Peroxynitrite-Triggered Carbon Monoxide Donor Improves Ischemic Stroke Outcome by Inhibiting Neuronal Apoptosis and Ferroptosis.

Cerebral ischemia-reperfusion injury produces excessive reactive oxygen and nitrogen species, including superoxide, nitric oxide, and peroxynitrite (ONOO-). We recently developed a new ONOO--triggered metal-free carbon monoxide donor (PCOD585), exhibiting a notable neuroprotective outcome on the rat middle cerebral artery occlusion model and rendering an exciting intervention opportunity toward ischemia-induced brain injuries. However, its therapeutic mechanism still needs to be addressed. In the pharmacological study, we found PCOD585 inhibited neuronal Bcl2/Bax/caspase-3 apoptosis pathway in the peri-infarcted area of stroke by scavenging ONOO-. ONOO- scavenging further led to decreased Acyl-CoA synthetase long-chain family member 4 and increased glutathione peroxidase 4, to minimize lipoperoxidation. Additionally, the carbon monoxide release upon the ONOO- reaction with PCOD585 further inhibited the neuronal Iron-dependent ferroptosis associated with ischemia-reperfusion. Such a synergistic neuroprotective mechanism of PCOD585 yields as potent a neuroprotective effect as Edaravone. Additionally, PCOD585 penetrates the blood-brain barrier and reduces the degradation of zonula occludens-1 by inhibiting matrix metalloproteinase-9, thereby protecting the integrity of the blood-brain barrier. Our study provides a new perspective for developing multi-functional compounds to treat ischemic stroke.

Insights into the mechanism of action of pterostilbene against influenza A virus-induced acute lung injury.

BACKGROUND: Severe respiratory system illness caused by influenza A virus infection is associated with excessive inflammation and abnormal apoptosis in alveolar epithelial cells (AEC). However, there are limited therapeutic options for influenza-associated lung inflammation and apoptosis. Pterostilbene (PTE, trans-3,5-dimethoxy-4-hydroxystilbene) is a dimethylated analog of resveratrol that has been reported to limit influenza A virus infection by promoting antiviral innate immunity, but has not been studied for its protective effects on virus-associated inflammation and injury in AEC. PURPOSE: Our study aimed to investigate the protective effects and underlying mechanisms of PTE in modulating inflammation and apoptosis in AEC, as well as its effects on macrophage polarization during influenza virus infection. STUDY DESIGN AND METHODS: A murine model of influenza A virus-mediated acute lung injury was established by intranasal inoculation with 5LD50 of mouse-adapted H1N1 viruses. Hematoxylin and eosin staining, immunofluorescence, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, Luminex and flow cytometry were performed. RESULTS: PTE effectively mitigated lung histopathological changes and injury induced by H1N1 viruses in vivo. These beneficial effects of PTE were attributed to the suppression of inflammation and apoptosis in AEC, as well as the modulation of M1 macrophage polarization. Mechanistic investigations revealed that PTE activated the phosphorylated AMP-activated protein kinase alpha (P-AMPKα)/sirtui1 (Sirt1)/PPARγ coactivator 1-alpha (PGC1α) signal axis, leading to the inhibition of nuclear factor kappa-B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling induced by H1N1 viruses, thereby attenuating inflammation and apoptosis in AEC. PTE also forced activation of the P-AMPKα/Sirt1/PGC1α signal axis in RAW264.7 cells, counteracting the activation of phosphorylated signal transducer and activator of transcription 1 (P-STAT1) induced by H1N1 viruses and the augment of P-STAT1 activation in RAW264.7 cells with interferon-gamma (IFN-γ) pretreatment before viral infection, thereby reducing H1N1 virus-mediated M1 macrophage polarization as well as the enhancement of macrophages into M1 phenotypes elicited by IFN-γ pretreatment. Additionally, the promotion of the transition of macrophages towards the M2 phenotype by PTE was also related to activation of the P-AMPKα/Sirt1/PGC1α signal axis. Moreover, co-culturing non-infected AEC with H1N1 virus-infected RAW264.7 cells in the presence of PTE inhibited apoptosis and tight junction disruption, which was attributed to the suppression of pro-inflammatory mediators and pro-apoptotic factors in an AMPKα-dependent manner. CONCLUSION: In conclusion, our findings suggest that PTE may serve as a promising novel therapeutic option for treating influenza-associated lung injury. Its ability to suppress inflammation and apoptosis in AEC, modulate macrophage polarization, and preserve alveolar epithelial cell integrity highlights its potential as a therapeutic agent in influenza diseases.

