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.

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.

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.

Disruption of De Novo Serine Synthesis in Müller Cells Induced Mitochondrial Dysfunction and Aggravated Oxidative Damage.

De novo serine synthesis plays important roles in normal mitochondrial function and cellular anti-oxidative capacity. It is reported to be mainly activated in glial cells of the central nervous system, but its role in retinal Müller glia remains unclear. In this study, we inhibited de novo serine synthesis using CBR-5884, a specific inhibitor of phosphoglycerate dehydrogenase (PHGDH, a rate limiting enzyme in de novo serine metabolism) in MIO-M1 cells (immortalized human Müller cells) and huPMCs (human primary Müller cells) under mild oxidative stress. Alamar blue and LDH (lactate dehydrogenase) assays showed significantly reduced metabolic activities and increased cellular damage of Müller cells, when exposed to CBR-5884 accompanied by mild oxidative stress; however, CBR-5884 alone had little effect. The increased cellular damage was partially reversed by supplementation with exogenous serine/glycine. HSP72 (an oxidative stress marker) and reactive oxygen species (ROS) levels were significantly increased; glutathione and NADPH/NADP+ levels were pronouncedly reduced under PHGDH inhibition accompanied by oxidative stress. JC-1 staining and Seahorse respiration experiments showed that inhibition of de novo serine synthesis in Müller cells can also increase mitochondrial stress and decrease mitochondrial ATP production. qPCR and Western blot demonstrated an increased expression of HSP60 (a key mitochondrial stress-related gene), and this was further validated in human retinal explants. Our study suggests that de novo serine synthesis is important for Müller cell survival, particularly when they are exposed to mild oxidative stress, possibly by maintaining mitochondrial function and generating glutathione and NADPH to counteract ROS.

670nm light treatment following retinal injury modulates Müller cell gliosis: Evidence from in vivo and in vitro stress models.

Photobiomodulation (PBM) with 670 nm light has been shown to accelerate wound healing in soft tissue injuries, and also to protect neuronal tissues. However, little data exist on its effects on the non-neuronal components of the retina, such as Müller cells (MCs), which are the principal macroglia of the retina that play a role in maintaining retinal homeostasis. The aim of this study was to explore the effects of 670 nm light on activated MCs using in vivo and in vitro stress models. Adult Sprague-Dawley rats were exposed to photo-oxidative damage (PD) for 24 h and treated with 670 nm light at 0, 3 and 14 days after PD. Tissue was collected at 30 days post-PD for analysis. Using the in vitro scratch model with a human MC line (MIO-M1), area coverage and cellular stress were analysed following treatment with 670 nm light. We showed that early treatment with 670 nm light after PD reduced MC activation, lowering the retinal expression of GFAP and FGF-2. 670 nm light treatment mitigated the production of MC-related pro-inflammatory cytokines (including IL-1ß), and reduced microglia/macrophage (MG/MΦ) recruitment into the outer retina following PD. This subsequently decreased photoreceptor loss, slowing the progression of retinal degeneration. In vitro, we showed that 670 nm light directly modulated MC activation, reducing rates of area coverage by suppressing cellular proliferation and spreading. This study indicates that 670 nm light treatment post-injury may have therapeutic benefit when administered shortly after retinal damage, and could be useful for retinal degenerations where MC gliosis is a feature of disease progression.

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.

Advances in TRH signaling.

The activity of the hypothalamus-pituitary-thyroid axis (HPT) is coordinated by hypophysiotropic thyrotropin releasing hormone (TRH) neurons present in the paraventricular nucleus of the hypothalamus. Hypophysiotropic TRH neurons act as energy sensors. TRH controls the synthesis and release of thyrotropin, which activates the synthesis and secretion of thyroid hormones; in target tissues, transporters and deiodinases control their local availability. Thyroid hormones regulate many functions, including energy homeostasis. This review discusses recent evidence that covers several aspects of TRH role in HPT axis regulation. Knowledge about the mechanisms of TRH signaling has steadily increased. New transcription factors engaged in TRH gene expression have been identified, and advances made on how they interact with signaling pathways and define the dynamics of TRH neurons response to acute and/or long-term influences. Albeit yet incomplete, the relationship of TRH neurons activity with positive energy balance has emerged. The importance of tanycytes as a central relay for the feedback control of the axis, as well as for HPT responses to alterations in energy balance, and other stimuli has been reinforced. Finally, some studies have started to shed light on the interference of prenatal and postnatal stress and nutrition on HPT axis programing, which have confirmed the axis susceptibility to early insults.

Voluntary exercise induces neurogenesis in the hypothalamus and ependymal lining of the third ventricle.

In the adult hypothalamus and ependymal lining of the third ventricle, tanycytes function as multipotential progenitor cells that enable continuous neurogenesis, suggesting that tanycytes may be able to mediate the restoration of homeostatic function after stroke. Voluntary wheel running has been shown to alter neurochemistry and neuronal function and to increase neurogenesis in rodents. In the present study, we found that voluntary exercise improved the survival rate and energy balance of stroke-prone spontaneously hypertensive rats (SHRSP/Kpo). We also investigated the effect of exercise on the proliferation and differentiation of hypothalamic cells using immunoreactivity for tanycytes and neural markers. The proliferation of elongated cells, which may be the tanycytes, was enhanced in exercising SHRSP compared to sedentary rats before and after stroke. In addition, the proliferation of cells was correlated with the induction of fibroblast growth factor-2 in the subependymal cells of the third ventricle and in the cerebrospinal fluid. Some of the newborn cells of exercising SHRSP showed differentiation into mature neurons after stroke. Our results suggest that voluntary exercise correlates with hypothalamic neurogenesis, leading to recovery of homeostatic functions in the adult brain after stroke.

Sustained alterations of hypothalamic tanycytes during posttraumatic hypopituitarism in male mice.

Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hypothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome.