Objective To explore the regulatory role of hederagenin(HG)on glutamate(Glu)-in-duced ferroptosis and corresponding inflammatory responses in mouse hippocampal neuron HT22 cells and investigate its potential mechanisms.Methods HT22 cells were randomly divided into control,Glu and HG groups(n=3).The cells of the control group received no treatment,the cells of the Glu group were treated with 35 mmol/L Glu for 24 h to establish a cellular model of ferroptosis in Alzheimer's disease,and the cells of the HG group were treated with 0.5 μmol/L HG and 35 mmol/L Glu for 24 h simultaneously.FerroOrange fluorescent probe was used to de-tect intracellular Fe2+.The production of reactive oxygen species(ROS),mitochondrial membrane potential,and levels of inflammatory factors TNF-α,IL-1β and IL-6 in the cells were assessed.Finally,the expression of the key regulator of iron death,glutathione peroxidase 4(GPX4)was measured.Results Compared to the control group,the levels of intracellular Fe2+,ROS,TNF-α,IL-1β,and IL-6 were significantly elevated,while the mitochondrial membrane potential was obvi-ously reduced in the Glu group(P＜0.05,P＜0.01).The HG group had significantly decreased Fe2+,ROS,TNF-α,IL-1β,and IL-6 and enhanced mitochondrial membrane potential than the Glu group(P＜0.05,P＜0.01).The GPX4 expression was significantly lower in the Glu group than the control group(1.00±0.02 vs 0.46±0.04,P＜0.01),and was notably higher in the 0.5 and 1.0 μmol/L HG groups when compared to the Glu group(0.64±0.03 and 0.59±0.05 vs 0.46±0.04,P＜0.01).Conclusion HG inhibits ferroptosis by regulating GPX4 expression,and thereby effec-tively alleviates the inflammatory response.

Objective:To investigate the effects of cattle encephalon glycoside and ignotin(CEGI)on the expression of glial fibrillary acidic protein(GFAP)and neuronal nuclear antigen(NeuN)in the brain of APP/PS1 model mice of Alzheimer's disease.Methods:A total of 36 5-month-old APP/PS1 dual-transgenic model mice were randomly divided into 3 groups: the model group(normal saline 6.6 ml·kg -1·d -1), CEGI group(CEGI 6.6 ml·kg -1·d -1)and donepezil group(donepezil 2 mg·kg -1·d -1), with 12 in each group.Twelve C57BL/6J mice of the same age were used as the normal control group.All mice were given drugs for 6 weeks consecutively.Brain tissue was collected for immunohistochemical staining to detect the expression of amyloid β-protein(Aβ), GFAP and NeuN, which were then analyzed quantitatively. Results:The results of immunohistochemical staining indicated that levels of Aβ and GFAP were higher and levels of NeuN were lower in the model group than in the normal control group(0.147±0.068% vs.0%, 61.750±22.020 vs.26.000±4.598, 0.021±0.002 vs.0.032±0.003, P<0.05). Levels of Aβ and GFAP were lower and levels of NeuN were higher in the CEGI group and the donepezil group than in the model group(0.058±0.055 % vs.0.057±0.045 %, 38.250±5.418 vs.36.130±5.963, 0.029±0.004 vs.0.027±0.003, P<0.05). There was no significant difference in the expression of Aβ, GFAP and NeuN between the CEGI group and the donepezil group( P>0.05). Conclusions:CEGI has multi-target neuroprotective effects via down-regulating the expression of Aβ and GFAP and up-regulating the expression of NeuN.

Objective To study the pathological features of argyrophilic grains in normal aging brain, AD, PD and progressive superior nuclear paralysis patients. Methods Brain tissue samples taken from 5 AD, 3 PD, 2 progressive superior nuclear paralysis patients with complete clinico-pathological data and 4 normal aging brain subjects were stained with HE, Luxol fast blue and Gallyas-Braak silver respectively. Aβ, tau, α-synuclein and P62 antibodies were detected by microscopy with immunohistochemical staining. The pathological features of argyrophilic grains were recorded. Results The Gallyas-Braak silver staining showed argyrophilic grain structure in 4 out of the 14 amygdaloid nucleus tissue samples (2 from AD patients, 1 from progressive superior nuclear paralysis patients and 1 from normal aging brain patients) with a positive rate of 28.6％. The immunohistochemical staining showed positive tau and P62 antibodies. Conclusion Argyrophilic grain lesion is not uncommon in aging-related neurodegenerative diseases such as normal aging brain, AD and progressive superior nuclear paralysis and can thus produce its superposition effect on the clinical symptoms of cognitive impairment in AD and progressive superior nuclear paralysis patients.

Objective: To search for biomarkers for human familial prion disease. Methods: Two-dimensional differential gel electrophoresis (2D-DIGE) proteomic analysis has been performed in frontal lobe tissues of 3 patients suffering from human familial prion disease (PrP) and 3 age-and sex-matched patients suffering from sudden death due to heart failure without neurological disease. Results: The maps revealed 14 polypeptide chains differentially modulated in the PrP samples, among those, 7 could be identified upon digestion and MALDI-TOF/MS analysis, of which 6 appeared to be up-regulated, 1 being down-regulated. Conclusions We highlight Galectin-1(Gal-1), ryanodine receptor 2 (RyR2), ubiquitin, Rab-interacting lysosomes protein-like protein 1 (RILPL-1) profillin 2 (PFN2), in the differential map. These proteins are related to neurogenesis, the clearance of misfolded proteins, stasis of calium channel, myoclonus and so on. These proteins are potential biomarkers or targets for treatment of prion disease.

OBJECTIVETo investigate histopathology and proteinopathy in the spinal cord of patients with common neurodegenerative diseases.

METHODSSpinal cord tissues from clinically and neuropathologically confirmed neruodegnerative diseases were enrolled in this study, including 3 cases of multiple system strophy, 4 cases of amyotrophic lateral sclerosis, 5 cases of Alzheimer's disease (AD, included 2 cases of AD combined with Parkinson's disease), 2 cases of progressive supranuclear palsy, 1 case of dementia with lewy body and 1 case of corticobasal degeneration from 1955 to 2013 at Chinese People's Liberation Army General Hospital. Four normal control cases were also included. Routine HE and Gallyas-Braak staining, and immunohistochemical stainings for anti-PHF tau (AT8), anti-α-synuclein, anti-TDP-43 and anti-ubiquitin were performed.

RESULTSExamination of the spinal cord in 3 cases with multiple system strophy revealed severe neuron loss in the intermediolateral nucleus of thoracic segment and Onuf's nucleus of the sacral segment, along with moderate neuron loss in the anterior horn of the cervical segment and mild myelin pallor in the anterior funiculus and anterolateral funiculus in the cervical and thoracic segments. Large amount of argentophilic, ubiquitin and synuclein positive oligodendroglial cytoplasmic inclusions were found widely distributed in the anterior horn and the anterior funiculus and anterolateral funiculus of the full spinal cord. Severe neuron loss and several morphological changes with gliosis in the anterior horn and severe loss of myelin in the anterior funiculus and anterolateral funiculus of the full spinal cord were observed in 4 cases of amyotrophic lateral sclerosis, 2 of which were found with Bunina bodies in neurons of the anterior horn. Three amyotrophic lateral sclerosis cases had ubiquitin-positive neuronal inclusions and TDP-43 positive neuronal and glial inclusions in the anterior horn at cervical and lumbar segments. A few argentophilic, tau positive neurofibrillary tangles (NFTs) and neuropil threads in the anterior horn at cervical and lumbar segments were found in 4 AD cases. Examination of spinal cord in 2 cases with Parkinson's disease combined with AD and 1 case with dementia with lewy body revealed severe neuron loss in the intermediolateral nucleus of thoracic segment, and a few synuclein positive lewy bodies and neuritis were also observed. There was mild neuron loss in the anterior horn at cervical and lumbar segments, along with some argentophilic, tau positive globous NFTs and many argentophilic, tau positive neutrophil threads were observed in 2 progressive supranuclear palsy cases and 1 corticobasal degeneration case.

CONCLUSIONEach common neurodegenerative diseases of the spinal cord including multiple system strophy, amyotrophic lateral sclerosis and Parkinson's disease has its own specific histopathology and proteinopathy characteristics.

Alzheimer Disease ; pathology ; Amyotrophic Lateral Sclerosis ; pathology ; DNA-Binding Proteins ; metabolism ; Humans ; Immunohistochemistry ; Inclusion Bodies ; pathology ; Neurodegenerative Diseases ; pathology ; Neurofibrillary Tangles ; pathology ; Neurons ; pathology ; Parkinson Disease ; pathology ; Spinal Cord ; pathology ; Ubiquitin ; metabolism ; alpha-Synuclein ; metabolism

Objective To construct and identify lentiviral vector pGC-FU-CXCR4 gene. Methods CXCR4 gene amplification was used by real-time polymerase chain reaction. The target gene fragments with the digested plasmids were exchange. Then the lentiviral vector pGC-FU-CXCR4 was constructed successfully. Use the constructed lentiviral vector to infect the competent escherichia coli cells. Polymerase chain reaction analysis was used to identify the cultural clones and DNA sequencing and comparative analysis were used to positive fragments. The successfully constructed plasmids had the same sequence with the target gene. Results Polymerase chain reaction tests showed that am-plified target genes were inserted in pGC-FU vectors. The electrophoresis results,digestion showed that the reconstructed plasmid was consist-ent with the theoretical fragment and the sequence result of the positive fragments were exactly the same with the target gene. Conclusion Lentiviral vectors of CXCR4 gene over-expression were successfully constructed.

