Regulating effect of miR-132-3p on the changes of MAPK pathway in rat brains and SH-SY5Y cells exposed to excessive fluoride by targeting expression of MAPK1.

BACKGROUND: Although the changes of mitogen-activated protein kinase (MAPK) pathway in the central nervous system (CNS) induced by excessive fluoride has been confirmed by our previous findings, the underlying mechanism(s) of the action remains unclear. Here, we investigate the possibility that microRNAs (miRNAs) are involved in the aspect. METHODS: As a model of chronic fluorosis, SD rats received different concentrations of fluoride in their drinking water for 3 or 6 months and SH-SY5Y cells were exposed to fluoride. Literature reviews and bioinformatics analyses were used to predict and real-time PCR to measure the expression of 12 miRNAs; an algorithm-based approach was applied to identify multiply potential target-genes and pathways; the dual-luciferase reporter system to detect the association of miR-132-3p with MAPK1; and fluorescence in situ hybridization to detect miR-132-3p localization. The miR-132-3p inhibitor or mimics or MAPK1 silencing RNA were transfected into cultured cells. Expression of protein components of the MAPK pathway was assessed by immunofluorescence or Western blotting. RESULTS: In the rat hippocampus exposed with high fluoride, ten miRNAs were down-regulated and two up-regulated. Among these, miR-132-3p expression was down-regulated to the greatest extent and MAPK1 level (selected from the 220 genes predicted) was corelated with the alteration of miR-132-3p. Furthermore, miR-132-3p level was declined, whereas the protein levels MAPK pathway components were increased in the rat brains and SH-SY5Y cells exposed to high fluoride. MiR-132-3p up-regulated MAPK1 by binding directly to its 3'-untranslated region. Obviously, miR-132-3p mimics or MAPK1 silencing RNA attenuated the elevated expressions of the proteins components of the MAPK pathway induced by fluorosis in SH-SY5Y cells, whereas an inhibitor of miR-132-3p just played the opposite effect. CONCLUSION: MiR-132-3p appears to modulate the changes of MAPK signaling pathway in the CNS associated with chronic fluorosis.

LiCl attenuates impaired learning and memory of APP/PS1 mice, which in mechanism involves α7 nAChRs and Wnt/ß-catenin pathway.

We examined the mechanism by which lithium chloride (LiCl) attenuates the impaired learning capability and memory function of dual-transgenic APP/PS1 mice. Six- or 12-month-old APP/PS1 and wild-type (WT) mice were randomized into four groups, namely WT, WT+Li (100 mg LiCl/kg body weight, gavage once daily), APP/PS1 and APP/PS1+Li. Primary rat hippocampal neurons were exposed to ß-amyloid peptide oligomers (AßOs), LiCl and/or XAV939 (inhibitor of Wnt/ß-catenin) or transfected with small interfering RNA against the ß-catenin gene. In the cerebral zone of APP/PS1 mice, the level of Aß was increased and those of α7 nicotinic acetylcholine receptors (nAChR), phosphor-GSK3ß (ser9), ß-catenin and cyclin D1 (protein and/or mRNA levels) reduced. Two-month treatment with LiCl at ages of 4 or 10 months weakened all of these effects. Similar expression variations were observed for these proteins in primary neurons exposed to AßOs, and these effects were attenuated by LiCl and aggravated by XAV939. Inhibition of ß-catenin expression lowered the level of α7 nAChR protein in these cells. LiCl attenuates the impaired learning capability and memory function of APP/PS1 mice via a mechanism that might involve elevation of the level of α7 nAChR as a result of altered Wnt/ß-catenin signalling.

An inexpensive anaerobic chamber for the genetic manipulation of strictly anaerobic bacteria.

Strictly anaerobic bacteria are important to both human health and industrial usage. These bacteria are sensitive to oxygen, therefore, it is preferable to manipulate these microbes in an anaerobic chamber. However, commercial anaerobic chambers (CACs) are expensive, making them less accessible to scientists with a limited budget, especially to those in developing countries. The high price of commercial chambers has hindered, at least partially, the progress of research on anaerobes in developing countries. In the research presented here, we developed an inexpensive and reliable anaerobic chamber and successfully achieved routine maintenance of eleven strictly anaerobic bacterial strains. Furthermore, genetic manipulation examples have been set for both Clostridioidesdifficile 630 and Clostridiumbeijerinckii NCIMB 8052 strains to validate that the chamber could applied to advanced genetic engineering of strictly anaerobes. C. difficile and C. beijerinckii were both genetically manipulated in this chamber, showing it's utility for the genetic engineering of anaerobes. Most importantly, the anaerobic chamber was 76% - 88% less expensive than a CACs and has similar functionality with regards to the cultivation and manipulation of strictly anaerobic bacteria. The anaerobic chamber described in this study will promote the research of anaerobes in developing counties and scientists who have limited research budgets.

