Study of the isomeric Maillard degradants, glycosylamine and Amadori rearrangement products, and their differentiation via MS2 fingerprinting from collision-induced decomposition of protonated ions.

RATIONALE: The focus of this work was to study glycosylamine and Amadori rearrangement products (ARPs), the two major degradants in the Maillard reactions of pharmaceutical interest, and utilize their MS2 fingerprints by liquid chromatography/high-resolution tandem mass spectrometry (LC/HRMS2 ) to quickly distinguish the two isomeric degradants. These two types of degradants are frequently encountered in the compatibility and stability studies of drug products containing primary or secondary amine active pharmaceutical ingredients (APIs), which are formulated with excipients consisting of reducing sugar functionalities. METHODS: Vortioxetine was employed as the primary model compound to react with lactose to obtain the glycosylamine and ARP degradants of the Maillard reaction, and their MS2 spectra (MS2 fingerprints) were obtained by LC/MS2 . Subsequently, the two degradants were isolated via preparative HPLC and their structures were confirmed by one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) determination. RESULTS: The MS2 fingerprints of the two degradants display significantly different profiles, despite the fact that many common fragments are observed. Specifically, protonated glycosylamine shows a prominent characteristic fragment of [Mvort + C2 H3 O]+ at m/z 341 (Mvort is the vortioxetine core), while protonated ARP shows a prominent characteristic fragment of [Mvort + CH]+ at m/z 311. Further study of the Maillard reactions between several other structurally diverse primary/secondary amines and lactose produced similar patterns. CONCLUSIONS: The study suggests that the characteristic MS2 fragment peaks and their ratios may be used to differentiate the glycosylamine and ARP degradants, the two isomeric degradants of the Maillard reaction, which are commonly encountered in finished dosage forms of pharmaceutical products containing primary and secondary amine APIs.

Enhanced Electrocatalytic Performance through Body Enrichment of Co-Based Bimetallic Nanoparticles In Situ Embedded Porous N-Doped Carbon Spheres.

Extending available body space loading active species and controllably tailoring the d-band center to Fermi level of catalysts are of paramount importance but extremely challenging for the enhancement of electrocatalytic performance. Herein, a melamine-bridged self-construction strategy is proposed to in situ embed Co-based bimetallic nanoparticles in the body of N-doped porous carbon spheres (CoM-e-PNC), and achieve the controllable tailoring of the d-band center position by alloying of Co and another transition metal M (M = Ni, Fe, Mn, and Cu). The enrichment and exposure of the active sites in the body interior of porous carbon spheres, and the best balance between the adsorption of OH species and the desorption of O2 induced by optimizing the d-band center position, collectively enhance the oxygen evolution reaction (OER) performance. Meanwhile, the relationship of d-band center position and OER activity is found to exhibit the volcano curve rule, where the CoNi-e-PNC catalyst shows optimal OER performance with an overpotential of 0.24 V at 10 mA cm-2 in alkaline media, outperforming those of the ever-reported CoNi-based catalysts. Besides, CoNi-e-PNC catalyst also demonstrates high OER stability with slight current decrease after 100 h.

Statins Attenuate Activation of the NLRP3 Inflammasome by Oxidized LDL or TNFα in Vascular Endothelial Cells through a PXR-Dependent Mechanism.

