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1.
Nat Commun ; 14(1): 8282, 2023 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-38092772

RESUMO

Structural variants (SVs), accounting for a larger fraction of the genome than SNPs/InDels, are an important pool of genetic variation, enabling environmental adaptations. Here, we perform long-read sequencing data of 320 Tibetan and Han samples and show that SVs are highly involved in high-altitude adaptation. We expand the landscape of global SVs, apply robust models of selection and population differentiation combining SVs, SNPs and InDels, and use epigenomic analyses to predict enhancers, target genes and biological functions. We reveal diverse Tibetan-specific SVs affecting the regulatory circuitry of biological functions, including the hypoxia response, energy metabolism and pulmonary function. We find a Tibetan-specific deletion disrupts a super-enhancer and downregulates EPAS1 using enhancer reporter, cellular knock-out and DNA pull-down assays. Our study expands the global SV landscape, reveals the role of gene-regulatory circuitry rewiring in human adaptation, and illustrates the diverse functional roles of SVs in human biology.


Assuntos
Altitude , Genoma , Humanos , Hipóxia/genética , Análise de Sequência de DNA , Adaptação Fisiológica/genética
2.
Front Microbiol ; 13: 944006, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35992649

RESUMO

Exogenous pathogen infection can induce autophagy in cells. Autophagy is essential for cell survival, development, and homeostasis. It not only regulates cell defense and stress, but also has a close relationship with innate and adaptive immunity. Complement is an important part of innate immunity, which could be activated by three approaches, including classic, alternative, and lectin pathways. All the three pathways result in the activation of C3, and generate anaphylatoxin fragments C3a and C5a, and formation of the membrane attack complex. Either C3a or C5a induces the inflammatory cytokines through binding to C3aR or C5aR, respectively. However, it is still unknown whether the complement could regulate the autophagy of intracellular microorganisms or not. In this study, we constructed a Toxoplasma gondii (T. gondii) and macrophages co-culture experimental model using T. gondii expressing enhanced green fluorescence protein (EGFP) fluorescence and C3-/-C57BL/6 J mice for that T. gondii invaded peritoneal macrophages in mice. Western blot, laser confocal microscopy (LCM), and transmission electron microscopy (TEM) were used to observe the changes of autophagy between the macrophages from wild-type (WT) and C3-/- mice. Flow cytometry and LCM were used to investigate the effect of autophagy on the killing ability of macrophages against T. gondii. Here, we found that local C3 could suppress not only the canonical autophagy of macrophage, but also the xenophagy to T. gondii. Interestingly, the inhibition of C3 on host cell autophagy could significantly suppress the clearance of T. gondii by the IFN-γ-primed macrophage. Finally, we investigated the mechanism of the autophagy regulation of C3 that the effect of C3 on the macrophage-specific autophagy against T. gondii depends on mTOR. And, there is C3a but not C5a/C5aR involved in regulating macrophage xenophagy against T. gondii. Collectively, our findings suggest locally generated C3 regulates the clearance of T. gondii by Macrophage through the regulation of the non-canonical IFN-γ-dependent autophagy pathway, and paint a clearer picture in the regulation of autophagy by innate immune components.

3.
Commun Biol ; 5(1): 548, 2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35668171

RESUMO

Ascending to high-altitude by non-high-altitude natives is a well-suited model for studying acclimatization to extreme environments. Acute mountain sickness (AMS) is frequently experienced by visitors. The diagnosis of AMS mainly depends on a self-questionnaire, revealing the need for reliable biomarkers for AMS. Here, we profiled 22 AMS symptom phenotypes, 65 clinical indexes, and plasma proteomic profiles of AMS via a combination of proximity extension assay and multiple reaction monitoring of a longitudinal cohort of 53 individuals. We quantified 1069 proteins and validated 102 proteins. Via differential analysis, machine learning, and functional association analyses. We found and validated that RET played an important role in the pathogenesis of AMS. With high-accuracies (AUCs > 0.9) of XGBoost-based models, we prioritized ADAM15, PHGDH, and TRAF2 as protective, predictive, and diagnostic biomarkers, respectively. Our findings shed light on the precision medicine for AMS and the understanding of acclimatization to high-altitude environments.