Membrane permeability-guided identification of neuroprotective components from Polygonum cuspidatun.

CONTEXT: Polygonum cuspidatum Sieb et Zucc. (Polygonaceae) possesses various pharmacological activities and has been widely using as one of the most popular and valuable Chinese herbal medicines in clinics. Its usage has increasingly attracted much of our attention and urges investigation on its bioactive components. OBJECTIVE: To establish a rapid and valid approach for screening potential neuroprotective components from P. cuspidatum. MATERIALS AND METHODS: Potential neuroprotective components from P. cuspidatum were screened utilizing liposome equilibrium dialysis followed by high-performance liquid chromatography (HPLC) analysis. Their neuroprotective effects on modulation of protein expression of α7 nAChR, α3 nAChR and synaptophysin (SPY) on SH-SY5Y human neuroblastoma cell line (SH-SY5Y) were evaluated by means of Western blotting. RESULTS: Two potential compounds, polydatin (C1) and emodin-8-O-ß-D-glucoside (C2), were detected and identified in our study. The biological tests showed that both compounds C1 and C2, respectively, at concentrations of 0.1 and 0.25 mg/mL significantly increased protein expression of α7 and α3 nicotinic acetylcholine receptors (nAChRs) in SH-SY5Y cells. Moreover, C1 and C2 at 0.1 mg/mL significantly reversed the Aß1₋42-induced decrease of α7 and α3 nAChRs protein expression in SH-SY5Y cells. In addition, C2 at 0.1 mg/mL significantly increased protein expression of SPY in SH-SY5Y cells and Aß11₋42-induced SH-SY5Y cells whereas C1 did not provide any positive effects. DISCUSSION AND CONCLUSION: In conclusion, our approach utilizing liposome equilibrium dialysis combined with HPLC analysis and cell-based assays is a prompt and useful method for screening neuroprotective agents.

Myricetin ameliorates cognitive impairment in 3×Tg Alzheimer's disease mice by regulating oxidative stress and tau hyperphosphorylation.

BACKGROUND: Alzheimer's disease is characterized by abnormal ß-amyloid (Aß) plaque accumulation, tau hyperphosphorylation, reactive oxidative stress, mitochondrial dysfunction and synaptic loss. Myricetin, a dietary flavonoid, has been shown to exert neuroprotective effects in vitro and in vivo. Here, we aimed to elucidate the mechanism and pathways involved in the protective effect of myricetin. METHODS: The effect of myricetin was assessed on Aß42 oligomer-treated neuronal SH-SY5Y cells and in 3×Tg mice. Behavioral tests were performed to assess the cognitive effects of myricetin (14 days, ip) in 3×Tg mice. The levels of beta-amyloid precursor protein (APP), synaptic and mitochondrial proteins, glycogen synthase kinase3ß (GSK3ß) and extracellular regulated kinase (ERK) 2 were assessed via Western blotting. Flow cytometry assays, immunofluorescence staining, and transmission electron microscopy were used to assess mitochondrial dysfunction and reactive oxidative stress. RESULTS: We found that, compared with control treatment, myricetin treatment improved spatial cognition and learning and memory in 3×Tg mice. Myricetin ameliorated tau phosphorylation and the reduction in pre- and postsynaptic proteins in Aß42 oligomer-treated neuronal SH-SY5Y cells and in 3×Tg mice. In addition, myricetin reduced reactive oxygen species generation, lipid peroxidation, and DNA oxidation, and rescued mitochondrial dysfunction via the associated GSK3ß and ERK 2 signalling pathways. CONCLUSIONS: This study provides new insight into the neuroprotective mechanism of myricetin in vitro in cell culture and in vivo in a mouse model of Alzheimer's disease.

Gastrodin alleviates mitochondrial dysfunction by regulating SIRT3-mediated TFAM acetylation in vascular dementia.

