Relationship between β-amyloid protein 1-42, thyroid hormone levels and the risk of cognitive impairment after ischemic stroke

BACKGROUND Post-stroke cognitive impairment(PSCI)is not only a common consequence of stroke but also an important factor for adverse prognosis of patients.Biochemical indicators such as blood lipids and blood pressure are affected by many factors,and the ability of evaluating the progress of patients with PSCI is insufficient.Therefore,it is necessary to find sensitive markers for predicting the progress of patients and avoiding PSCI.Recent studies have shown thatβ-amyloid protein 1-42(Aβ1-42)and thyroid hormone levels are closely related to PSCI,which may be the influencing factors of PSCI,but there are few related studies.AIM To investigate the relationship between serum levels of Aβand thyroid hormones in acute stage and PSCI and its predicted value.METHODS A total of 195 patients with acute cerebral infarction confirmed from June 2016 to January 2018 were enrolled in this study.Baseline data and serological indicators were recorded to assess cognitive function of patients.All patients were followed up for 1 year.Their cognitive functions were evaluated within 1 wk,3 mo,6 mo and 1 yr after stroke.At the end of follow-up,the patients were divided into PSCI and non-PSCI according to Montreal cognitive assessment score,and the relationship between biochemical indexes and the progression of PSCI was explored.RESULTS Compared with patients with non-PSCI,the levels of Aβ1-42,triiodothyronine(T3)and free thyroxin were lower in the patients with PSCI.Repeated measures analysis of variance showed that the overall content of Aβ1-42 and T3 in PSCI was also lower than that of the non-PSCI patients.Further analysis revealed that Aβ1-42(r=0.348),T3(r=0.273)and free thyroxin(r=0.214)were positively correlated with disease progression(P<0.05),suggesting that these indicators have the potential to predict disease progression and outcome.Cox regression analysis showed that Aβ1-42 and T3 were important factors of PSCI.Then stratified analysis showed that the lower the Aβ1-42 and T3,the higher risk of PSCI in patients who were aged over 70,female and illiterate.CONCLUSION Aβ1-42 and T3 have the ability to predict the progression of PSCI,which is expected to be applied clinically to reduce the incidence of PSCI and improve the quality of life of patients.

Effect of Panax notoginseng saponins on the expression of beta-amyloid protein in the cortex of the parietal lobe and hippocampus, and spatial learning and memory in a mouse model of senile dementia

BACKGROUND： The pharmacological actions of Panax notoginseng saponins （PNS） lie in removing free radicals, anti-inflammation and anti-oxygenation. It can also improve memory and behavior in rat models of Alzheimer＇s disease. OBJECTIVE： Using the Morris water maze, immunohistochemistry, real-time PCR and RT-PCR, this study aimed to measure improvement in spatial learning, memory, expression of amyloid precursor protein （App） and β -amyloid （A β ）, to investigate the mechanism of action of PNS in the treatment of AD in the senescence accelerated mouse-prone 8 （SAMP8） and compare the effects with huperzine A. DESIGN, TIME AND SETTING： A completely randomized grouping design, controlled animal experiment was performed in the Center for Research ＆ Development of New Drugs, Guangxi Traditional Chinese Medical University from July 2005 to April 2007. MATERIALS： Sixty male SAMP8 mice, aged 3 months, purchased from Tianjin Chinese Traditional Medical University of China, were divided into four groups： PNS high-dosage group, PNS low-dosage group, huperzine A group and control group. PNS was provided by Weihe Pharmaceutical Co., Ltd. （batch No.： Z53021485, Yuxi, Yunan Province, China）. Huperzine A was provided by Zhenyuan Pharmaceutical Co., Ltd. （batch No.： 20040801, Zhejiang, China）. METHODS： The high-dosage group and low-dosage group were treated with 93.50 and 23.38 mg/kg PNS respectively per day and the huperzine A group was treated with 0.038 6 mg/kg huperzine A per day, all by intragastric administration, for 8 consecutive weeks. The same volume of double distilled water was given to the control group. MAIN OUTCOME MEASURES： After drug administration, learning and memory abilities were assessed by place navigation and spatial probe tests. The recording indices consisted of escape latency （time-to-platform）, and the percentage of swimming time spent in each quadrant. The number of A β 1-40, A β 1-42 and App immunopositive neurons in the brains of SAMP8 mice was analyzed by immunohistochemistry. The mRNA content ofApp, tau, acetylcholinesterase, and synaptophysin （Syp） was tested by real time PCR and RT-PCR. RESULTS： The PCR results show that PNS can downregulate the expression of the App gene and upregulate the expression of the Syp gene in the parietal cortex and hippocampus of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than those of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. The results of the Morris water maze and immunohistochemistry indicated that PNS can improve the capacity for spatial learning and memory in SAMP8 mice, and reduce the content of A β 1-40, A β 1-42 and expression of App in the brains of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than that of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. CONCLUSION： These results support the hypothesis that PNS plays a therapeutic and protective role on the pathological lesions and learning dysfunction of Alzheimer＇s disease. The therapeutic effects of PNS for Alzheimer＇s disease are possibly achieved through downregulating the expression of the App gene and upregulating the expression of the Syp gene. The therapeutic effects of PNS are dose-dependent and are greater than the effect of huperzine A.