Objective To investigate the change of genomic DNA of liver and spleen tissue for different age of the elderly,and provide the experimental data for aging-related research. Methods 35 livers and 33 spleens of autopsied samples preserved in refrigerator at-80 ℃ were divided into 3 groups according to age:age 65y to 79y,age 80y to 89y,age≥90y. The content of DNA in liver and spleen was determined by ultraviolet absorbent method. Results Compaired with age 80y to 89y (0. 310 ± 0. 286)mg/mL,the content of DNA in liver was significant higher at age 65y to 79y (1.464 ±0.488)mg/mL and age ≥90y(1.147 ±0.333)mg/mL(P<0.05);Compared with age 80y to 89y(0. 938 ± 0. 589)mg/mL,the content of DNA in spleen was significant higher at age 65y to 79y(1. 723 ± 0. 726)mg/mL and age≥90y(1. 688 ± 0. 963)mg/mL(P<0. 05). The content of DNA was significant lower in liver (0. 856 ± 0. 658)mg/mL than that in spleen (1. 414 ± 0. 852)mg/mL. Conclusion The content of DNA in human liver and spleen tissue may be decrease along with aging. The content of DNA in the group at age≥90y may be increase. There were some differences between different viscera tissue in content of DNA.

Objective To investigate histopathology and proteinopathy in the spinal cord of patients with common neurodegenerative diseases. Methods Spinal cord tissues from clinically and neuropathologically confirmed neruodegnerative diseases were enrolled in this study , including 3 cases of multiple system strophy , 4 cases of amyotrophic lateral sclerosis , 5 cases of Alzheimer′s disease ( AD, included 2 cases of AD combined with Parkinson′s disease ) , 2 cases of progressive supranuclear palsy , 1 case of dementia with lewy body and 1 case of corticobasal degeneration from 1955 to 2013 at Chinese People′s Liberation Army General Hospital.Four normal control cases were also included.Routine HE and Gallyas-Braak staining , and immunohistochemical stainings for anti-PHF tau ( AT8 ) , anti-α-synuclein , anti-TDP-43 and anti-ubiquitin were performed.Results Examination of the spinal cord in 3 cases with multiple system strophy revealed severe neuron loss in the intermediolateral nucleus of thoracic segment and Onuf′s nucleus of the sacral segment , along with moderate neuron loss in the anterior horn of the cervical segment and mild myelin pallor in the anterior funiculus and anterolateral funiculus in the cervical and thoracic segments.Large amount of argentophilic , ubiquitin and synuclein positive oligodendroglial cytoplasmic inclusions were found widely distributed in the anterior horn and the anterior funiculus and anterolateral funiculus of the full spinal cord.Severe neuron loss and several morphological changes with gliosis in the anterior horn and severe loss of myelin in the anterior funiculus and anterolateral funiculus of the full spinal cord were observed in 4 cases of amyotrophic lateral sclerosis , 2 of which were found with Bunina bodies in neurons of the anterior horn.Three amyotrophic lateral sclerosis cases had ubiquitin-positive neuronal inclusions and TDP-43 positive neuronal and glial inclusions in the anterior horn at cervical and lumbar segments.A few argentophilic , tau positive neurofibrillary tangles ( NFTs) and neuropil threads in the anterior horn at cervical and lumbar segments were found in 4 AD cases.Examination of spinal cord in 2 cases with Parkinson′s disease combined with AD and 1 case with dementia with lewy body revealed severe neuron loss in the intermediolateral nucleus of thoracic segment , and a few synuclein positive lewy bodies and neuritis were also observed.There was mild neuron loss in the anterior horn at cervical and lumbar segments, along with some argentophilic , tau positive globous NFTs and many argentophilic , tau positive neutrophil threads were observed in 2 progressive supranuclear palsy cases and 1 corticobasal degeneration case.Conclusion Each common neurodegenerative diseases of the spinal cord including multiple system strophy,amyotrophic lateral sclerosis and Parkinson′s disease has its own specific histopathology and proteinopathy characteristics .

OBJECTIVETo recognize relationship of protein related neurodegeneration abnormal aggregation in the aged brains with their cognitive and motor functions.

