Investigation on the Q-markers of Bushen Huoxue Prescriptions for DR treatment based on chemometric methods and spectrum-effect relationship.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetic retinopathy (DR) is a kind of complex complication of late diabetes mellitus with high incidence and risk of blindness. Bushen Huoxue Prescription (BHP), which consists of Rehmanniae radix (RR), Salviae miltiorrhizae radix et rhizoma (SMRR), Ginseng radix et rhizome (GRR) and Puerariae lobatae radix (PLR), has an active effect on the treatment of DR. However, the quality markers (Q-markers) of BHP are not entirely clear. PURPOSE: This study aimed to screen the Q-markers of BHP for DR treatment based on the establishment of spectrum-effect relationship and verified experiment. MATERIALS AND METHODS: In this study, 12 BHP samples (S1-S12) for fingerprint analysis and pharmacological evaluation were prepared according to a four-factor and twelve-level uniform design. High performance liquid chromatography-ultraviolet detector-evaporative light scattering detector (HPLC-UV-ELSD) was employed to analyze the fingerprint on the basis of the characteristics of BHP components. The evaluation of sample similarity was carried out by similarity analysis (SA) and hierarchical cluster analysis (HCA). The pharmacological indicators, including expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in the retina of Sprague Dawley (SD) rats induced by streptozotocin (STZ), were detected by enzyme-linked immunosorbent assay (ELISA). Besides, the spectrum-effect relationship between common peaks of fingerprints and the pharmacological results was investigated by partial least squares regression (PLSR) and canonical correlation analysis (CCA). The results of spectrum-effect relationship were verified by the expression of VEGF and HIF-1α on primary culture retinal Müller cells induced by hyperglycemia and hypoxia. RESULTS: In the HPLC-UV-ELSD fingerprint, 23 common peaks in UV and 14 common peaks in ELSD were identified. The pharmacological results indicated that the expression of VEGF and HIF-1α in the retina of SD rats was inhibited by 12 BHP samples to varying degrees compared with the model group. Based on SA and heatmap of HCA, S4 and S8 were clearly distinguished from other samples. The results of PLSR and CCA revealed that the contents of puerarin, daidzin, salvianolic acid B and ginsenoside Rb1 were inversely correlated with the expression of VEGF and HIF-1α. Hence, the four compounds may be the main active components to prevent and treat DR. The results of intervention on primary culture retinal Müller cells showed that puerarin, daidzin, salvianolic acid B, and ginsenoside Rb1 can significantly inhibit the expression of VEGF and HIF-1α. CONCLUSIONS: The spectrum-effect relationship of BHP was successfully established, and the Q-markers of BHP for the prevention and treatment of DR were preliminarily confirmed. It provides a feasible method for the research of quality control.

670nm photobiomodulation modulates bioenergetics and oxidative stress, in rat Müller cells challenged with high glucose.

Diabetic retinopathy (DR), the most common complication of diabetes mellitus, is associated with oxidative stress, nuclear factor-κB (NFκB) activation, and excess production of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1). Muller glial cells, spanning the entirety of the retina, are involved in DR inflammation. Mitigation of DR pathology currently occurs via invasive, frequently ineffective therapies which can cause adverse effects. The application of far-red to near-infrared (NIR) light (630-1000nm) reduces oxidative stress and inflammation in vitro and in vivo. Thus, we hypothesize that 670nm light treatment will diminish oxidative stress preventing downstream inflammatory mechanisms associated with DR initiated by Muller cells. In this study, we used an in vitro model system of rat Müller glial cells grown under normal (5 mM) or high (25 mM) glucose conditions and treated with a 670 nm light emitting diode array (LED) (4.5 J/cm2) or no light (sham) daily. We report that a single 670 nm light treatment diminished reactive oxygen species (ROS) production and preserved mitochondrial integrity in this in vitro model of early DR. Furthermore, treatment for 3 days in culture reduced NFκB activity to levels observed in normal glucose and prevented the subsequent increase in ICAM-1. The ability of 670nm light treatment to prevent early molecular changes in this in vitro high glucose model system suggests light treatment could mitigate early deleterious effects modulating inflammatory signaling and diminishing oxidative stress.

Interleukin-6 actions in the hypothalamus protects against obesity and is involved in the regulation of neurogenesis.