Key gene and protein changes in the beta-amyloid pathway following Longyanshen polysaccharides treatment in a mouse model of Alzheimer's disease

BACKGROUND： During onset and development of Alzheimer＇s disease, β-amyloid （Aβ） precursor protein （APP）, β-site amyloid precursor protein cleaving enzyme （BACE）, and β-amyloid are key genes and proteins in the Aβ pathway, and over-expression of these genes can lead to Aβ deposit/on in the brain. OBJECTIVE： To observe the influence of Longyanshen polysaccharides on expression of BACE, APP, and Aβ in the senescence-accelerated mouse prone/8 （SAMP8） brain, and to compare these effects with huperzine A treatment. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiochemical experiment was performed at the Department of Pharmacology and Scientific Experimental Center of Guangxi Medical University from September 2005 to January 2008. MATERIALS： Longyanshen polysaccharfdes powder was extracted from the dried slices of the medicinal plant Longyanshen. The active component, Longyanshen polysaccharides, was provided by the Department of Pharmacology, Guangxi Medical University; huperzine A was purchased from Yuzhong Drug Manufactory, China. METHODS： Healthy SAMP8 mice were used to establish a model of Alzheimer＇s disease. A total of 50 SAMP8 mice were randomly assigned to 5 groups （n = 10）： SAMP8, huperzine A, low-, middle-, and high-dose polysaccharides. In addition, 10 senescence-accelerated mouse resistant 1 （SAMR1） mice were selected as normal controls. SAMP8 and SAMR1 mice were administered 30 mL/kg normal saline; the huperzine A group was administered 0.02 mg/kg huperzine A; the low-, middle-, and high-dose polysaccharides groups were respectively administered 45, 90, and 180 mg/kg Longyanshen polysaccharides. Each group was treated by intragastric administration, once per day, for 50 consecutive days. MAIN OUTCOME MEASURES： One hour after the final administration, immunohistochemical analysis was used to determine Aβ expression in the cortex and hippocampus of SAMP8 mice. Reverse-transcription polymerase chain reaction was used to determine mRNA levels of BACE and APP in SAMP8 brain tissue. RESULTS： Compared with the SAMR1 group, Aβ expression in the cerebral cortex and hippocampus, as well as expression of BACE, APP mRNA in the brain was significantly increased in the SAMP8 group （P 〈 0.05-0.01）. Compared with the SAMP8 group, Aβ expression, as well as BACE and APP mRNA expression, were significantly decreased in the cerebral cortex and hippocampus of huperzine A and low-, middle-, and high-dose polysaccharides groups （P 〈 0.05-0.01）. In particular, the effect of high-dose polysaccharides was the most significant （P 〈 0.05-0.01 ）. CONCLUSION： Longyanshen polysaccharides reduced or inhibited over-expression of BACE, APP, and Aβ in SAMP8 mice in a dose-dependent manner, and the effect was not worse than huperzine A.

Mutations of beta-amyloid precursor protein alter the consequence of Alzheimer's disease pathogenesis

Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β(Aβ). Approximately 25 mutations in β-amyloid precursor protein(APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations(A673T, A673 V, E682 K, E693 G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ_(1–40) and Aβ_(1–42) levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673 T mutation decreases Aβ_(42)/Aβ_(40) rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673 V, E682 K, and E693 Q mutations promote Aβ_(42)/Aβ_(40) rate by increasing levels of CTF99, Aβ_(42), Aβ_(40), and IAT, and decreasing VVIA levels. Pathogenic E693 G mutation shows no significant change in Aβ_(42)/Aβ_(40) ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ_(42/40) rate, thereby resulting in Alzheimer's disease.