Assuntos
Doença da Altitude , Proteínas ADAM , Doença Aguda , Altitude , Doença da Altitude/diagnóstico , Biomarcadores , Humanos , Proteínas de Membrana , Proteômica
4.
Artigo em Inglês | MEDLINE | ID: mdl-34567219

RESUMO

Depressive symptoms have been found to be highly prevalent among patients with coronary heart disease (CHD) and seriously affect the patients' quality of life. However, most psychotropic drugs have warnings about potential side effects. Accordingly, safer effective alternatives are urgently demanded. Angina pectoris of CHD is considered as "chest stuffiness and heartache syndrome" in traditional Chinese medicine, with the major syndrome type named Qi stagnation and blood stasis. Qi-regulating and blood circulation-promoting therapy has increasingly shown unique advantages in CHD patients. This study investigated the efficacy of Xuefu Zhuyu decoction, a representative prescription of Qi-regulating and blood circulation-promoting therapy, on angina pectoris patients with depressive symptoms. Depressive symptoms were stratified at baseline in 30 patients with stable angina pectoris who participated in both baseline and 12-week follow-up studies. After performing a stratified analysis, the angina pectoris-specific health status and traditional Chinese medicine "chest stuffiness and heartache syndrome" were evaluated by self-reports using the associated questionnaire scales, respectively. We measured serum concentrations of serotonin, brain-derived neurotrophic factor, and ATP, which are associated with the development of depression. We found that the Xuefu Zhuyu granule significantly improved the angina pectoris-specific health status in patients after 12 weeks of treatment; specifically, it had a better curative effect on patients with depressive symptoms. Xuefu Zhuyu granule also significantly improved the chest stuffiness and heartache syndrome in patients with depressive symptoms (efficacy index is 61.24%, P < 0.05 versus baseline). Interestingly, Xuefu Zhuyu granule has been found to be more susceptible to improving ATP levels in patients with depressive symptoms, indicating that the improvement in serum ATP levels might account for the better efficacy of Xuefu Zhuyu granule in patients with depressive symptoms. Our data provide prospective evidence that Xuefu Zhuyu granule improves angina pectoris-specific health status through regulating Qi and promoting blood circulation. This trial is registered with ChiCTR-IOR-15006989.

5.
J Tradit Chin Med ; 40(6): 974-982, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33258349

RESUMO

OBJECTIVE: To investigate the efficacy of Chinese medicines on Qi stagnation and blood stasis in rats with myocardial ischemia. METHODS: Fifty male Wistar rats were randomly divided into five groups (n = 10) as follows: (a) sham operation (Sham), (b) myocardial ischemia (Model), (c) treatment that regulates Qi (Qi), (d) treatment that promotes blood circulation (Blood), (e) treatment that both regulates Qi and promotes blood circulation (QB). The rat model was established via activities restriction for 6 h followed by tail clamp stimulation for 5 mins every day for 7 d and occlusion left coronary anterior descending artery. Afterwards rats were treated with medicines that regulate Qi and/or promote blood circulation via gavage for 14 d. Behavioral parameters were evaluated using open field and elevated plus-maze tests. The tongue color and sublingual vein were visually examined. Blood flow perfusion of tongue and auricle were detected using PIM Ⅱ. The mesenteric microcirculation was examined via capillaroscopy, and hemodynamics was assessed using a polygraph system. Serum homocysteine (Hcy), creatine kinase isoenzyme (CKMB) levels and endothelin-1 (ET-1) were measured. Hematoxylin and eosin staining and transmission electron microscopy were employed to detect the myocardial morphology and ultrastructure, respectively. RESULTS: Compared with findings in Sham group, rats in model group had coarse hair, dark mucosa of the lips and claw, low activity, and increased anxiety. Compared with findings in Model group, rats in the three treatment groups exhibited a lighter tongue color without an extended and varicose sublingual vein. There were significant increases of auricle blood flow perfusion in the Qi group and tongue bottom blood flow perfusion in the QB group. Compared with findings in Model rats, rats in Blood group exhibited improved mesenteric microcirculation associated with increased mesenteric blood flow and a larger arteriole diameter. Moreover, compared with findings in Model rats, Qi and QB rats exhibited increased left ventricular ± dp/dtmax, decreased serum CKMB, Hcy, ET-1 levels, and reduced myocardial ultrastructural damage. CONCLUSION: Myocardial ischemia damage was suppressed by Traditional Chinese Medicines that regulate Qi and promote blood circulation.


Assuntos
Circulação Sanguínea/efeitos dos fármacos , Medicamentos de Ervas Chinesas/administração & dosagem , Isquemia Miocárdica/tratamento farmacológico , Qi , Animais , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Masculino , Isquemia Miocárdica/fisiopatologia , Ratos , Ratos Wistar
6.
Ann Transl Med ; 8(6): 322, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32355766