BACKGROUND: Interleukin-6 (IL6) produced in the context of exercise acts in the hypothalamus reducing obesity-associated inflammation and restoring the control of food intake and energy expenditure. In the hippocampus, some of the beneficial actions of IL6 are attributed to its neurogenesis-inducing properties. However, in the hypothalamus, the putative neurogenic actions of IL6 have never been explored, and its potential to balance energy intake can be an approach to prevent or attenuate obesity. METHODS: Wild-type (WT) and IL6 knockout (KO) mice were employed to study the capacity of IL6 to induce neurogenesis. We used cell labeling with Bromodeoxyuridine (BrdU), immunofluorescence, and real-time PCR to determine the expression of markers of neurogenesis and neurotransmitters. We prepared hypothalamic neuroprogenitor cells from KO that were treated with IL6 in order to provide an ex vivo model to further characterizing the neurogenic actions of IL6 through differentiation assays. In addition, we analyzed single-cell RNA sequencing data and determined the expression of IL6 and IL6 receptor in specific cell types of the murine hypothalamus. RESULTS: IL6 expression in the hypothalamus is low and restricted to microglia and tanycytes, whereas IL6 receptor is expressed in microglia, ependymocytes, endothelial cells, and astrocytes. Exogenous IL6 reduces diet-induced obesity. In outbred mice, obesity-resistance is accompanied by increased expression of IL6 in the hypothalamus. IL6 induces neurogenesis-related gene expression in the hypothalamus and in neuroprogenitor cells, both from WT as well as from KO mice. CONCLUSION: IL6 induces neurogenesis-related gene expression in the hypothalamus of WT mice. In KO mice, the neurogenic actions of IL6 are preserved; however, the appearance of new fully differentiated proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons is either delayed or disturbed.

Dietary Supplement Enriched in Antioxidants and Omega-3 Promotes Glutamine Synthesis in Müller Cells: A Key Process against Oxidative Stress in Retina.

To prevent ocular pathologies, new generation of dietary supplements have been commercially available. They consist of nutritional supplement mixing components known to provide antioxidative properties, such as unsaturated fatty acid, resveratrol or flavonoids. However, to date, only one preclinical study has evaluated the impact of a mixture mainly composed of those components (Nutrof Total®) on the retina and demonstrated that in vivo supplementation prevents the retina from structural and functional injuries induced by light. Considering the crucial role played by the glial Müller cells in the retina, particularly to regulate the glutamate cycle to prevent damage in oxidative stress conditions, we questioned the impact of this ocular supplement on the glutamate metabolic cycle. To this end, various molecular aspects associated with the glutamate/glutamine metabolism cycle in Müller cells were investigated on primary Müller cells cultures incubated, or not, with the commercially mix supplement before being subjected, or not, to oxidative conditions. Our results demonstrated that in vitro supplementation provides guidance of the glutamate/glutamine cycle in favor of glutamine synthesis. These results suggest that glutamine synthesis is a crucial cellular process of retinal protection against oxidative damages and could be a key step in the previous in vivo beneficial results provided by the dietary supplementation.

Moderate Prenatal Ethanol Exposure Stimulates CXCL12/CXCR4 Chemokine System in Radial Glia Progenitor Cells in Hypothalamic Neuroepithelium and Peptide Neurons in Lateral Hypothalamus of the Embryo and Postnatal Offspring.

BACKGROUND: Prenatal exposure to ethanol (EtOH) has lasting effects on neuropeptide and neuroimmune systems in the brain alongside detrimental alcohol-related behaviors. At low-to-moderate doses, prenatal EtOH stimulates neurogenesis in lateral hypothalamus (LH) and increases neurons that express the orexigenic peptides hypocretin/orexin (Hcrt/OX) and melanin-concentrating hormone (MCH), and the proinflammatory chemokine CCL2, which through its receptor CCR2 stimulates cell differentiation and movement. Our recent studies demonstrated that CCL2 and CCR2 colocalize with MCH neurons and are involved in EtOH's stimulatory effect on their development but show no relation to Hcrt/OX. Here, we investigated another chemokine, CXCL12, and its receptor, CXCR4, which promote neurogenesis and neuroprogenitor cell proliferation, to determine if they also exhibit peptide specificity in their response to EtOH exposure. METHODS: Pregnant rats were intraorally administered a moderate dose of EtOH (2 g/kg/d) from embryonic day 10 (E10) to E15. Their embryos and postnatal offspring were examined using real-time quantitative PCR and immunofluorescence histochemistry, to determine if EtOH affects CXCL12 and CXCR4 and the colocalization of CXCR4 with Hcrt/OX and MCH neurons in the LH and with radial glia neuroprogenitor cells in the hypothalamic neuroepithelium (NEP). RESULTS: Prenatal EtOH strongly stimulated CXCL12 and CXCR4 in LH neurons of embryos and postnatal offspring. This stimulation was significantly stronger in Hcrt/OX than MCH neurons in LH and also occurred in radial glia neuroprogenitor cells dense in the NEP. These effects were sexually dimorphic, consistently stronger in females than males. CONCLUSIONS: While showing prenatal EtOH exposure to have a sexually dimorphic, stimulatory effect on CXCL12 and CXCR4 in LH similar to CCL2 and its receptor, these results reveal their distinct relationship to the peptide neurons, with the former closely related to Hcrt/OX and the latter to MCH, and they link EtOH's actions in LH to a stimulatory effect on neuroprogenitor cells in the NEP.

n-3 Fatty Acid and Its Metabolite 18-HEPE Ameliorate Retinal Neuronal Cell Dysfunction by Enhancing Müller BDNF in Diabetic Retinopathy.