Remote Ischemic Postconditioning-Mediated Neuroprotection against Stroke by Promoting Ketone Body-Induced Ferroptosis Inhibition.

Neuronal death resulting from ischemic stroke is the primary cause of adult mortality and disability, and effective neuroprotective agents for poststroke intervention are still lacking. Remote ischemic postconditioning (RIPostC) has demonstrated significant protective effects against ischemia in various organs; however, the specific mechanisms are not fully understood. This study investigated the potential neuroprotective mechanisms of RIPostC in the context of ischemic stroke. Using a rat model of middle cerebral artery occlusion, we found that RIPostC mitigated neurological damage, improved movement in the open-field test, and protected against neuronal apoptosis. In terms of energy metabolism, RIPostC enhanced ATP levels, suppressed lactate content, and increased the production of ketone bodies (KBs). In the ferroptosis assay, RIPostC protected against lipoperoxidation, reversed the reduction of glutathione peroxidase 4 (GPX4), and mitigated the excessive expression of long-chain acyl-CoA synthetase family member 4 (ACSL4). In oxygen-glucose deprivation/reoxygenation-treated HT22 cells, KBs maintained GPX4 levels, suppressed ACSL4 expression, and preserved the mitochondrial cristae number. However, the effect of KBs on the expression of GPX4, ACSL4, and the number of mitochondrial cristae was blocked by erastin. Moreover, both RIPostC and KBs reduced total iron and ferrous ion content by repressing iron transporters both in vitro and in vivo. In conclusion, KBs-induced mitigation of ferroptosis could represent a new therapeutic mechanism for RIPostC in treating stroke.

Spexin2 Is a Novel Food Regulator in Gallus gallus.

Spexin2 (SPX2), a paralog of SPX1, is a newly identified gene in non-mammalian vertebrates. Limited studies in fish have evidenced its important role in food intake and energy balance modulation. However, little is known about its biological functions in birds. Using the chicken (c-) as a model, we cloned the full-length cDNA of SPX2 by using RACE-PCR. It is 1189 base pair (bp) in length and predicted to generate a protein of 75 amino acids that contains a 14 amino acids mature peptide. Tissue distribution analysis showed that cSPX2 transcripts were detected in a wide array of tissues, with abundant expression in the pituitary, testis, and adrenal gland. cSPX2 was also observed to be ubiquitously expressed in chicken brain regions, with the highest expression in the hypothalamus. Its expression was significantly upregulated in the hypothalamus after 24 or 36 h of food deprivation, and the feeding behavior of chicks was obviously suppressed after peripheral injection with cSPX2. Mechanistically, further studies evidenced that cSPX2 acts as a satiety factor via upregulating cocaine and amphetamine regulated transcript (CART) and downregulating agouti-related neuropeptide (AGRP) in hypothalamus. Using a pGL4-SRE-luciferase reporter system, cSPX2 was demonstrated to effectively activate a chicken galanin II type receptor (cGALR2), a cGALR2-like receptor (cGALR2L), and a galanin III type receptor (cGALR3), with the highest binding affinity for cGALR2L. Collectively, we firstly identified that cSPX2 serves as a novel appetite monitor in chicken. Our findings will help clarify the physiological functions of SPX2 in birds as well as its functional evolution in vertebrates.

Corticosterone stage-dependently inhibits progesterone production presumably via impeding the cAMP-StAR cascade in granulosa cells of chicken preovulatory follicles.

Stress can suppress reproduction capacity in either wild or domestic animals, but the exact mechanism behind it, especially in terms of steroidogenesis, remains under-investigated so far. Considering the important roles of progesterone in avian breeding, we investigated the modulation of corticosterone on progesterone production in cultured granulosa cells of chicken follicles at different developmental stages. Using enzyme immunoassays, our study showed that corticosterone could only inhibit progesterone synthesis in granulosa cells from F5-6, F4, and F3 follicles, but not F2 and F1 follicles. Coincidentally, both quantitative real-time PCR and western blotting revealed that corticosterone could downregulate steroidogenic acute regulatory protein (StAR) expression, suggesting the importance of StAR in corticosterone-related actions. Using the dual-luciferase reporter system, we found that corticosterone can potentially enhance, rather than inhibit, the activity of StAR promoter. Of note, combining high-throughput transcriptomic analysis and quantitative real-time PCR, phosphodiesterase 10A (PDE10A), protein kinase cAMP-dependent type II regulatory subunit alpha (PRKAR2A) and cAMP responsive element modulator (CREM) were identified to exhibit the differential expression patterns consistent with cAMP blocking in granulosa cells from F5-6, F4, and F3, but not F2 and F1 follicles. Afterward, the expression profiles of these genes in granulosa cells of distinct developmental-stage follicles were examined by quantitative real-time PCR, in which all of them expressed correspondingly with progesterone levels of granulosa cells during development. Collectively, these findings indicate that corticosterone can stage-dependently inhibit progesterone production in granulosa cells of chicken preovulatory follicles, through impeding cAMP-induced StAR activity presumptively.

