RESUMO
Fetal growth restriction (FGR) is a common outcome in human suboptimal gestation and is related to prenatal origins of cardiovascular dysfunction in offspring. Despite this, therapy of human translational potential has not been identified. Using human umbilical and placental vessels and the chicken embryo model, we combined cellular, molecular, and functional studies to determine whether N-acetylcysteine (NAC) and hydrogen sulphide (H2S) protect cardiovascular function in growth-restricted unborn offspring. In human umbilical and placental arteries from control or FGR pregnancy and in vessels from near-term chicken embryos incubated under normoxic or hypoxic conditions, we determined the expression of the H2S gene CTH (i.e. cystathionine γ-lyase) (via quantitative PCR), the production of H2S (enzymatic activity), the DNA methylation profile (pyrosequencing) and vasodilator reactivity (wire myography) in the presence and absence of NAC treatment. The data show that FGR and hypoxia increased CTH expression in the embryonic/fetal vasculature in both species. NAC treatment increased aortic CTH expression and H2S production and enhanced third-order femoral artery dilator responses to the H2S donor sodium hydrosulphide in chicken embryos. NAC treatment also restored impaired endothelial relaxation in human third-to-fourth order chorionic arteries from FGR pregnancies and in third-order femoral arteries from hypoxic chicken embryos. This NAC-induced protection against endothelial dysfunction in hypoxic chicken embryos was mediated via nitric oxide independent mechanisms. Both developmental hypoxia and NAC promoted vascular changes in CTH DNA and NOS3 methylation patterns in chicken embryos. Combined, therefore, the data support that the effects of NAC and H2S offer a powerful mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy. KEY POINTS: Gestation complicated by chronic fetal hypoxia and fetal growth restriction (FGR) increases a prenatal origin of cardiovascular disease in offspring, increasing interest in antenatal therapy to prevent against a fetal origin of cardiovascular dysfunction. We investigated the effects between N-acetylcysteine (NAC) and hydrogen sulphide (H2S) in the vasculature in FGR human pregnancy and in chronically hypoxic chicken embryos. Combining cellular, molecular, epigenetic and functional studies, we show that the vascular expression and synthesis of H2S is enhanced in hypoxic and FGR unborn offspring in both species and this acts to protect their vasculature. Therefore, the NAC/H2S pathway offers a powerful therapeutic mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy.
Assuntos
Acetilcisteína , Epigênese Genética , Retardo do Crescimento Fetal , Sulfeto de Hidrogênio , Hipóxia , Animais , Sulfeto de Hidrogênio/metabolismo , Acetilcisteína/farmacologia , Embrião de Galinha , Humanos , Feminino , Gravidez , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/fisiopatologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Metilação de DNA , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Vasodilatação/efeitos dos fármacos , Placenta/metabolismo , Placenta/irrigação sanguínea , Artérias Umbilicais/metabolismoRESUMO
During estrus, the poll glands of male Bactrian Camels (Camelus Bactrianus) become slightly raised, exuding a large amount of pale yellow watery secretion with a characteristic odor that may contain hydrogen sulfide (H2S). However, whether H2S can be synthesized in the poll glands of male Bactrian Camels and its role in inducing camel estrus remains unclear. This study aimed to identify differentially expressed proteins (DEPs) and signaling pathways in the poll gland tissues of male Bactrian Camels using data independent acquisition (DIA) proteomics. Additionally, gas chromatography-mass spectrometry (GC-MS) was performed to identify differentially expressed metabolites (DEMs) in the neck hair containing secretions during estrus in male Bactrian Camels, to explore the specific expression patterns and mechanisms in the poll glands of camels during estrus. The results showed that cystathionine-γ-lyase (CTH) and cystathionine-ß-synthase (CBS), which are closely related to H2S synthesis in camel poll glands during estrus, were mainly enriched in glycine, serine, and threonine metabolism, amino acid biosynthesis, and metabolic pathways. In addition, both enzymes were widely distributed and highly expressed in the acinar cells of poll gland tissues in camels during estrus. Meanwhile, the neck hair secretion contains high levels of amino acids, especially glycine, serine, threonine, and cystathionine, which are precursors for H2S biosynthesis. These results demonstrate that the poll glands of male Bactrian Camels can synthesize and secrete H2S during estrus. This study provides a basis for exploring the function and mechanism of H2S in the estrus of Bactrian Camels.