Excessive activation of the NLRP3 inflammasome is implicated in cardiovascular diseases. Statins exert an anti-inflammatory effect independent of their cholesterol-lowering effect. This study investigated the potential role of statins in the activation of the NLRP3 inflammasome in endothelial cells (ECs). Western blotting and quantitative reverse-transcription polymerase chain reaction showed that oxidized low-density lipoprotein (ox-LDL) or tumor necrosis factor α (TNFα) activated the NLRP3 inflammasome in ECs. Simvastatin or mevastatin significantly suppressed the effects of ox-LDL or TNFα Promoter reporter assays and small interfering RNA knockdown revealed that statins inhibit ox-LDL-mediated NLRP3 inflammasome activation via the pregnane X receptor (PXR). In addition, PXR agonists (rifampicin and SR12813) or overexpression of a constitutively active PXR markedly suppressed the NLRP3 inflammasome activation. Conversely, PXR knockdown abrogated the suppressive effect of rifampicin on NLRP3 inflammasome activation. Knockdown of lectin-like ox-LDL receptor or overexpression of IκBα-attenuated ox-LDL- or TNFα-triggered activation of the NLRP3 inflammasome. Chromatin immunoprecipitation assays indicated that mevastatin inhibited nuclear factor-κB binding to the promoter regions of the human NLRP3 gene. Collectively, these results demonstrate that the statin activation of PXR inhibits the activation of NLRP3 inflammasome in response to atherogenic stimuli such as ox-LDL and TNFα in ECs, providing a new mechanism for the cardiovascular benefit of statins.

Xenobiotic pregnane X receptor (PXR) regulates innate immunity via activation of NLRP3 inflammasome in vascular endothelial cells.

Pregnane X receptor (PXR) is a member of nuclear receptor superfamily and responsible for the detoxification of xenobiotics. Our previously study demonstrated that PXR is expressed in endothelial cells (ECs) and acts as a master regulator of detoxification genes to protect ECs against xenobiotics. Vascular endothelial cells are key sentinel cells to sense the pathogens and xenobiotics. In this study, we examined the potential function of PXR in the regulation of innate immunity in vasculatures. Treatments with PXR agonists or overexpression of a constitutively active PXR in cultured ECs increased gene expression of the key pattern recognition receptors, including Toll-like receptors (TLR-2, -4, -9) and NOD-like receptors (NOD-1 and -2 and NLRP3). In particular, PXR agonism triggered the activation of NLRP3 inflammasome and the ensuing cleavage and maturation of caspase-1 and interleukin-1ß (IL-1ß). Conversely, selective antagonism or gene silencing of PXR abrogated NLRP3 inflammasome activation. In addition, we identified NLRP3 as a transcriptional target of PXR by using the promoter-reporter and ChIP assays. In summary, our findings revealed a novel regulatory mechanism of innate immune by PXR, which may act as a master transcription factor controlling the convergence between the detoxification of xenobiotics and the innate immunity against them.

Carbon cloth supporting spinel CuMn0.5Co2O4 nanoneedles with the regulated electronic structure by multiple metal elements as catalysts for efficient oxygen evolution reaction.

Developing transition metal oxide catalysts to replace the noble metal oxide catalysts for efficient oxygen evolution reaction (OER) is essential to promote the practical application of water splitting. Herein, we designed and constructed the carbon cloth (CC) supporting spinel CuMn0.5Co2O4 nanoneedles with regulated electronic structure by multiple metal elements with variable chemical valences in the spinel CuMn0.5Co2O4. The carbon cloth not only provided good conductivity for the catalytic reaction but also supported the well-standing spinel CuMn0.5Co2O4 nanoneedles arrays with a large special surface area. Meanwhile, the well-standing nanoneedles arrays and mesoporous structure of CuMn0.5Co2O4 nanoneedles enhanced their wettability and facilitated access for electrolyte to electrochemical catalysis. Besides, the regulated electronic structure and generated oxygen vacancies of CuMn0.5Co2O4/CC by multiple metal elements improved the intrinsic catalytic activity and the durability of OER activity. Profiting from these merits, the CuMn0.5Co2O4/CC electrode exhibited superior OER activity with an ultralow overpotential of 189 mV at the current density of 10 mA⋅cm-2 and a smaller Tafel slope of 64.1 mV⋅dec-1, which was competitive with the noble metal oxides electrode. And the CuMn0.5Co2O4/CC electrode also exhibited long-term durability for OER with 95.3% of current retention after 1000 cycles. Therefore, the competitive OER activity and excellent cycling durability suggested that the CuMn0.5Co2O4/CC electrode is a potential candidate catalyst for efficient OER.

