A rapid and non-invasive fluorescence method for quantifying coenzyme Q10 in blood and urine in clinical analysis.

BACKGROUND: Coenzyme Q10 (CoQ10) supplementation can improve cognition in patients with Alzheimer's disease (AD) and AD transgenic model mice. To ameliorate the discomfort that patients with AD suffer after several blood extractions, a non-invasive method for detecting urine CoQ10 levels needs to be established. METHODS: Here, we developed a new technique of fluorescence spectrophotometry with ethyl cyanoacetate (FS-ECA), on the basis of the principle that the chemical derivative obtained from the interaction between CoQ10 and ECA was detected by a fluorescence detector at λex/em  = 450/515 nm. As a standard reference method, the same batches of the clinical samples were analyzed by high-performance liquid chromatography with an ultraviolet detector (HPLC-UV) at 275 nm. RESULTS: The limits of detection (LOD) and limits of quantization (LOQ) (serum: 0.021 and 0.043 mg/L; urine: 0.012 and 0.025 mg/L) determined by the FS-ECA method were similar to that obtained through HPLC-UV (serum: 0.017 and 0.035 mg/L; urine: 0.012 and 0.025 mg/L). More importantly, this new FS-ECA technique as well as the conventional HPLC-UV method could detect a marked difference in urine CoQ10 levels between AD and controls. CONCLUSION: Our findings suggest that this non-invasive method for quantifying urine CoQ10 potentially replaces HPLC to detect blood CoQ10.

New insight into Alzheimer's disease: Light reverses Aß-obstructed interstitial fluid flow and ameliorates memory decline in APP/PS1 mice.

INTRODUCTION: Pharmacological therapies to treat Alzheimer's disease (AD) targeting "Aß" have failed for over 100 years. Low levels of laser light can disassemble Aß. In this study, we investigated the mechanisms that Aß-blocked extracellular space (ECS) induces memory disorders in APP/PS1 transgenic mice and addressed whether red light (RL) at 630 nm rescues cognitive decline by reducing Aß-disturbed flow of interstitial fluid (ISF). METHODS: We compared the heating effects on the brains of rats illuminated with laser light at 630, 680, and 810 nm for 40 minutes, respectively. Then, a light-emitting diode with red light at 630 nm (LED-RL) was selected to illuminate AD mice. The changes in the structure of ECS in the cortex were examined by fluorescent double labeling. The volumes of ECS and flow speed of ISF were quantified by magnetic resonance imaging. Spatial memory behaviors in mice were evaluated by the Morris water maze. Then, the brains were sampled for biochemical analysis. RESULTS: RL at 630 nm had the least heating effects than other wavelengths associated with ~49% penetration ratio into the brains. For the molecular mechanisms, Aß could induce formaldehyde (FA) accumulation by inactivating FA dehydrogenase. Unexpectedly, in turn, FA accelerated Aß deposition in the ECS. However, LED-RL treatment not only directly destroyed Aß assembly in vitro and in vivo but also activated FA dehydrogenase to degrade FA and attenuated FA-facilitated Aß aggregation. Subsequently, LED-RL markedly smashed Aß deposition in the ECS, recovered the flow of ISF, and rescued cognitive functions in AD mice. DISCUSSION: Aß-obstructed ISF flow is the direct reason for the failure of the developed medicine delivery from superficial into the deep brain in the treatment of AD. The phototherapy of LED-RL improves memory by reducing Aß-blocked ECS and suggests that it is a promising noninvasive approach to treat AD.

Hypomagnetic fields cause anxiety in adult male mice.

Hypomagnetic fields (HMF), that is, the elimination of the geomagnetic field (GMF), are a risk factor to the health of astronauts in outer space. It has been established that continuous HMF exposure affects cytoskeleton assembly, cell proliferation, embryonic development, and even learning and memory. In addition, although there were some previous studies that focused on the effects of long-term HMF-exposure, so far very limited investigations have been conducted to examine the short-term HMF effect in animals. In this study, we exposed adult male C57BL/6 mice to a 3-axis Helmholtz-coil HMF-simulation system for 72 h and found that short-term HMF-exposure induced a significant increase in anxiety-related behaviors. And our findings provide important information for both psychological intervention and the health care of astronauts. Bioelectromagnetics. 40:27-32, 2019. © 2018 Wiley Periodicals, Inc.

Illumination with 630 nm Red Light Reduces Oxidative Stress and Restores Memory by Photo-Activating Catalase and Formaldehyde Dehydrogenase in SAMP8 Mice.