BACKGROUND: Mitochondrial dysfunction is key to the pathogenesis of vascular dementia (VaD). Sirtuin-3 (SIRT3), an essential member of the sirtuins family, has been proven to be a critical sirtuin in regulating mitochondrial function. The phenolic glucoside gastrodin (GAS), a bioactive ingredient from Gastrodiae Rhizome (known in Chinese as Tian ma) demonstrates significant neuroprotective properties against central nervous system disorders; however, the precise mechanisms through which GAS modulates VaD remain elusive. PURPOSE: This study aims to investigate whether GAS confers a protective role against VaD, and to figure out the underlying molecular mechanisms. METHODS: A bilateral common carotid artery occlusion (BCCAO)-mediated chronic cerebral hypoperfusion (CCH) VaD rat model and a hypoxia model using HT22 cells were employed to investigate pharmacological properties of GAS in mitigating mitochondrial dysfunction. A SIRT3 agonist resveratrol (RES), a SIRT3 inhibitor 3-TYP and SIRT3-knockdown in vitro were used to explore the mechanism of GAS in association with SIRT3. The ability of SIRT3 to bind and deacetylate mitochondrial transcription factor A (TFAM) was detected by immunoprecipitation assay, and TFAM acetylation sites were further validated using mass spectrometry. RESULTS: GAS increased SIRT3 expression and ameliorated mitochondrial structure, mitochondrial respiration, mitochondrial dynamics along with upregulated TFAM, mitigating oxidative stress and senescence. Comparable results were noted with the SIRT3 agonist RES, indicating an impactful neuroprotection played by SIRT3. Specifically, the attenuation of SIRT3 expression through knockdown techniques or exposure to the SIRT3 inhibitor 3-TYP in HT22 cells markedly abrogated GAS-mediated mitochondrial rescuing function. Furthermore, our findings elucidate a novel facet: SIRT3 interacted with and deacetylated TFAM at the K5, K7, and K8 sites. Decreased SIRT3 is accompanied by hyper-acetylated TFAM. CONCLUSION: The present results were the first to demonstrate that the SIRT3/TFAM pathway is a protective target for reversing mitochondrial dysfunction in VaD. The findings suggest that GAS-mediated modulation of the SIRT3/TFAM pathway, a novel mechanism, could ameliorate CCH-induced VaD, offering a potentially beneficial therapeutic strategy for VaD.

Exposure to fluoride exacerbates the cognitive deficit of diabetic patients living in areas with endemic fluorosis, as well as of rats with type 2 diabetes induced by streptozotocin via a mechanism that may involve excessive activation of the poly(ADP ribose) polymerase-1/P53 pathway.

Epidemiology has shown that fluoride exposure is associated with the occurrence of diabetes. However, whether fluoride affects diabetic encephalopathy is unclear. Elderly diabetic patients in areas with endemic (n = 169) or no fluorosis (108) and controls (85) underwent Montreal Cognitive Assessment. Sprague-Dawley rats receiving streptozotocin and/or different fluoride doses were examined for spatial learning and memory, brain morphology, blood-brain barrier, fasting blood glucose and insulin. Cultured SH-SY5Y cells were treated with 50 mM glucose and/or low- or high-dose fluoride, and P53-knockdown or poly-ADP-ribose polymerase-1 (PARP-1) inhibition. The levels of PARP-1, P53, poly-ADP-ribose (PAR), apoptosis-inducing factor (AIF), and phosphorylated-histone H2A.X (ser139) were measured by Western blotting. Reactive oxygen species (ROS), 8-hydroxydeguanosine (8-OHdG), PARP-1 activity, acetyl-P53, nicotinamide adenine dinucleotide (NAD+), activities of mitochondrial hexokinase1 (HK1) and citrate synthase (CS), mitochondrial membrane potential and apoptosis were assessed biochemically. Cognition of diabetic patients in endemic fluorosis areas was poorer than in other regions. In diabetic rats, fasting blood glucose, insulin resistance and blood-brain barrier permeability were elevated, while spatial learning and memory and Nissl body numbers in neurons declined. In these animals, expression and activity of P53 and PARP-1 and levels of NAD+, PAR, ROS, 8-OHdG, p-histone H2A.X (ser139), AIF and apoptosis content increased; whereas mitochondrial HK1 and CS activities and membrane potential decreased. SH-SY5Y cells exposed to glucose exhibited changes identical to diabetic rats. The changes in diabetic rats and cells treated with glucose were aggravated by fluoride. P53-knockout or PARP-1 inhibition mitigated the effects of glucose with/without low-dose fluoride. Elevation of diabetic encephalopathy was induced by exposure to fluoride and the underlying mechanism may involve overactivation of the PARP-1/P53 pathway.

