Skeletal muscle cystathionine γ-lyase deficiency promotes obesity and insulin resistance and results in hyperglycemia and skeletal muscle injury upon HFD in mice.

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.

Mitochondrial ROS Accumulation Contributes to Maternal Hypertension and Impaired Remodeling of Spiral Artery but Not IUGR in a Rat PE Model Caused by Maternal Glucocorticoid Exposure.

Increased maternal glucocorticoid levels have been implicated as a risk factor for preeclampsia (PE) development. We found that pregnant rats exposed to dexamethasone (DEX) showed hallmarks of PE features, impaired spiral artery (SA) remodeling, and elevated circulatory levels of sFlt1, sEng IL-1ß, and TNFα. Abnormal mitochondrial morphology and mitochondrial dysfunction in placentas occurred in DEX rats. Omics showed that a large spectrum of placental signaling pathways, including oxidative phosphorylation (OXPHOS), energy metabolism, inflammation, and insulin-like growth factor (IGF) system were affected in DEX rats. MitoTEMPO, a mitochondria-targeted antioxidant, alleviated maternal hypertension and renal damage, and improved SA remodeling, uteroplacental blood flow, and the placental vasculature network. It reversed several pathways, including OXPHOS and glutathione pathways. Moreover, DEX-induced impaired functions of human extravillous trophoblasts were associated with excess ROS caused by mitochondrial dysfunction. However, scavenging excess ROS did not improve intrauterine growth retardation (IUGR), and elevated circulatory sFlt1, sEng, IL-1ß, and TNFα levels in DEX rats. Our data indicate that excess mitochondrial ROS contributes to trophoblast dysfunction, impaired SA remodeling, reduced uteroplacental blood flow, and maternal hypertension in the DEX-induced PE model, while increased sFlt1 and sEng levels and IUGR might be associated with inflammation and an impaired energy metabolism and IGF system.

Reduced hydrogen sulfide production contributes to adrenal insufficiency induced by hypoxia via modulation of NLRP3 inflammasome activation.

Objective: Adrenocortical responsiveness is critical for maintaining glucocorticoids production and homeostasis during stress. We sought to investigate adrenocortical responsiveness during hypoxia in mice and the mechanisms responsible for the regulation of adrenal responsiveness.Methods: (1) Adult male WT mice were randomly divided into four groups: normoxia, hypoxia (24h), hypoxia (72h), hypoxia (72h) + GYY4137(hydrogen sulfide (H2S) donor, 133mmol/kg/day); (2) WT mice were randomly divided into four groups: sham, adrenalectomy (ADX), sham+hypoxia, ADX+hypoxia; (3) Cse-/- mice were randomly divided into two groups: Cse-/-, Cse-/- +GYY4137.Results: The circulatory level of corticosteroid induced by ACTH stimulation was significantly reduced in the mice with hypoxia compared with control mice. The mortality rate induced by lipopolysaccharide (LPS) increased during hypoxia. Cystathionine-γ-lyase (CSE) expression was significantly reduced in adrenal glands during hypoxia. GYY4137 treatment significantly increased adrenal responsiveness and attenuated NLRP3 inflammasome activation in mice treated by hypoxia and Cse-/- mice. Furthermore, The sulfhydrated level of PSMA7 in adrenal gland was decreased in the mice with hypoxia and Cse-/- mice. PSMA7 was S-sulfhydrated at cysteine 70. Blockage of S-sulfhydration of PSMA7 increased NLRP3 expression in adrenocortical cells.Conclusion: Reduced H2S production mediated hypo-adrenocortical responsiveness and NLRP3 inflammasome activation via PAMA7 S-sulfhydration during hypoxia.

Term and Preterm Birth Initiation Is Associated with the Macrophages Shifting to M1 Polarization in Gestational Tissues in Mice.