Transcriptomics profile of human bronchial epithelial cells exposed to ambient fine particles and influenza virus (H3N2).

Fine particulate matter (PM2.5) pollution remains a major threat to public health. As the physical barrier against inhaled air pollutants, airway epithelium is a primary target for PM2.5 and influenza viruses, two major environmental insults. Recent studies have shown that PM2.5 and influenza viruses may interact to aggravate airway inflammation, an essential event in the pathogenesis of diverse pulmonary diseases. Airway epithelium plays a critical role in lung health and disorders. Thus far, the mechanisms for the interactive effect of PM2.5 and the influenza virus on gene transcription of airway epithelial cells have not been fully uncovered. In this present pilot study, the transcriptome sequencing approach was introduced to identify responsive genes following individual and co-exposure to PM2.5 and influenza A (H3N2) viruses in a human bronchial epithelial cell line (BEAS-2B). Enrichment analysis revealed the function of differentially expressed genes (DEGs). Specifically, the DEGs enriched in the xenobiotic metabolism by the cytochrome P450 pathway were linked to PM2.5 exposure. In contrast, the DEGs enriched in environmental information processing and human diseases, such as viral protein interaction with cytokines and cytokine receptors and epithelial cell signaling in bacterial infection, were significantly related to H3N2 exposure. Meanwhile, co-exposure to PM2.5 and H3N2 affected G protein-coupled receptors on the cell surface. Thus, the results from this study provides insights into PM2.5- and influenza virus-induced airway inflammation and potential mechanisms.

Multifunctional effects of hollow flower-like CoTiO3 microspheres wrapped by reduced graphene as sulfur host in Li-S battery.

The poor conductivity of sulfur, the shuttle effect and sluggish redox reaction kinetics of lithium polysulfides (LiPSs) are considered the main obstacles to the practical application of Lithium-sulfur (Li-S) batteries. Thus, it is urgent to design multifunctional host materials to eliminate these obstacles. Herein, we designed a hollow flower-like CoTiO3 wrapped by reduced graphene oxide (h-CoTiO3@rGO) as sulfur host materials. The hollow structure of h-CoTiO3@rGO not only endows sufficient space for high sulfur loading, but also physically and chemically confines the shuttle effect of LiPSs through the formation of Co-S chemical bonding. The large specific surface area and excellent electrocatalytic ability of h-CoTiO3@rGO provide amounts of active sites to accelerate the redox reaction of LiPSs. Meanwhile, the conductive reduced graphene oxide (rGO) covered on the surface of CoTiO3 microspheres offers an interconnected conductive network to support the fast electron/ion transfer. Profit from these merits, the battery employing the multifunctional h-CoTiO3@rGO as sulfur host exhibited excellent cycling stability with an ultralow capacity fading of 0.0127 % per cycle after 500 cycles at 1C. Even the battery with high sulfur loading of 5.2 mg/cm2 still delivered a high area capacity of 5.02 mAh/cm2, which was competitive with the commercial Li-ion batteries. Therefore, the competitive capacity and superior cycling stability suggest that the h-CoTiO3@rGO/S cathode is a potential candidate for high-performance Li-S batteries.

Microglial Activation and Oxidative Stress in PM2.5-Induced Neurodegenerative Disorders.

Fine particulate matter (PM2.5) pollution remains a prominent environmental problem worldwide, posing great threats to human health. The adverse effects of PM2.5 on the respiratory and cardiovascular systems have been extensively studied, while its detrimental effects on the central nervous system (CNS), specifically neurodegenerative disorders, are less investigated. Neurodegenerative disorders are characterized by reduced neurogenesis, activated microglia, and neuroinflammation. A variety of studies involving postmortem examinations, epidemiological investigations, animal experiments, and in vitro cell models have shown that PM2.5 exposure results in neuroinflammation, oxidative stress, mitochondrial dysfunction, neuronal apoptosis, and ultimately neurodegenerative disorders, which are strongly associated with the activation of microglia. Microglia are the major innate immune cells of the brain, surveilling and maintaining the homeostasis of CNS. Upon activation by environmental and endogenous insults, such as PM exposure, microglia can enter an overactivated state that is featured by amoeboid morphology, the over-production of reactive oxygen species, and pro-inflammatory mediators. This review summarizes the evidence of microglial activation and oxidative stress and neurodegenerative disorders following PM2.5 exposure. Moreover, the possible mechanisms underlying PM2.5-induced microglial activation and neurodegenerative disorders are discussed. This knowledge provides certain clues for the development of therapies that may slow or halt the progression of neurodegenerative disorders induced by ambient PM.