Preventing expression of the nicotinic receptor subunit α7 in SH-SY5Y cells with interference RNA indicates that this receptor may protect against the neurotoxicity of Aß.

The present aim was to characterize the influence of the α7 nicotinic acetylcholine receptor (nAChR) on BACE, the enzyme that cleaves the amyloid precursor protein (APP) at the ß-site, as well as on the oxidative stress induced by amyloid-ß peptide (Aß). To this end, human neuroblastoma SH-SY5Y cells were transfected with siRNAs targeting the α7 nAChR subunit and/or exposed to Aß1-42. For α7 nAChR, BACE1 (cleaving at the ß-site of APP) and BACE2 (cleaving within the Aß domain), α-secretase (ADAM10), and the two components of γ-secretase, PS and NCT, the mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. The level of Aß1-42 in the cell culture medium was determined by an ELISA procedure. The extent of lipid peroxidation and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were assayed spectrophotometrically. In the transfected SH-SY5Y cells, expression of α7 nAChR was reduced; the level of BACE1 increased and that of BACE2 decreased; the amount of ADAM10 lowered; and the level of PS raised. Moreover, the level of Aß1-42 in the culture medium was elevated. Treatment of non-transfected cells with Aß elevated the level of malondialdehyde (MDA) and lowered the activities of SOD and GSH-Px and these changes were potentiated by inhibiting expression of α7 nAChR. These results indicate that α7 nAChR plays a significant role in amyloidogenic metabolism of APP and the oxidative stress evoked by Aß, suggesting that this receptor might help protect against the neurotoxicity of Aß.

[Effects of α3 neuronal nicotinic acetylcholine receptor on cell apoptosis and p38 MAPK signal transduction pathway in SH-SY5Y cells].

OBJECTIVE: To investigate the effects of α3 neuronal nicotinic acetylcholine receptor (nAChR) on apoptosis and p38 signal transduction pathway in SH-SY5Y cells and to assess the roles of α3 nAChR in the pathogenesis of Alzheimer's disease (AD). METHODS: The levels of α3 nAChR mRNA and protein were measured by real-time PCR and Western blot, respectively, in SH-SY5Y cells transfected with α3 nAChR siRNA. The mRNA level of bcl-2 and bax was measured by the real-time PCR. The siRNA transfected SH-SY5Y cells and control were then treated with 10 µmol/L Aß25-35 for another 48 h, and the change in apoptotic rate and the levels of p-p38 and p38 were measured by flow cytometry and Western blot. Subsequently these SH-SY5Y cells were exposed to a blocker of p38 protein, and the apoptotic rate was measured again. RESULTS: Compared to the controls, the expression of α3 nAChR at mRNA and protein levels in the SH-SY5Y cells transfected with α3 nAChR siRNA decreased by 95% and 86%, respectively; the mRNA levels of bax increased 2.11 times and that for bcl-2 decreased 0.53 times. The apoptotic rate was unaffected (3.40% ± 0.20%); but it increased after Aß25-35 treatment (24.52% ± 1.59%); the level of p-p38 protein also increased by 178% in the α3 nAChR inhibited cells treated with Aß25-35. Compared to controls, the Aß25-35-treated SH-SY5Y cells and the Aß25-35-treated and siRNA-transfected cells both showed a reduction in apoptosis after treatment with p38 blocker, especially in the former. CONCLUSION: The siRNA silencing of α3 nAChR mRNA may enhance the effect of Aß25-35 on the cell apoptosis by increasing the levels of p38 protein and bax mRNA and decreasing the level of bcl-2 mRNA, which may play a role in the pathogenesis of AD.

Resveratrol Attenuates the Disruption of Lipid Metabolism Observed in Amyloid Precursor Protein/Presenilin 1 Mouse Brains and Cultured Primary Neurons Exposed to Aß.