Assuntos
Camelus , Sulfeto de Hidrogênio , Proteômica , Animais , Sulfeto de Hidrogênio/metabolismo , Camelus/metabolismo , Masculino , Proteômica/métodos , Cistationina beta-Sintase/metabolismo , Metabolômica/métodos , Cistationina gama-Liase/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Estro/metabolismo , FemininoRESUMO
Endogenous hydrogen sulfide (H2S) plays an important role in bone metabolism. However, the exact role of H2S in intestinal calcium and phosphorus absorption and its potential in preventing and treating primary osteoporosis remains unknown. Therefore, this study aimed to investigate the potential of H2S in promoting intestinal calcium and phosphorus absorption and alleviating primary osteoporosis. We measured the apparent absorptivity of calcium, femoral bone density, expression and sulfhydration of the duodenal endoplasmic reticulum protein of 57 kDa (ERp57), duodenal cystathionine γ-lyase (CSE) expression, and serum H2S content in adult and old CSE-knockout and wild-type mice. We also assessed intracellular reactive oxygen species (ROS) and Ca2+ content in CSE-overexpressing or knockout intestinal epithelial cell (IEC)-6 cells. In senile mice, CSE knockout decreased endogenous H2S, ERp57 sulfhydration, and intestinal calcium absorption and worsened osteoporosis, which were partially reversed by GYY4137, an H2S donor. CSE overexpression in IEC-6 cells increased ERp57 sulfhydration, protein kinase A and C activity, and intracellular Ca2+, whereas CSE knockout exerted the opposite effects. Furthermore, hydrogen peroxide (H2O2) stimulation had similar effects as in CSE knockout, which were reversed by pretreatment with sodium hydrosulfide before H2O2 stimulation and restored by DL-dithiothreitol. These findings suggest that H2S attenuates primary osteoporosis by preventing ROS-induced ERp57 damage in intestinal epithelial cells by enhancing ERp57 activity and promoting intestinal calcium absorption, thereby aiding in developing therapeutic interventions to prevent osteoporosis.
Assuntos
Cálcio , Sulfeto de Hidrogênio , Osteoporose , Isomerases de Dissulfetos de Proteínas , Animais , Masculino , Camundongos , Cálcio/metabolismo , Linhagem Celular , Cistationina gama-Liase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Absorção Intestinal/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoporose/metabolismo , Osteoporose/prevenção & controle , Isomerases de Dissulfetos de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
In chronic liver injury, quiescent hepatic stellate cells (HSCs) transdifferentiate into activated myofibroblast-like cells and produce large amounts of extracellular matrix components, e.g. collagen type 1. Cellular senescence is characterized by irreversible cell-cycle arrest, arrested cell proliferation and the acquisition of the senescence-associated secretory phenotype (SASP) and reversal of HSCs activation. Previous studies reported that H2S prevents induction of senescence via its antioxidant activity. We hypothesized that inhibition of endogenous H2S production induces cellular senescence and reduces activation of HSCs. Rat HSCs were isolated and culture-activated for 7 days. After activation, HSCs treated with H2S slow-releasing donor GYY4137 and/or DL-propargylglycine (DL-PAG), an inhibitor of the H2S-producing enzyme cystathionine γ-lyase (CTH), as well as the PI3K inhibitor LY294002. In our result, CTH expression was significantly increased in fully activated HSCs compared to quiescent HSCs and was also observed in activated stellate cells in a in vivo model of cirrhosis. Inhibition of CTH reduced proliferation and expression of fibrotic markers Col1a1 and Acta2 in HSCs. Concomitantly, DL-PAG increased the cell-cycle arrest markers Cdkn1a (p21), p53 and the SASP marker Il6. Additionally, the number of ß-galactosidase positive senescent HSCs was increased. GYY4137 partially restored the proliferation of senescent HSCs and attenuated the DL-PAG-induced senescent phenotype. Inhibition of PI3K partially reversed the senescence phenotype of HSCs induced by DL-PAG. Inhibition of endogenous H2S production reduces HSCs activation via induction of cellular senescence in a PI3K-Akt dependent manner. Our results show that cell-specific inhibition of H2S could be a novel target for anti-fibrotic therapy via induced cell senescence.
Assuntos
Alcinos , Senescência Celular , Glicina , Células Estreladas do Fígado , Sulfeto de Hidrogênio , Morfolinas , Compostos Organotiofosforados , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/metabolismo , Animais , Senescência Celular/efeitos dos fármacos , Morfolinas/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Alcinos/farmacologia , Compostos Organotiofosforados/farmacologia , Ratos , Masculino , Cistationina gama-Liase/metabolismo , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Colágeno Tipo I/metabolismo , Ratos Sprague-Dawley , Fosfatidilinositol 3-Quinases/metabolismo , Células Cultivadas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fenótipo Secretor Associado à Senescência , Proteína Supressora de Tumor p53/metabolismoRESUMO
Chinese hamster ovary (CHO) cells require cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of cysteine at high concentrations is a challenge due to its limited solubility and instability in solution. Methionine can be converted to cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer cysteine prototrophy, i.e., the ability to grow in a cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (ßME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and ßME levels, were able to proliferate in the HCY-free, ßME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn't completely adapt to ßME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, 'omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.