Regional pattern and signatures of gut microbiota in rural residents with coronary heart disease: A metagenomic analysis.

Coronary heart disease (CHD) is tightly associated with gut microbiota, but microbiota heterogeneity limits the application of microbial biomarkers and personalized interventions demand regional-specific features. The purpose of this study was to comprehensively characterize the regional pattern of gut microbiota in rural residents with CHD and assess the predictive value and clinical correlations of local microbial signatures. We profiled the gut microbiota by shotgun metagenomic sequencing from 19 CHD and 19 healthy residents in rural Xinxiang, China, and tested the physiological parameters. The results indicated that microbial diversity, as well as KEGG orthology (KO) and carbohydrate-active enzymes (CAZymes) functions, deserved no significant disparities between CHD and healthy residents. The relative abundance of Bacteroidetes phylum was significantly lower and unclassified Lachnospiraceae genus, and Eubacterium rectale species were markedly higher in CHD residents compared with the healthy control. Co-occurrence network revealed a more diverse and scattered ecology in CHD residents. LEfSe identified 39 potential biomarkers and butanoate metabolism and glycosyltransferases families were the enhanced KO and CAZymes in CHD residents, respectively. Twenty key signatures were determined by the random forest algorithm and most of them belonged to the Clostridium cluster. These key signatures harbored a superior accuracy of 83.9% to distinguish CHD and healthy residents and, fasting serum insulin, diastolic blood pressure, and body mass index were the top three clinical parameters influencing the gut bacterial community. Furthermore, we also found that low-density lipoprotein and waist circumference had significantly positive correlations with the members of the Clostridium cluster. These findings expand our knowledge in the regional-specific pattern of gut microbiota for rural CHD residents and highlight the non-invasive diagnostic value and clinical correlations of microbial signatures.

[Pregnane X receptor promotes programmed cell death protein 4 expression in HepG2 cells].

OBJECTIVE: To investigate the role of pregnane X receptor (PXR) in the regulation of programmed cell death proteins (PDCDs) in HepG2 cells and explore the underlying molecular mechanism. METHODS: HepG2 cells were treated with PXR agonist rifampicin (10 µmol/L) or SR12813 (1 µmol/L) for 24 h, using DMSO as the negative control. HepG2 cells were infected with constitutively activated PXR adenovirus (VP-PXR) for 36 h, with the cells infected with Mock as the negative control. The mRNA levels of PDCD2, PDCD4, PDCD5, and PDCD6 and the expression of miRNA21 were detected using qRT-PCR, and the protein level of PDCD4 was detected with Western blotting. Bioinformatic analysis was performed to predict the potential PXRresponsive elements (PXREs) motifs in the promotor region of human PDCD4. RESULTS: The expressions of PDCD5 and PDCD6 mRNA did not differ significantly between rifampicin-treated and the control cells, while PDCD4 mRNA expression increased (t=4.209, P=0.008) and PDCD2 mRNA decreased significantly (t=-2.875, P=0.017) in rifampicin-treated cells. The mRNA expressions of PDCD2, PDCD5 and PDCD6 showed no significant difference between SR12813-treated cells and the control cells, while PDCD4 mRNA expression increased obviously in SR12813-treated cells (t=4.574, P=0.006). The PXR target gene MDR1 also increased significantly in the rifampicin- and SR12813-treated cells compared with the control cells (P=0.020 and 0.01, respectively). Infection of the cells with VP-PXR adenovirus resulted in significantly increased expression of PDCD4 and MDR1 mRNA as compared with Mock group (t=3.343, P=0.000; t=3.343, P=0.024, respectively) without causing obvious changes in PDCD2 and PDCD6 mRNA expressions. The protein level of PDCD4 increased significantly in both rifampicin (t= 2.779, P=0.025) group and VP- PXR group (t=3.066, P=0.012). The expression of miRNA21, the negative regulatory factor of PDCD4, did not differ significantly between PXR agonist group and the control group. Informatic analysis revealed the presence of putative PXREs in the 5'-flanking region of PDCD4 gene. CONCLUSIONS: Our findings demonstrate that PXR agonism in HepG2 cells increases the expression of PDCD4, which is independent of miRNA21 pathway, and PDCD4 may be a target gene of PXR in HepG2 cells.