To examine whether resveratrol (RSV), an activator of silent mating-type information regulation 2 homolog 1 (SIRT1), can reverse the disruption of lipid metabolism caused by ß-amyloid peptide (Aß), APP/PS1 mice or cultured primary rat neurons were treated with RSV, suramin (inhibitor of SIRT1), ZLN005, a stimulator of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), or PGC-1α silencing RNA. In the brains of the APP/PS1 mice, expressions of SIRT1, PGC-1α, low-density lipoprotein receptor (LDLR) and very LDLR (VLDLR) were reduced at the protein and, in some cases, mRNA levels; while the levels of the proprotein convertase subtilisin/kexin type 9 (PCSK9), apolipoprotein E (ApoE), total cholesterol and LDL were all elevated. Interestingly, these changes were reversed by administration of RSV, while being aggravated by suramin. Furthermore, activation of PGC-1α, but inhibition of SIRT1, decreased the levels of PCSK9 and ApoE, while increased those of LDLR and VLDLR in the neurons exposed to Aß, and silencing PGC-1α, but activation of SIRT1, did not influence the levels of any of these proteins. These findings indicate that RSV can attenuate the disruption of lipid metabolism observed in the brains of APP mice and in primary neurons exposed to Aß by activating SIRT1, in which the mechanism may involve subsequently affecting PGC-1α.

The influence of NQO2 on the dysfunctional autophagy and oxidative stress induced in the hippocampus of rats and in SH-SY5Y cells by fluoride.

INTRODUCTION: For investigating the mechanism of brain injury caused by chronic fluorosis, this study was designed to determine whether NRH:quinone oxidoreductase 2 (NQO2) can influence autophagic disruption and oxidative stress induced in the central nervous system exposed to a high level of fluoride. METHODS: Sprague-Dawley rats drank tap water containing different concentrations of fluoride for 3 or 6 months. SH-SY5Y cells were either transfected with NQO2 RNA interference or treated with NQO2 inhibitor or activator and at the same time exposed to fluoride. The enrichment of gene signaling pathways related to autophagy was evaluated by Gene Set Enrichment Analysis; expressions of NQO2 and autophagy-related protein 5 (ATG5), LC3-II and p62, and mammalian target of rapamycin (mTOR) were quantified by Western-blotting or fluorescent staining; and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) assayed biochemically and reactive oxygen species (ROS) detected by flow cytometry. RESULTS: In the hippocampal CA3 region of rats exposed to high fluoride, the morphological characteristics of neurons were altered; the numbers of autophagosomes in the cytoplasm and the levels of NQO2 increased; the level of p-mTOR was decreased, and the levels of ATG5, LC3-II and p62 were elevated; and genes related to autophagy enriched. In vitro, in addition to similar changes in NQO2, p-mTOR, ATG5, LC3 II, and p62, exposure of SH-SY5Y cells to fluoride enhanced MDA and ROS contents and reduced SOD activity. Inhibition of NQO2 with RNAi or an inhibitor attenuated the disturbance of the autophagic flux and enhanced oxidative stress in these cells exposed to high fluoride. CONCLUSION: Our findings indicate that NQO2 may be involved in regulating autophagy and oxidative stress and thereby exerts an impact on brain injury caused by chronic fluorosis.

Extract of Ginkgo biloba leaves attenuates neurotoxic damages in rats and SH-SY5Y cells exposed to a high level of fluoride.

BACKGROUND: Potential protection against the neurotoxic damages of high levels of fluoride on rats and SH-SY5Y cells by extract of Ginkgo biloba leaves, as well as underlying mechanisms, were examined. METHODS: The rats were divided randomly into 4 groups, i.e., control, treatment with the extract (100 mg/kg body weight, gavage once daily), treatment with fluoride (50 ppm F- in drinking water) and combined treatment with both; SH-SY5Y cells exposed to fluoride and fluoride in combination with the extract or 4-Amino-1,8-naphthalimide (4-ANI), an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1). Spatial learning and memory in the rats were assessed employing Morris water maze test; the contents of fluoride in brains and urine by fluoride ion-selective electrode; cytotoxicity of fluoride was by CCK-8 kit; the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) by appropriate kits; the level of 8-hydroxydeoxyguanosine (8-OHdG) was by ELISA; the content of ROS and frequency of apoptosis by flow cytometry; the expressions of phospho-histone H2A.X(Ser139), PARP-1, poly (ADP-ribose) (PAR) and Sirtuin-1 (SIRT1) by Western blotting or immunofluorescence. RESULTS: The rats with prolong treatment of fluoride exhibited dental fluorosis, the increased contents of fluoride in brains and urine and the declined ability of learning and memory. In the hippocampus of the rats and SH-SY5Y cells exposed to fluoride, the levels of ROS, MDA, apoptosis, 8-OHdG and the protein expressions of histone H2A.X(Ser139), PARP-1 and PAR were all elevated; the activities of SOD and GSH-Px and the protein expression of SIRT1 reduced. Interestingly, the treatment of Ginkgo biloba extract attenuated these neurotoxic effects on rats and SH-SY5Y cells exposed to fluoride and the treatment of 4-ANI produced a neuroprotective effect against fluoride exposure. CONCLUSION: Ginkgo biloba extract attenuated neurotoxic damages induced by fluoride exposure to rats and SH-SY5Y cells and the underlying mechanism might involve the inhibition of PARP-1 and the promotion of SIRT1.