Evidence for progesterone acting as an inhibitor of stress axis via stimulating pituitary neuropeptide B/W receptor 2 (NPBWR2) expression in chickens.

In vertebrates, the hypothalamus-pituitary-adrenal gland (HPA) axis is the main endocrine pathway regulating the stress response, thus also called the stress axis. It has been well-accepted that the stress axis is tightly controlled by both hypothalamic stimulators and inhibitors [e.g. corticotropin (ACTH)-releasing inhibitory factor (CRIF)]. However, the identity of authentic CRIF remains unclear for decades. Recently, neuropeptide W (NPW) was proved to be the physiological CRIF in chickens. Together with its functional receptor (NPBWR2), they play critical roles in attenuating the activity of the chicken stress axis. Because increasing pieces of evidence suggested that sex steroids could regulate the stress axis, using chicken as a model, we investigated whether the newly identified CRIF and its receptor are under the control of sex steroids in this study. Our results showed that: (1) expression of NPW-NPBWR2 in the hypothalamus-pituitary axis was sexually dimorphic and developmental stage-dependent; (2) progesterone (P4), rather than 17ß-estradiol (E2) and dihydrotestosterone (DHT), could dose- and time-dependently upregulate NPBWR2 expression, which was accompanied with the decrease of ACTH synthesis and secretion, in cultured pituitary cells; (3) intraperitoneal injection of P4 could elevate the mRNA level of pituitary NPBWR2; (4) P4-stimulated NPBWR2 expression was relevant to both nPR-mediated genomic action and mPRs-triggered nongenomic route associated with MEK/ERK, PI3K/AKT cascade, and calcium influx. To our knowledge, our results discover a novel route of sex steroids in modulating the stress axis of chickens, which lays a foundation to reveal the complicated interaction network between reproduction and stress axes in chickens.

Characterization of spexin (SPX) in chickens: molecular cloning, functional analysis, tissue expression and its involvement in appetite regulation.

Spexin (SPX) is a conservative tetradecapeptide which has been proven to participate in multiple physiological processes, including anxiety, feed intake, and energy metabolism in fish and mammals. However, whether SPX exists and functions in birds remain largely unknown. Using chicken (c-) as a model, the full-length cDNA encoding cSPX precursor was cloned, and it was predicted to generate a mature peptide with 14 amino acids conserved across vertebrates. The pGL4-SRE-luciferase reporter system-based functional analysis demonstrated that cSPX was effective in activating chicken galanin type Ⅱ receptor (cGALR2), cGALR2-like receptor (cGALR2L) and galanin type Ⅲ receptor (cGALR3), thus to stimulate intracellular MAPK/ERK signaling pathway. Quantitative real-time PCR revealed that SPX was widely expressed in chicken tissues, especially abundant in the central nervous system, pituitary, testes, and pancreas. Interestingly, it was noted that chicken hypothalamic SPX mRNA could be up-regulated by 24-h and 36-h fasting, heralding its latent capacity in appetite regulation. In accordance with this speculation, peripheral injection of cSPX was proved to be functional in reducing feed intake of 3-wk-old chicks. Furthermore, we found that cSPX could reduce the expression of AgRP and MCH, with a concurrent rise in CART1 mRNA level in the hypothalamic of chicks. Collectively, our findings not only provide the evidences that SPX can act as a satiety factor by orchestrating the expression of key feeding regulators in the chicken hypothalamus but also help to facilitate a better understanding of its functional evolution across vertebrates.

Ischemic accumulation of succinate induces Cdc42 succinylation and inhibits neural stem cell proliferation after cerebral ischemia/reperfusion.

Ischemic accumulation of succinate causes cerebral damage by excess production of reactive oxygen species. However, it is unknown whether ischemic accumulation of succinate affects neural stem cell proliferation. In this study, we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery. We found that succinate levels increased in serum and brain tissue (cortex and hippocampus) after ischemia/reperfusion injury. Oxygen-glucose deprivation and reoxygenation stimulated primary neural stem cells to produce abundant succinate. Succinate can be converted into diethyl succinate in cells. Exogenous diethyl succinate inhibited the proliferation of mouse-derived C17.2 neural stem cells and increased the infarct volume in the rat model of cerebral ischemia/reperfusion injury. Exogenous diethyl succinate also increased the succinylation of the Rho family GTPase Cdc42 but repressed Cdc42 GTPase activity in C17.2 cells. Increasing Cdc42 succinylation by knockdown of the desuccinylase Sirt5 also inhibited Cdc42 GTPase activity in C17.2 cells. Our findings suggest that ischemic accumulation of succinate decreases Cdc42 GTPase activity by induction of Cdc42 succinylation, which inhibits the proliferation of neural stem cells and aggravates cerebral ischemia/reperfusion injury.

Pyrogallol enhances therapeutic effect of human umbilical cord mesenchymal stem cells against LPS-mediated inflammation and lung injury via activation of Nrf2/HO-1 signaling.

The main challenges in clinical applications of mesenchymal stem cells (MSCs) are attributed to their heterogeneity. It is believed that preconditioning of MSCs with active compounds may enhance the expression of potentially therapeutic molecules and thus achieve stable and effective therapeutic outcomes. In the present study, we investigated the mechanism by which pyrogallol increased the therapeutic efficacy of human umbilical cord mesenchymal stem cells (hUCMSCs) against LPS-induced acute lung injury (ALI). hUCMSCs with pyrogallol treatment increased expression of HO-1 at both mRNA and protein levels, accompanied by Kelch-Like ECH-Associated Protein 1 (Keap1) degradation, and upregulation of the Nrf2 protein levels as well as nuclear translocation of Nrf2. Moreover, the modulation of Keap1 and Nrf2 as well as HO-1 upregulation by pyrogallol was reversed by pretreatment with N-acetylcysteine (NAC) and a P38 kinase inhibitor (SB203580). Whereas, NAC pretreatment abrogated pyrogallol-mediated activation of P38 kinase, indicating that pyrogallol-derived ROS led to P38 kinase activation, thus promoting Nrf2/HO-1 signaling. Additionally, we found that the induction of p62 by the pyrogallol-mediated ROS/P38/Nrf2 axis interacted with Keap1 and resulted in autophagic degradation of Keap1, which created a positive feedback loop to further release of Nrf2. Furthermore, the increased expression of HO-1 in pyrogallol-pretreated hUCMSCs led to enhanced inhibitory effects on LPS-mediated TLR4/P-P65 signaling in BEAS-2B cells, resulting in increasing suppression of LPS-indued expression of a series of pro-inflammatory mediators. Compared to untreated hUCMSCs, Sprague-Dawley (SD) rats with pyrogallol-primed hUCMSCs transplantation showed enhanced improvements in LPS-mediated lung pathological alterations, the increased lung index (lung/body ratio), apoptosis of epithelial cells, the activation of TLR4/NF-κB signaling as well as the release of pro-inflammatory mediators. Together, these results suggested that hUCMSCs with pyrogallol pretreatment enhanced the therapeutic efficacy of hUCMSCs, which may provide a promising therapeutic strategy to maximize the therapeutic efficacy of hUCMSC-based therapy for treating LPS-associated ALI.

Momordica charantia Exosome-Like Nanoparticles Exert Neuroprotective Effects Against Ischemic Brain Injury via Inhibiting Matrix Metalloproteinase 9 and Activating the AKT/GSK3ß Signaling Pathway.