Structure elucidation and formation mechanistic study of a methylene-bridged pregabalin dimeric degradant in pregabalin extended-release tablets.

During the pharmaceutical development of pregabalin extended-release tablets, an unknown degradant at a relative retention time (RRT) of 11.7 was observed and its nominal amount exceeded the ICH identification threshold in an accelerated stability study. The aim of this study is to identify the structure and investigate the formation mechanism of this impurity for the purpose of developing a chemically stable pharmaceutical product. By utilizing multi-stage LC-MS analysis in conjunction with mechanism-based stress study, the structure of the RRT 11.7 impurity was rapidly identified as a dimeric degradant that is caused by dimerization of two pregabalin molecules with a methylene bridging the two pregabalin moieties. The structure of the dimer was confirmed by 1D and 2D NMR measurement. The formation pathway of the dimeric degradant was also inferred from the mechanism-based stress study, which implicated that the bridging methylene could originate from formaldehyde which might be the culprit that triggers the dimerization in the first place. The subsequent API-excipients compatibility study indicated that the degradant was indeed formed in the compatibility experiments between pregabalin API and two polymeric excipients (PEO and PVPP) that are known to contain residual formaldehyde, but only in the co-presence of another excipient, colloidal silicon dioxide (SiO2). The kinetic behavior of the degradant formation was also investigated and two kinetic models were utilized based on the Arrhenius and Eyring equations, respectively, to calculate the activation energy (Ea) as well as the enthalpy of activation (△H‡), entropy of activation (△S‡), and Gibbs free energy (△G‡) of the degradation reaction. The results of this study would be useful for the understanding of similar dimeric degradant formation in finished products of drug substances containing primary or secondary amine moieties.

Facile fabrication of Ni0.85Se nanowires by the composite alkali salt method as a novel cathode material for asymmetric supercapacitors.

The search for Earth-abundant and efficient electrode materials is significant for advanced supercapacitors. Here we introduce a facile strategy for one-step synthesis of Ni0.85Se nanowires via a composite alkali salt method. When used as an electrode material in supercapacitors, the as-prepared Ni0.85Se nanowires exhibit a high specific capacitance of 1354 F g-1 at a current density of 1 A g-1, and still retain 671 F g-1 at 30 A g-1. The superior electrochemical performance of the Ni0.85Se electrode can be attributed to the metallic conductivity of nickel selenides and fast electrical transport along the axial direction due to the nanowire morphology. For practical applications, an asymmetric supercapacitor was assembled by using Ni0.85Se and activated carbon, which delivered a high energy density of 40.7 W h kg-1 at a power density of 800 W kg-1 and 12.1 W h kg-1 at 16 kW kg-1. Moreover, the device retained 92.4% specific capacitance after 20 000 cycles at a high current density of 5 A g-1, showing its promising application prospects.

Heteroatom-doped MoSe2 Nanosheets with Enhanced Hydrogen Evolution Kinetics for Alkaline Water Splitting.