[Influence of inhibited α7 nicotinic acetylcholine receptor gene expression on the production of ß-amyloid peptide in SH-SY5Y cells].

OBJECTIVE: To investigate the influence of inhibited α7 neuronal nicotinic acetylcholine receptor (nAChR) by small interference RNA (siRNA) in SH-SY5Y cells and to explore the connection of these changes with the ß-amyloid precursor protein (APP) metabolism and the pathogenesis of Alzheimer's disease (AD). METHODS: The siRNA of α7 nAChR was transfected into SH-SY5Y cells, and the expression of α7 nAChR and two subtypes of ß-secretases (BACE1 and BACE2) at mRNA and protein levels was studied by real-time PCR and Western blot, respectively. The variation of Aß(1-42) content was detected by ELISA. RESULTS: As compared with controls, the expression of α7 nAChR at mRNA and protein levels in the SH-SY5Y cells transfected with the α7 nAChR siRNA were decreased by 84% and 79% (P < 0.01), respectively. The expressions of BACE1 mRNA and protein levels was increased by 527% and 71% (P < 0.01), respectively, while the expression of BACE2 decreased by 58% and 75% (P < 0.01), respectively. The Aß(1-42) content increased by 208% (P < 0.01). CONCLUSIONS: An inhibited α7 nAChR mRNA induced by siRNA may markedly stimulate the production of Aß through the mechanism of increased expression of BACE1 and inhibited expression of BACE2, which may be related to the pathogenesis of AD.

Integrated transcriptomic and proteomic analysis indicated that neurotoxicity of rats with chronic fluorosis may be in mechanism involved in the changed cholinergic pathway and oxidative stress.

BACKGROUND: To reveal the underling molecular mechanism in brain damage induced by chronic fluorosis, the neurotoxicity and its correlation were investigated by transcriptomics and proteomics. METHODS: Sprague-Dawley rats were treated with fluoride at different concentrations (0, 5, 50 and 100 ppm, prepared by NaF) for 3 months. Spatial learning and memory were evaluated by Morris water maze test; neuronal morphological change in the hippocampus was observed using Nissl staining; and the level of oxidative stress including reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by biological methods. The high-throughput transcriptome sequencing (RNA-Seq) and tandem mass tag (TMT) proteomic sequencing were performed to detect the expression of differentially expressed genes and proteins, respectively. RESULTS: The results showed that compared with control group, rats exposed to high-dose fluoride exhibited declined abilities of learning and memory, decreased SOD activity and increased ROS and MDA levels, with lighter colored Nissl bodies. A total of 28 important differentially expressed genes (DEGs) were screened out by transcriptomics. Then, functional enrichment analyses showed that upregulated proteins enriched in cellular transport, while downregulated proteins enriched in synapse-related pathways. Thirteen corresponding DEGs and DAPs (cor-DEGs-DAPs) were identified by differential expressions selected with positively correlated genes/proteins, most of which were related to neurodegenerative changes and oxidative stress response. CONCLUSION: These results provide new omics evidence that rats chronically exposed to high-dose fluoride can induce neurotoxicity in the brains through changes in the cholinergic pathway and oxidative stress.

Rapid screening and identification of caffeic acid and its esters in Erigeron breviscapus by ultra-performance liquid chromatography/tandem mass spectrometry.

Caffeic acid and its esters (CAEs) are widely distributed in the plant kingdom and have been reported to elicit a wide range of exceptional biological activities. Present methods for screening and characterization of CAEs normally need the use of liquid chromatography diode-array detection/multistage mass spectrometry (LC-DAD/MS(n)). In this report, a rapid and efficient method coupling ultra-performance liquid chromatography (UPLC) with fragment-targeted multi-reaction monitoring (MRM) has been developed for screening CAEs in a crude extract of Erigeron breviscapus, while a UPLC/quasi-MS(n) method has been applied in the structural identification of these compounds. Furthermore, a simple quasi-UPLC/MS/MS method based on in-source collision-induced dissociation (CID) has been proposed for rapid identification of the CAEs. As a result, a total of more than 34 CAEs were detected and their structures characterized. Nine of them were reported from E. breviscapus for the first time. Applications of these strategies in the chemical investigation of an injection of E. breviscapus resulted in the identifications of 16 CAEs. These strategies, if appropriate modifications are made, will be very useful in screening and characterization not only of CAEs, but of other structural types of compounds in various complex matrices.