Assuntos
Cricetulus , Cisteína , Engenharia Metabólica , Células CHO , Animais , Cisteína/metabolismo , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cricetinae , Homocisteína/metabolismo , Homocisteína/genéticaRESUMO
BACKGROUND: Acute kidney injury (AKI) causes distant liver injury, to date, which causes poor outcomes of patients with AKI. Many studies have been performed to overcome AKI-associated liver injury. However, those studies have mainly focused on hepatocytes, and AKI-induced liver injury still remains a clinical problem. Here, we investigated the implication of cholangiocytes and their primary cilia which are critical in final bile secretion. Cholangiocyte, a lining cell of bile ducts, are the only liver epithelial cell containing primary cilium (a microtubule-based cell surface signal-sensing organelle). METHODS: Cystathione γ-lyase (CSE, a transsulfuration enzyme) deficient and wild-type mice were subjected to kidney ischemia followed by reperfusion (KIR). Some mice were administered with N-acetyl-cysteine (NAC). RESULTS: KIR damaged hepatocytes and cholagiocytes, disrupted cholangiocytes primary cilia, released the disrupted ciliary fragments into the bile, and caused abnormal bile secretion. Glutathione (GSH) and H2S levels in the livers were significantly reduced by KIR, resulting in increased the ratio oxidized GSH to total GSH, and oxidation of tissue and bile. CSE and cystathione ß-synthase (CBS) expression were lowered in the liver after KIR. NAC administration increased total GSH and H2S levels in the liver and attenuated KIR-induced liver injuries. In contrast, Cse deletion caused the reduction of total GSH levels and worsened KIR-induced liver injuries, including primary cilia damage and abnormal bile secretion. CONCLUSIONS: These results indicate that KIR causes cholangiocyte damage, cholangiocytes primary cilia disruption, and abnormal bile secretion through reduced antioxidative ability of the liver.
Assuntos
Bile , Cílios , Traumatismo por Reperfusão , Animais , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Cílios/metabolismo , Cílios/patologia , Camundongos , Bile/metabolismo , Masculino , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Camundongos Endogâmicos C57BL , Glutationa/metabolismo , Camundongos Knockout , Fígado/patologia , Fígado/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/genética , Rim/metabolismo , Rim/patologia , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Ductos Biliares/patologia , Ductos Biliares/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologiaRESUMO
Antibiotic resistance is one of the most serious global health threats. Therefore, there is a need to develop antimicrobial agents with new mechanisms of action. Targeting of bacterial cystathionine γ-lyase (bCSE), an enzyme essential for bacterial survival, is a promising approach to overcome antibiotic resistance. Here, we described a series of (heteroarylmethyl)benzoic acid derivatives and evaluated their ability to inhibit bCSE or its human ortholog hCSE using known bCSE inhibitor NL2 as a lead compound. Derivatives bearing the 6-bromoindole group proved to be the most active, with IC50 values in the midmicromolar range, and highly selective for bCSE over hCSE. Furthermore, none of these compounds showed significant toxicity to HEK293T cells. The obtained data were rationalized by ligand-based and structure-based molecular modeling analyses. The most active compounds were also found to be an effective adjunct to several widely used antibacterial agents against clinically relevant antibiotic-resistant strains of such bacteria as Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The most potent compounds, 3h and 3i, also showed a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. Finally, compound 3i manifested potentiating activity in pneumonia, sepsis, and infected-wound in vivo models.
