S-9-PAHSA's neuroprotective effect mediated by CAIII suppresses apoptosis and oxidative stress in a mouse model of type 2 diabetes.

BACKGROUND: With the rapidly increasing prevalence of metabolic diseases such as type 2 diabetes mellitus (T2DM), neuronal complications associated with these diseases have resulted in significant burdens on healthcare systems. Meanwhile, effective therapies have remained insufficient. A novel fatty acid called S-9-PAHSA has been reported to provide metabolic benefits in T2DM by regulating glucose metabolism. However, whether S-9-PAHSA has a neuroprotective effect in mouse models of T2DM remains unclear. METHODS: This in vivo study in mice fed a high-fat diet (HFD) for 5 months used fasting blood glucose, glucose tolerance, and insulin tolerance tests to examine the effect of S-9-PAHSA on glucose metabolism. The Morris water maze test was also used to assess the impact of S-9-PAHSA on cognition in the mice, while the neuroprotective effect of S-9-PAHSA was evaluated by measuring the expression of proteins related to apoptosis and oxidative stress. In addition, an in vitro study in PC12 cells assessed apoptosis, oxidative stress, and mitochondrial membrane potential with or without CAIII knockdown to determine the role of CAIII in the neuroprotective effect of S-9-PAHSA. RESULTS: S-9-PAHSA reduced fasting blood glucose levels significantly, increased insulin sensitivity in the HFD mice and also suppressed apoptosis and oxidative stress in the cortex of the mice and PC12 cells in a diabetic setting. By suppressing oxidative stress and apoptosis, S-9-PAHSA protected both neuronal cells and microvascular endothelial cells in in vivo and in vitro diabetic environments. Interestingly, this protective effect of S-9-PAHSA was reduced significantly when CAIII was knocked down in the PC12 cells, suggesting that CAIII has a major role in the neuroprotective effect of S-9-PAHSA. However, overexpression of CAIII did not significantly enhance the protective effect of S-9-PAHSA. CONCLUSION: S-9-PAHSA mediated by CAIII has the potential to exert a neuroprotective effect by suppressing apoptosis and oxidative stress in neuronal cells exposed to diabetic conditions. Furthermore, S-9-PAHSA has the capability to reduce fasting blood glucose and LDL levels and enhance insulin sensitivity in mice fed with HFD.

Effect of acupuncture with donepezil based on syndrome differentiation on cognitive function in patients with mild-to-moderate Alzheimer's disease: a study protocol for a multicenter randomized controlled trial.

BACKGROUND: There has been a rapid increase in the worldwide prevalence of Alzheimer's disease (AD). Previous studies have shown that acupuncture can improve neurological and cognitive function; however, the utility of applying acupuncture in patients with AD remains unclear. This study protocol describes a clinical trial for evaluating the efficacy and safety of acupuncture based on syndrome differentiation with donepezil hydrochloride on cognitive function in patients with AD. METHODS/DESIGN: This multicenter randomized controlled trial commenced on February 1, 2019, at the Shanghai Longhua Hospital of TCM, Shanghai Huashan Hospital of Fudan University, and Shanghai Mental Health Center, and will conclude on June 30, 2022. The study will recruit 184 patients randomly divided into an acupuncture group or a control group at a 1:1 ratio. All participants will receive donepezil hydrochloride (5 mg/day), and those in the acupuncture group will receive acupuncture based on syndrome differentiation with donepezil for 12 weeks. The primary outcome will be the post-treatment change in the Alzheimer's Disease Assessment Scale-cognition score at 12 weeks. The secondary outcomes will be the efficacy scores of the Minimum Mental State Examination, Alzheimer's Disease Cooperative Research Activity-Daily Life, and Quality of Life-Alzheimer's Disease. All assessments will be performed at baseline, after treatment (week 12), and at follow-up (weeks 24 and 36). DISCUSSION: This trial may provide high-quality evidence for the efficacy of acupuncture in the treatment of AD. The results of this study will be published in peer-reviewed journals. TRIAL REGISTRATION: ClinicalTrials.gov NCT03810794 . Registered on 17 January 2019.

High glucose and palmitic acid induces neuronal senescence by NRSF/REST elevation and the subsequent mTOR-related autophagy suppression.