RESUMO

BACKGROUND: Psychological stress can provoke and aggravate myocardial ischemia, and this stress can even trigger acute coronary syndromes or sudden cardiac death. Therefore, for the first time, this study aimed to investigate the method for establishing a rat model of myocardial ischemia with psychological stress and its evaluation. METHODS: Forty male Wistar rats were randomly divided into the sham (S, n=10), myocardial infarct (MI, n=10), psychological stress (MODEL, n=10), and myocardial infarct with psychological stress (MI + MODEL, n=10) groups. The rat model of psychological stress was established by measuring the data from activity restriction for 6 hours and followed by tail clamp stimulation for 5 minutes every day for 14 days. The rat model of the myocardial infarct with psychological stress was established by occluding the left coronary anterior descending artery in the MODEL rats. The body weight of rats was measured daily, the behavior parameters were evaluated via open-field test and elevated plus-maze, tongue color and sublingual vein were observed, rats' acral blood flow perfusion was detected by PIM II (Perfusion Imager II), mesenteric microcirculation was measured by capillaroscopy, and hemodynamics was measured by a polygraph system. An automatic biochemical analyzer determined the content of serum cTnT (cardiac troponin T), Hcy (homocysteine), and activity of LDH (lactate dehydrogenase). Myocardial infarct size was measured with TTC (triphenyhetrazolium chloride) staining. RESULTS: We found that rats in the psychological stress (MODEL) group were characterized by coarse hair, dark mucosa of the lips and claw, low spirit, decreased body weight, and increased anxiety. Compared with rats in the sham group, rats in the MODEL + MI group showed decreased mesenteric blood flow, narrowed arteriole and venule diameter, reduced acral blood flow perfusion, and LV ±dp/dtmax (the maximal rate of the increased and decrease of left ventricular pressure), as well as increased serum content of cTnT, Hcy, and LDH activity. Compared with the MI group, rats in the MODEL + MI group showed deteriorated microcirculation dysfunction manifested as a dark tongue color of deep purple, prominently extended and varicose sublingual vein, and aggravated myocardial damage in the form of increased infarct size and LDH leakage. CONCLUSIONS: In conclusion, the rat model of myocardial ischemia with psychological stress was successfully established, and manifested as aggravating behavioral disorder, mesenteric microcirculation and left ventricular dysfunction, and myocardial damage.

7.
Shock ; 53(3): 363-372, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31135707

RESUMO

BACKGROUND: Impaired autophagic flux contributes to cardiomyocyte death in ischemia/reperfusion (I/R) injury. Restoring the impaired autophagic flux by using agents may be a promising strategy that alleviates myocardial I/R injury. The present study aimed to evaluate the effect of exogenous calreticulin (CRT) postconditioning on impaired autophagic flux induced by hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: Rat myocardial I/R injury model was prepared. CRT postconditionging was fulfilled by an intraperitoneal injection of CRT (0.5 mg/kg body weight) 5 min before reperfusion. Hemodynamics, serum lactate dehydrogenase (LDH) activity and Cardiac troponin T (TnT) content, and infarct size were measured. The H/R injury model of H9c2 cells was prepared. CRT postconditioning was performed by adding 25 pg/mL CRT to the medium at the onset of reoxygenation. Cell death rate, lactate dehydrogenase (LDH) leakage, intracellular reactive oxygen species (ROS), and malondialdehyde (MDA) were assessed. Autophagic flux was monitored by mRFP-GFP-LC3 adenovirus infection. The number of autophagosomes and autolysosomes in cells were determined by counting the fluorescence dots. Western blot assay was used to determine the expression of autophagy-related proteins. RESULTS: CRT postconditionging improved cardiac function, reduced serum LDH activity and TnT content, and limited myocardial infarct size after myocardial I/R injury in rat. H/R induced H9c2 cells injury and autophagosomes accumulation in cells. CRT postconditioning attenuated H/R-induced cell death, LDH leakage, and the increase of intracellular ROS and MDA. Meanwhile, CRT postconditioning suppressed H/R-induced excessive formation of autophagosomes, as shown by a decrease of autophagosomes and the ratio of LC3-II/LC3-I, LC3-II, and Beclin1. It also improved H/R-induced impaired autophagosomes clearance, as shown by an increase of autolysosomes and the level of LAMP-2, and a decrease of the level of p62. CONCLUSION: These findings suggested that CRT postconditioning reduced myocardial I/R injury. CRT postconditioning also inhibited the excessive formation of autophagosomes, promoted the clearance of autophagosomes, and resorted the autophagic flux, consequently reduced the H/R injury in H9c2 cells.


Assuntos
Calreticulina/uso terapêutico , Pós-Condicionamento Isquêmico/métodos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Autofagia , Técnicas de Cultura de Células , Modelos Animais de Doenças , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Ratos , Ratos Sprague-Dawley
8.
Life Sci ; 237: 116944, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31604108

RESUMO

AIMS: Endoplasmic reticulum stress (ERS) is an evolutionarily conserved cell stress response. Recently, it was found that ERS induces not only apoptosis but also endoplasmic reticulophagy (ER-phagy). A previous study demonstrated that inhibition of ER-phagy alleviates cell injury. The purpose of this study was to investigate the involvement of the protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in ERS-induced ER-phagy in H9c2 cardiomyoblasts. To address this aim, cells were treated with ERS inhibitors and a Nrf2 inhibitor before establishment of thapsigargin (TG)- or tunicamycin (TM)-induced ERS models in H9c2 cardiomyoblasts. MAIN METHODS: Transmission electron microscopy and immunofluorescence staining were used to detect ER-phagy. Western blotting was employed to detect the levels of calreticulin (CRT), total and phosphorylated PERK, nuclear Nrf2, activated transcription factor 4 (ATF4), light chain 3B (LC3B)-II and Beclin 1. Immunofluorescence staining was used to assess subcellular location of Nrf2. KEY FINDING: TG or TM induced H9c2 cell injury and ER-phagy and upregulated CRT expression, PERK phosphorylation, Nrf2 nuclear translocation, and expression of ATF4, Beclin 1, and LC3B-II compared with control cells. Treatment with ERS inhibitors decreased TG- or TM-induced ER-phagy, downregulated CRT expression, PERK phosphorylation, Nrf2 nuclear translocation and the expression of ATF4, Beclin 1 and LC3B-II. Moreover, a Nrf2 inhibitor downregulated the expression of ATF4, Beclin 1 and LC3B-II and alleviated TG- or TM-induced ER-phagy and H9c2 cell injury. SIGNIFICANCE: These findings suggest that the PERK/Nrf2 pathway mediates upregulation of ER-phagy, thereby inducing cell injury in H9c2 cardiomyoblasts.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/patologia , Miócitos Cardíacos/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteína Beclina-1/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Transdução de Sinais
9.
Front Pharmacol ; 10: 985, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31551786

RESUMO

Coronary heart disease (CHD) remains the leading cause of morbidity and mortality worldwide. Traditional Chinese medicine (TCM) is one of the effective complementary and alternative therapies used to improve the prognosis of CHD patients. Xuefu Zhuyu (XFZY) decoction, a classical traditional Chinese medication for regulating Qi and promoting blood circulation, has a clinical benefit in CHD; however, the underlying mechanism is not clear. Recently, it was found that the metabolites involved in amino acid metabolism and the tricarboxylic acid cycle were altered in CHD patients with Qi and Yin deficiency syndrome. To understand the material foundation of Qi, it is of great significance to study the differential metabolites involved in Qi during treatment of CHD with Qi-regulating and blood-promoting herbs. In this study, we investigated the metabolic profiles of serum in CHD patients by nontargeted metabolomics analysis to detect differential metabolites between the XFZY decoction group and placebo group. Ten CHD patients were enrolled and treated with placebo granules or XFZY decoction granules in a random and double-blind manner. Serum samples of all patients were evaluated by untargeted high-performance liquid chromatography with tandem mass spectrometry-based metabolomics. In total, 513 metabolites were detected in the serum of CHD patients, and six of these metabolites participating in seven metabolic pathways were significantly different between CHD patients treated with XFZY decoction and the placebo group. Among the six differential metabolites, FA (20:2)-H and tetracarboxylic acid (24:0), involved in fatty acid metabolism; cis-aconitic acid, which participates in the tricarboxylic acid cycle; 2-deoxy-D-glucose, involved in glucose metabolism; and N-acetylglycine, involved in amino acid metabolism, were decreased, whereas spermine, which participates in amino acid metabolism, was increased as compared with the placebo group. Our findings, combined with the perspective of biological functions, indicate that 2-deoxy-D-glucose and spermine might constitute the partial material foundation of Qi in CHD patients treated with XFZY decoction.

10.
Life Sci ; 232: 116591, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31228513

RESUMO

AIMS: Calreticulin (CRT) is a multifunctional protein that protects endothelial cells by alleviating actin cytoskeleton injury, but the underlying mechanism remains unclear. CRT was recently identified as a novel acyltransferase; acetylation at the N-terminus of actin monomers strengthens actin polymerization. This study was undertaken to determine whether CRT protects human microvascular endothelial cells (HMECs) against microwave radiation through actin acetylation. MATERIALS AND METHODS: We prepared a eukaryotic-derived recombinant CRT and incubated the HMECs with it prior to microwave exposure. We then assessed cell injury and endothelial function, detected actin polymerization and acetylation after HMECs exposure to S-band high-power microwaves. Coimmunoprecipitation, pull-down, and ex vitro acetylation reaction were performed to determine whether actin is a novel substrate of CRT acyltransferase. Finally, we employed the mutant experiments to demonstrate the acetylation sites contributing to CRT acetyltransferase activity. KEY FINDINGS: Microwave radiation induced severe cell injury and endothelial contact dysfunction, reduced the polymerization of actin filaments, and destroyed the actin arrangement, ultimately reducing acetylated actin expression. CRT treatment upregulated actin acetylation levels, promoted polymerization, and facilitated thicker and longer F-actin stress fibre formation. Pre-incubation with CRT rescued microwave-induced cell injury, decreased actin acetylation, and rendered the actin cytoskeleton radiation-retardant. The level of acetyl-actin was positively correlated with actin polymerization. Actin was identified as a novel substrate of CRT, being acetylated mainly through the CRT P-domain at lys-206 and -207. SIGNIFICANCE: This work provides a better understanding of the underlying mechanism of CRT-induced cytoprotection, and suggests a novel therapeutic target for microwave radiation-related diseases with endothelial dysfunction.


Assuntos
Actinas/metabolismo , Calreticulina/farmacologia , Microvasos/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Linhagem Celular , Citoproteção , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Microvasos/efeitos da radiação , Micro-Ondas , Substâncias Protetoras , Processamento de Proteína Pós-Traducional , Transdução de Sinais
11.
Shock ; 46(5): 531-540, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27219857

RESUMO

To investigate whether myofibrillogenesis regulator 1 (MR-1) attenuates renal ischemia/reperfusion (I/R) injury via inhibiting phosphorylated Akt (p-Akt) mitochondrial translocation-mediated opening of the mitochondrial permeability transition pore (mPTP), we injected adenovirus containing MR-1 gene or its siRNAs to the left kidney subcapsular areas of Sprague-Dawley rats, which subsequently underwent experimental renal I/R injury. Renal functions and the severity of the tubular injury were evaluated by the serum creatinine and blood urea nitrogen levels and the pathological scores. We also examined the mitochondrial morphology and functions. Total/p-Akt were assessed by western blot using the mitochondrial and the cytosolic fractions of cortex of renal tissue, respectively. We found that mitochondrial and cytosolic MR-1 levels and mitochondrial p-Akt decreased, and cytosolic p-Akt increased after reperfusion. Subcapsular injection of adenovirus led to higher MR-1 expression in the mitochondria/cytosol, inhibited mPTP opening, and alleviated renal I/R injury; adenovirus injection also upregulated mitochondrial total and p-Akt levels more prominently compared with the normal saline (NS) group. Subcapsular injection of MR-1 siRNAs significantly lowered MR-1 expression and induced renal injury, with increased mPTP opening and mitochondrial damage, similar to I/R injury. MR-1 interacted with Akt in renal cortex homogenate. Wortmannin, a phosphatidylinositol 3 kinase (PI3K) inhibitor, abolished both mitochondrial p-Akt recruitment and the protective effect of MR-1 overexpression on I/R injury. To conclude, MR-1 protects kidney against I/R injury through inhibiting mPTP opening and maintaining mitochondrial integrity, through the recruitment of PI3K-dependent p-Akt to the mitochondria. MR-1 could be a new therapeutic strategy for renal I/R injury.


Assuntos
Rim/metabolismo , Rim/patologia , Proteínas Musculares/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Citocromos c/metabolismo , Modelos Animais de Doenças , Masculino , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Musculares/genética , Traumatismo por Reperfusão Miocárdica/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley
12.
J Geriatr Cardiol ; 12(5): 540-6, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26512246

RESUMO

BACKGROUND: Endoplasmic reticulum (ER) stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin (PQS) is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured cardiomyocytes were stimulated with thapsigargin (TG), which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration. METHODS: Primary cultured cardiomyocytes from neonatal rats were exposed to TG (1 µmol/L) treatment for 24 h, following PQS pre-treatment (160 µg/mL) for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase (Si-PERK) for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 (GRP78), calreticulin, PERK, eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) were assayed by western blotting. RESULTS: Both PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect. CONCLUSIONS: Our data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings provide novel data regarding the molecular mechanisms by which PQS inhibits cardiomyocyte apoptosis.