Diabetic retinopathy (DR) is a widespread vision-threatening disease, and neuroretinal abnormality should be considered as an important problem. Brain-derived neurotrophic factor (BDNF) has recently been considered as a possible treatment to prevent DR-induced neuroretinal damage, but how BDNF is upregulated in DR remains unclear. We found an increase in hydrogen peroxide (H2O2) in the vitreous of patients with DR. We confirmed that human retinal endothelial cells secreted H2O2 by high glucose, and H2O2 reduced cell viability of MIO-M1, Müller glia cell line, PC12D, and the neuronal cell line and lowered BDNF expression in MIO-M1, whereas BDNF administration recovered PC12D cell viability. Streptozocin-induced diabetic rats showed reduced BDNF, which is mainly expressed in the Müller glia cell. Oral intake of eicosapentaenoic acid ethyl ester (EPA-E) ameliorated BDNF reduction and oscillatory potentials (OPs) in electroretinography (ERG) in DR. Mass spectrometry revealed an increase in several EPA metabolites in the eyes of EPA-E-fed rats. In particular, an EPA metabolite, 18-hydroxyeicosapentaenoic acid (18-HEPE), induced BDNF upregulation in Müller glia cells and recovery of OPs in ERG. Our results indicated diabetes-induced oxidative stress attenuates neuroretinal function, but oral EPA-E intake prevents retinal neurodegeneration via BDNF in Müller glia cells by increasing 18-HEPE in the early stages of DR.

Tanycytic TSPO inhibition induces lipophagy to regulate lipid metabolism and improve energy balance.

Hypothalamic glial cells named tanycytes, which line the 3rd ventricle (3V), are components of the hypothalamic network that regulates a diverse array of metabolic functions for energy homeostasis. Herein, we report that TSPO (translocator protein), an outer mitochondrial protein, is highly enriched in tanycytes and regulates homeostatic responses to nutrient excess as a potential target for an effective intervention in obesity. Administration of a TSPO ligand, PK11195, into the 3V, and tanycyte-specific deletion of Tspo reduced food intake and elevated energy expenditure, leading to negative energy balance in a high-fat diet challenge. Ablation of tanycytic Tspo elicited AMPK-dependent lipophagy, breaking down lipid droplets into free fatty acids, thereby elevating ATP in a lipid stimulus. Our findings suggest that tanycytic TSPO affects systemic energy balance through macroautophagy/autophagy-regulated lipid metabolism, and highlight the physiological significance of TSPO in hypothalamic lipid sensing and bioenergetics in response to overnutrition. ABBREVIATIONS: 3V: 3rd ventricle; ACAC: acetyl-Coenzyme A carboxylase; AGRP: agouti related neuropeptide; AIF1/IBA1: allograft inflammatory factor 1; AMPK: AMP-activated protein kinase; ARC: arcuate nucleus; Atg: autophagy related; Bafilo: bafilomycin A1; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CNS: central nervous system; COX4I1: cytochrome c oxidase subunit 4I1; FFA: free fatty acid; GFAP: glial fibrillary acidic protein; HFD: high-fat diet; ICV: intracerebroventricular; LAMP2: lysosomal-associated membrane protein 2; LD: lipid droplet; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MBH: mediobasal hypothalamus; ME: median eminence; MEF: mouse embryonic fibroblast; NCD: normal chow diet; NEFM/NFM: neurofilament medium; NPY: neuropeptide Y; OL: oleic acid; POMC: pro-opiomelanocortin-alpha; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Rax: retina and anterior neural fold homeobox; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RER: respiratory exchange ratio; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TG: triglyceride; TSPO: translocator protein; ULK1: unc-51 like kinase 1; VCO2: carbon dioxide production; VMH: ventromedial hypothalamus; VO2: oxygen consumption.

Thiamine transporter 2 is involved in high glucose-induced damage and altered thiamine availability in cell models of diabetic retinopathy.