Inflammation in gestational tissues plays critical role in parturition initiation. We sought to investigate the leukocyte infiltration and cytokine profile in uterine tissues to understand the inflammation during term and preterm labor in the mouse model. Preterm birth was induced by the administration of lipopolysaccharide (LPS) or RU38486. The populations of leukocytes were determined by flow cytometry. Macrophages were the largest population in the myometrium and decidua in late gestation. The macrophage population was significantly changed in the myometrium and decidua from late pregnancy to term labor and significantly changed at LPS- and RU386-induced preterm labor. Neutrophils, T cells, and NKT cells were increased in LPS- and RU38486-induced preterm labor. The above changes were accompanied by the increased expression of cytokines and chemokines. In late gestation, M2 macrophages were the predominant phenotype in gestational tissues. M1 macrophages significantly increased in these tissues at term and preterm labor. IL-6 and NLRP3 expression was significantly increased in macrophages at labor, supporting that macrophages exhibit proinflammatory phenotypes. NLRP3 inflammasome inhibitor MCC950 mainly suppressed macrophage infiltration in the myometrium at term labor and preterm labor. Our data suggest that the M1 polarization of macrophages contributes to inflammation linked to term and preterm labor initiation in gestational tissues.

Mitochondria Targeted Antioxidant Significantly Alleviates Preeclampsia Caused by 11ß-HSD2 Dysfunction via OPA1 and MtDNA Maintenance.

We have previously demonstrated that placental 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) dysfunction contributes to PE pathogenesis. We sought to elucidate molecular mechanisms underlying 11ß-HSD2 dysfunction-induced PE and to seek potential therapeutic targets using a 11ß-HSD2 dysfunction-induced PE-like rat model as well as cultured extravillous trophoblasts (EVTs) since PE begins with impaired function of EVTs. In 11ß-HSD2 dysfunction-induced PE-like rat model, we revealed that placental mitochondrial dysfunction occurred, which was associated with mitDNA instability and impaired mitochondrial dynamics, such as decreased optic atrophy 1 (OPA1) expression. MitoTEMPO treatment significantly alleviated the hallmark of PE-like features and improved mitDNA stability and mitochondrial dynamics in the placentas of rat PE-like model. In cultured human EVTs, we found that 11ß-HSD2 dysfunction led to mitochondrial dysfunction and disrupted mtDNA stability. MitoTEMPO treatment improved impaired invasion and migration induced by 11ß-HSD2 dysfunction in cultured EVTs. Further, we revealed that OPA1 was one of the key factors that mediated 11ß-HSD2 dysfunction-induced excess ROS production, mitochondrial dysfunction and mtDNA reduction. Our data indicates that 11ß-HSD2 dysfunction causes mitochondrial dysfunctions, which impairs trophoblast function and subsequently results in PE development. Our study immediately highlights that excess ROS is a potential therapeutic target for PE.

Endogenous Hydrogen Sulfide Is an Important Factor in Maintaining Arterial Oxygen Saturation.

The gasotransmitter H2S is involved in various physiological and pathophysiological processes. The aim of this study was to investigate the physiological functions of H2S in the lungs. In the model of mouse with genetic deficiency in a H2S natural synthesis enzyme cystathionine-γ-lyase (CSE), we found that arterial oxygen saturation (SaO2) was decreased compared with wild type mice. Hypoxyprobe test showed that mild hypoxia occurred in the tissues of heart, lungs and kidneys in Cse -/- mice. H2S donor GYY4137 treatment increased SaO2 and ameliorated hypoxia state in cardiac and renal tissues. Further, we revealed that lung blood perfusion and airway responsiveness were not linked to reduced SaO2 level. Lung injury was found in Cse -/- mice as evidenced by alveolar wall thickening, diffuse interstitial edema and leukocyte infiltration in pulmonary tissues. IL-8, IL-1ß, and TNF-α levels were markedly increased and oxidative stress levels were also significantly higher with increased levels of the pro-oxidative biomarker, MDA, decreased levels of the anti-oxidative biomarkers, T-AOC and GSH/GSSG, and reduced superoxide dismutase (SOD) activity in lung tissues of Cse -/- mice compared with those of wild type mice. GYY4137 treatment ameliorated lung injury and suppressed inflammatory state and oxidative stress in lung tissues of Cse -/- mice. A decrease in SaO2 was found in normal mice under hypoxia. These mice displayed lung injury as evidenced by alveolar wall thickening, interstitial edema and leukocyte infiltration. Increased levels of inflammatory cytokines and oxidative stress were also found in lung tissues of the mice with hypoxia insult. GYY4137 treatment increased SaO2 and ameliorated lung injury, inflammation and oxidative stress. Our data indicate that endogenous H2S is an important factor in maintaining normal SaO2 by preventing oxidative stress and inflammation in the lungs.

Furin is involved in uterine activation for labor.