13.
Exp Cell Res ; 337(2): 234-42, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26051880

RESUMO

Hypoxia/reoxygenation (H/R) injury is characterized by microfilament reorganization in cardiomyocytes. Previous studies have shown that myofibrillogenesis regulator-1 (MR-1) is expressed in the myocardium and promotes actin organization in cardiomyocytes. The purpose of this study was to investigate the role of MR-1 in attenuating hypoxia/reoxygenation injury in cardiomyocytes through promoting restoration of the microfilament. To address this aim, an H/R model of cultured neonatal cardiomyocytes was used to assess filamentous actin (F-actin) and α-actinin organization through immunofluorescence microscopy analysis. RT-PCR was used to detect mRNA levels of MR-1 and myosin regulatory light chain-2 (MLC-2). Western blot analysis was used to detect protein levels of MR-1 and filamentous actin/globular actin (F-/G-actin) as well as MLC-2 and myosin light chain kinase (MLCK) phosphorylation and protein expression. We also explored the effects of overexpressing or knocking down MR-1 on H/R injury and the MLCK/MLC-2/F-actin pathway. We found that H/R induced cardiomyocyte injury and disruption of F-actin and α-actinin with a decrease in the F-/G-actin ratio compared with controls. Compared with the H/R group, MR-1 overexpression attenuated H/R-induced injury and disruption of F-actin and α-actinin in cardiomyocytes with an increase in the F-/G-actin ratio. MR-1 overexpression also up-regulated H/R-induced MLCK and MLC-2 phosphorylation. However, MR-1 knockdown aggravated H/R injury by further disrupting F-actin and α-actinin, as well as decreasing the F-/G-actin ratio. MR-1 knockdown also down-regulated MLCK and MLC-2 phosphorylation induced by H/R injury. These findings suggest that MR-1 attenuates H/R-induced cardiomyocyte injury by promoting microfilament reorganization through the activation of the MLCK/MLC-2 pathway.


Assuntos
Citoesqueleto de Actina/fisiologia , Hipóxia/complicações , Proteínas Musculares/metabolismo , Miócitos Cardíacos/citologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Animais Recém-Nascidos , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Hipóxia/patologia , Técnicas Imunoenzimáticas , Proteínas Musculares/genética , Miócitos Cardíacos/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Apoptosis ; 20(3): 285-97, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25542256

RESUMO

The purpose of this study was to investigate the role of myofibrillogenesis regulator-1 (MR-1) in cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R), through protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. To address this aim, an H/R model of neonatal rat cardiomyocytes was used. MR-1 was overexpressed using an adenoviral vector system and knocked down using MR-1 specific siRNA. Apoptosis was assessed by using Annexin V/PI double staining, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay, and the Bcl-2/Bax ratio. Western blotting was used to detect the protein levels of MR-1, glucose-regulated protein 78 (GRP78), total and phosphorylated PERK, Nrf2, activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), Bcl-2 and Bax. Immunofluorescence staining was used to assess the subcellular location of Nrf2. We found that H/R induced significant apoptosis in neonatal rat cardiomyocytes. MR-1 overexpression attenuated H/R-induced apoptosis, decreased GRP78 (P < 0.01) and CHOP expression (P < 0.05), and increased the Bcl-2/Bax ratio (P < 0.01). MR-1 overexpression suppressed H/R-induced PERK phosphorylation, Nrf2 nuclear translocation, and ATF4 expression (P < 0.01). While MR-1 knockdown aggravated H/R-induced apoptosis, increased expression of GRP78 and CHOP (P < 0.05), and decreased the Bcl-2/Bax ratio (P < 0.01). MR-1 knockdown significantly increased H/R-induced PERK phosphorylation (P < 0.05), Nrf2 nuclear translocation, and ATF4 expression (P < 0.01). These findings suggest that MR-1 alleviates H/R-induced cardiomyocyte apoptosis through inhibition of the PERK/Nrf2 pathway.


Assuntos
Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/genética , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Hipóxia Celular , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Oxigênio/farmacologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Transgenes , eIF-2 Quinase/genética
15.
Microcirculation ; 21(8): 717-29, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24930861

RESUMO

OBJECTIVE: To determine the effects of cytosolic CRT on MR-induced MMEC injury, and the underlying mechanism. METHODS: MMECs were randomized into eight groups: control, AdCRT (infected with pAdCMV/V5-DEST-CRT adenovirus), stCRT (transfected with rCRT-siRNAs), Mock (transfected with scrambled siRNAs), MR (exposed to MR for six minutes), AdCRT + MR, stCRT + MR, and Mock + MR. The magnitude of cell injury were assessed by Annexin V-PI staining, LDH activity in culture medium, MMEC migration ability, ultrastructure and cytoskeletal stability. Subcellular colocalization of CRT and ConA or integrin were evaluated by immunocytochemistry. The mRNA and protein expression levels of target genes were examined by qRT-PCR and western blotting, respectively. RESULTS: MR-induced cytotoxicity was dose-dependent. Overexpression of cytosolic CRT suppressed MR injury, shown as decreased cell apoptosis, reduced LDH activity, enhanced cell migration capability, and maintenance of ultrastructure and cytoskeleton integrity. Conversely, CRT deficiency aggravated MR-induced injury. Exposure of AdCRT MMECs to MR promoted membrane translocation of CRT and the interaction of CRT-integrin-α. Correlation analysis revealed that integrin-α expression or FAK phosphorylation was positively associated with cytosolic CRT expression. CONCLUSIONS: Cytosolic CRT inhibits MR-induced MMEC injury through activation of the integrin-FAK pathway.


Assuntos
Calbindina 2/biossíntese , Células Endoteliais/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Cadeias alfa de Integrinas/metabolismo , Micro-Ondas/efeitos adversos , Animais , Calbindina 2/genética , Citosol/metabolismo , Células Endoteliais/patologia , Quinase 1 de Adesão Focal/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos da radiação , Cadeias alfa de Integrinas/genética , Masculino , Fosforilação/genética , Fosforilação/efeitos da radiação , Ratos , Ratos Sprague-Dawley
16.
Am J Nephrol ; 39(4): 279-87, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24714450

RESUMO

BACKGROUND/AIMS: Ischemia/reperfusion (I/R) injury is characterized by cytoskeletal reorganization and loss of polarity in proximal tubule epithelial cells. Previously, we showed that myofibrillogenesis regulator (MR)-1 promoted actin organization in cardiomyocytes. MR-1 is also expressed in the kidney. METHODS: In this study, we investigated MR-1 expression in acute renal failure induced by I/R in Sprague-Dawley rats. We determined the MR-1 expression and the ratio of fibrous actin (F-actin) to globular actin (G-actin). HK-2 cells were treated with or without hypoxia/reoxygenation (H/R), and MR-1 levels were increased by adenoviral overexpression or silenced by RNA interference. RESULTS: I/R and H/R resulted in cellular injury and decreases of MR-1, the F-/G-actin ratio, and myosin light chain (MLC)-2. MR-1 overexpression attenuated H/R-induced cell injury and loss of surface membrane polarity of actin. MR-1 overexpression also increased the expression and phosphorylation of MLC-2 and MLC kinase, which were decreased in MR-1-silenced and H/R-treated cells. CONCLUSION: Together, these data show that MR-1 promoted actin polarity on the membrane surface and protected HK-2 cells from H/R injury. The mechanism might involve the rapid organization of F-actin through the upregulation and phosphorylation of MLC-2.


Assuntos
Actinas/metabolismo , Injúria Renal Aguda/metabolismo , Hipóxia/metabolismo , Proteínas Musculares/metabolismo , Traumatismo por Reperfusão/metabolismo , Injúria Renal Aguda/prevenção & controle , Animais , Miosinas Cardíacas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Regulação para Baixo , Terapia Genética , Humanos , Masculino , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/prevenção & controle
17.
Microcirculation ; 2014 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-24635541

RESUMO

OBJECTIVE: The study aimed to investigate the effect of microwave radiation on microvasculature as well as the underlying mechanisms. METHODS: Sprague Dawley rats were exposed to microwave radiation. Microvascular diameters, flow velocity, blood perfusion and permeability were measured. Cultured endothelial cells from microvessels were subjected to microwave radiation. Cytoskeleton, apoptosis, protein synthesis and the markers of endoplasmic reticulum stress including 78-kDa glucose-regulated protein and calreticulin in endothelial cells were examined. RESULTS: Microwave radiation decreased microvascular diameters and blood perfusion, increased the permeability of microvessles. And microwave radiation induced the formation of stress fibers, apoptosis, and LDH leakage from microvascular endothelial cells. Also, when microvascular endothelial cells were exposed to microwaves, protein synthesis was significantly elevated. We found that upon microwave radiation, the expression of 78-kDa glucose-regulated protein and calreticulin were greatly upregulated in microvascular endothelial cells. We also investigated possible signaling pathways for endoplasmic reticulum stress-initiated apoptosis. C/EBP homologous protein (CHOP) pathway was activated in microvascular endothelial cells exposed to microwaves. CONCLUSIONS: Microwave radiation induces microvascular injury by triggering the apoptotic pathway of endoplasmic reticulum stress. This article is protected by copyright. All rights reserved.

18.
Microcirculation ; 21(6): 506-15, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24589181

RESUMO

OBJECTIVE: This study was designed to evaluate whether exogenous CRT was beneficial for alleviating MR-induced injury by suppressing ER stress in rat MMECs. METHODS: MMECs were pretreated with CRT (25 pg/mL) for 12 hours, followed by the exposure to 2.856 GHz radiation at a mean power density of 30 mW/cm(2) for six minutes. MR-induced injury in MMECs was evaluated by LDH leakage, apoptosis, and cell viability analysis. The expression of GRP78, CRT, CHOP, Bcl-2, and Bax were examined by Western blot analysis to reflect ER stress response and ER stress-related apoptosis. RESULTS: MR induced marked MMECs injury, as shown by increased LDH leakage and apoptosis rate and decreased cell viability. MR also induced excessive ER stress, characterized by increased expression of GRP78 and CRT, and ER stress-related apoptotic signaling as well, as shown by the upregulation of CHOP and Bax and the downregulation of Bcl-2. Exogenous CRT pretreatment remarkably attenuated MR-induced cell apoptosis and LDH leakage, ER stress, and activation of the ER stress-related apoptotic signaling. CONCLUSIONS: Exogenous CRT attenuates MR-induced ER stress-related apoptosis by suppressing CHOP-mediated apoptotic signaling pathways in MMECs.