METHODSBrain tissues from the consecutive autopsy cases of the aged from January 2005 to December 2006 in PLA General Hospital were carried out for immunohistochemical staining with beta amyloid, tau, α-synuclein and ubiquitin antibodies. The consortium to establish a registry for Alzheimer's disease (CERAD) was used to semi-quantitatively analyze Aβ positive core plaques density and Braak staging for tau positive neurofibrillary tangles (NFTs) and α-synuclein positive Lewy bodies. In addition, Aβ positive cerebral amyloid angiopathy (CAA), neuritic plaques and various ubiquitin positive structures were also observed. The relationship of these protein abnormal depositions in the aged brains with cognitive and motor functions were analyzed.

RESULTSIn brain tissues of 16 consecutive autopsy cases of the aged from 78 to 95 years, there were 13 cases with Aβ positive core plaques, their density was 2 cases with sparse, 2 cases with moderate and 9 cases with frequent, respectively, according to CREAD.Eight cases with Aβ positive CAA were found, including 6 cases of mild CAA and 2 cases of severe CAA. There were 12 cases with tau positive NFTs, including 6 cases with Braak stageI-II, 4 cases with stage III-IV and 2 cases with stage V-VI. There were 5 cases with frequent Aβ core plaques, meanwhile existing numerous tau/ubiquitin positive neuritic plaques and Braak stage IV-VI of tau positive NFTs, all of them presented cognitive dysfunction. Among 4 other cases with frequent Aβ core plaques, only one case coexisted α-synuclein positive Lewy bodies showed moderate cognitive impairment, remaining 3 cases did not present cognitive dysfunction. There were 4 cases with α-synuclein positive Lewy bodies in the brainstem, and all of these cases presented parkinsonian motor dysfunction. 13 cases with ubiquitin positive structures were found.

CONCLUSIONSBeta amyloid protein positive deposit in the aged brain is an important marker of normal brain aging and cognitive impairment; frequent Aβ core plaques in the neocortex plus Braak IV and above tau positive NFTs are closely related to cognitive dysfunction of Alzheimer's disease; α-synuclein positive Lewy bodies in the brainstem is one of the important pathological markers of parkinsonian motor disorders; ubiquitin deposition involves the development of some characteristic structures of several neurodegenerative diseases.

Aged ; Alzheimer Disease ; metabolism ; pathology ; Amyloid beta-Peptides ; analysis ; Autopsy ; Brain ; pathology ; Brain Chemistry ; Cerebral Amyloid Angiopathy ; Humans ; Neurofibrillary Tangles ; chemistry ; pathology ; Plaque, Amyloid ; Ubiquitin ; analysis ; alpha-Synuclein ; analysis ; tau Proteins ; analysis

OBJECTIVETo assess the association of vitamin D receptor (VDR) gene Fok I and Bsm I polymorphisms with dyslipidemia in elderly male patients with type 2 diabetes of Han nationality.

METHODSA total of 328 elderly male residents of Han nationality in Beijing, including 237 type 2 diabetic patients and 91 healthy control subjects, were enrolled in this study. The diabetic patients were divided into non-dyslipidemia group (DO group, n=134) and dyslipidemia group (DH group, n=103). All the participants were genotyped for Fok I and Bsm I polymorphisms in VDR gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing technology, and the results were compared with their clinical characteristics.

RESULTSFor Fok I, the frequency of F allele was significantly higher in the diabetic patients than in the control group (Χ(2)=3.873, P=0.049, OR=1.439, 95% CI: 1.001-2.071). In the dominant model, the frequency of FF genotype was significantly higher in the diabetic group (Χ(2)=5.057, P=0.025, OR=1.756, 95% CI: 1.072-2.875) as well as in DH group (Χ(2)=6.168, P=0.013, OR=2.06, 95% CI: 1.161-3.663) than in the control group. There was no significant differences in the genotype frequency or allele distribution in other paired groups (P>0.05). Compared with Ff + ff genotype, FF genotype was associated with a significantly decreased average diastolic blood pressure (P=0.039) but significantly increased postprandial blood glucose (P=0.035), triglycerides (P=0.049) and uric acid (P=0.031). No significant difference was detected in genotype frequency or allele distribution of Bsm I polymorphisms between the groups (P>0.05); serum creatinine levels were significantly higher in bb genotype than in BB + Bb genotype group (P=0.011).

CONCLUSIONVDR gene Fok I polymorphisms may be a risk factor for dyslipidemia in elderly male patients with type 2 diabetes among Chinese Han population, where Bsm I polymorphisms are not associated with diabetic dyslipdiemia.

Aged ; Alleles ; Blood Glucose ; Blood Pressure ; Case-Control Studies ; Diabetes Mellitus, Type 2 ; genetics ; Dyslipidemias ; genetics ; Ethnic Groups ; Genotype ; Humans ; Male ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Receptors, Calcitriol ; genetics ; Risk Factors ; Triglycerides ; blood