Electrochemical water splitting for hydrogen generation is a vital part for the prospect of future energy systems, however, the practical utilization relies on the development of highly active and earth-abundant catalysts to boost the energy conversion efficiency as well as reduce the cost. Molybdenum diselenide (MoSe2 ) is a promising nonprecious metal-based electrocatalyst for hydrogen evolution reaction (HER) in acidic media, but it exhibits inferior alkaline HER kinetics in great part due to the sluggish water adsorption/dissociation process. Herein, the alkaline HER kinetics of MoSe2 is substantially accelerated by heteroatom doping with transition metal ions. Specifically, the Ni-doped MoSe2 nanosheets exhibit the most impressive catalytic activity in terms of lower overpotential and larger exchange current density. The density functional theory (DFT) calculation results reveal that Ni/Co doping plays a key role in facilitating water adsorption as well as optimizing hydrogen adsorption. The present work paves a new way to the development of low-cost and efficient electrocatalysts towards alkaline HER.

Melting and freezing behaviors of prototype ionic liquids, 1-butyl-3-methylimidazolium bromide and its chloride, studied by using a nano-Watt differential scanning calorimeter.

1-Butyl-3-methylimidazolium bromide ([bmim]Br) and its chloride ([bmim]Cl) are representative prototypes of ionic liquids. We investigated the melting and freezing behaviors of [bmim]Br and [bmim]Cl by using a homemade differential scanning calorimeter (DSC) with nano-Watt stability and sensitivity. The measurements were carried out at heating and cooling rates slow enough to mimic quasi-static processes. Their thermal behaviors of melting and freezing show characteristic features such as a wide pre-melting range and excessive supercooling and individual behaviors of single crystals even for the same substance. The melting temperatures of [bmim]Br and [bmim]Cl were determined from the broad DSC traces and discussed in relation to the crystal structure. We suggest that the observed characteristics are due to the dynamics of the cooperative change between gauche-trans (GT) and trans-trans (TT) conformations of the butyl group in the [bmim]+ cation.

17ß-Estradiol Protects the Retinal Nerve Cells Suppressing TLR2 Mediated Immune-Inflammation and Apoptosis from Oxidative Stress Insult Independent of PI3K.

The excessive apoptosis of retinal nerve cells (RNCs) could cause a variety of retinal neurodegenerative diseases which could result in the irreversible blindness. In this study, the experiment models of H2O2 and light-induced oxidative insult in the retina of Sprague-Dawley (SD) rat were used. We demonstrated the role of toll-like receptor 2 (TLR2) in apoptosis and immune-inflammation induced by oxidative stress insult. Meanwhile, we also tried to elucidate the modulating mechanism of 17ß-estradiol (E2) resistant to TLR2 induced by oxidative stress insult. The cell apoptosis induced by oxidative stress was examined by annexin V-FITC/propidium iodide (PI) assay using flow cytometry and the expressions of TLR2 and inflammatory cytokines were determined by real-time PCR and western blotting. Peptidoglycan (PGN) as the ligand of TLR2 and small interfering RNAs of TLR2 (siTLR2) were used to determine the role of TLR2. From the results, firstly, we demonstrated that E2 could reduce the expressions of TLR2 and inflammatory cytokines including TNF-ɑ, IFN-γ, and IL-1ß induced by oxidative stress; secondly, the phosphoinositide 3-kinase (PI3K) could not influence the effect of E2 on reducing TLR2 expression induced by H2O2 in RNCs; thirdly, PGN could promote the damage effect of H2O2 by transforming RNCs from late apoptosis to necrosis, however, E2 could decrease the cell apoptosis mediated by PGN; and finally, the apoptosis of RNCs and the expressions of the inflammatory cytokines induced by H2O2 administration were significantly inhibited after TLR2 interference. In summary, E2 reduces the TLR2-mediated immune-inflammation, thereby protecting RNCs against oxidative stress-induced apoptosis via a PI3K-independent signaling pathway. The present results provide evidence that inhibiting of TLR2-mediated immune-inflammation might be a possible therapeutic way to exert auxiliary role on E2 neuroprotection.