[Influence of APP(SWE) transfection on nicotinic receptors in cultured neuronal cells].

OBJECTIVE: To investigate the influence of APP(SWE) on the expression of neuronal acetylcholine receptors (nAChRs) and its relationship with Alzheimer's disease (AD). METHODS: APP(SWE), carried the Swedish family AD double mutants, were transfected into SH-SY5Y cells and primary cultured neurons from rat brains to build a cellular model of AD. The mRNA levels of APP and nAChRs, and the protein levels of total APP, αAPPs and nAChRs in the cultured cells were measured using real-time PCR and Western blot, respectively. The numbers of α3 nAChR were determined by receptor-[³H]epibatidine binding assay. RESULTS: Increased expressions of Swedish 670/671 APP at mRNA and protein levels, and down-regulation of αAPPs were observed in both of the cultured neuronal cells transfected with APP(SWE). A significant increase of α7 nAChR expression at protein and mRNA levels was detected in the APP(SWE) transfected SH-SY5Y cells. On the other hand, after transfection with APP(SWE), the expressions of α3 nAChR at protein and mRNA levels in SH-SY5Y cells, and α4 nAChR at mRNA level in primary cultured neurons were inhibited. In addition, the numbers of receptor binding sites were deceased in SH-SY5Y cells overexpressing with APP(SWE). CONCLUSION: Overexpression of APP(SWE) can decrease αAPPs and modify nAChRs by increasing expression of α7 nAChR and decreasing α3 and α4 nAChRs, which might play an important role in the pathogenesis of AD.

Expression levels of the α7 nicotinic acetylcholine receptor in the brains of patients with Alzheimer's disease and their effect on synaptic proteins in SH-SY5Y cells.

Alzheimer's disease (AD) is a chronic neurodegenerative, and abnormal aggregation of the neurotoxic ß amyloid (Aß) peptide is an early event in AD. The present study aimed to determine the correlation between the nicotinic acetylcholine receptor α7 subunit (α7 nAChR) and Aß in the brains of patients with AD, and to investigate whether the increased expression levels of the α7 nAChR could alter the neurotoxicity of Aß. The expression levels of α7 nAChR and Aß in the brains of patients with AD and healthy brains were analyzed using immunofluorescence. Moreover, SH­SY5Y cells were used to stably overexpress or silence α7 nAChR expression levels, prior to the treatment with or without 1 µmol/l Aß1­42 oligomer (AßO). The mRNA and protein expression levels of α7 nAChR, synaptophysin (SYP), postsynaptic density of 95 kDa (PSD­95) and synaptosomal­associated protein of 25 kDa (SNAP­25) were subsequently analyzed using reverse transcription­quantitative PCR and western blotting. In addition, the concentration of acetylcholine (ACh) and the activity of acetylcholinesterase (AChE) were analyzed using spectrophotometry, while the cell apoptotic rate was determined using flow cytometry. The expression of Aß in the brains of patients with AD was found to be significantly increased, whereas the expression of α7 nAChR was significantly decreased compared with the healthy control group. In vitro, the expression levels of α7 nAChR were significantly increased or decreased following the overexpression or silencing of the gene, respectively. Consistent with these observations, the mRNA and protein expression levels of SYP, PSD­95 and SNAP­25 were also significantly increased following the overexpression of α7 nAChR and decreased following the genetic silencing of the receptor. In untransfected or negative control cells, the expression levels of these factors and the apoptotic rate were significantly reduced following the exposure to AßO, which was found to be attenuated by α7 nAChR overexpression, but potentiated by α7 nAChR RNA silencing. However, no significant differences were observed in either the ACh concentration or AChE activity following transfection. Collectively, these findings suggested that α7 nAChR may protect the brains of patients with AD against Aß, as α7 nAChR overexpression increased the expression levels of SYP, SNAP­25 and PSD­95, and attenuated the inhibitory effect of Aß on the expression of these synaptic proteins and cell apoptosis. Overall, this indicated that α7 nAChR may serve an important neuroprotective role in AD.