Cell senescence is a basic aging mechanism. Previous studies have found that the cellular senescence in adipose tissue and other tissues, such as the pancreas, muscle and liver, is associated with the pathogenesis and progression of type 2 diabetes; however, strong evidence of whether diabetes directly causes neuronal senescence in the brain is still lacking. In this study, we constructed a high glucose and palmitic acid (HGP) environment on PC12 neuronal cells and primary mouse cortical neurons to simulate diabetes. Our results showed that after HGP exposure, neurons exhibited obvious senescence-like phenotypes, including increased NRSF/REST level, mTOR activation and cell autophagy suppression. Downregulation of NRSF/REST could remarkably alleviate p16, p21 and γH2A.X upregulations induced by HGP treatment, and enhance mTOR-autophagy of neurons. Our results suggested that the diabetic condition could directly induce neuronal senescence, which is mediated by the upregulation of NRSF/REST and subsequent reduction of mTOR-autophagy.

Knockdown of NRSF Alleviates Ischemic Brain Injury and Microvasculature Defects in Diabetic MCAO Mice.

Diabetes is one of the well-established risk factors of stroke and is associated with a poor outcome in patients with stroke. Previous studies have shown that the expression of neuron restrictive silencer factor (NRSF) is elevated in diabetes as well as ischemic stroke. However, the role of NRSF in regulating an outcome of diabetic ischemic stroke has not been completely understood. Here, we hypothesized that diabetes-induced NRSF elevation can aggravate brain injury and cognition impairment in ischemic stroke. The diabetic ischemic stroke mice model was established by 8 weeks of high-fat-diet feeding and 5 days of streptozotocin injection followed by 30 min of middle cerebral artery occlusion (MCAO). We found that diabetes enhanced the MCAO-induced elevation of NRSF in the hippocampus in accompany with an elevation of its corepressors, HDAC1, and mSin3A, and decrease of ß-TrCP. By using histological/immunofluorescence staining and neurobehavioral testing, our results showed that the brain damage and learning/memory impairment were aggravated in diabetic ischemic mice but significantly attenuated after stereotaxic injection of NRSF-shRNA. Meanwhile, by performing whole-brain clearing with PEGASOS, microvascular reconstruction, western blotting, and ELISA, we found that NRSF-shRNA markedly alleviated the vasculature disorders and rescued the suppression of NRP-1, VEGF, and VEGFR2 in the hippocampus of diabetic ischemic mice. Therefore, our results demonstrated for the first time that the elevation of hippocampal NRSF plays an important role in alleviating brain injury and cognitive disabilities in diabetic ischemic mice, potentially via the reduction of NRP-1/VEGF signaling.

9-PAHSA Improves Cardiovascular Complications by Promoting Autophagic Flux and Reducing Myocardial Hypertrophy in Db/Db Mice.

Atherosclerotic cardiovascular disease is a common and severe complication of diabetes. There is a large need to identify the effective and safety strategies on diabetic cardiovascular disease (DCVD). 9-PAHSA is a novel endogenous fatty acid, and has been reported to reduce blood glucose levels and attenuate inflammation. We aim to evaluate the effects of 9-PAHSA on DCVD and investigate the possible mechanisms underlying it. Firstly, serum 9-PAHSA levels in human were detected by HPLC-MS/MS analysis. Then 9-PAHSA was synthesized and purified. The synthesized 9-PAHSA was gavaged to db/db mice with 50 mg/kg for 4 weeks. The carotid arterial plaque and cardiac structure was assessed by ultrasound. Cardiac autophagy was tested by western blot analysis, electron microscope and iTRAQ. The results showed that 9-PAHSA, in patients with type 2 diabetes mellitus (T2DM), was significantly lower than that in non-diabetic subjects. Administration of 9-PAHSA for 2 weeks reduced blood glucose levels. Ultrasound observed that continue administration of 9-PAHSA for 4 weeks ameliorated carotid vascular calcification, and attenuated myocardial hypertrophy and dysfunction in db/db mice. Electron microscopy showed continue 9-PAHSA treatment significantly increased autolysosomes, while dramatically decreased greases in the myocardial cells of the db/db mice. Moreover, iTRAQ analysis exhibited that continue 9-PAHSA treatment upregulated BAG3 and HSPB8. Furthermore, western blot analysis confirmed that 9-PAHSA down-regulated Akt/mTOR and activated PI3KIII/BECN1 complex in diabetic myocardium. Thus, 9-PAHSA benefits DCVD in diabetic mice by ameliorating carotid vascular calcification, promoting autophagic flux and reducing myocardial hypertrophy.

The role of carbonic anhydrase III and autophagy in type 2 diabetes with cardio-cerebrovascular disease.

Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases among the elderly people. The T2DM increases the risk of cardio-cerebrovascular disease (CCD), and the main pathological change of the CCD is atherosclerosis (AS). Meanwhile, the carbonic anhydrases (CAs) are involved in the formation and progression of plaques in AS. However, the exact physiological mechanism of carbonic anhydrase III (CAIII) has not been clear yet, and there are also no correlation study between CAIII protein and T2DM with CCD. The 8-week old diabetic mice (db/db-/- mice) and wild-type mice (wt mice) were feed by a normal diet till 32 weeks, and detected the carotid artery vascular opening angle using the method of biomechanics; The changes of cerebral cortex and myocardium were watched by the ultrastructure, and the autophagy were observed by electron microscope; The tissue structure, inflammation and cell injury were observed by Hematoxylin and eosin (HE) staining; The apoptosis of cells were observed by TUNEL staining; The protein levels of CAIII, IL-17, p53 were detected by immunohistochemical and Western Blot, and the Beclin-1, LC3, NF-κB were detected by Western Blot. All statistical analysis is performed using PRISM software. Compared with wt mice, db/db-/- mice' carotid artery open angle increased significantly. Electron microscope results indicated that autophagy in db/db-/- mice cerebral cortex and heart tissue decreased and intracellular organelle ultrastructure were damaged. HE staining indicated that, db/db-/- mice' cerebral cortex and heart tissue stained lighter, inflammatory cells infiltration, cell edema were obvious, myocardial fibers were disorder, and myocardial cells showed different degrees of degeneration. Compared with wt mice, TUNEL staining showed that there was obviously increase in db/db-/- mice cortex and heart tissue cell apoptosis. The results of immunohistochemistry and Western Blot indicated that CAIII, Beclin-1 and LC3II/I expression levels conspicuously decreased in cortex and heart tissue of db/db-/- mice, and the expression level of IL-17, NF-κB and p53 obviously increased. The carotid artery' vascular stiffness was increased and which was probably related with formation of AS in diabetic mice. And the autophagy participated in the occurrence and development of diabetic CCD. CAIII protein might somehow be involved in the regulation of autophagy probably through affecting cell apoptosis and inflammation, but the underlying mechanism remains to be further studied.

A novel palmitic acid hydroxy stearic acid (5-PAHSA) plays a neuroprotective role by inhibiting phosphorylation of the m-TOR-ULK1 pathway and regulating autophagy.

AIMS: Type 2 diabetes mellitus (T2DM) can lead to brain dysfunction and a series of neurological complications. Previous research demonstrated that a novel palmitic acid (5-PAHSA) exerts effect on glucose tolerance and chronic inflammation. Autophagy was important in diabetic-related neurodegeneration. The aim of the present study was to investigate whether 5-PAHSA has specific therapeutic effects on neurological dysfunction in diabetics, particularly with regard to autophagy. METHODS: 5-PAHSA was successfully synthesized according to a previously described protocol. We then carried out a series of in vitro and in vivo experiments using PC12 cells under diabetic conditions, and DB/DB mice, respectively. PC12 cells were treated with 5-PAHSA for 24 h, while mice were administered with 5-PAHSA for 30 days. At the end of each experiment, we analyzed glucolipid metabolism, autophagy, apoptosis, oxidative stress, cognition, and a range of inflammatory factors. RESULTS: Although there was no significant improvement in glucose metabolism in mice administered with 5-PAHSA, ox-LDL decreased significantly following the administration of 5-PAHSA in serum of DB/DB mice (p < 0.0001). We also found that the phosphorylation of m-TOR and ULK-1 was suppressed in both PC12 cells and DB/DB mice following the administration of 5-PAHSA (p < 0.05 and p < 0.01), although increased levels of autophagy were only observed in vitro (p < 0.05). Following the administration of 5-PAHSA, the concentration of ROS decreased in PC12 cells and the levels of CRP increased in high-dose group of 5-PAHSA (p < 0.01). There were no significant changes in terms of apoptosis, other inflammatory factors, or cognition in DB/DB mice following the administration of 5-PAHSA. CONCLUSION: We found that 5-PAHSA can enhance autophagy in PC12 cells under diabetic conditions. Our data demonstrated that 5-PAHSA inhibits phosphorylation of the m-TOR-ULK1 pathway and suppressed oxidative stress in PC12 cells, and exerted influence on lipid metabolism in DB/DB mice.

Corrigendum: 9-PAHSA Improves Cardiovascular Complications by Promoting Autophagic Flux and Reducing Myocardial Hypertrophy in Db/Db Mice.

[This corrects the article DOI: 10.3389/fphar.2021.754387.].

EGb761 improves the cognitive function of elderly db/db-/- diabetic mice by regulating the beclin-1 and NF-κB signaling pathways.

To assess whether EGb761 could protect elderly diabetic mice with cognitive disorders and explore the role of beclin-1-mediated autophagy in these protective effects. Two-month-old male db/db-/- mice and wild-type C57/BL6 mice were randomly divided into six groups: db/db-/- control, db/db-/- 50 mg, db/db-/- 100 mg, wild-type (WT) control, WT 50 mg, and WT 100 mg. EGb761 (50 mg/kg or 100 mg/kg of bodyweight) was given by gavage once a day for 1 month from the age of 6 months. Y-maze and social choice tests were performed at 8th months. The blood pressure was measured. The imaging changes in the brain were measured using magnetic resonance imaging (MRI). The expression and distribution of beclin-1, LC3, and NF-κB were detected using immunohistochemistry staining and western blotting. Ultrastructure alterations in the hippocampus were observed using transmission electron microscopy. Compared with WT mice, the learning ability, memory and overall cognitive function of db/db-/- mice decreased (P < 0.05), and EGb761 could significantly improve the learning and memory function of db/db-/- mice (P < 0.05). EGb761 significantly improved systolic blood pressure in db/db-/- mice (P < 0.01). In addition, fMRI-bold showed a decline in the hippocampus of mice in the db/db-/- group compared with WT. EGb761 could improve these above changes. Immunohistochemistry staining and western blotting confirmed that EGb761 significantly increased beclin-1 and reduced LC3-II/I levels in the brains of db/db-/- mice (P < 0.05). NF-κB levels were obviously higher in the db/db-/- group than that in the WT group, and EGb761 significantly reduced NF-κB levels in db/db-/- mice (P < 0.05). There was a trend of increased autophagosomes in db/db-/- mice, but EGb761 did not change obviously the number of autophagosomes. Compared with normal aged WT mice, aging db/db-/- mice had more common complications of cerebral small vessel disease and cognitive dysfunction. EGb761 could significantly improve the cognitive function of aging db/db-/- mice via a mechanism that may involve the regulation of beclin-1, LC3, and NF-κB.

G-CSF and cognitive dysfunction in elderly diabetic mice with cerebral small vessel disease: Preventive intervention effects and underlying mechanisms.

AIMS: Although cognitive dysfunction is a common neurological complication in elderly patients with diabetes, the mechanisms underlying this relationship remain unclear, and effective preventive interventions have yet to be developed. Thus, this study investigated the preventive effects and mechanisms of action associated with granulocyte colony-stimulating factor (G-CSF) on cognitive dysfunction in elderly diabetic mice with cerebral small vessel disease. METHODS: This study included 40 male db/db diabetic and wild-type (WT) mice that were categorized into the following four groups at the age of 3 weeks: db/db group (DG), db/db+G-CSF group (DGG), WT group (WG), and WT+G-CSF group (WGG). The mice were fed normal diets for 4 months and then given G-CSF (75 µg/kg) via intraperitoneal injections for 1 month. At 7.5 months of age, the cognitive abilities of the mice were assessed with the Y-maze test and the Social Choice Test; body weight, blood pressure (BP), and blood glucose measurements were obtained throughout the study. Brain imaging and blood oxygen level-dependent (BOLD) contrast imaging analyses were performed with a small animal magnetic resonance imaging (MRI) system, autophagosome levels were detected with a transmission electron microscope (TEM), hippocampal neurons were assessed with hematoxylin and eosin (HE) staining, and protein expressions and distributions were evaluated using immunohistochemistry and Western blot analyses. RESULTS: (i) The body weight and blood glucose levels of the DG and DGG mice were significantly higher than those of the WG and WGG mice; (ii) social choice and spatial memory capabilities were significantly reduced in DG mice but were recovered by G-CSF in DGG mice; (iii) the MRI scans revealed multiple lacunar lesions and apparent hippocampal atrophy in the brains of DG mice, but G-CSF reduced the number of lacunar lesions and ameliorated hippocampal atrophy; (iv) the MRI-BOLD scans showed a downward trend in whole-brain activity and reductions in the connectivities of the hippocampus and amygdala with subcortical structures in DG mice, but G-CSF clearly improved the altered brain activity as well as the connectivity of the hippocampus in DGG mice; (v) HE staining revealed fewer neurons in the hippocampus in DG mice; (vi) TEM analyses revealed significantly fewer autophagosomes in the hippocampi of DG mice, but G-CSF did not increase these numbers; (vii) there were significant reductions in mechanistic target of rapamycin (mTOR) and LC3-phosphatidylethanolamine conjugate (LC3)-II/I levels in the hippocampi of DG mice, whereas p62 was upregulated, and G-CSF significantly enhanced the levels of Beclin1, mTOR, and LC-II/I in DGG mice; and (viii) G-CSF significantly reversed increases in nuclear factor κB (NF-κB) protein levels in DG but not in WG mice. CONCLUSIONS: In this study, aged diabetic mice were prone to cognitive dysfunction and cerebral small vessel disease. However, administration of G-CSF significantly improved cognitive function in elderly db/db diabetic mice, and this change was likely related to the regulation of autophagy and NF-κB signaling pathways.

Beclin-1- mediated autophagy may be involved in the elderly cognitive and affective disorders in streptozotocin-induced diabetic mice.

BACKGROUND: Diabetes is the most common metabolic disease with many chronic complications, and cognitive disorders are one of the common complications in patients with diabetes. Previous studies have showed that autophagy played important roles in the progression of metabolic syndrome, diabetes and other diseases. So we investigated whether aged diabetic mice are prone to be associated with the cognitive and affective disorders and whether Beclin-1-mediated autophagy might be involved in thepahological process. METHODS: High-fat diet/streptozotocin (STZ) injection-induced diabetic C57 mice were adopted in this study. Cognitive disorders were detected by Morris water maze and fear conditional test. Affective disorders were detected by tail suspension test and forced swimming test. Magnetic resonance imaging was applied to observe changes of morphology and metabolism in the brain. The 18 F-fluorodeoxyglucose positron emission tomography (FDG-PET) was used to assess metabolism changes in the brain of aged diabetic mice. Autophagy were evaluated by Beclin- 1, LC3II/I and P62, which were detected by western blot analysis and observed by electron microscopy. RESULTS: 1. Compared with control group, diabetes mice showed significantly decreasing abilities in spatial memory and conditioned fear memory (all P < 0.05), and increasing tendency of depression (P < 0.05). 2. MRI showed that the majority of elderly diabetic mice were associated with multiple cerebral small vessel disease. Some even showed hippocampal atrophy, ventricular dilatation and leukoaraiosis. 3. FDG-PET-CT discovered that the glucose metabolism in the amygdala and hippocampus was significantly decreased compared with normal aged mice (P < 0.05). 4. Electron microscopy found that, although autophagy bodies was not widespread, and there was no significant difference between the two groups, yet compared with normal aged mice, apparent cell edema, myelinated tow reduction and intracellular lipofuscin augmentation existed in elderly diabetic mice brain. 5. The level of p62 was increased in the STZ-induced diabetic mice hippocampus and striatum, and beclin1 protein expression were significantly decreased in diabetic mice hippocampus compared with normal aged mice (P < 0.05). There was a upward trend of the ratio of LC3II/I in hippocampus, cortex and striatum, but no statistically difference between the two groups. CONCLUSION: Compared with normal aged mice, diabetic aged mice were apt to cerebral small vessel disease and associated with cognitive and affective disorders, which may be related to the significantly reduced glucose metabolism in hippocampus and amygdala. Beclin1 mediated autophagy in hippocampus probably played an important role in cognitive and affective disorders of STZ-induced aged diabetic mice.

Enhanced Neuroprotective Effects of Coadministration of Tetrandrine with Glutathione in Preclinical Model of Parkinson's Disease.

Aim. In this study we examined the influence of tetrandrine (Tet) on the neuroprotective effects of glutathione (GSH) in the 6-hydroxydopamine- (6-OHDA-) lesioned rat model of Parkinson's disease (PD). Methods. Levels in the redox system, dopamine (DA) metabolism, dopaminergic neuronal survival, and apoptosis of the substantia nigra (SN) and striatum, as well as the rotational behavior of animals were examined after a 50-day administration of GSH + Tet (or GSH) and/or L-3,4-dihydroxyphenylalanine (L-dopa) to PD rats. Ethics Committee of Huashan Hospital, Fudan University approved the protocol (number SYXK2009-0082). Results. Administration of GSH or Tet alone did not show any significant effects on the factors evaluated in the PD rats. However, in the GSH + Tet group, we observed markedly decreased oxidative damage, inhibition of DA metabolism and enhanced DA synthesis, increased tyrosine hydroxylase- (TH-) immunopositive neuronal survival, and delayed apoptosis of dopaminergic neurons in the SN. Animal rotational behavior was improved in the GSH + Tet group. Additionally, coadministration of GSH + Tet appeared to offset the possible oxidative neurotoxicity induced by L-dopa. Conclusion. In this study, we demonstrated that tetrandrine allowed occurrence of the neuroprotective effect of glutathione probably due to inhibition of P-glycoprotein on 6-hydroxydopamine-lesioned rat models of Parkinson's disease, including rats undergoing long-term L-dopa treatment.

NF-κB Signaling is Involved in the Effects of Intranasally Engrafted Human Neural Stem Cells on Neurofunctional Improvements in Neonatal Rat Hypoxic-Ischemic Encephalopathy.

AIM: Hypoxic-ischemic encephalopathy (HIE) is a common neurological disease in infants with persistent neurobehavioral impairments. Studies found that neural stem cell (NSC) therapy benefits HIE rats; however, the mechanisms underlying are still unclear. The current study investigated the efficacy and molecular events of human embryonic neural stem cells (hNSCs) in neonatal hypoxic-ischemic (HI) rats. METHODS: PKH-26-labeled hNSCs were intranasally delivered to P7 Sprague Dawley rats 24 h after HI. Neurobehavioral tests were performed at the indicated time after delivery: righting reflex and gait testing at D1, 3, 5, and 7; grid walking at D7 and 14; social choice test (SCT) at D28; and Morris water maze from D35 to 40. Protein expression was determined by Western blot analysis. Brain damage was assessed by cresyl violet staining and MBP staining. hNSC distribution and differentiation were observed by in vivo bioluminescence imaging and immunofluorescence staining. RESULTS: (1) hNSCs migrated extensively into brain areas within 24 h after the delivery, survived even at D42 with the majority in ipsi-hemisphere, and could be co-labeled with NeuN or GFAP. (2) hNSCs reduced the upregulation in cytosolic IL-1ß, p-IκBα, and NF-κB p65 levels, whereas enhanced nuclear p65 expression in HI rats at D3 after the delivery. (3) hNSCs decreased HI-induced brain tissue loss and white matter injury at D42 after the delivery. (4) hNSCs improved neurological outcomes in HI rats in the tests of righting reflex (within 3 days), gait (D5), grid (D7), SCT (D28), and water maze (D42). CONCLUSION: Intranasal delivery of hNSCs could prevent HI-induced brain injury and improve neurobehavioral outcomes in neonatal HI rats, which is possibly related to the modulation of NF-κB signaling.

Acid-sensing ion channels activation and hypoxia upregulate Homer1a expression.

BACKGROUND: Recent studies have indicated that dynamic alterations in the structure of postsynaptic density (PSD) are involved in the pathogenesis of many central nervous system disorders, including ischemic stroke. Homer is the newly identified scaffolding protein located at PSD and regulates synaptic function. Homer1a, an immediate early gene, has been shown to be induced by several stimulations, such as glutamate, brain-derived neurotrophic factor, and trauma. However, whether acidosis mediated by acid-sensing ion channels (ASICs) and hypoxia during cerebral ischemia can change Homer1a expression remains to be determined. RESULTS: We investigated that acidosis and hypoxia selectively and rapidly upregulated Homer1a expression, but not Homer1b/c in cultured cortical neurons. We also found that Homer1a exhibited induction expression in brain cortex of the middle cerebral artery occlusion (MCAO) rats. Additionally, acid-evoked Homer1a mRNA induction depended on extracellular signal-regulated kinase1/2 (ERK1/2) and Akt activity, and ASIC1a-mediated calcium influx whereas hypoxia depended only on ERK1/2 activity. Also, we demonstrated that continuous acidosis and hypoxia resulted in pronounced cell injury and Homer1a knockdown with small interfering RNA aggravated this damage induced by 3 h acid and hypoxia incubation in neuro-2a cells. CONCLUSION: Homer1a might act as an activity-dependent regulator responding to extracellular stimuli during cerebral ischemia.

[Synchrotron radiation-based FTIR microspectroscopy study of 6-hydroxydopamine induced Parkinson's disease cell model].

SH-SY5Y cell line treated with 6-hydroxydopamine (6-OHDA) is a classical Parkinson's disease (PD). In the present study, synchrotron-based Fourier transform infrared (FTIR) microspectroscopy was used to analyze the biochemical composition of SH-SY5Y cell line treated with 6-OHDA. The detailed spectral analyses show the significant changes in cellular compositions such as lipids, proteins and nucleic acids in SH-SY5Y cells treated with 6-OHDA compared to control SH-SY5Y cells. As a result, the unsaturation levels of phospholipids decrease in SH-SY5Y cells treated with 6-OHDA compared to control cells, the analysis of protein secondary structure shows the significantly higher ratio of beta-sheet in PD cells compared to that of control cells, and the content of nuclear acid is highly decreased compared to that of control cells, suggesting that 6-OHDA induces the serious oxidative damage in SH-SY5Y cells. These findings suggest that SR-FTIR is an effective and precise technical tool to probe the biochemical changes of cells and then evaluate the pathological damage in cells.

Glutathione prevents free fatty acids-induced oxidative stress and apoptosis in human brain vascular endothelial cells through Akt pathway.

AIMS: The damage of human brain vascular endothelial cells (HBVECs) is the key pathogenesis of diabetes-associated cerebral vascular complications. The aim of this study was to elucidate the effects of glutathione (GSH) on free fatty acids (FFAs)-induced HBVECs apoptosis, oxidative stress, and the involved possible signaling pathway. METHODS: After culturing HBVECs for 72 h with GSH and FFAs, we determined cell proliferation by CCK8, detected apoptosis by caspase-3 and Annexin V-FITC/PI staining, and judged oxygen stress by determining the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP). We investigated whether the Akt pathway was involved in FFAs-induced signaling pathway alteration and whether GSH influenced the above effects. RESULTS: After being cultured in 200 µM FFAs for 72 h, the HBVECs proliferation significantly decreased; HBVECs apoptosis increased; the ROS levels increased; and the HBVECs MMP subsequently decreased. FFAs induced a significant decrease in phosphorylated active Akt. These alterations were obviously prevented when 1 mM GSH was added to culture medium containing FFAs, and the above effects of GSH were blocked by Akt inhibitor. CONCLUSION: GSH may prevent FFAs-induced HBVECs damage, oxidative stress, and apoptosis through activating the Akt pathway.

The bradykinin B2 receptor mediates hypoxia/reoxygenation induced neuronal cell apoptosis through the ERK1/2 pathway.

The bradykinin B2 receptor (B2R) mediates many physiological processes such as hypotension, inflammation and blood-vessel permeability. Hypoxia/reoxygenation (H/R) induces neuronal cell apoptosis. It was found that B2R expression was enhanced in primary cultured cortical neurons after H/R treatment. Addition of bradykinin (BK) alleviated the neuronal damage from H/R. This protective effect of BK was inhibited by the B2R antagonist, HOE140, and the ERK1/2 antagonist, PD98059. The phosphorylation of ERK1/2 was increased under H/R, and the addition of BK enhanced this effect. These results indicate that B2R plays an important role in protecting neurons from damage induced by H/R and this effect may function through the ERK1/2 pathway.