Beta-amyloid precursor protein cleavage enzyme-1 expression in adult rat retinal neurons in the early period after lead exposure

Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein （Aβ）,and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 （BACE-1） in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer＇s disease.Adult rats were intraocularly injected with different doses of lead acetate （10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L）.The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer＇s disease.

THE PROTECTIVE EFFECTS OF THE TOTAL SAPONIN OF DIPSACUS ASPEROIDES ON THE APOPTOSIS OF HIPPOCAMPAL NEURONS INDUCED BY β-AMYLOID PROTEIN

Objective To investigate the effects of the total saponin of Dipsacus asperoides (tSDA) and ginsenoside Rb1 (GRb1) on the apoptosis of primary cultured hippocampal neurons induced by β-amyloid protein (Aβ). Methods Primary cultured hippocampal neurons, the cultures were pretreated with tSDA and GRb1 on 10d for 24 hours respectively. Then the cultures were treated with 35 μmol·L -1 Aβ25-35 for 24 hours, observed the changing of survival rate of neurons and the apoptosis of neurons with biochemical analysis combining immunofluorescent cytochemical double-staining technique. Results Hippocampal neurons were treated with 35 μmol·L -1 Aβ for 24 hours, and survival rate of neurons downed to 52.6%. When neurons were pretreated by tSDA and GRb1, survival rate of neurons increased 11% to 15%. The findings of immunofluorescent cytochemical double-staining indicated that apoptotic neurons were obviously more than that of the blank group, reaching 43.9%.When neurons were pretreated by tSDA and GRb1, apoptotic neurons were downed to 16.6%, 10.8% respectively. Conclusion tSDA had the same effects as GRb1, protecting the neurons, antagonizing neurotoxicity of Aβ, increasing survival rate of neurons, and reducing apoptotic neurons induced by Aβ.

Dynamic changes of beta-amyloid protein deposition in hippocampus of female ovariectomized rats

BACKGROUND： To evaluate and summarize the effects of cerebral perfusion and vascular reserve on the treatment of SICAS. Recently, research on β-amyloid protein has focused on the regulatory effects of estrogen or phytoestrogen on its deposition. However, there have been only a few reports on dynamic changes of β -amyloid protein deposition in hippocampus of ovariectomized rats. OBJECTIVE： To measure β -amyloid protein deposition in the hippocampal formation of ovariectomized rats by using immunohistochemistry; to observe time-dependent dynamic changes. DESIGN： Randomized controlled animal study. SETTING： Third Xiangya Hospital of Central South University. MATERIALS： The experiment was carried out in the Central Laboratory of the Third Xiangya Hospital of Central South University from November 2005 to December 2006. Fifty healthy female Sprague Dawley （SD） rats, weighing （293 ± 10） g, were provided by the Animal Laboratory of Xiangya Medical College, Central South University. All rats had neither a childbearing history nor hepatic or renal disease, or skeletal deformity. β-amyloid protein immunohistochemical kit was provided by Wuhan Boster Company. The experiment was in accordance with animal ethics standards. METHODS： All rats were randomly divided into five groups, including normal control group （n = 10）, sham operation group （n = 10）, and ovariectomized group （n = 30）. After anesthesia in the ovariectomized group, the bilateral ovaries were separated and resected. The same volume of fat was resected in the sham operation group. Rats from the normal control group, however, did not receive any surgical treatments. Rats in the normal control group and sham operation group were sacrificed by anesthesia 7 weeks after surgery. Every ten rats from the ovariectomized group was respectively sacrificed at 7, 15, and 30 weeks after surgery. lmmunohistochemistry was used to detect β-amyloid protein deposition in hippocampal sections. Cell counting and gray value measurements served to record the dynamic changes in β-amyloid protein deposition. MAIN OUTCOME MEASURES： （1） Morphological changes. （2） Positive cell counts from β -amyloid protein stainings and gray value measurements. RESULTS： All 50 rats were involved in the final analysis. （1） Morphological changes. β -amyloid-positive cells were detected in the hippocampus of all rats. Biebrich scarlet stained neurites with a swollen cytoplasm. A few β -amyloid-positive cells were observed in all groups 7 weeks after surgery, and plasma and neurites were slightly stained. By 15 weeks after surgery, a number of β -amyloid-positive cells were observed in the ovariectomized group, and plasma and neurites were also slightly stained. By 30 weeks after surgery, however, many β-amyloid-positive cells were observed in the ovariectomized group. These cells were partially aggregated and darkly stained. （2） Positive cell counts and gray value of β-amyloid protein in hippocampus. At 7 weeks after surgery, cell counts and gray value measurements were not significantly different in the ovariectomized group compared to the sham operation group and normal control group （P 〉 0.05）. Cell counts and gray value measurements were higher in the ovariectomized group by 15 weeks compared to those by 7 weeks in the normal control group, sham operation group and ovariectomized group （P 〈 0.05）. At 30 weeks after surgery, cell counts and gray value measurements were higher in the ovariectomized group compared to the normal control group. In addition, there were significant differences between sham operation group and ovariectomized group, at 7 and 15 weeks after operation （P 〈 0.05-0.01 ）. Cell counts and gray value measurements increased in all groups over time. CONCLUSION： Extended estrogen deficiency in rats can increase β-amyloid protein deposition in the hippocampus and the deposition increases over time.