Thiamine prevents high glucose-induced damage in microvasculature, and progression of retinopathy and nephropathy in diabetic animals. Impaired thiamine availability causes renal damage in diabetic patients. Two single-nucleotide polymorphisms in SLC19A3 locus encoding for thiamine transporter 2 are associated with absent/minimal diabetic retinopathy and nephropathy despite long-term type 1 diabetes. We investigated the involvement of thiamine transporter 1 and thiamine transporter 2, and their transcription factor specificity protein 1, in high glucose-induced damage and altered thiamine availability in cells of the inner blood-retinal barrier. Human endothelial cells, pericytes and Müller cells were exposed to hyperglycaemic-like conditions and/or thiamine deficiency/over-supplementation in single/co-cultures. Expression and localization of thiamine transporter 1, thiamine transporter 2 and transcription factor specificity protein 1 were evaluated together with intracellular thiamine concentration, transketolase activity and permeability to thiamine. The effects of thiamine depletion on cell function (viability, apoptosis and migration) were also addressed. Thiamine transporter 2 and transcription factor specificity protein 1 expression were modulated by hyperglycaemic-like conditions. Transketolase activity, intracellular thiamine and permeability to thiamine were decreased in cells cultured in thiamine deficiency, and in pericytes in hyperglycaemic-like conditions. Thiamine depletion reduced cell viability and proliferation, while thiamine over-supplementation compensated for thiamine transporter 2 reduction by restoring thiamine uptake and transketolase activity. High glucose and reduced thiamine determine impairment in thiamine transport inside retinal cells and through the inner blood-retinal barrier. Thiamine transporter 2 modulation in our cell models suggests its major role in thiamine transport in retinal cells and its involvement in high glucose-induced damage and impaired thiamine availability.

Characterisation of endogenous players in fibroblast growth factor-regulated functions of hypothalamic tanycytes and energy-balance nuclei.

The mammalian hypothalamus regulates key homeostatic and neuroendocrine functions ranging from circadian rhythm and energy balance to growth and reproductive cycles via the hypothalamic-pituitary and hypothalamic-thyroid axes. In addition to its neurones, tanycytes are taking centre stage in the short- and long-term augmentation and integration of diverse hypothalamic functions, although the genetic regulators and mediators of their involvement are poorly understood. Exogenous interventions have implicated fibroblast growth factor (FGF) signalling, although the focal point of the action of FGF and any role for putative endogenous players also remains elusive. We carried out a comprehensive high-resolution screen of FGF signalling pathway mediators and modifiers using a combination of in situ hybridisation, immunolabelling and transgenic reporter mice, aiming to map their spatial distribution in the adult hypothalamus. Our findings suggest that ß-tanycytes are the likely focal point of exogenous and endogenous action of FGF in the third ventricular wall, utilising FGF receptor (FGFR)1 and FGFR2 IIIc isoforms, but not FGFR3. Key IIIc-activating endogenous ligands include FGF1, 2, 9 and 18, which are expressed by a subset of ependymal and parenchymal cells. In the parenchymal compartment, FGFR1-3 show divergent patterns, with FGFR1 being predominant in neuronal nuclei and expression of FGFR3 being associated with glial cell function. Intracrine FGFs are also present, suggestive of multiple modes of FGF function. Our findings provide a testable framework for understanding the complex role of FGFs with respect to regulating the metabolic endocrine and neurogenic functions of hypothalamus in vivo.

Notoginsenoside R1 Ameliorates Diabetic Retinopathy through PINK1-Dependent Activation of Mitophagy.

: Accumulating evidence has indicated that inflammation, oxidative stress, apoptosis, and autophagy in retinal Müller cells are involved in diabetic retinopathy (DR). Notoginsenoside R1 (NGR1), a novel saponin extracted from Panax notoginseng, posesses pharmacological properties, including treating diabetic encephalopathy and improving microcirculatory disorders. Nevertheless, its beneficial effects on DR and the potential mechanism remain to be elucidated. In this study, we found retinal vascular degeneration, reduced retinal thickness, and impaired retinal function in db/db mice were all dramatically attenuated by oral treatment with NGR1 (30 mg/kg) for 12 weeks. NGR1 pretreatment also significantly inhibited apoptosis, markedly suppressed the VEGF expression, markedly increased PEDF expression and markedly inhibited oxidative stress and inflammation in rat retinal Müller cells (rMC-1) subjected to high glucose (HG) and in the retinas of db/db mice. Furthermore, NGR1 pre-treatment upregulated the level of PINK1 and Parkin, increased the LC3-II/LC3-I ratio, and downregulated the level of p62/SQSTM1 in rMC-1 cells induced by HG and in the retinas of db/db mice. Moreover, NGR1 administration enhanced the co-localization of GFP-LC3 puncta and MitoTracker in rMC-1 cells. Importantly, knockdown of PINK1 abolished the protective effects of NGR1. In conclusion, these phenomena suggested that NGR1 prevented DR via PINK1-dependent enhancement of mitophagy.