Assuntos
Calbindina 2/metabolismo , Permeabilidade Capilar/efeitos da radiação , Estresse do Retículo Endoplasmático/efeitos da radiação , Células Endoteliais/metabolismo , Microvasos/metabolismo , Micro-Ondas/efeitos adversos , Animais , Apoptose/efeitos da radiação , Células Endoteliais/patologia , Feminino , Proteínas de Choque Térmico/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Microvasos/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos da radiação , Proteína X Associada a bcl-2/metabolismo
19.
Shock ; 40(4): 339-44, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23856922

RESUMO

Panax quinquefolium saponin (PQS) alleviates hypoxia-reoxygenation injury of cardiomyocytes in vitro by inhibiting excessive endoplasmic reticulum stress (ERS)-related apoptosis. We hypothesized that inhibition of excessive ERS-related apoptosis contributes to cardioprotection in ventricular remodeling after acute myocardial infarction (AMI). Sprague-Dawley rats subjected to AMI were randomly treated with water, PQS (50 mg/kg per day, 100 mg/kg per day, or 200 mg/kg per day), or taurine (300 mg/kg per day), an ERS inhibitor, for 4 weeks. Left ventricular (LV) fractional shortening, ejection fraction, and structure were then evaluated using echocardiography. Myocardial infarct size was measured by Evans blue and 2,3,5-triphenyhetrazolium chloride staining. The hydroxyproline level was assayed using the colorimetric method. Cardiomyocyte apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling. In addition, expression of ERS molecules in the noninfarcted myocardium was detected using Western blotting. We found that PQS treatment significantly reduced infarct size and LV dilation and improved LV ejection fraction and fractional shortening in rat hearts. Panax quinquefolium saponin treatment also decreased hydroxyproline level in noninfarcted myocardium. Panax quinquefolium saponin treatment significantly decreased expression of glucose regulating protein 78, calreticulin, C/EBP homologous protein (CHOP), and Bax protein, as well as increased Bcl-2 protein expression in noninfarcted myocardium. Panax quinquefolium saponin treatment (200 mg/kg per day) mimicked the results achieved from the taurine-treated rats. Expression of CHOP positively correlated with the apoptosis index of cardiomyocytes in the noninfarcted myocardium (r = 0.797, P < 0.01). Taken together, PQS treatment significantly improves AMI-induced LV remodeling, and this may be attributed to inhibiting CHOP-mediated ERS-related apoptosis.


Assuntos
Saponinas/uso terapêutico , Animais , Apoptose , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Masculino , Infarto do Miocárdio , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Ratos , Ratos Sprague-Dawley , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/fisiologia
20.
Apoptosis ; 18(1): 85-98, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23108759

RESUMO

Endothelial cells (ECs) are directly exposed to hypoxia and contribute to injury during myocardial ischemia/reperfusion. Hypoxic preconditioning (HPC) protects ECs against hypoxia injury. This study aimed to explore whether HPC attenuates hypoxia/reoxygenation (H/R) injury by suppressing excessive endoplasmic reticulum stress (ERS) in cultured microvascular ECs (MVECs) from rat heart. MVECs injury was measured by lactate dehydrogenase (LDH) leakage, cytoskeleton destruction, and apoptosis. Expression of glucose regulating protein 78 (GRP78) and C/EBP homologous protein (CHOP), activation of caspase-12 (pro-apoptosis factors) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) were detected by western blot analysis. HPC attenuated H/R-induced LDH leakage, cytoskeleton destruction, and cell apoptosis, as shown by flow cytometry, Bax/Bcl-2 ratio, caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. HPC suppressed H/R-induced ERS, as shown by a decrease in expression of GRP78 and CHOP, and caspase-12 activation. HPC enhanced p38 MAPK phosphorylation but decreased that of protein kinase R-like ER kinase (PERK, upstream regulator of CHOP). SB202190 (an inhibitor of p38 MAPK) abolished HPC-induced cytoprotection, downregulation of GRP78 and CHOP, and activation of caspase-12, as well as PERK phosphorylation. HPC may protect MVECs against H/R injury by suppressing CHOP-dependent apoptosis through p38 MAPK mediated downregulation of PERK activation.


Assuntos
Hipóxia Celular , Estresse do Retículo Endoplasmático/fisiologia , Células Endoteliais/fisiologia , Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose , Endotélio Vascular/citologia , Feminino , Proteínas de Choque Térmico/biossíntese , Masculino , Ratos , Ratos Sprague-Dawley , eIF-2 Quinase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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