The β-amyloid protein induces S100β expression in rat hippocampus through a mechanism that involves IL-1

Objective To explore the effect of β-amyloid protein (Aβ) on S100β expression in rat hippocampus and its mechanisms. Methods At 7 days after bilateral stereotaxis injection of different dose of fibrillar Aβ 25-35 and interluekin-1 receptor antagonist (IL-1ra) into the rat CA1 region, the learning and memory abilities of rats were tested with passive avoidance task. Amyloid deposition was detected by using Congo red staining technique. Nissl staining and immunohistochemical techniques were used to analyze the number of neurons, and GFAP and the S100β expression in hippocampal CA1 region , respectively. Results After fibrillar Aβ injection, the step-through latency of rats was significantly shortened compared to that of the control group. The GFAP positive astrocytes were found surrounding amyloid deposition. Neuronal loss occurred in the pyramidal cell layer of CA1 region. The number of S100β positive cells in Aβ-treated group was significantly increased compared with that in the control group. After IL-1ra injection, the number of S100β positive cells was significantly decreased. Conclusion Intrahippocampal injection of Aβ 25-35 could cause similar pathologic changes of Alzheimer’s disease. Aβ 25-35 was capable of up-regulating S100β expression in a dose-dependent manner. The injection of IL-1ra could attenuate the effect of Aβ on S100β expression.

BACE1 inhibitors:A promising therapeutic approach for the management of Alzheimer’s disease

Alzheimer’s disease is a neurological disorder marked by the accumulation of amyloid beta(Aβ)aggregates,resulting from mutations in the amyloid precursor protein.The enzymeβ-secretase,also known asβ-site amyloid precursor protein cleaving enzyme 1(BACE1),plays a crucial role in generating Aβpeptides.With no targeted therapy available for Alzheimer’s disease,inhibiting BACE1 aspartic protease has emerged as a primary treatment target.Since 1999,compounds demonstrating potential binding to the BACE1 receptor have advanced to human trials.Structural optimization of synthetically derived compounds,coupled with computational approaches,has offered valuable insights for developing highly selective leads with drug-like properties.This review highlights pivotal studies on the design and development of BACE1 inhibitors as anti-Alzheimer’s disease agents.It summarizes computational methods employed in facilitating drug discovery for potential BACE1 inhibitors and provides an update on their clinical status,indicating future directions for novel BACE1 inhibitors.The promising clinical results of Elenbecestat(E-2609)catalyze the development of effective,selective BACE1 inhibitors in the future.

Impact of Sub-chronic Aluminium-maltolate Exposure on Catabolism of Amyloid Precursor Protein in Rats

Objective To investigate the impact of sub-chronic Aluminium-maltolate [Al（mal）s] exposure on the catabolism of amyloid precursor protein （APP） in rats. Methods Forty adult male Sprague-Dawley （SD） rats were randomly divided into five groups： the control group, the maltolate group （7.56 mg/kg BW）, and the Al（mal）s groups （0.27, 0.54, and 1.08 mg/kg BW, respectively）. Control rats were administered with 0.9% normal saline through intraperitoneal （i.p.） injection. Maltolate and Al（mal）s were administered to the rats also through i.p. injections. Administration was conducted daily for two months. Rat neural behavior was examined using open field tests （OFT）. And the protein expressions and their mRNAs transcription related with APP catabolism were studied using enzyme-linked immunosorbent assay （ELISA） and real-time polymerase chain reaction （RT-PCR）. Results The expressions of APP, 13-site APP cleaving enzyme 1 （BACEI） and presenilin-1 （PSi） proteins and their mRNAs transcription increased gradually with the increase of Al（mal）3 doses （P〈0.05）. The enzyme activity of BACEI in the 0.54 and 1.08 mg/kg Al（mal）s groups increased significantly （P〈0.05）. The expression of 8-amyloid protein （AS） 1-40 gradually decreased while the protein expression of A81-42 increased gradually with the increase of Al（mal）s doses （P〈0.05）. Conclusion Result from our study suggested that one of the possible mechanisms that Al（mal）s can cause neurotoxicity is that Al（mal）s can increase the generation of A81-42 by facilitating the expressions of APP, β-, and γ-secretase.

