/uso terapêutico , Ribavirina/uso terapêutico , Ritonavir/uso terapêutico , Sulfonamidas/uso terapêutico , Uracila/análogos & derivados , Uracila/uso terapêutico , Valina/uso terapêutico
16.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208638

RESUMO

Fatty acids are derived from diet and fermentative processes by the intestinal flora. Two to five carbon chain fatty acids, termed short chain fatty acids (SCFA) are increasingly recognized to play a role in intestinal homeostasis. However, the characteristics of slightly longer 6 to 10 carbon, medium chain fatty acids (MCFA), derived primarily from diet, are less understood. Here, we demonstrated that SCFA and MCFA have divergent immunomodulatory propensities. SCFA down-attenuated host pro-inflammatory IL-1ß, IL-6, and TNFα response predominantly through the TLR4 pathway, whereas MCFA augmented inflammation through TLR2. Butyric (C4) and decanoic (C10) acid displayed most potent modulatory effects within the SCFA and MCFA, respectively. Reduction in TRAF3, IRF3 and TRAF6 expression were observed with butyric acid. Decanoic acid induced up-regulation of GPR84 and PPARγ and altered HIF-1α/HIF-2α ratio. These variant immune characteristics of the fatty acids which differ by just several carbon atoms may be attributable to their origins, with SCFA being primarily endogenous and playing a physiological role, and MCFA exogenously from the diet.


Assuntos
Ácidos Graxos Voláteis/metabolismo , Ácidos Graxos/metabolismo , Imunomodulação , Biomarcadores , Ácido Butírico/metabolismo , Candida/fisiologia , Citocinas/metabolismo , Dieta , Microbioma Gastrointestinal , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imunomodulação/genética , Mediadores da Inflamação/metabolismo , Receptor 4 Toll-Like/metabolismo
17.
ACS Appl Mater Interfaces ; 13(23): 27513-27521, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34086446

RESUMO

Successful gene therapy is highly dependent on the efficiency of gene delivery, which is mostly achieved by the carrier. Current gene carriers are generally nontherapeutic and take over most of the proportion in the delivery systems. Therefore, a library of polymerized and cationic photosensitive drugs (polyphotosensitizers, pPSs) with HIF-1α siRNA delivery capability is constructed to realize using "drug" to deliver "gene". The pPS component acts as both a therapeutic carrier for intracellular HIF-1α siRNA delivery and a photosensitive drug with photodynamic therapy (PDT). A reactive oxygen species (ROS)-cleavable linker is used to polymerize PS, allowing the successful segregation of PS monomers in space, avoiding the undesired aggregation-caused quenching (ACQ) effect and enhancing the in vitro and in vivo PDT effect. The complexes formed by pPSs and HIF-1α siRNA exhibited desired siRNA condensation and serum stability at the optimal conditions (pPSs with guanidines/siRNA weight ratio of 15), efficient intracellular internalization, and gene-silencing efficiency (60%) compared with commercial available transfection reagents (40%), as well as synergistic in vitro and in vivo phototoxicity for the combination PDT-gene therapy toward cancer treatment. This study provides a promising paradigm for the design of both the gene delivery carrier and the photosensitizer, as well as for broad utilities in the combination therapy toward cancer treatment.


Assuntos
Carcinoma Hepatocelular/terapia , Sistemas de Liberação de Medicamentos , Terapia Genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fotoquimioterapia , Porfirinas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Cátions/química , Proliferação de Células , Terapia Combinada , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos , Porfirinas/química , Espécies Reativas de Oxigênio , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 35(6): 761-768, 2021 Jun 15.
Artigo em Chinês | MEDLINE | ID: mdl-34142505

RESUMO

Objective: To investigate the effects of hypoxia inducible factor 1α (HIF-1α) overexpression on the differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) into vascular endothelial cells. Methods: SHED was isolated from the retained primary teeth donated by healthy children by using collagenase digestion method. The third generation cells were identified by flow cytometry and alizarin red and alkaline phosphatase (ALP) staining after osteogenic differentiation culture. The SHED were divided into blank control group (SHED without any treatment), empty group (SHED infected with empty lentivirus), HIF-1α overexpression group (SHED infected with HIF-1α overexpression lentivirus), Wnt inhibitor group (SHED interfered by IWR-1), and combination group (HIF-1α overexpressed SHED interfered by IWR-1). Real-time fluorescence quantitative PCR (qRT-PCR) and Western blot were used to analyze the expressions of HIF-1α mRNA and protein in the SHED of blank control group, empty group, and HIF-1α overexpression group. Then the SHED in 5 groups were induced differentiation into vascular endothelial cells for 14 days. The expressions of cell surface marker molecule [von Willebrand factor (vWF) and CD31] were detected by flow cytometry. The mRNA expressions of vascular cell adhesion protein 1 (VCAM-1), KDR (Kinase-inserted domain containing receptor), and VE-cadherin (VE) were analyzed by qRT-PCR. The protein expressions of phosphate-glycogen synthasc kinase 3ß (p-GSK3ß) and ß-catenin were analyzed by Western blot. The tube forming ability of induced cells was detected by Matrigel tube forming experiment. The ability of endothelial cells to phagocytic lipid after differentiation was detected by DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) phagocytosis. Results: After identification, the cells were SHED. After lentivirus transfection, compared with the blank control group and the empty group, the expressions of HIF-1α mRNA and protein in the HIF-1α overexpression group increased significantly ( P<0.05). Compared with the blank control group and the empty group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of ß-catenin protein were significantly higher ( P<0.05), the relative expression of p-GSK3ß protein was significantly lower ( P<0.05), the number of tubules formed and the ability to phagocytic lipids significantly increased ( P<0.05) in the HIF-1α overexpression group; while the indicators in the Wnt inhibitor group were opposite to those in the HIF-1α overexpression group ( P<0.05). Compared with the HIF-1α overexpression group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of ß-catenin protein were significantly lower ( P<0.05), the relative expression of p-GSK3ß protein was significantly higher, and the number of tubules formed and the ability of phagocytosis of lipids significantly reduced, showing significant differences between groups ( P<0.05). Conclusion: Overexpression of HIF-1α can promote SHED to differentiate into vascular endothelial cells by activating Wnt/ß-catenin signaling pathway.