Assuntos
Antibacterianos , Cistationina gama-Liase , Inibidores Enzimáticos , Humanos , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Animais , Testes de Sensibilidade Microbiana , Modelos Moleculares , Células HEK293 , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Benzoatos/farmacologia , Benzoatos/química , Benzoatos/síntese química , Camundongos , Staphylococcus aureus/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
MAIN CONCLUSION: PLDα1 promoted H2S production by positively regulating the expression of LCD. Stomatal closure promoted by PLDα1 required the accumulation of H2S under drought stress. Phospholipase Dα1 (PLDα1) acting as one of the signal enzymes can respond to drought stress. It is well known that hydrogen sulfide (H2S) plays an important role in plant responding to biotic or abiotic stress. In this study, the functions and relationship between PLDα1 and H2S in drought stress resistance in Arabidopsis were explored. Our results indicated that drought stress promotes PLDα1 and H2S production by inducing the expression of PLDα1 and LCD genes. PLDα1 and LCD enhanced plant tolerance to drought by regulating membrane lipid peroxidation, proline accumulation, H2O2 content and stomatal closure. Under drought stress, the H2O2 content of PLDα1-deficient mutant (pldα1), L-cysteine desulfhydrase (LCD)-deficient mutant (lcd) was higher than that of ecotype (WT), the stomatal aperture of pldα1 and lcd was larger than that of WT. The transcriptional and translational levels of LCD were lower in pldα1 than that in WT. Exogenous application of the H2S donor NaHS or GYY reduced the stomatal aperture of WT, pldα1, PLDα1-CO, and PLDα1-OE lines, while exogenous application of the H2S scavenger hypotaurine (HT) increased the stomatal aperture. qRT-PCR analysis of stomatal movement-related genes showed that the expression of CAX1, ABCG5, SCAB1, and SLAC1 genes in pldα1 and lcd were down-regulated, while ACA1 and OST1 gene expression was significantly up-regulated. Thus, PLDα1 and LCD are required for stomatal closure to improve drought stress tolerance.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Secas , Regulação da Expressão Gênica de Plantas , Sulfeto de Hidrogênio , Fosfolipase D , Estômatos de Plantas , Arabidopsis/genética , Arabidopsis/fisiologia , Estômatos de Plantas/fisiologia , Estômatos de Plantas/genética , Fosfolipase D/metabolismo , Fosfolipase D/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sulfeto de Hidrogênio/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Fisiológico/genética , Prolina/metabolismo , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Peroxidação de LipídeosRESUMO
OBJECTIVES: The objective of this study was to investigate whether skeletal muscle cystathionine γ-lyase (CTH) contributes to high-fat diet (HFD)-induced metabolic disorders using skeletal muscle Cth knockout (CthΔskm) mice. METHODS: The CthΔskm mice and littermate Cth-floxed (Cthf/f) mice were fed with either HFD or chow diet for 13 weeks. Metabolomics and transcriptome analysis were used to assess the impact of CTH deficiency in skeletal muscle. RESULTS: Metabolomics coupled with transcriptome showed that CthΔskm mice displayed impaired energy metabolism and some signaling pathways linked to insulin resistance (IR) in skeletal muscle although the mice had normal insulin sensitivity. HFD led to reduced CTH expression and impaired energy metabolism in skeletal muscle in Cthf/f mice. CTH deficiency and HFD had some common pathways enriched in the aspects of amino acid metabolism, carbon metabolism, and fatty acid metabolism. CthΔskm+HFD mice exhibited increased body weight gain, fasting blood glucose, plasma insulin, and IR, and reduced glucose transporter 4 and CD36 expression in skeletal muscle compared to Cthf/f+HFD mice. Impaired mitochondria and irregular arrangement in myofilament occurred in CthΔskm+HFD mice. Omics analysis showed differential pathways enriched between CthΔskm mice and Cthf/f mice upon HFD. More severity in impaired energy metabolism, reduced AMPK signaling, and increased oxidative stress and ferroptosis occurred in CthΔskm+HFD mice compared to Cthf/f+HFD mice. DISCUSSION: Our results indicate that skeletal muscle CTH expression dysregulation contributes to metabolism disorders upon HFD.
Assuntos
Cistationina gama-Liase , Dieta Hiperlipídica , Hiperglicemia , Resistência à Insulina , Músculo Esquelético , Obesidade , Animais , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Camundongos , Obesidade/metabolismo , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/genética , Cistationina gama-Liase/deficiência , Dieta Hiperlipídica/efeitos adversos , Hiperglicemia/metabolismo , Camundongos Knockout , Masculino , Metabolismo EnergéticoRESUMO
The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical L-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.
Assuntos
Domínio Catalítico , Cistationina gama-Liase , Sulfeto de Hidrogênio , Pseudomonas aeruginosa , Pseudomonas aeruginosa/enzimologia , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/química , Cristalografia por Raios X , Especificidade por Substrato , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/química , Modelos Moleculares , Cisteína/metabolismo , Cisteína/química , Conformação Proteica , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Homocisteína/metabolismo , Homocisteína/química , CatáliseRESUMO
BACKGROUND: Intracerebral hemorrhage (ICH) comprises primary and secondary injuries, the latter of which induces increased inflammation and apoptosis and is more severe. Activating transcription factor 6 (ATF6) is a type-II transmembrane protein in the endoplasmic reticulum (ER). ATF6 target genes could improve ER homeostasis, which contributes to cryoprotection. Hence, we predict that ATF6 will have a protective effect on brain tissue after ICH. METHOD: The ICH rat model was generated through autologous blood injection into the right basal ganglia, the expression of ATF6 after ICH was determined by WB and IF. The expression of ATF6 was effectively controlled by means of intervention, and a series of measures was used to detect cell death, neuroinflammation, brain edema, blood-brain barrier and other indicators after ICH. Finally, the effects on long-term neural function of rats were measured by behavioral means. RESULT: ATF6 was significantly increased in the ICH-induced brain tissues. Further, ATF6 was found to modulate the expression of cystathionine γ-lyase (CTH) after ICH. Upregulation of ATF6 attenuated neuronal apoptosis and inflammation in ICH rats, along with mitigation of ICH-induced brain edema, blood-brain barrier deterioration, and cognitive behavior defects. Conversely, ATF6 genetic knockdown induced effects counter to those aforementioned. CONCLUSIONS: This study thereby emphasizes the crucial role of ATF6 in secondary brain injury in response to ICH, indicating that ATF6 upregulation may potentially ameliorate ICH-induced secondary brain injury. Consequently, ATF6 could serve as a promising therapeutic target to alleviate clinical ICH-induced secondary brain injuries.
Assuntos
Fator 6 Ativador da Transcrição , Hemorragia Cerebral , Cistationina gama-Liase , Animais , Masculino , Ratos , Fator 6 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Apoptose , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/metabolismo , Lesões Encefálicas/metabolismo , Hemorragia Cerebral/metabolismo , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/genética , Modelos Animais de Doenças , Ratos Sprague-DawleyRESUMO
Diabetic cardiomyopathy (DCM) contributes significantly to the heightened mortality rate observed among diabetic patients, with myocardial fibrosis (MF) being a pivotal element in the disease's progression. Hydrogen sulfide (H2S) has been shown to mitigate MF, but the specific underlying mechanisms have yet to be thoroughly understood. A connection has been established between the evolution of DCM and the incidence of cardiomyocyte pyroptosis. Our research offers insights into H2S protective impact and its probable mode of action against DCM, analyzed through the lens of MF. In this study, a diabetic rat model was developed using intraperitoneal injections of streptozotocin (STZ), and hyperglycemia-stimulated cardiomyocytes were employed to replicate the cellular environment of DCM. There was a marked decline in the expression of cystathionine γ-lyase (CSE), a catalyst for H2S synthesis, in both the STZ-induced diabetic rats and hyperglycemia-stimulated cardiomyocytes. Experimental results in vivo indicated that H2S ameliorates MF and enhances cardiac functionality in diabetic rats by mitigating cardiomyocyte pyroptosis. In vitro assessments highlighted the induction of cardiomyocyte pyroptosis and the subsequent decline in cell viability under hyperglycemic conditions. However, the administration of sodium hydrosulfide (NaHS) curtailed cardiomyocyte pyroptosis and augmented cell viability. In contrast, propargylglycine (PAG), a CSE inhibitor, reversed the effects rendered by NaHS administration. Additional exploration indicated that the mitigating effect of H2S on cardiomyocyte pyroptosis is modulated through the ROS/NLRP3 pathway. In essence, our findings corroborate the potential of H2S in alleviating MF in diabetic subjects. This therapeutic effect is likely attributable to the regulation of cardiomyocyte pyroptosis via the ROS/NLRP3 pathway. This discovery furnishes a prospective therapeutic target for the amelioration and management of MF associated with diabetes.