Plant exosome-like nanoparticles (ELNs) have shown great potential in treating tumor and inflammatory diseases, but the neuroprotective effect of plant ELNs remains unknown. In the present study, we isolated and characterized novel ELNs from Momordica charantia (MC) and investigated their neuroprotective effects against cerebral ischemia-reperfusion injury. In the present study, MC-ELNs were isolated by ultracentrifugation and characterized. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) and MC-ELN injection intravenously. The integrity of the blood-brain barrier (BBB) was examined by Evans blue staining and with the expression of matrix metalloproteinase 9 (MMP-9), claudin-5, and ZO-1. Neuronal apoptosis was evaluated by TUNEL and the expression of apoptotic proteins including Bcl2, Bax, and cleaved caspase 3. The major discoveries include: 1) Dil-labeled MC-ELNs were identified in the infarct area; 2) MC-ELN treatment significantly ameliorated BBB disruption, decreased infarct sizes, and reduced neurological deficit scores; 3) MC-ELN treatment obviously downregulated the expression of MMP-9 and upregulated the expression of ZO-1 and claudin-5. Small RNA-sequencing revealed that MC-ELN-derived miRNA5266 reduced MMP-9 expression. Furthermore, MC-ELN treatment significantly upregulated the AKT/GSK3ß signaling pathway and attenuated neuronal apoptosis in HT22 cells. Taken together, these findings indicate that MC-ELNs attenuate ischemia-reperfusion-induced damage to the BBB and inhibit neuronal apoptosis probably via the upregulation of the AKT/GSK3ß signaling pathway.

KAP1 phosphorylation promotes the survival of neural stem cells after ischemia/reperfusion by maintaining the stability of PCNA.

AIMS: To explore the function of phosphorylation of KAP1 (p-KAP1) at the serine-824 site (S824) in the proliferation and apoptosis of endogenous neural stem cells (NSCs) after cerebral ischemic/reperfusion (I/R). METHODS: The apoptosis and proliferation of C17.2 cells transfected with the p-KAP1-expression plasmids and the expression of proliferation cell nuclear antigen (PCNA) and p-KAP1 were detected by immunofluorescence and Western blotting after the Oxygen Glucose deprivation/reperfusion model (OGD/R). The interaction of p-KAP1 and CUL4A with PCNA was analyzed by immunoprecipitation. In the rats MCAO model, we performed the adeno-associated virus (AAV) 2/9 gene delivery of p-KAP1 mutants to verify the proliferation of endogenous NSCs and the colocalization of PCNA and CUL4A by immunofluorescence. RESULTS: The level of p-KAP1 was significantly down-regulated in the stroke model in vivo and in vitro. Simulated p-KAP1(S824) significantly increased the proliferation of C17.2 cells and the expression of PCNA after OGD/R. Simulated p-KAP1(S824) enhanced the binding of p-KAP1 and PCNA and decreased the interaction between PCNA and CUL4A in C17.2 cells subjected to OGD/R. The AAV2/9-mediated p-KAP1(S824) increased endogenous NSCs proliferation, PCNA expression, p-KAP1 binding to PCNA, and improved neurological function in the rat MCAO model. CONCLUSIONS: Our findings confirmed that simulated p-KAP1(S824) improved the survival and proliferation of endogenous NSCs. The underlying mechanism is that highly expressed p-KAP1(S824) promotes binding to PCNA, and inhibits the binding of CUL4A to PCNA. This reduced CUL4A-mediated ubiquitination degradation to increase the stability of PCNA and promote the survival and proliferation of NSCs.

Association of the Coronavirus Disease 2019 Outbreak on the Diabetes Self-Management in Chinese Patients: An Analytical Cross-Sectional Study.

Background: The coronavirus disease 2019 (COVID-19) outbreak has seriously affected people's lives, especially those with chronic diseases. Diabetes self-management, which plays an important role in glycaemic control and reducing the risk of acute and long-term complications, may be discouraged by social distancing. Purpose: To evaluate the level of self-management activities in Chinese patients with type 2 diabetes mellitus (T2DM) during the COVID-19 pandemic. Patients and Methods: A survey of with 872 patients with T2DM in the inpatient and outpatient departments through face-to-face interviews was conducted from 1 July, 2020 to 30 September, 2020. The main outcome measures were glycaemic control status and level of self-management activities during the pandemic. Results: In terms of glycaemic control, the data showed that patients with fasting plasma glucose (FPG) < 7.0 mmol/L (36.4%), postprandial plasma glucose (PPG) < 10.0 mmol/L (26.3%), or glycosylated haemoglobin (HbA1c) < 7.0% (18.6%) in our investigation has well-controlled blood glucose level, and 11.9% of patients experienced blood glucose <3.9 mmol/L during the outbreak. The diabetes self-management of Chinese patients decreased and the final diabetes self-management score of the Chinese patients was 3.4 ± 1.45. Patients with higher education, diabetes education, comorbidities, and online consultations had higher diabetes self-management scores (P <0.05). Adherence to diabetes self-management in the normal glycaemic control group was higher than that in the substandard glycaemic control group (P<0.05). Among all participants, 72.1% of the patients reduced the frequency of hospital visits, and 44.8% considered that they had diabetes-related stress during the pandemic. The mean anxiety level score rated by 286 patients was 5.3±2.8. Conclusion: The COVID-19 pandemic has affected diabetes self-management, including substandard glycemic control, increased diabetes-related stress, limited exercise range and medical visits. Therefore, future interventions should focus on the online management of chronic diseases and support online consultation' development and promotion, which can overcome physical distance and provide personalized services conveniently.