Assuntos
Células Endoteliais , Subunidade alfa do Fator 1 Induzível por Hipóxia , Osteogênese , Diferenciação Celular , Criança , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células-Tronco , Dente Decíduo
19.
FASEB J ; 35(7): e21645, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34105824

RESUMO

Peripheral arterial disease (PAD) is one of the major complications of diabetes due to an impairment in angiogenesis. Since there is currently no drug with satisfactory efficacy to enhance blood vessel formation, discovering therapies to improve angiogenesis is critical. An imidazolinone metabolite of the metformin-methylglyoxal scavenging reaction, (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl) guanidine (IMZ), was recently characterized and identified in the urine of type-2 diabetic patients. Here, we report the pro-angiogenesis effect of IMZ (increased aortic sprouting, cell migration, network formation, and upregulated multiple pro-angiogenic factors) in human umbilical vein endothelial cells. Using genetic and pharmacological approaches, we showed that IMZ augmented angiogenesis by activating the endothelial nitric oxide synthase (eNOS)/hypoxia-inducible factor-1 alpha (HIF-1α) pathway. Furthermore, IMZ significantly promoted capillary density in the in vivo Matrigel plug angiogenesis model. Finally, the role of IMZ in post-ischemic angiogenesis was examined in a chronic hyperglycemia mouse model subjected to hind limb ischemia. We observed improved blood perfusion, increased capillary density, and reduced tissue necrosis in mice receiving IMZ compared to control mice. Our data demonstrate the pro-angiogenic effects of IMZ, its underlying mechanism, and provides a structural basis for the development of potential pro-angiogenic agents for the treatment of PAD.


Assuntos
Membro Posterior/fisiopatologia , Hiperglicemia/complicações , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/complicações , Metformina/metabolismo , Neovascularização Patológica/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Hipoglicemiantes/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imidazolinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Aldeído Pirúvico/metabolismo
20.
Life Sci ; 281: 119763, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34186050

RESUMO

AIMS: Beclin1(BECN1) is known as an autophagy-related protein and the expression is promoted by apelin in lung adenocarcinoma cells, suggesting that apelin activates autophagy in lung adenocarcinoma. However, the functions of apelin-induced autophagy in lung adenocarcinoma tumorigenesis and deterioration are still unknown. Thus, this study aims to investigate the effects of apelin-induced autophagy on lung adenocarcinoma tumorigenesis and deterioration. MAIN METHODS: Protein expression of exogenous genes were detected by Western blotting analysis. Lung adenocarcinoma cell migration was assessed with cell migration assays. Autophagy was measured with quantification of GFP-LC3 or RFP-GFP-LC3 puncta using fluorescence microscopy in cells by an observed blinded to experimental condition and by western blot analysis of LC3 and p62 in cell lysates as well as autophagy flux. Immunofluorescence staining was performed in human lung adenocarcinoma A549 cells with p-cofilin antibody. The proteins expression in cancer specimens were examined with immunohistochemistry. KEY FINDINGS: Here, we reveal that apelin induces autophagy activation in lung adenocarcinoma. Apelin/APJ regulates BECN1 transcription via HIF1A. Apelin/APJ-activated autophagy promotes lung adenocarcinoma cell migration. Moreover, treatment with autophagy inhibitors significantly decreases apelin/APJ-induced lung adenocarcinoma cell migration. Evaluation of patient samples of lung adenocarcinoma reveals an association between APJ with BECN1 expression and a poor prognosis. SIGNIFICANCE: Our studies demonstrate that apelin-induced autophagy promotes lung adenocarcinoma cell migration which suggests a potential therapeutic target for lung adenocarcinoma.


Assuntos
Adenocarcinoma/patologia , Receptores de Apelina/metabolismo , Apelina/metabolismo , Autofagia , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Transdução de Sinais , Células A549 , Fatores de Despolimerização de Actina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Autofagia/genética , Proteína Beclina-1/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosforilação
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