Improving cognitive impairment by Tongxinluo via inhibiting expression of beta-secretase 1/beta-amyloid peptide in experimental vascular dementia

BACKGROUND： Tongxinluo has been clinically proven to be effective in improving memory and cognitive function in patients with post-stroke vascular dementia. Is the mechanism related to the deposition of beta-amyloid peptide （A β ） in hippocampus？ OBJECTIVE： To observe the effect of Tongxinluo on cognitive impairment in a mouse model with vascular dementia and the changes of A β deposition and β -secretase 1 （BACE1） expression. DESIGN： Randomized controlled study. SETTING： State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University and Affiliated Drum Tower Hospital of Nanjing University Medical School. MATERIALS： The experiment was carried out in the State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University and Affiliated Drum Tower Hospital of Nanjing University Medical School from March 2006 to January 2007. A total of 36 healthy Kunming mice, 18 of each gender, were chosen. The study was conducted in accordance with the National Regulations of Experimental Animal Administration, and all animal experiments were approved by the Committee of Experimental Animal Administration of Nanjing University. Tongxinluo was provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd. METHODS： All mice were randomly divided into 6 groups, including naive control （n=6）, sham-operated control （n=6） and experimental groups treated with different doses of Tongxinluo （0.2, 0.4, and 0.6 g/kg/d; n=6 for each group） or vehicle （n=6）. Five groups were subjected to bilateral common carotid arteries （2-VO） occlusion to produce a vascular dementia model （no occlusion was performed in sham-operated group）. The mice in the Tongxinluo treatment groups were intragastricly administered daily with a Tongxinluo suspension （40 g/L in distilled water） at doses of 0.2, 0.4 or 0.6 g/kg/d from day 1 to day 30 post-surgery. The animals in vehicle, sham-operated and naive groups were administered an equal volume of distilled water. MAIN OUTCOME MEASURES： ①Escape latency time determined in all groups of mice before and after 2-VO occlusion by Morris water maze. ②Changes in BACEI mRNA expression in the hippocampi of mice among the six groups by RT-PCR assay, and BACEI and A β protein expression in the hippocampi of mice by Western blot. RESULTS： All 36 mice were involved in the final analysis.① No difference was detected in escape latency time to a hidden platform among all groups in water maze test before surgery （P 〉 0.05） At 30 days after 2-VO occlusion, the vehicle animals exhibited a significantly longer latency in finding the hidden platform compared to that of sham-operated and naive animals （P 〈 0.01）. The prolonged escape latency was significantly reduced by oral administration of 0.4 g or 0.6 kg/day （P 〈 0.01, P 〈 0.05）. BACEI mRNA and protein expression in vehicle animals were much higher than in sham-operated and naive animals （P 〈 0.01）. The ischemia-induced increases in BACE1 mRNA and protein level were attenuated by all three doses of Tongxinluo treatment （P 〈 0.01）, and the 0.4 g/kg/d treatment was the most effective. A β protein expression in vehicle animals after 2-VO occlusion were much higher than in sham-operated and naive animals （P 〈 0.01）. 2-VO occlusion-induced A β generation was significantly attenuated by all doses of Tongxinluo treatment, with the most effective dose being 0.4 g/kg/d （P 〈 0.01）. CONCLUSION： BACE1 mRNA levels and protein levels of BACEI and A β are reduced in the hippocampi of vascular dementia model mice by all three doses of Tongxinluo treatment, with the most effective dose being 0.4 g/kg/d. The results suggest that inhibition of post-ischemia BACEI expression and A β generation in brain might underlie Tongxinluo＇s effects in improving cognitive impairment.