The uterus undergoes distinct molecular and functional changes during pregnancy and parturition. These processes are associated with the dramatic changes in various proteins. Given that the maturation and activation of many proteins require proteolytic processing by proprotein convertases (PCs), we sought to explore the role of PCs in uterine activation for labor. First, we found that furin was the most dramatically increased PC member in myometrial tissues from the pregnant women after onset of labor at term. Using the model of cultured human myometrial smooth muscle cells (HMSMCs), we showed that furin inhibitor CMK, D6R treatment and furin siRNA transfection suppressed contractility. Inhibition of furin activity or interfering furin expression decreased connexin 43 (CX43), prostaglandin (PG) endoperoxide synthase-2 (COX-2) and PGF2α receptor (FP) expression and NF-κB activation. In mouse model, administration of furin inhibitors prolonged gestational length. However, D6R treatment did not affect RU38486- and lipopolysaccharides (LPS)-induced preterm birth. Furthermore, D6R and furin siRNA treatment reduced the release of soluble form of tumor necrosis factor (TNF)-related weak inducer of apoptosis (TWEAK), while furin overexpression led to an increase in soluble TWEAK release in cultured HMSMCs. D6R treatment decreased TWEAK level in blood of pregnant mice. TWEAK treatment promoted contractility and NF-κB activation, while TWEAK receptor fibroblast growth factor-inducible 14 (FN14) antagonist treatment inhibited contractility and NF-κB activation in HMSMCs. In pregnant mice, administration of FN14 antagonist prolonged gestational length. Our data suggest that furin can act as a stimulator for uterine activation for labor at term. TWEAK is one of the potential substrates which mediate furin regulation of parturition initiation.

Spatial learning and memory deficits induced by prenatal glucocorticoid exposure depend on hippocampal CRHR1 and CXCL5 signaling in rats.

BACKGROUND: Prenatal synthetic glucocorticoid (sGC) exposure increases the susceptibility to cognitive and affective disorders in postnatal life. We previously demonstrated that prenatal sGC exposure results in an increase in corticotropin-releasing hormone (CRH) receptor type 1 (CRHR1) expression in the hippocampus of rats, and CRHR1 is involved in synapse formation via regulation of C-X-C chemokine ligand 5 (CXCL5) in hippocampus. We sought to investigate that the roles of CRHR1 and CXCL5 in learning and memory impairment caused by prenatal sGC exposure. METHODS: Pregnant rats were administered with saline or dexamethasone (DEX) from gestational day (GD) 14 to GD21. DEX offspring at 2-day old were treated with saline and CRHR1 antagonists (antalarmin and CP154526) for 7 days. Some DEX offspring received intra-hippocampal injection of AAV9 carrying CXCL5 gene. Spatial learning and memory was assessed by Morris water maze test. Immunofluorescence analysis was applied to show synapsin I and PSD95 signals in hippocampus. Synapsin I and PSD95 protein level and CXCL5 concentration were determined by western blotting and ELISA, respectively. Organotypic hippocampal slice cultures were used to investigate the effect of DEX on CXCL5 production in vitro. RESULTS: Both male and female DEX offspring displayed impairment of spatial learning and memory in adulthood. Synapsin I and PSD95 signals and CXCL5 levels were decreased in DEX offspring. DEX offspring with antalarmin and CP154526 treatment showed improved spatial learning and memory. Antalarmin and CP154526 treatment increased synapsin I and PSD95 signals and CXCL5 concentration in hippocampus. Bilaterally hippocampal injection of AAV9 carrying CXCL5 gene improved the spatial learning and memory and increased CXCL5 concentration and synapsin I and PSD95 levels in hippocampus. DEX dose-dependently suppressed CXCL5 production in cultured hippocammpal slices, which was prevented by antalarmin treatment. CONCLUSION: CRHR1 and CXCL5 signaling in the hippocampus are involved in spatial learning and memory deficits caused by prenatal DEX exposure. CRHR1 activation contributes to decreased CXCL5 production in hippocampus induced by prenatal DEX treatment. Our study provides a molecular basis of prenatal GC exposure programming spatial learning and memory.

Contribution of placental 11ß-HSD2 to the pathogenesis of preeclampsia.

Preeclampsia, a major human pregnancy-specific disorder, leads to maternal and fetal morbidity and mortality. Here we reported that 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), an enzyme that degrades active glucocorticoids, is one of the key factors that contributes to preeclampsia development. In the pregnant rat model, we firstly confirmed that administration of 11ß-HSD2 inhibitor carbenoxolone (CBX) subcutaneously or by placenta-targeted delivery system could lead to a decrease in placental 11ß-HSD2 expression and activity and an increase in corticosterone level in placenta and maternal circulation. Then, we showed that subcutaneous administration and placenta-targeted delivery of CBX resulted in the hallmark of preeclampsia-like features including hypertension, proteinuria, renal damages as well as elevated circulatory soluble fms-like tyrosine kinase 1 (sFlt1) and increased sFlt1/placental growth factor (PlGF) ratio in pregnant rats. These animals displayed decreased trophoblast invasion in uterus, impaired spiral artery remodeling, and reduced placental blood flow. Preeclampsia-like features could also be induced by administration of dexamethasone in pregnant rats. In the cultured human trophoblast models, we found that cortisol only inhibited migration and invasion of the extravillous trophoblasts with 11ß-HSD2 knockdown, and promoted sFlt1 release in the cultured syncytiotrophoblasts with 11ß-HSD2 knockdown. Furthermore, we elucidated that cortisol stimulated a disintegrin and metalloprotease (ADAM)17 expression in placentas, thereby promoting sFlt1 release in placenta. Collectively, our study provided the evidence that placental 11ß-HSD2 dysfunction plays a key role in the development of preeclampsia and immediately identified innovative target to counteract preeclampsia.

Antitumor Drug Combretastatin-A4 Phosphate Aggravates the Symptoms of Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice.

Ulcerative colitis (UC) is an idiopathic inflammatory bowel disease (IBD) that causes long-lasting inflammation and ulcers in the innermost lining of the colon and rectum. Previous studies demonstrated that resveratrol suppresses colitis and colon cancer associated with colitis by improving glucose metabolism, but resveratrol use is limited by its low oral bioavailability. Combretastatin-A4 phosphate (CA4P) is a vascular-disrupting agent with antitumor activity. CA4P is structurally similar to resveratrol, but whether CA4P has the same effect as resveratrol on UC is not clear. In this study, we examined the pharmacological effects of CA4P administration on dextran sulfate sodium (DSS)-induced inflammation in a mouse model of UC. C57BL/6 mice were administered 2.5% DSS in the drinking water to induce acute UC. CA4P (11 mg/kg/d) was injected intraperitoneally daily. The Disease Activity Index (DAI) score and histological score were evaluated to determine the severity of UC. Colon tissues and blood samples were collected for histological analyses. The results show that CA4P plus DSS significantly decreased colon length (P < 0.05 versus DSS+PBS group) and body weight (P < 0.001 versus PBS group), while increased spleen weight (P < 0.01 versus DSS+PBS group), DAI score (P < 0.01 versus DSS+PBS group), and histological score (P < 0.01 versus DSS+PBS group). Moreover, CA4P exacerbated the pathological features of colitis and significantly increased proinflammatory cytokines (IL-1ß, IL-6, TNF-α) and inflammatory cells (neutrophil, lymphocyte, monocyte). These findings reveal that CA4P aggravates the symptoms of DSS-induced UC and provide a key reference for the potential of CA4P as an anticancer drug.

WDR79 mediates the proliferation of non-small cell lung cancer cells by regulating the stability of UHRF1.

WD repeat protein 79 (WDR79) is a member of the WD-repeat protein family characterized by the presence of a series of WD-repeat domains and is a scaffold protein that participates in telomerase assembly, Cajal body formation and DNA double strand break repair. Although previous studies have revealed that WDR79 is frequently overexpressed in non-small cell lung cancer (NSCLC) and promotes the proliferation of NSCLC cells, the underlying mechanism responsible for WDR79-mediated NSCLC proliferation is not fully understood. In this study, we report a novel molecular function of WDR79 that mediates NSCLC cell proliferation by controlling the stability of UHRF1. In the nucleus, WDR79 colocalized and interacted with UHRF1. As a result, overexpression of WDR79 stabilized UHRF1, whereas ablation of WDR79 decreased the level of UHRF1. Meanwhile, we showed that WDR79 can protect UHRF1 from poly-ubiquitination-mediated proteolysis, which facilitated the stabilization of UHRF1. We further demonstrated that WDR79 exerts a proliferation effect on NSCLC cells by stabilizing UHRF1. These findings reveal that WDR79 is a novel UHRF1 regulator by maintaining UHRF1 stability, and they also provide a clue as to how to explore WDR79 for potential therapeutic application in NSCLC.