Activation of α7 nAChR by PNU-282987 improves synaptic and cognitive functions through restoring the expression of synaptic-associated proteins and the CaM-CaMKII-CREB signaling pathway.

Ligands of nicotinic acetylcholine receptors (nAChRs) are widely considered as potential therapeutic agents. The present study used primary hippocampus cells and APPswe/PSEN1dE9 double-transgenic mice models to study the possible therapeutic effect and underlying mechanism of the specific activation of α7 nAChR by PNU-282987 in the pathogenesis of Alzheimer's disease. The results indicated that activation of α7 nAChR attenuated the Aß-induced cell apoptosis, decreased the deposition of Aß, increased the expression of synaptic-associated proteins, and maintained synaptic morphology. Furthermore, in the APP/PS1_DT mice model, activation of α7 nAChR attenuated Aß-induced synaptic loss, reduced the deposition of Aß in the hippocampus, maintained the integral structure of hippocampus-derived synapse, and activated the calmodulin (CaM)-calmodulin-dependent protein kinase II (CaMKII)-cAMP response element-binding protein signaling pathway by upregulation of its key signaling proteins. In addition, activation of α7 nAChR improved the learning and memory abilities of the APP/PS1_DT mice. Collectively, the activation of α7 nAChR by PNU-282987 attenuated the toxic effect of Aß in vivo and in vitro, which including reduced deposition of Aß in the hippocampus, maintained synaptic morphology by partially reversing the expression levels of synaptic-associated proteins, activation of the Ca2+ signaling pathway, and improvement of the cognitive abilities of APP/PS1_DT mice.

The neuroprotective effects of SIRT1 in mice carrying the APP/PS1 double-transgenic mutation and in SH-SY5Y cells over-expressing human APP670/671 may involve elevated levels of α7 nicotinic acetylcholine receptors.

The aim was to determine whether the neuroprotective effect of SIRT1 in Alzheimer's disease (AD), due to inhibition of aggregation of the ß-amyloid peptide (Aß), involves activation of α7 nAChR. In present study, four-month-old APP/PS1 mice were administered resveratrol (RSV) or suramin once daily for two months, following which their spatial learning and memory were assessed using the Morris water maze test. Deposits of Aß in vivo were detected by near-infrared imaging (NIRI) and confocal laser scanning. SH-SY5Y/APPswe cells were treated with RSV, suramin, U0126 or methyllycaconitine (MLA). Levels of proteins and mRNA were determined by Western blotting and qRT-PCR, respectively. The results show that activation of SIRT1 improved their spatial learning and memory and reduced the production and aggregation of Aß in the hippocampus and cerebral cortex; whereas inhibition of SIRT1 had the opposite effects. In addition, activation of SIRT1 increased the levels of both α7 nAChR and αAPP in the brains these animals. Finally, activation of SIRT1 elevated the levels of pERK1/2, while inhibition of ERK1/2 counteracted the increase in α7 nAChR caused by RSV. These findings indicate that neuroprotection by SIRT1 may involve increasing levels of α7 nAChR through activation of the MAPK/ERK1/2 signaling pathway.

Lowered levels of nicotinic acetylcholine receptors and elevated apoptosis in the hippocampus of brains from patients with type 2 diabetes mellitus and db/db mice.

Cognitive impairment caused by diabetes has been gradually recognized. Generally, nicotinic acetylcholine receptors (nAChRs) play an important role in the pathogenesis in dementia disorders including Alzheimer's disease (AD). However, the expression of nAChRs in the brains of type 2 diabetes mellitus (T2DM) is unexplored. This study explored the alterations of nAChRs in the postmortem brains of patients with T2DM and brains of db/db mice. Morris water maze test was used to appraise the ability of spatial learning and memory; Western blotting and RT-qPCR were performed to determine the expressions of target protein and mRNA, respectively; TUNEL was used to detect the apoptosis of neurons. We found that the protein levels of nAChR α7 and α4 subunits were significantly decreased and the apoptosis rates in neurons elevated in the hippocampus of T2DM patients and db/db mice as comparison to controls. Furthermore, the db/db mice exhibited the impaired cognition, the elevated level of pro-apoptotic protein and the reduced level of anti-apoptotic and synaptic proteins. This study shows the lowered level of nAChR α7 and α4 subunits and the elevated apoptosis in the hippocampus of T2DM patients and db/db mice, which might help explain the impaired cognition in T2DM.