AIMS: Pharmacological treatments for Alzheimer's disease (AD) have not resulted in desirable clinical efficacy over 100 years. Hydrogen peroxide (H2O2), a reactive and the most stable compound of reactive oxygen species, contributes to oxidative stress in AD patients. In this study, we designed a medical device to emit red light at 630 ± 15 nm from a light-emitting diode (LED-RL) and investigated whether the LED-RL reduces brain H2O2 levels and improves memory in senescence-accelerated prone 8 mouse (SAMP8) model of age-related dementia. RESULTS: We found that age-associated H2O2 directly inhibited formaldehyde dehydrogenase (FDH). FDH inactivity and semicarbazide-sensitive amine oxidase (SSAO) disorder resulted in endogenous formaldehyde (FA) accumulation. Unexpectedly, excess FA, in turn, caused acetylcholine (Ach) deficiency by inhibiting choline acetyltransferase (ChAT) activity in vitro and in vivo. Interestingly, the 630 nm red light can penetrate the skull and the abdomen with light penetration rates of ∼49% and ∼43%, respectively. Illumination with LED-RL markedly activated both catalase and FDH in the brains, cultured cells, and purified protein solutions, all reduced brain H2O2 and FA levels and restored brain Ach contents. Consequently, LED-RL not only prevented early-stage memory decline but also rescued late-stage memory deficits in SAMP8 mice. INNOVATION: We developed a phototherapeutic device with 630 nm red light, and this LED-RL reduced brain H2O2 levels and reversed age-related memory disorders. CONCLUSIONS: The phototherapy of LED-RL has low photo toxicity and high rate of tissue penetration and noninvasively reverses aging-associated cognitive decline. This finding opens a promising opportunity to translate LED-RL into clinical treatment for patients with dementia. Antioxid. Redox Signal. 00, 000-000.

Icariin protects SH-SY5Y cells from formaldehyde-induced injury through suppression of Tau phosphorylation.

OBJECTIVE: To investigate the neuroprotective effects of icariin on formaldehyde (FA)-treated human neuroblastoma SH-SY5Y cells and the possible mechanisms involved. METHODS: SH-SY5Y cells were divided into FA treatment group, FA treatment group with icariin, and the control group. Cell viability, apoptosis, and morphological changes were determined by cell counting kit-8 (CCK 8), flow cytometry, and confocal microscopy, respectively. The phosphorylation of Tau protein was examined by western blotting. RESULTS: FA showed a half lethal dose (LD50) of 0.3 mmol/L in SH-SY5Y cells under the experimental conditions. Icariin (1-10 µmol/L) prevented FA-induced cell death in SH-SY5Y cells in a dose-dependent manner, with the optimal effect observed at 5 µmol/L. After FA treatment, the absorbance in FA group was 1.31±0.05, while in the group of icariin (5 µmol/L) was 1.63±0.05. Examination of cell morphology by confocal microscopy demonstrated that 5 µmol/L icariin significantly attenuated FA-induced cell injury (P <0.05). Additionally, Icariin inhibited FA-induced cell apoptosis in SH-SY5Y cells. Results from western blotting showed that icariin suppressed FA-induced phosphorylation at Thr 181 and Ser 396 of Tau protein, while having no effect on the expression of the total Tau protein level. Furthermore, FA activated Tau kinase glycogen synthase kinase 3 beta (GSK-3ß) by enhancement of Y216 phosphorylation, but icariin reduced Y216 phosphorylation and increased Ser 9 phosphorylation. CONCLUSION: Icariin protects SH-SY5Y cells from FA-induced injury poßsibly through the inhibition of GSK-3ß-mediated Tau phosphorylation.

Neuroprotective effects of geniposide in SH-SY5Y cells and primary hippocampal neurons exposed to Aß42.

Our former studies have suggested that TongLuoJiuNao (TLJN) is clinically efficacious in the treatment of dementia and improving learning and memory in AD models. When Aß aggregated with oligomer, it is known to be able to induce cellular toxicity as well as cognitive impairment. We tested the possibility that TLJN affects the formation of Aß oligomers. In our experiment, TLJN improved cell viability, inhibited LDH release, and promoted the outgrowth of neurites of neurons treated with Aß. Geniposide, the main component of TLJN, could increase the cell viability of SY5Y-APP695sw cells. The cytotoxicity of pretreated Aß with geniposide was decreased in a dose-dependent manner. SDS-PAGE and Western blotting showed that geniposide and TLJN stimulated Aß oligomer assembly. Compared with the control, more and longer fibrils of Aß in the presence of geniposide were observed under electron microscope though the fibrils became less sensitive to thioflavin T staining. In sum, geniposide is able to protect neurons from Aß-induced damage by remodeling Aß.

The herbal compound geniposide rescues formaldehyde-induced apoptosis in N2a neuroblastoma cells.