Effects of Ginsenoside Rg1 on nuclear factor-kappa B activity in beta amyloid protein-treated neural cells

BACKGROUND： Modern pharmacological studies have shown that Ginsenoside Rgl is one of the active components of ginseng that promote intelligence in the nervous system. Ginsenoside Rgl can improve memory and learning in mouse models of β-amyloid protein （Aβ）-induced dementia. OBJECTIVE： To investigate whether effects of Ginsenoside Rgl against Aβ are associated with activity of nuclear factor-kappa B （NF-κB）. DESIGN, TIME AND SETTING： The randomized performed at the DME Center, Institute of Clinica controlled, cell biological experiment was Pharmacology, Guangzhou University of Chinese Medicine, China from July 2005 to May 2006. MATERIALS： Beta-amyloid fragment 25-35 （Aβ25-35） was supplied by the Neural Biochemical Laboratory, Xuanwu Hospital, Capital Medical University, China. Ginsenoside Rgl was obtained from National Institute for the Control of Pharmaceutical and Biological Products, China. Rabbit anti-rat NF-κB p65 antibody was purchased from Santa Cruz Biotechnology, USA. METHODS： Hippocampal neurons and cortical astrocytes of neonatal Sprague Dawley rats were harvested and treated with various concentrations （0, 5, 10, 20, and 40 μmol/L） of Aβ for 6, 12, and 24 hours to establish cellular models of Alzheimer＇s disease. Cellular models were pretreated with various concentrations of Ginsenoside Rgl （1,2, 4, 8, and 16 μmol/L）. According to cell morphology and activity, the following conditions were selected： 40 μmol/L Aβ for 24 hours, as well as 2, 4, and 8 μmol/L Ginsenoside Rg1. NF-κB activity was observed using immunofluorescence and cytochemical staining. MAIN OUTCOME MEASURES： Morphology and viability of hippocampal neurons and cortical astrocytes, and activities of NF-κB were measured. RESULTS： Hippocampal neuron activity was significantly greater in the normal and 2 and 4 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.05）. Astrocyte activity was significantly greater in the normal, 1,2, 4, 8, and 16 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.05）. NF-κB activity of hippocampal neurons was significantly greater in the normal, 2, 4, and 8 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.01）. NF-κB activity of astrocytes was significantly less in the normal, 2, 4, and 8 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.01 or P 〈 0.05）. No significant difference in NF-κB activity was determined between the 2 μmol/L Ginsenoside Rgl and normal groups （P 〉 0.05）. CONCLUSION： Ginsenoside Rgl protected neural cells by upregulating NF-κB activity in neurons and downregulating NF-κB activity in astrocytes. Ginsenoside Rgl （2 μmol/L） maintained cell activity and NF-κB activity at normal levels.

Effect of Bak Foong Pills on the expression of β-amyloid in rat retina with optic nerve transection

AIM:To investigate the effect of Bak Foong Pills(BFP) on the expression of β-amyloid(Aβ) in rats retina with optic nerve transaction,and its roles and possible mechanisms in protecting optic nerve damage.· METHODS:Seventy-two healthy,Sprague-Dawley,adult rats were randomly assigned to three groups:negative control group(control group),optic nerve transection group(model group) and BFP treatment group(BFP group,100μg/mL) followed by establishing optic nerve transection model.The expression of Aβ was measured at 48 hours by Western-blotting.Moreover,the expressions of Bcl-2,Bax and Caspase-3 mRNA were evaluated at 48 hours by reverse transcriptase polymerase chain reaction(RT-PCR).RESULTS:There were significant differences among the control,model and BFP groups in the expression of Aβ(all P <0.01).Aβ expression was significantly higher in the model and BFP groups than that in the control group(P < 0.01),with a more significant reduction in the BFP group than that in the model group(P <0.01).Moreover,there were also significant differences among the three groups in the expressions of Bcl-2/Bax(Bcl-2:anti-apoptotic;Bax:proapoptotic) and Caspase-3 mRNA(proapoptotic)(all P<0.01).Bcl-2/Bax ratio was significantly lower and Caspase-3 mRNA expression was significantly higher in the model and BFP groups than those in the control group(P <0.01),with a significant growing of Bcl-2/Bax and reduction of Caspase-3 in the BFP group than those in the model group(P<0.01).· CONCLUSION:BFP can down-regulate Aβ expression in retina and may inhibit apoptosis and protect optic nerve by enhancing Bcl-2/Bax ratio and inhibiting Caspase-3 pathway.