Momordica. charantia-Derived Extracellular Vesicles-Like Nanovesicles Protect Cardiomyocytes Against Radiation Injury via Attenuating DNA Damage and Mitochondria Dysfunction.

Thoracic radiotherapy patients have higher risks of developing radiation-induced heart disease (RIHD). Ionizing radiation generates excessive reactive oxygens species (ROS) causing oxidative stress, while Momordica. charantia and its extract have antioxidant activity. Plant-derived extracellular vesicles (EVs) is emerging as novel therapeutic agent. Therefore, we explored the protective effects of Momordica. charantia-derived EVs-like nanovesicles (MCELNs) against RIHD. Using density gradient centrifugation, we successfully isolated MCELNs with similar shape, size, and markers as EVs. Confocal imaging revealed that rat cardiomyocytes H9C2 cells internalized PKH67 labeled MCELNs time-dependently. In vitro assay identified that MCELNs promoted cell proliferation, suppressed cell apoptosis, and alleviated the DNA damage in irradiated (16 Gy, X-ray) H9C2 cells. Moreover, elevated mitochondria ROS in irradiated H9C2 cells were scavenged by MCELNs, protecting mitochondria function with re-balanced mitochondria membrane potential. Furthermore, the phosphorylation of ROS-related proteins was recovered with increased ratios of p-AKT/AKT and p-ERK/ERK in MCELNs treated irradiated H9C2 cells. Last, intraperitoneal administration of MCELNs mitigated myocardial injury and fibrosis in a thoracic radiation mice model. Our data demonstrated the potential protective effects of MCELNs against RIHD. The MCELNs shed light on preventive regime development for radiation-related toxicity.

Corticosterone triggers anti-proliferative and apoptotic effects, and downregulates the ACVR1-SMAD1-ID3 cascade in chicken ovarian prehierarchical, but not preovulatory granulosa cells.

The coordinated proliferation and apoptosis of granulosa cells plays a critical role in follicular development. To identify the exact mechanisms of how stress-driven glucocorticoid production suppresses reproduction, granulosa cells were isolated from chicken follicles at different developmental stages and then treated with corticosterone. Using CCK-8, EDU and TUNEL assays, we showed that corticosterone could trigger both anti-proliferative and pro-apoptotic effects in granulosa cells from 6 to 8 mm follicles only, while depicting no influence on granulosa cells from any preovulatory follicles. High-throughput transcriptomic analysis identified 1362 transcripts showing differential expression profiles in granulosa cells from 6 to 8 mm follicles after corticosterone treatment. Interestingly, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that 17 genes were enriched in the TGF-ß signaling pathway, and 13 showed differential expression patterns consistent with corticosterone-induced effects. The differential expression profiles of these 13 genes were examined by quantitative real-time PCR in cultured chicken ovarian granulosa cells at diverse developmental stages following corticosterone challenge for a short (8 h) or long period (24 h). After 24 h of treatment, INHBB, FST, FMOD, NOG, ACVR1, SMAD1 and ID3 were the genes that responded consistently with corticosterone-induced proliferative and apoptotic events in all granulosa cells detected. However, only ACVR1, SMAD1 and ID3 could initiate coincident expression patterns after being treated for 8 h, suggesting their significance in corticosterone-mediated actions. Collectively, these findings indicate that corticosterone can inhibit proliferation and cause apoptosis in chicken ovarian prehierarchical, but not preovulatory granulosa cells, through impeding ACVR1-SMAD1-ID3 signaling presumptively.

Enhanced autophagy promotes radiosensitivity by mediating Sirt1 downregulation in RM-1 prostate cancer cells.