The herbal medicine Tong Luo Jiu Nao (TLJN) contains geniposide (GP) and ginsenoside Rg1 at a molar ratio of 10:1. Rg1 is the major component of another herbal medicine, panax notoginseng saponin (PNS). TLJN has been shown to strengthen brain function in humans, and in animals it improves learning and memory. We have previously shown that TLJN reduces amyloidogenic processing in Alzheimer's disease (AD) mouse models. Together this suggests TLJN may be a potential treatment for patients with dementia. Because chronic damage of the central nervous system by formaldehyde (FA) has been presented as a risk factor for age-associated cognitive dysfunction, in the present study we investigated the protective effect of both TLJN and GP in neuron-like cells exposed to FA. FA-exposed murine N2a neuroblastoma cells were incubated with TLJN, its main ingredient GP, as well as PNS, to measure cell viability and morphology, the rate of apoptosis and expression of genes encoding Akt, FOXO3, Bcl2 and p53. The CCK-8 assay, cytoskeletal staining and flow cytometry were used to test cell viability, morphology and apoptosis, respectively. Fluorescent quantitative real-time PCR (qRT-PCR) was used to monitor changes in gene expression, and HPLC to determine the rate of FA clearance. Treatment of N2a cells with 0.09 mmol L(-1) FA for 24 h significantly reduced cell viability, changed cell morphology and promoted apoptosis. Both TLJN and GP conferred neuroprotection to FA-treated N2a cells, whereas PNS, which had to be used at lower concentrations because of its toxicity, did not. Our data demonstrate that TLJN can rescue neuronal damage caused by FA and that its main ingredient, GP, has a major role in this efficacy. This presents purified GP as a drug or lead compound for the treatment of AD.

The protective effect of geniposide on human neuroblastoma cells in the presence of formaldehyde.

BACKGROUND: Formaldehyde can induce misfolding and aggregation of Tau protein and ß amyloid protein, which are characteristic pathological features of Alzheimer's disease (AD). An increase in endogenous formaldehyde concentration in the brain is closely related to dementia in aging people. Therefore, the discovery of effective drugs to counteract the adverse impact of formaldehyde on neuronal cells is beneficial for the development of appropriate treatments for age-associated cognitive decline. METHODS: In this study, we assessed the neuroprotective properties of TongLuoJiuNao (TLJN), a traditional Chinese medicine preparation, against formaldehyde stress in human neuroblastoma cells (SH-SY5Y cell line). The effect of TLJN and its main ingredients (geniposide and ginsenoside Rg1) on cell viability, apoptosis, intracellular antioxidant activity and the expression of apoptotic-related genes in the presence of formaldehyde were monitored. RESULTS: Cell counting studies showed that in the presence of TLJN, the viability of formaldehyde-treated SH-SY5Y cells significantly recovered. Laser scanning confocal microscopy revealed that the morphology of formaldehyde-injured cells was rescued by TLJN and geniposide, an effective ingredient of TLJN. Moreover, the inhibitory effect of geniposide on formaldehyde-induced apoptosis was dose-dependent. The activity of intracellular antioxidants (superoxide dismutase and glutathione peroxidase) increased, as did mRNA and protein levels of the antiapoptotic gene Bcl-2 after the addition of geniposide. In contrast, the expression of the apoptotic-related gene - P53, apoptotic executer - caspase 3 and apoptotic initiator - caspase 9 were downregulated after geniposide treatment. CONCLUSIONS: Our results indicate that geniposide can protect SH-SY5Y cells against formaldehyde stress through modulating the expression of Bcl-2, P53, caspase 3 and caspase 9, and by increasing the activity of intracellular superoxide dismutase and glutathione peroxidase.

Neuroprotective effects of tongluojiunao in neurons exposed to oxygen and glucose deprivation.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongluojiunao (TLJN) is an herb extract that mainly contains ginsenoside Rg1 and geniposide, which are clinically used for treating ischemic damages in the brain. AIM OF THE STUDY: In the stroke, cerebral ischemia followed by oxygen reperfusion induced apoptosis in hippocampal neurons, while extension of axons and dendrites in neurons may compensate for and repair damages of neuronal network in the hypoxia brain. In this study, we investigated whether TLJN can protect neurons against damages by ischemia in brain vasculature. MATERIALS AND METHODS: We measured cell viability and lactate dehydrogenase (LDH) release from primary culture of rat hippocampal neurons before and after the neurons were deprived of oxygen and glucose (OGD). In addition, the effects were evaluated with cell viability and neurite outgrowth before or after OGD. RESULTS: We found that TLJN could play a neuroprotective role to cultured primary rat hippocampal neurons under both normal and oxygen/glucose-deprivation (OGD) conditions. TLJN could protect both cultured primary rat hippocampal neurons and brain microvascular endothelial cells (BMECs) from cell death under both normal and oxygen/glucose-deprivation (OGD) conditions. Moreover, under the same conditions, BMECs-conditioned media pretreated by TNJN could also promote neuron viability and neurite outgrowth, indicating that TLJN stimulated BMECs to secret some neuroprotective/neurotrophic factors. CONCLUSION: These findings suggest that TLJN has a marked neuroprotective and neurotrophic roles by either direct or indirect operation, and provide insight into the mechanism of clinical efficacy of this drug against stroke.