Müller glial dysfunction during diabetic retinopathy in rats is reduced by the acrolein-scavenging drug, 2-hydrazino-4,6-dimethylpyrimidine.

AIMS/HYPOTHESIS: Recent studies suggest that abnormal function in Müller glial cells plays an important role in the pathogenesis of diabetic retinopathy. This is associated with the selective accumulation of the acrolein-derived advanced lipoxidation end-product, Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine), on Müller cell proteins. The aim of the current study was to identify more efficacious acrolein-scavenging drugs and determine the effects of the most potent on Müller cell FDP-lysine accumulation and neuroretinal dysfunction during diabetes. METHODS: An ELISA-based in vitro assay was optimised to compare the acrolein-scavenging abilities of a range of drugs. This identified 2-hydrazino-4,6-dimethylpyrimidine (2-HDP) as a new and potent acrolein scavenger. The ability of this agent to modify the development of diabetic retinopathy was tested in vivo. Male Sprague Dawley rats were divided into three groups: (1) non-diabetic; (2) streptozotocin-induced diabetic; and (3) diabetic treated with 2-HDP in their drinking water for the duration of diabetes. Liquid chromatography high-resolution mass spectrometry was used to detect 2-HDP reaction products in the retina. Immunohistochemistry, real-time quantitative (q)RT-PCR and electroretinography were used to assess retinal changes 3 months after diabetes induction. RESULTS: 2-HDP was the most potent of six acrolein-scavenging agents tested in vitro (p < 0.05). In vivo, administration of 2-HDP reduced Müller cell accumulation of FDP-lysine at 3 months in rats rendered diabetic with streptozotocin (p < 0.001). A 2-HDP adduct was identified in the retinas of diabetic animals treated with this compound. 2-HDP supplementation was associated with reduced Müller cell gliosis (p < 0.05), reduced expression of the oxidative stress marker haem oxygenase-1 (p < 0.001) and partial normalisation of inwardly rectifying K+ channel 4.1 (Kir4.1) expression (p < 0.001 for staining in perivascular regions and the innermost region of the ganglion cell layer). Diabetes-induced retinal expression of inflammatory markers, inflammatory signalling compounds and activation of retinal microglial cells were all reduced in 2-HDP-treated animals. Retinal neurophysiological defects in diabetic animals, as indicated by changes in the electroretinogram 7 weeks after induction of diabetes, were also reduced by 2-HDP (p < 0.05-0.01 for b-wave amplitudes at flash intensities from -10 to +10 dB; p < 0.01 for time to peak of summed oscillatory potentials at +10 dB). CONCLUSIONS/INTERPRETATION: These findings support the hypothesis that Müller cell accumulation of FDP-lysine plays an important role in the development of diabetic retinopathy. Our results also suggest that 2-HDP may have therapeutic potential for delaying or treating this sight-threatening complication.

Protective effects of Erigeron breviscapus Hand.- Mazz. (EBHM) extract in retinal neurodegeneration models.