17ß-estradiol ameliorates light-induced retinal damage in Sprague-Dawley rats by reducing oxidative stress.

Oxidative stress is considered as a major cause of light-induced retinal neurodegeneration. The protective role of 17ß-estradiol (ßE2) in neurodegenerative disorders is well known, but its underlying mechanism remains unclear. Here, we utilized a light-induced retinal damage model to explore the mechanism by which ßE2 exerts its neuroprotective effect. Adult male and female ovariectomized (OVX) rats were exposed to 8,000 lx white light for 12 h to induce retinal light damage. Electroretinogram (ERG) assays and hematoxylin and eosin (H&E) staining revealed that exposure to light for 12 h resulted in functional damage to the rat retina, histological changes, and retinal neuron loss. However, intravitreal injection (IVI) of ßE2 significantly rescued this impaired retinal function in both female and male rats. Based on the level of malondialdehyde (MDA) production (a biomarker of oxidative stress), an increase in retinal oxidative stress followed light exposure, and ßE2 administration reduced this light-induced oxidative stress. Quantitative reverse-transcriptase (qRT)-PCR indicated that the messenger RNA (mRNA) levels of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (Gpx) were downregulated in female OVX rats but were upregulated in male rats after light exposure, suggesting a gender difference in the regulation of these antioxidant enzyme genes in response to light. However, ßE2 administration restored or enhanced the SOD and Gpx expression levels following light exposure. Although the catalase (CAT) expression level was insensitive to light stimulation, ßE2 also increased the CAT gene expression level in both female OVX and male rats. Further examination indicated that the antioxidant proteins thioredoxin (Trx) and nuclear factor erythroid 2-related factor 2 (Nrf2) are also involved in ßE2-mediated antioxidation and that the cytoprotective protein heme oxygenase-1 (HO-1) plays a key role in the endogenous defense mechanism against light exposure in a ßE2-independent manner. Taken together, we provide evidence that ßE2 protects against light-induced retinal damage via its antioxidative effect, and its underlying mechanism involves the regulation of the gene expression levels of antioxidant enzymes (SOD, CAT, and Gpx) and proteins (Trx and Nrf2). Our study provides conceptual evidence in support of estrogen replacement therapy for postmenopausal women to reduce the risk of age-related macular degeneration.

The involvement of PI3K-mediated and L-VGCC-gated transient Ca2+ influx in 17ß-estradiol-mediated protection of retinal cells from H2O2-induced apoptosis with Ca2+ overload.

Intracellular calcium concentration ([Ca(2+)]i) plays an important role in regulating most cellular processes, including apoptosis and survival, but its alterations are different and complicated under diverse conditions. In this study, we focused on the [Ca(2+)]i and its control mechanisms in process of hydrogen peroxide (H2O2)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17ß-estradiol (ßE2) anti-apoptosis. Fluo-3AM was used as a Ca(2+) indicator to detect [Ca(2+)]i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 µM H2O2-induced retinal cell apoptosis occurred at 4 h after H2O2 stress and increased in a time-dependent manner, but [Ca(2+)]i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H2O2 stress; b) 10 µM ßE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca(2+)]i, which appeared only at 0.5 h after ßE2 application; c) increased [Ca(2+)]i under 100 µM H2O2 treatment for 2 hrs or 10 µM ßE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca(2+) stores; d) importantly, the transiently increased [Ca(2+)]i induced by 10 µM ßE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca(2+) channels (L-VGCC), but the increased [Ca(2+)]i induced by 100 µM H2O2 treatment for 2 hrs was not affected; and e) pretreatment with 10 µM ßE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H2O2, which was also associated with its transient [Ca(2+)]i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of ßE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.

17ß-estradiol impedes Bax-involved mitochondrial apoptosis of retinal nerve cells induced by oxidative damage via the phosphatidylinositol 3-kinase/Akt signal pathway.