Assuntos
Diabetes Mellitus Experimental , Cardiomiopatias Diabéticas , Fibrose , Sulfeto de Hidrogênio , Miócitos Cardíacos , Piroptose , Ratos Sprague-Dawley , Animais , Piroptose/efeitos dos fármacos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/tratamento farmacológico , Cardiomiopatias Diabéticas/prevenção & controle , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Masculino , Ratos , Cistationina gama-Liase/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estreptozocina , Miocárdio/patologia , Miocárdio/metabolismo , Glicina/farmacologia , Glicina/análogos & derivados , Sobrevivência Celular/efeitos dos fármacosRESUMO
Hydrogen sulfide (H2S), recognized as the third gasotransmitter, plays a pivotal role in the pathophysiological processes of various diseases. Cystathionine γ-lyase (CSE) is the main enzyme for H2S production in the skin. However, effects and mechanisms of H2S in diabetic skin wound healing remain unclear. Our findings revealed a decrease in plasma H2S content in diabetic patients with skin wounds. CSE knockout (KO) diabetic mice resulted in delayed wound healing, reduced blood perfusion, and CD31 expression around the wounds. It also led to increased infiltration of inflammatory cells and M1-type macrophages, decreased collagen levels, α-smooth muscle actin (α-SMA), and proliferating cell nuclear antigen (PCNA) expression. Additionally, there were enhanced expressions of necroptosis related proteins, including receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain like protein (MLKL). In comparison, sodium hydrosulfide (NaHS), H2S donor, accelerated skin wound healing in leptin receptor deficiency (db/db) mice. This acceleration was accompanied by increased blood perfusion and CD31 expression, reduced infiltration of inflammatory cells and M1-type macrophages, elevated collagen levels, α-SMA, and PCNA expressions, and decreased necroptosis-related protein expressions together with nuclear factor-κB (NF-κB) p65 phosphorylation. In conclusion, H2S regulates macrophage polarization and necroptosis, contributing to the acceleration of diabetic skin wound healing. These findings offer a novel strategy for the treatment of diabetic skin wounds.
Assuntos
Cistationina gama-Liase , Diabetes Mellitus Experimental , Sulfeto de Hidrogênio , Macrófagos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necroptose , Pele , Sulfetos , Cicatrização , Animais , Sulfeto de Hidrogênio/metabolismo , Cicatrização/efeitos dos fármacos , Pele/patologia , Pele/metabolismo , Pele/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Diabetes Mellitus Experimental/metabolismo , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/genética , Masculino , Camundongos , Humanos , Necroptose/efeitos dos fármacos , Receptores para Leptina/genética , Receptores para Leptina/metabolismoRESUMO
Acute inflammatory responses often involve the production of reactive oxygen and nitrogen species by innate immune cells, particularly macrophages. How activated macrophages protect themselves in the face of oxidative-inflammatory stress remains a long-standing question. Recent evidence implicates reactive sulfur species (RSS) in inflammatory responses; however, how endogenous RSS affect macrophage function and response to oxidative and inflammatory insults remains poorly understood. In this study, we investigated the endogenous pathways of RSS biogenesis and clearance in macrophages, with a particular focus on exploring how hydrogen sulfide (H2S)-mediated S-persulfidation influences macrophage responses to oxidative-inflammatory stress. We show that classical activation of mouse or human macrophages using lipopolysaccharide and interferon-γ (LPS/IFN-γ) triggers substantial production of H2S/RSS, leading to widespread protein persulfidation. Biochemical and proteomic analyses revealed that this surge in cellular S-persulfidation engaged â¼2% of total thiols and modified over 800 functionally diverse proteins. S-persulfidation was found to be largely dependent on the cystine importer xCT and the H2S-generating enzyme cystathionine γ-lyase and was independent of changes in the global proteome. We further investigated the role of the sulfide-oxidizing enzyme sulfide quinone oxidoreductase (SQOR), and found that it acts as a negative regulator of S-persulfidation. Elevated S-persulfidation following LPS/IFN-γ stimulation or SQOR inhibition was associated with increased resistance to oxidative stress. Upregulation of persulfides also inhibited the activation of the macrophage NLRP3 inflammasome and provided protection against inflammatory cell death. Collectively, our findings shed light on the metabolism and effects of RSS in macrophages and highlight the crucial role of persulfides in enabling macrophages to withstand and alleviate oxidative-inflammatory stress.
Assuntos
Sulfeto de Hidrogênio , Ativação de Macrófagos , Macrófagos , Estresse Oxidativo , Estresse Oxidativo/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Animais , Camundongos , Humanos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Lipopolissacarídeos , Inflamação/metabolismo , Cistationina gama-Liase/metabolismo , Sulfetos/farmacologia , Interferon gama/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oxirredução , Proteômica/métodosRESUMO
The engineered human cystathionine-γ-lyase (hCGL) resulting in enhanced activity toward both cysteine and cystine unveils a potential robust antitumor activity. However, the presence of cysteine residues has the potential to induce oligomerization or incorrect disulfide bonding, which may decrease the bioavailability of biopharmaceuticals. Through a meticulous design process targeting the cysteine residues within engineered hCGL, a set of potential beneficial mutants were obtained by virtual screening employing Rosetta and ABACUS. Experimental measurements have revealed that most of the mutants showed increased activity toward both substrates l-Cys and CSSC. Furthermore, mutants C109V and C229D demonstrated Tm value increases of 8.2 and 1.8 °C, respectively. After an 80 min incubation at 60 °C, mutant C229D still maintained high residual activity. Unexpectedly, mutant C109V, displaying activity approximately 2-fold higher than the activity of wild type (WT) for both substrates, showed disappointing instability in plasma, which suggests that computational design still requires further consideration. Analysis of their structure and molecular dynamics (MD) simulation revealed the impact of hydrophobic interaction, hydrogen bonds, and near-attack conformation (NAC) stability on activity and stability. This study acquired information about mutants that exhibit enhanced activity or thermal resistance and serve as valuable guidance for subsequent specific cysteine modifications.