IGF-1对PC12细胞PRNP表达及APP代谢的影响

目的·探讨胰岛素样生长因子-1(IGF-1)对肾上腺嗜铬细胞瘤细胞(PC12)中朊蛋白编码基因(PRNP)表达及淀粉样蛋白前体(APP)代谢的影响。方法·建立阿尔茨海默病(AD)细胞模型;用不同浓度的IGF-1(20、40、80 ng/mL)作用于PC12细胞24 h,采用real-time PCR检测PRNP m RNA的表达水平;Western blotting检测AKT/p AKT、ERK/p ERK蛋白的表达水平;ELISA检测细胞上清液中β-淀粉样蛋白42(Aβ42)的水平。结果·与空白对照组相比,AD模型组PRNP m RNA的表达量明显升高(P<0.01);不同浓度IGF-1处理细胞24 h后,40 ng/mL及80 ng/mL IGF-1组PRNP m RNA的表达量显著升高(P<0.01)。模型组及IGF-1各浓度组中APP蛋白的表达均无显著变化(P>0.05)。与对照组相比,AD模型组上清液中Aβ42的水平显著下降,其他各组随着IGF-1浓度的增加而降低,其中40 ng/mL及80 ng/mL IGF-1组Aβ42的表达水平降低显著(P<0.05)。与对照组相比,各组AKT/p AKT、ERK/p ERK蛋白的表达量均明显升高,且随着IGF-1剂量的增加而上升(P<0.05)。结论·IGF-1降低PRNP基因表达的同时影响APP代谢,此过程可能与PI3K/AKT和MAPK/ERK1/2信号通路有关。

中药呆聪液对痴呆大鼠模型脑内β-淀粉样前体蛋白基因表达的影响

目的探讨呆聪液对痴呆大鼠模型脑内β-淀粉样前体蛋白(β-APP)基因表达的影响。方法选用22月龄的老龄大鼠,用海人酸破坏脑基底核法,造成学习和记忆功能障碍的痴呆动物模型,通过水迷宫法和反转录聚合酶链反应(RT-PCR)观察了呆聪液对痴呆模型大鼠学习记忆能力和脑内β-APP基因表达的影响。结果老龄痴呆模型大鼠学习记忆能力下降,脑内β-APP基因表达增强。呆聪液的3个不同剂量组与痴呆模型组比较差异有显著性(P<0.05),都能明显提高学习记忆能力,降低脑内β-APP mRNA含量,并有一定的量效关系。结论呆聪液能改善老龄痴呆大鼠的学习和记忆功能并下调β-淀粉样前体蛋白基因的表达。

地黄饮子含药脑脊液对受损PC12细胞β-淀粉样前体蛋白mRNA表达的干预

目的:探讨地黄饮子含药脑脊液对β-淀粉样蛋白(Aβ25-35)所致PC12细胞损伤的β-淀粉样前体蛋白(APP)mRNA的表达及地黄饮子对其的干预作用。方法:运用中药脑脊液药理学方法把PC12细胞分为空白组,模型组,西药对照组,地黄饮子脑脊液低,中,高剂量组6组,分别加入正常培养液,正常脑脊液及含药脑脊液,应用实时定量PCR法检测受损PC12细胞β-淀粉样前体蛋白mRNA表达(用2-ΔΔC t表示,Ct为阈循环值,ΔΔCt=ΔCt各干预组-ΔCt空白组,ΔCt=CtAPP-CtGAPDH)。结果:地黄饮子能明显降低PC12细胞受β-淀粉样蛋白刺激时APP的过度反应,降低APP mRNA的表达,与模型组比较,差异有统计学意义(P<0.01)。结论:地黄饮子脑脊液可以抑制β-淀粉样蛋白损伤对PC12细胞的损伤。

糖尿病肾病时血浆基质金属蛋白酶3及血栓前体蛋白的变化

目的:探讨基质金属蛋白酶3(MMP-3)和血栓前体蛋白(TpP)在2型糖尿病肾病(DN)中的变化。方法:用ELISA双抗体夹心法测定57例DN患者及35例正常对照者的血浆MMP-3和TpP水平,DM组根据24h尿白蛋白排泄量(UAE)分DN0(UAE<30mg/24h)、DN1(30mg/24h≤UAE<300mg/24h)、DN2(UAE≥300mg/24h)3组。结果:DN0组血浆MMP-3和TpP水平与正常对照组间无显著差异。DN1组血浆MMP-3水平高于正常对照组(P<0.05),但TpP水平无显著差异。DN2组血浆MMP-3水平显著高于正常对照组(P<0.01)、DN0组(P<0.05)及DN1组(P<0.05),TpP水平也高于正常对照组(P<0.01)。DN患者血浆MMP-3和TpP水平呈正相关(P<0.01)。结论:MMP-3及TpP可能参与DN发生,MMP-3水平变化与DN病变程度相关。