Purpose: To investigate the neuroprotective effects of scutellarin, an active component of the multifunctional traditional Chinese herb Erigeron breviscapus (vant.) Hand.-Mazz. (EBHM), which has been used as a neuroprotective therapy for cerebrovascular diseases. We performed the experiments using in vitro and in vivo models of retinal neurodegeneration. Methods: In the in vitro experiments, we exposed BV-2 cells to low oxygen levels in an incubator for 24 and 48 h to generate hypoxia models. We then treated these cells with scutellarin at concentrations of 2, 10, and 50 µM. Cell viability was measured using an enzyme-linked immunosorbent assay (ELISA). The levels of the components of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome signaling pathway, including NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cleaved caspase-1, interleukin-18 (IL-18), and IL-1ß were analyzed using western blots and ELISAs. In the in vivo study, we raised the intraocular pressure of Brown Norway rats to 60 mmHg for 30 min to generate a high intraocular pressure (HIOP) model, that is, an acute glaucoma model. The rats were then treated with scutellarin via oral gavage for 2 consecutive weeks. The relevant components of the NLRP3 inflammasome signaling pathway were analyzed with western blots and ELISAs. Retinal ganglion cells (RGCs) were retrogradely labeled using 4% Fluoro-Gold, and then the numbers of cells were calculated. Retinal microglial cells were labeled using immunofluorescence, and then the morphological changes were observed. Results: In the in vitro cell viability experiments, 50 µM scutellarin statistically significantly enhanced the viability rate when compared to 2 µM and 10 µM scutellarin (hypoxia + 50 µM EBHM group: 94.01±2.130% and 86.02±2.520% after 24 and 48 h, respectively; hypoxia model group: 74.98±3.860% and 64.41±4.890% after 24 and 48 h, respectively; for all when compared to normal control, p<0.001). Scutellarin inhibited the expression of NLRP3 in vitro (the hypoxia + EBHM group/normal control group ratio versus the hypoxia model group/normal control group ratio: 2.30±0.12 versus 4.06±0.19, p<0.01) and in vivo (the HIOP + EBHM group/normal control group ratio versus the HIOP model group/normal control ratio: 3.39±0.42 versus 6.07±0.22, p<0.01). Scutellarin administration also reduced the upregulation of ASC, cleaved caspase-1, IL-18, and IL-1ß in vitro and in vivo. In the in vivo study, the RGC survival rate was statistically significantly improved following scutellarin administration (p<0.001 versus the HIOP group), and the number of impaired retinal microglial cells was statistically significantly reduced following scutellarin treatment when compared with the HIOP model group. Conclusions: EBHM extract scutellarin exhibits protective effects in retinal hypoxia models by inhibiting NLRP3 inflammasome-mediated inflammatory reactions. Thus, EBHM extract scutellarin may be an appropriate therapeutic option for disorders related to retinal neurodegeneration, such as glaucoma.

Endothelin-2 Injures the Blood-Retinal Barrier and Macroglial Müller Cells: Interactions with Angiotensin II, Aldosterone, and NADPH Oxidase.

Although increasing evidence indicates that endothelin-2 (Edn2) has distinct roles in tissue pathology, including inflammation, glial cell dysfunction, and angiogenesis, its role in the retina and the factors that regulate its actions are not fully understood. We hypothesized that Edn2 damages the blood-retinal barrier (BRB) and that this is mediated by interactions with the renin-angiotensin-aldosterone system and reactive oxygen species derived from NADPH oxidase (Nox). C57BL/6J mice received an intravitreal injection of Edn2 or control vehicle to examine the blood pressure-independent effects of Edn2. Mice administered Edn2 were randomized to receive by intraperitoneal injection treatments that inhibited the Edn type a receptor, Edn type b receptor, angiotensin type 1 receptor, mineralocorticoid receptor, or Nox isoforms 1 to 4. One month later, mice administered Edn2 exhibited breakdown of the BRB with increased vascular leakage, vascular endothelial growth factor expression, and infiltrating macrophages (Ly6C+CD45highCD11b+). Further, macroglial Müller cells, which influence the integrity of the BRB and prevent retinal edema, became gliotic and expressed increased levels of water (aquaporin-4) and ion (Kir4.1) channels. This Edn2-mediated retinopathy was reduced by all treatments. Complementary in vitro studies in cultured Müller cells supported these findings and demonstrated the importance of reactive oxygen species in mediating these events. In conclusion, Edn2 has detrimental effects on the BRB and Müller cells that involve interactions with the renin-angiotensin aldosterone system and Nox1/4.

Cholesterol regulates polymodal sensory transduction in Müller glia.

Over- and underexposure to cholesterol activates glia in neurodegenerative brain and retinal diseases but the molecular targets of cholesterol in glial cells are not known. Here, we report that disruption of unesterified membrane cholesterol content modulates the transduction of chemical, mechanical and temperature stimuli in mouse Müller cells. Activation of TRPV4 (transient receptor potential vanilloid type 4), a nonselective polymodal cation channel was studied following the removal or supplementation of cholesterol using the methyl-beta cyclodextrin (MßCD) delivery vehicle. Cholesterol extraction disrupted lipid rafts and caveolae without affecting TRPV4 trafficking or membrane localization protein. However, MßCD suppressed agonist (GSK1016790A)- and temperature-evoked elevations in [Ca2+ ]i , and suppressed transcellular propagation of Ca2+ waves. Lowering the free membrane cholesterol content markedly prolonged the time-course of the glial swelling response, whereas MßCD:cholesterol supplementation enhanced agonist- and temperature-induced Ca2+ signals and shortened the swelling response. Taken together, these data show that membrane cholesterol modulates polymodal transduction of agonists, swelling and temperature stimuli in retinal radial glia and suggest that dyslipidemic retinas might be associated with abnormal glial transduction of ambient sensory inputs.

In vitro studies on nobiletin isolated from citrus plants and the bioactive metabolites, inhibitory action against gelatinase enzymatic activity and the molecular mechanisms in human retinal Müller cell line.