Oxidative stress leading to retinal nerve cells (RNCs) apoptosis is a major cause of neurodegenerative disorders of the retina. 17ß-Estradiol (E2) has been suggested to be a neuroprotective agent in the central nervous system; however, at present, the underlying mechanisms are not well understood, and the related research on the RNCs is less reported. Here, in order to investigate the protective role and mechanism of E2 against oxidative stress-induced damage on RNCs, the transmission electron microscopy and annexin V-FITC/propidium iodide assay were applied to detect the RNCs apoptosis. Western blot and real-time PCR were used to determine the expression of the critical molecules in Bcl-2 and caspase family associated with apoptosis. The transmission electron microscopy results showed that H(2)O(2) could induce typical features of apoptosis in RNCs, including formation of the apoptosome. E2 could, however, suppress the H(2)O(2)-induced morphological changes of apoptosis. Intriguingly, we observed E2-mediated phagocytic scavenging of apoptosome. In response to H(2)O(2)-induced apoptosis, Bax, acting as one of the pivotal pro-apoptotic members of Bcl-2 family, increased significantly, which directly resulted in an increased ratio of Bax to anti-apoptotic protein Bcl-2 (Bax/Bcl-2). Additionally, caspases 9 and 3, which are the critical molecules of the mitochondrial apoptosis pathway, were activated by H(2)O(2). In contrast, E2 exerted anti-apoptotic effects by reducing the expression of Bax to decrease the ratio of Bax/Bcl-2 and impeded the caspases 9/3 activation. Moreover, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, could sharply block the effect of E2 in reducing the percentage of apoptotic cells resistance to H(2)O(2). And the attenuation of Bax, the reduced activities of caspases 9/3 and the impeded release of mitochondrial cytochrome c mediated by E2 resistance to H(2)O(2) damage were significantly retrieved by LY294002 administration. Taken together, E2 protects the RNCs against H(2)O(2)-induced apoptosis by significantly inhibiting the Bax-involved mitochondrial apoptosis via the activation of PI3K/Akt signal pathway.

Mitogen-activated protein kinase pathways are involved in the upregulation of calcitonin gene-related peptide of rat trigeminal ganglion after organ culture.

The trigeminal ganglion (TG) can express and release calcitonin gene-related peptide (CGRP), an important neuropeptide that plays a crucial role in migraine attack and cluster headache. Activation of rat TG increases CGRP expression. However, the regulatory mechanism of CGRP expression in TG neurons remains to be explored. This study aims to evaluate the involvement of mitogen-activated protein kinase (MAPK) pathways in CGRP upregulation after rat TG organ culture. Rat TG was cultured alone for 24 h or cultured in combination with MAPK inhibitors, tumor necrosis factor α (TNF-α), or interleukin 1ß (IL-1ß) for 24 h. CGRP protein was determined using immunohistochemistry. The mRNA levels of CGRP, TNF-α, and IL-1ß were analyzed through real-time quantitative polymerase chain reaction. MAPK phosphorylation was detected via western blot. After rat TG organ culture, the expressions of CGRP, TNF-α, and IL-1ß were upregulated at 24 h. The phosphorylation of extracellular signal-regulated kinases (ERK1/2), P38, and c-jun N-terminal kinases (JNK) significantly increased at 30 min compared with fresh rat TG. In addition, both CGRP expression and phosphorylation of ERK1/2, P38, and JNK were enhanced obviously after rat TG treatment with TNF-α or IL-1ß compared with fresh rat TG. However, they decreased markedly after rat TG pretreatment with PD98059 (ERK1/2 inhibitor), SB203580 (P38 inhibitor), or SP600125 (JNK inhibitor) compared with rat TG co-culture with TNF-α or IL-1ß. In conclusion, the elevated CGRP expression after rat TG organ culture can be regulated via MAPK pathways. The findings provide insight into the molecular mechanisms and experimental evidence for therapeutic targets of migraine.