胆碱能前体细胞移植入β-淀粉样前体蛋白转基因小鼠脑内的存活和迁移

目的观察新生小鼠海马原代培养的胆碱能神经前体细胞移植入淀粉样前体蛋白(amyloid precusor protein,APP)转基因小鼠脑内以后的存活和迁移状况,以探索胆碱能神经前体细胞对阿尔茨海默病的治疗前景。方法取新生24h以内增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因小鼠,以原代细胞分离培养方法获得海马胆碱能神经前体细胞,以前体细胞特异性标记抗巢蛋白(Nestin)和胆碱能细胞特异性标记胆碱乙酰基转移酶(choline acetyltransferase,Ch AT)做细胞免疫组化鉴定细胞特性。细胞培养成功后,收集细胞,在小鼠脑立体定位仪的固定下,参照小鼠脑立体定位图谱对APP转基因小鼠施行脑内注射。移植1周以后灌注取移植鼠脑组织行冰冻切片,在共聚焦显微镜下通过外源细胞的绿色自发荧光观察移植细胞的存活和迁移情况。结果在移植位点,外源性绿色荧光细胞存活良好并以移植点为中心向外迁移,且呈继续迁移的趋势。结论新生小鼠海马原代细胞培养以后呈现神经前体细胞的特性并且胆碱能阳性,移植入APP转基因小鼠脑内以后,这些胆碱能阳性的神经前体细胞存活良好并呈现较强迁移能力,为进一步探索该细胞对阿尔茨海默病的治疗可能性提供了依据。

miR-185与Apba-1在大鼠脑缺血再灌注损伤的表达变化及调控关系

目的探讨miR-185与Apba-1在脑缺血再灌注损伤的表达差异及调控关系。方法将雄性SD大鼠60只随机选择10只为假手术组,其余大鼠给予线栓法构建大脑中动脉梗死再灌注模型,将存活并且按Garcia评分法进行神经功能评分差值≤3分的30只大鼠随机分成3个实验组(术后1 d组、3 d组、7 d组),每组10只。定量反转录酶-聚合酶链反应检测各组大鼠脑组织miR-185和Apba-1 mRNA的表达。蛋白免疫印迹法检测Apba-1蛋白的表达水平。双荧光素酶报告基因实验检测miR-185与Apba-1的调控关系。结果与假手术组比较,miR-185 mRNA在各实验组脑组织的表达量明显升高(P<0.05),且表达量随再灌注时间延长在各实验组间呈逐渐上升的趋势(P<0.05);Apba-1 mRNA在各实验组脑组织表达量明显降低(P<0.05,P<0.01);且表达量随再灌注时间延长在各实验组间呈逐渐下降的趋势(P<0.05)。并且miR-185能直接作用于Apba-1的3'非翻译区预计靶位点从而调控Apba-1的表达。结论 miR-185和Apba-1在脑缺血再灌注损伤后均存在表达差异,且miR-185可负性调控Apba-1的表达。

脑胰岛素信号在阿尔兹海默症模型小鼠渐进过程中的改变

目的:探讨阿尔兹海默症(Alzheimer’s disease,AD)形成过程中脑胰岛素信号及葡萄糖转运体(glucose transporters,GLUTs)表达的改变,为AD早期诊断提供依据。方法:借助APP/PS1转基因AD模型小鼠,运用Western blot检测该模型鼠皮层和海马区域胰岛素信号通路相关蛋白的表达。结果:与对照组相比,AD模型鼠在3月龄时,表现为脑胰岛素信号应激性激活,下游AKT/GSK3β等胰岛素信号分子磷酸化水平升高,而GLUTs此时表达变化不明显;5月龄时,该模型表现为脑胰岛素信号磷酸化水平显著下降,其下游AKT/GSK3β等信号分子磷酸化水平表达下调,同时观察到海马中GLUT3、GLUT4表达下调,AD病理性蛋白(APP)、Tau蛋白磷酸化水平显著增加。结论:胰岛素信号紊乱和葡萄糖转运体在AD形成过程中发生紊乱,且随年龄增长,具有紊乱加剧的趋势。