Autophagy is a double-edged sword that affects tumor progression by promoting cell survival or death depending on different living contexts. The concrete mechanism by which autophagy modulates the efficacy of radiotherapy for prostate cancer (PC) remains unclear. We exposed RM-1 PC cells to X-ray and explored the role of autophagy in radiation injury. Our results showed increased apoptosis and autophagy levels in RM-1 cells after radiation. Pharmacological inhibition of autophagy by chloroquine significantly mitigated radiation-induced apoptosis, while the enhancement of autophagy by rapamycin aggravated apoptosis. Sirt1, a member of sirtuin family, deacetylates various transcription factors to trigger cell survival in response to radiation injury. We found that radiation led to Sirt1 downregulation, which was reversed by the inhibition of autophagy. On the contrary, enhanced autophagy further diminished protein level of Sirt1. Notably, overexpression of Sirt1 by plasmid significantly alleviated radiation-induced apoptosis, but silenced Sirt1 by siRNA further induced apoptosis, indicating the radioprotective effect of Sirt1 on RM-1 cells. In summary, our findings suggested that autophagy-mediated Sirt1 downregulation might be a promising therapeutic target for PC.

Healthy Serum-Derived Exosomes Improve Neurological Outcomes and Protect Blood-Brain Barrier by Inhibiting Endothelial Cell Apoptosis and Reversing Autophagy-Mediated Tight Junction Protein Reduction in Rat Stroke Model.

Blood-brain barrier (BBB) dysfunction causing edema and hemorrhagic transformation is one of the pathophysiological characteristics of stroke. Protection of BBB integrity has shown great potential in improving stroke outcome. Here, we assessed the efficacy of exosomes extracted from healthy rat serum in protection against ischemic stroke in vivo and in vitro. Exosomes were isolated by gradient centrifugation and ultracentrifugation and exosomes were characterized by transmission electron microscopy (TEM) and nanoparticle tracking video microscope. Exosomes were applied to middle cerebral artery occlusion (MCAO) rats or brain microvascular endothelial cell line (bEnd.3) subjected to oxygen-glucose deprivation (OGD) injury. Serum-derived exosomes were injected intravenously into adult male rats 2 h after transient MCAO. Infarct volume and gross cognitive function were assessed 24 h after reperfusion. Poststroke rats treated with serum-derived exosomes exhibited significantly reduced infarct volumes and enhanced neurological function. Apoptosis was assessed via terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining and the expression of B-cell lymphoma-2 (Bcl-2), Bax, and cleaved caspase-3 24 h after injury. Our data showed that serum exosomes treatment strikingly decreased TUNEL+ cells in the striatum, enhanced the ratio of Bcl-2 to Bax, and inhibited cleaved caspase-3 production in MCAO rats and OGD/reoxygenation insulted bEnd.3 cells. Under the consistent treatment, the expression of microtubule-associated protein 1 light chain 3B-II (LC3B-II), LC3B-I, and Sequestosome-1 (SQSTM1)/p62 was detected by Western blotting. Autolysosomes were observed via TEM. We found that serum exosomes reversed the ratio of LC3B-II to LC3B-I, prevented SQSTM1/p62 degradation, autolysosome formation, and autophagic flux. Together, these results indicated that exosomes isolated from healthy serum provided neuroprotection against experimental stroke partially via inhibition of endothelial cell apoptosis and autophagy-mediated BBB breakdown. Intravenous serum-derived exosome treatment may, therefore, provide a novel clinical therapeutic strategy for ischemic stroke.

The protein adduction derived from reactive metabolites of multiple furanoids in cortex Dictamni-treated mice.

The association of herb medicine Cortex Dictamni (CD) with severe even fatal hepatotoxicity has been widely reported. Recently, we demonstrated that the metabolic activation of at least ten furanoids in CD was responsible for the liver injury caused by the ethanol extract of CD (ECD) in mice. Protein adduction by reactive metabolites is considered to initiate the process of liver injury. Unlike single chemicals, the mode of and the details of protein modification by multiple components in an herb is unclear. This study aimed to characterize protein adductions derived from the reactive metabolite of furanoids in ECD-treated mice and define the association of protein adduction with liver injury. The hepatic cysteine- and lysine-based protein adducts derived from epoxide or cis-enedione of at least six furanoids were identified in mice. The furanoids with an earlier serum content Tmax were mainly to bind with hepatic glutathione and no protein adducts were formed except for dictamnine. The hepatic proteins were modified by the later absorbed furanoids. The levels of hepatic protein adduct were correlated with the degree of liver injury. In addition, the reactive metabolites of different furanoids can simultaneously bind to the model peptide by the identical reactive moiety, indicating the additive effects of the individual furanoids in the modification of hepatic proteins. In conclusion, hepatic protein adduction by multiple furanoids may play a role in ECD-induced liver injury. The earlier absorbed furanoids were mainly to bind with glutathione whereas the hepatic proteins were modified by the later furanoids.