Diabetic retinopathy (DR) is the most common cause of vision loss in patients with diabetes mellitus. Despite the presence of effective therapy, DR is still a significant health burden. A recent research suggests that matrix metalloproteinases (MMPs) could be promising targets, which exert multiple actions on early- and late-stage pathogenesis of DR. Among the MMP family, gelatinases (MMP-2 and MMP-9) act as potent proinflammatory, proangiogenic, and pro-apoptotic factors. Therefore, the pharmacological inhibitory effect of gelatinases on retinal MMP-producing cells may be useful in the treatment or prevention of DR. Nobiletin isolated from citrus plants is a multi-functional polymethoxylated flavone, which exerts biological effects including inhibitory action against MMP activity in several cancer cells. In the present study, we demonstrated that nobiletin isolated from citrus plants attenuated MMP-9 enzymatic activity through the suppression of transcription for MMP-9 gene expression and augmentation of TIMP-1 production in retinal Müller cells. Nobiletin regulated MMP-9 gene expression and TIMP-1 by inhibiting the PI3K/Akt signaling pathway. In addition, we observed the augmentation of inhibitory action against MMP-9 enzymatic activity by 4'-demethylated nobiletin, which is a major metabolite of nobiletin. We believe that the enhancement of inhibitory action against MMP-9 enzymatic activity by 4'-demethylated nobiletin is through the dual inhibition on Erk1/2 and Akt phosphorylation. The structure-activity relationship analysis revealed that, for the enhancement of inhibitory action against MMP-9 enzymatic activity, demethylation at position 4' in B-ring was a key structural modification in Müller cells, which are an important source of MMPs found in vitreous fluid and retinal tissues in retinal proliferative diseases. These results suggested that nobiletin, derived from a natural source, may serve as a novel MMP inhibitor with minimal side effects, and lead compound for the design of more efficacious drugs.

Melatonin delays photoreceptor degeneration in a mouse model of autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) comprises a group of incurable inherited retinal degenerations. Targeting common processes, instead of mutation-specific treatment, has proven to be an innovative strategy to combat debilitating retinal degeneration. Growing evidence indicates that melatonin possesses a potent activity against neurodegenerative disorders by mitigating cell damage associated with apoptosis and inflammation. Given the pleiotropic role of melatonin in central nervous system, the aim of the present study was to investigate whether melatonin would afford protection against retinal degeneration in autosomal recessive RP (arRP). Rd10, a well-characterized murine model of human arRP, received daily intraperitoneal injection of melatonin (15 mg/kg) between postnatal day (P) 13 and P30. Retinas treated with melatonin or vehicle were harvested for analysis at P30 and P45, respectively. The findings showed that melatonin could dampen the photoreceptors death and delay consequent retinal degeneration. We also observed that melatonin weakened the expression of glial fibrillary acidic protein (GFAP) in Müller cells. Additionally, melatonin could alleviate retinal inflammatory response visualized by IBA1 staining, which was further corroborated by downregulation of inflammation-related genes, such as tumor necrosis factor alpha (Tnf-α), chemokine (C-C motif) ligand 2 (Ccl2), and chemokine (C-X-C motif) ligand 10 (Cxcl10). These data revealed that melatonin could ameliorate retinal degeneration through potentially attenuating apoptosis, reactive gliosis, and microglial activation in rd10 mice. Moreover, these results suggest melatonin as a promising agent improving photoreceptors survival in human RP.

A seasonal switch in histone deacetylase gene expression in the hypothalamus and their capacity to modulate nuclear signaling pathways.

Seasonal animals undergo changes in physiology and behavior between summer and winter conditions. These changes are in part driven by a switch in a series of hypothalamic genes under transcriptional control by hormones and, of recent interest, inflammatory factors. Crucial to the control of transcription are histone deacetylases (HDACs), generally acting to repress transcription by local histone modification. Seasonal changes in hypothalamic HDAC transcripts were investigated in photoperiod-sensitive F344 rats by altering the day-length (photoperiod). HDAC4, 6 and 9 were found to change in expression. The potential influence of HDACs on two hypothalamic signaling pathways that regulate transcription, inflammatory and nuclear receptor signaling, was investigated. For inflammatory signaling the focus was on NF-κB because of the novel finding made that its expression is seasonally regulated in the rat hypothalamus. For nuclear receptor signaling it was discovered that expression of retinoic acid receptor beta was regulated seasonally. HDAC modulation of NF-κB-induced pathways was examined in a hypothalamic neuronal cell line and primary hypothalamic tanycytes. HDAC4/5/6 inhibition altered the control of gene expression (Fos, Prkca, Prkcd and Ptp1b) by inducers of NF-κB that activate inflammation. These inhibitors also modified the action of nuclear receptor ligands thyroid hormone and retinoic acid. Thus seasonal changes in HDAC4 and 6 have the potential to epigenetically modify multiple gene regulatory pathways in the hypothalamus that could act to limit inflammatory pathways in the hypothalamus during long-day summer-like conditions.

Central injection of fibroblast growth factor 1 induces sustained remission of diabetic hyperglycemia in rodents.

Type 2 diabetes (T2D) is among the most common and costly disorders worldwide. The goal of current medical management for T2D is to transiently ameliorate hyperglycemia through daily dosing of one or more antidiabetic drugs. Hypoglycemia and weight gain are common side effects of therapy, and sustained disease remission is not obtainable with nonsurgical approaches. On the basis of the potent glucose-lowering response elicited by activation of brain fibroblast growth factor (FGF) receptors, we explored the antidiabetic efficacy of centrally administered FGF1, which, unlike other FGF peptides, activates all FGF receptor subtypes. We report that a single intracerebroventricular injection of FGF1 at a dose one-tenth of that needed for antidiabetic efficacy following peripheral injection induces sustained diabetes remission in both mouse and rat models of T2D. This antidiabetic effect is not secondary to weight loss, does not increase the risk of hypoglycemia, and involves a novel and incompletely understood mechanism for increasing glucose clearance from the bloodstream. We conclude that the brain has an inherent potential to induce diabetes remission and that brain FGF receptors are potential pharmacological targets for achieving this goal.

Glucosamine induces REDD1 to suppress insulin action in retinal Müller cells.

Resistance to insulin action is a key cause of diabetic complications, yet much remains unknown about the molecular mechanisms that contribute to the defect. Glucose-induced insulin resistance in peripheral tissues such as the retina is mediated in part by the hexosamine biosynthetic pathway (HBP). Glucosamine (GAM), a leading dietary supplement marketed to relieve the discomfort of osteoarthritis, is metabolized by the HBP, and in doing so bypasses the rate-limiting enzyme of the pathway. Thus, exogenous GAM consumption potentially exacerbates the resistance to insulin action observed with diabetes-induced hyperglycemia. In the present study, we evaluated the effect of GAM on insulin action in retinal Müller cells in culture. Addition of GAM to Müller cell culture repressed insulin-induced activation of the Akt/mTORC1 signaling pathway. However, the effect was not recapitulated by chemical inhibition to promote protein O-GlcNAcylation, nor was blockade of O-GlcNAcylation sufficient to prevent the effects of GAM. Instead, GAM induced ER stress and subsequent expression of the protein Regulated in DNA Damage and Development (REDD1), which was necessary for GAM to repress insulin-stimulated phosphorylation of Akt on Thr308. Overall, the findings support a model whereby GAM promotes ER stress in retinal Müller cells, resulting in elevated REDD1 expression and thus resistance to insulin action.

Increased Thyroid Hormone Activation Accompanies the Formation of Thyroid Hormone-Dependent Negative Feedback in Developing Chicken Hypothalamus.

The hypothalamic-pituitary-thyroid axis is governed by hypophysiotropic TRH-synthesizing neurons located in the hypothalamic paraventricular nucleus under control of the negative feedback of thyroid hormones. The mechanisms underlying the ontogeny of this phenomenon are poorly understood. We aimed to determine the onset of thyroid hormone-mediated hypothalamic-negative feedback and studied how local hypothalamic metabolism of thyroid hormones could contribute to this process in developing chicken. In situ hybridization revealed that whereas exogenous T4 did not induce a statistically significant inhibition of TRH expression in the paraventricular nucleus at embryonic day (E)19, T4 treatment was effective at 2 days after hatching (P2). In contrast, TRH expression responded to T3 treatment in both age groups. TSHß mRNA expression in the pituitary responded to T4 in a similar age-dependent manner. Type 2 deiodinase (D2) was expressed from E13 in tanycytes of the mediobasal hypothalamus, and its activity increased between E15 and P2 both in the mediobasal hypothalamus and in tanycyte-lacking hypothalamic regions. Nkx2.1 was coexpressed with D2 in E13 and P2 tanycytes and transcription of the cdio2 gene responded to Nkx2.1 in U87 glioma cells, indicating its potential role in the developmental regulation of D2 activity. The T3-degrading D3 enzyme was also detected in tanycytes, but its level was not markedly changed before and after the period of negative feedback acquisition. These findings suggest that increasing the D2-mediated T3 generation during E18-P2 could provide the sufficient local T3 concentration required for the onset of T3-dependent negative feedback in the developing chicken hypothalamus.