Assuntos
Cistationina gama-Liase , Cisteína , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Cisteína/química , Cisteína/metabolismo , Humanos , Cistationina gama-Liase/genética , Cistationina gama-Liase/química , Cistationina gama-Liase/metabolismo , Estabilidade Enzimática , Cistina/química , Ligação de Hidrogênio , Mutação , CinéticaRESUMO
Cardiac hypertrophy is the most prevalent compensatory heart disease that ultimately leads to spontaneous heart failure. Mounting evidence suggests that microRNAs (miRs) and endogenous hydrogen sulfide (H2S) play a crucial role in the regulation of cardiac hypertrophy. In this study, we aimed to investigate whether inhibition of miR-27a could protect against cardiac hypertrophy by modulating H2S signaling. We established a model of cardiac hypertrophy by obtaining hypertrophic tissue from mice subjected to transverse aortic constriction (TAC) and from cells treated with angiotensin-II. Molecular alterations in the myocardium were quantified using quantitative real time PCR (qRT-PCR), Western blotting, and ELISA. Morphological changes were characterized by hematoxylin and eosin (HE) staining and Masson's trichrome staining. Functional myocardial changes were assessed using echocardiography. Our results demonstrated that miR-27a levels were elevated, while H2S levels were reduced in TAC mice and myocardial hypertrophy. Further luciferase and target scan assays confirmed that cystathionine-γ-lyase (CSE) was a direct target of miR-27a and was negatively regulated by it. Notably, enhancement of H2S expression in the heart was observed in mice injected with recombinant adeno-associated virus vector 9 (rAAV9)-anti-miR-27a and in cells transfected with a miR-27a inhibitor during cardiac hypertrophy. However, this effect was abolished by co-transfection with CSE siRNA and the miR-27a inhibitor. Conversely, injecting rAAV9-miR-27a yielded opposite results. Interestingly, our findings demonstrated that glucagon-like peptide-1 (GLP-1) agonists could mitigate myocardial damage by down-regulating miR-27a and up-regulating CSE. In summary, our study suggests that inhibition of miR-27a holds therapeutic promise for the treatment of cardiac hypertrophy by increasing H2S levels. Furthermore, our findings unveil a novel mechanism of GLP-1 agonists involving the miR-27a/H2S pathway in the management of cardiac hypertrophy.
Assuntos
Estenose da Valva Aórtica , Insuficiência Cardíaca , MicroRNAs , Animais , Camundongos , Cardiomegalia/genética , Peptídeo 1 Semelhante ao Glucagon , MicroRNAs/genética , Cistationina gama-LiaseRESUMO
Gastric cancer (GC), known for its high incidence and poor prognosis, urgently necessitates the identification of reliable prognostic biomarkers to enhance patient outcomes. We scrutinized data from 375 GC patients alongside 32 non-cancer controls, sourced from the TCGA database. A univariate Cox Proportional Hazards Model (COX) regression was employed to evaluate expressions of ferroptosis-related genes. This was followed by the application of Least Absolute Shrinkage and Selection Operator (LASSO) and multivariate COX regression for the development of prognostic models. The composition of immune cell subtypes was quantified utilizing CIBERSORT, with their distribution in GC versus control samples being comparatively analyzed. Furthermore, the correlation between the expressions of Cystathionine Gamma-Lyase (CTH) and Microtubule Associated Protein 1 Light Chain 3 Beta (MAP1LC3B) and the abundance of immune cell subtypes was explored. Our bioinformatics findings underwent validation through immunohistochemical analysis. Our prognostic models integrated CTH and MAP1LC3B. Survival analysis indicated that patients categorized as high-risk, as defined by the model, exhibited significantly lower survival rates compared to their low-risk counterparts. Notably, CTH expression inversely correlated with monocyte levels, while MAP1LC3B expression showed an inverse relationship with the abundance of M2 macrophages. Immunohistochemical validation corroborated lower expressions of CTH and MAP1LC3B in GC tissues relative to control samples, in concordance with our bioinformatics predictions. Our study suggests that the dysregulation of CTH, MAP1LC3B, and the accompanying monocyte-macrophage dynamics could be pivotal in the prognosis of GC. These elements present potential targets for prognostic assessment and therapeutic intervention.
Assuntos
Ferroptose , Neoplasias Gástricas , Humanos , Biomarcadores , Cistationina gama-Liase/metabolismo , Proteínas Associadas aos Microtúbulos , Prognóstico , Neoplasias Gástricas/genéticaRESUMO
A major challenge in treating patients with glioblastoma is the inability to eliminate highly invasive cells with chemotherapy, radiation, or surgical resection. As cancer cells face the issue of replicating or invading neighboring tissue, they rewire their metabolism in a concerted effort to support necessary cellular processes and account for altered nutrient abundance. In this issue of the JCI, Garcia et al. compared an innovative 3D hydrogel-based invasion device to regional patient biopsies through a comprehensive multiomics-based approach paired with a CRISPR knockout screen. Their findings elucidate a role for cystathionine γ-lyase (CTH), an enzyme in the transsulfuration pathway, as a means of regulating the cellular response to oxidative stress. CTH-mediated conversion of cystathionine to cysteine was necessary for regulating reactive oxygen species to support invasion. Meanwhile, inhibition of CTH suppressed the invasive glioblastoma phenotype. However, inhibiting CTH resulted in a larger overall tumor mass. These findings suggest that targeting the transsulfuration pathway may serve as a means of redirecting glioblastoma to proliferate or invade.
Assuntos
Glioblastoma , Humanos , Glioblastoma/genética , Glioblastoma/patologia , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cistationina/metabolismo , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
The post-translational modification of cysteine through redox reactions, especially S-sulfhydration, plays a critical role in regulating protein activity, interactions, and spatial arrangement. This review focuses on the impact of protein S-sulfhydration on vascular function and its implications in vascular diseases. Dysregulated S-sulfhydration has been linked to the development of vascular pathologies, including aortic aneurysms and dissections, atherosclerosis, and thrombotic diseases. The H2S signaling pathway and the enzyme cystathionine γ-lyase (CSE), which is responsible for H2S generation, are identified as key regulators of vascular function. Additionally, potential therapeutic targets for the treatment of vascular diseases, such as the H2S donor GYY4137 and the HDAC inhibitor entinostat, are discussed. The review also emphasizes the antithrombotic effects of H2S in regulating platelet aggregation and thrombosis. The aim of this review is to enhance our understanding of the function and mechanism of protein S-sulfhydration modification in vascular diseases, and to provide new insights into the clinical application of this modification.
Assuntos
Aterosclerose , Sulfeto de Hidrogênio , Humanos , Sulfeto de Hidrogênio/metabolismo , Aterosclerose/tratamento farmacológico , Processamento de Proteína Pós-Traducional , Cistationina gama-Liase/metabolismoRESUMO
Environmental electrophiles modify thiol groups of proteins in organs, disrupting cellular functions carried out by the modified proteins and increasing the risk of various diseases. The transcription factor NF-E2-related factor 2 (Nrf2) plays a crucial role in detoxifying electrophiles by forming glutathione adducts and subsequently excreting them into extracellular spaces. Supersulfides such as cysteine persulfides (CysSSH) produced by cystathionine γ-lyase (CSE) capture environmental electrophiles through sulfur adduct formation. However, the Nrf2 and CSE contributions to blocking environmental electrophile-mediated toxicity have yet to be evaluated. Therefore, we assessed the individual and combined roles of Nrf2 and CSE in suppressing toxicity induced by environmental electrophiles using Nrf2 knockout (KO), CSE KO, and Nrf2/CSE double KO (DKO) mice. Our findings indicate that CSE/Nrf2 DKO mice are more sensitive to environmental electrophiles compared to their single KO counterparts, highlighting the distinct mechanisms through which both pathways mitigate the toxic effects of reactive electrophiles. Moreover, diverse metabolites produced by symbiotic gut bacteria in the human body are known to exert various effects on host organ functions beyond the intestinal tract. We observed reduced blood supersulfide levels in mice lacking gut microflora compared to normal mice. Furthermore, we identified intestinal bacteria belonging to the families Ruminococcaceae and Lachnospiraceae as high CysSSH-producing bacteria. This suggests that the gut microbiota serves as a source of in vivo supersulfide molecules. These findings suggest that supersulfide derived from gut bacteria may act protectively against environmental electrophilic exposure in the host.