Development of a Water Matrix Certified Reference Material for Volatile Organic Compounds Analysis in Water.

Water matrix certified reference material (MCRM) of volatile organic compounds (VOCs) is used to provide quality assurance and quality control (QA/QC) during the analysis of VOCs in water. In this research, a water MCRM of 28 VOCs was developed using a "reconstitution" approach by adding VOCs spiking, methanol solution into pure water immediately prior to analysis. The VOCs spiking solution was prepared gravimetrically by dividing 28 VOCs into seven groups, then based on ISO Guide 35, using gas chromatography-mass spectrometry (GC-MS) to investigate the homogeneity and long-term stability. The studies of homogeneity and long-term stability indicated that the batch of VOCs spiking solution was homogeneous and stable at room temperature for at least 15 months. Moreover, the water MCRM of 28 VOCs was certified by a network of nine competent laboratories, and the certified values and expanded uncertainties of 28 VOCs ranged from 6.2 to 17 µg/L and 0.5 to 5.3 µg/L, respectively.

Momordica charantia polysaccharides modulate the differentiation of neural stem cells via SIRT1/Β-catenin axis in cerebral ischemia/reperfusion.

BACKGROUND: Stroke is the leading cause of long-term motor disability and cognitive impairment. Recently, neurogenesis has become an attractive strategy for the chronic recovery of stroke. It is important to understand the molecular mechanism that promotes neural stem cell (NSC) neurogenesis for future NSC-based therapies. Our previous study showed that Momordica charantia polysaccharides (MCPs) exerted neuroprotective effects on stroke via their anti-oxidant and anti-inflammation activities. However, it remains unknown whether MCPs promote NSC neurogenesis after cerebral ischemic/reperfusion injury (IRI). METHODS: We investigated MCPs' function in differentiation of neural stem cells (NSCs) in vivo and in vitro experiments. Based on a middle cerebral artery occlusion (MCAO) rat model, the effect of MCPs on neuronal differentiation after MCAO was analyzed. Primary NSCs and neural stem cell line C17.2 were cultured and subjected to glutamate stimulation to establish the cell model of IRI. We evaluated the effect of MCPs on NSC differentiation in IRI cell model by Western blot and immunofluorescence staining. The SIRT1 activity of NSCs post glutamate stimulation was also evaluated by CELL SIRT1 COLORIMETRY ASSAY KIT. In addition, molecular mechanism was clarified by employing the activator and inhibitor of SIRT1. RESULTS: MCPs had no effects on the differentiation of neural stem cells under physiological conditions while shifted NSC differentiation potential from the gliogenic to neurogenic lineage under pathological conditions. Activation of SIRT1 with MCPs was responsible for the neuronal differentiation of C17.2-NSCs. The neuronal differentiation effect of MCPs was attributed to upregulation SIRT1-mediated deacetylation of ß-catenin. MCP-induced deacetylation via SIRT1 promoted nuclear accumulation of ß-catenin in NSCs. CONCLUSION: Our findings indicate that the deacetylation of ß-catenin by SIRT1 represents a critical mechanism of action of MCPs in promoting NSC neuronal differentiation. It provides an improved understanding of molecular mechanism underlying neuroprotective effects of MCPs in IRI, indicating its potential role on treating ischemic stroke especially chronic recovery.

KAP1 silencing relieves OxLDL-induced vascular endothelial dysfunction by down-regulating LOX-1.

KRAB domain-associated protein 1 (KAP1) is highly expressed in atherosclerotic plaques. Here, we studied the role of KAP1 in atherosclerosis development using a cell model of endothelial dysfunction induced by oxidative low-density lipoprotein (OxLDL). The phosphorylation and protein levels of KAP1 were similar between OxLDL-treated and non-treated endothelial cells (ECs). KAP1 depletion significantly inhibited the production of OxLDL-enhanced reactive oxygen species and the expression of adhesion molecules in ECs. Treatment with OxLDL promoted the proliferation and migration of ECs, which was also confirmed by the elevated levels of the proliferative markers c-Myc and PCNA, as well as the migratory marker MMP-9. However, these effects were also abrogated by KAP1 depletion. Moreover, the depletion of KAP1 in OxLDL-treated ECs resulted in decreases in the LOX-1 level and increases in eNOS expression. Generally, the data suggest that strategies targeting KAP1 depletion might be particularly useful for the prevention or treatment of atherosclerosis.