The injury progression of T lymphocytes in a mouse model with subcutaneous injection of a high dose of sulfur mustard

Background: In clinical studies, the findings on sulfur mustard(SM) toxicity for CD3+CD4+ and CD3+CD8+ T lymphocyte subsets are contradictory. In animal experiments, the effect of SM on the T cell number and proliferation is incompatible and is even the opposite of the results in human studies. In this study, we observed the dynamic changes of T lymphocytes in the first week in a high-dose SM-induced model.Methods: Mice were exposed to SM by subcutaneous injection(20 mg/kg) and were sacrificed 4 h, 24 h, 72 h and 168 h later. Spleen T lymphocyte proliferation was evaluated by 3H-Td R. Flow cytometric analysis was used to observe the percentage of CD3+CD4+ and CD3+CD8+ T lymphocyte subsets. The IL-1e assayed using the Luminex method. DNA damage in bone marrow ceβ, IL-6, IL-10 and TNF-lls was observed with α levels in plasma werthe single cell gel electrophoresis technique(SCGE).Results: SM continuously inhibited the proliferation of lymphocytes for 7 days, and there was a significant rebound of Con A-induced T lymphocyte proliferation only at 24 h. The percentage of CD3+CD4+ and CD3+CD8+ lymphocytes was upregulated, which was accompanied by increased IL-1β and TNF-creased in the PG group at 4 h. The peak of lymphocytic apoptα and decreased IL-10. The IL-6 level was gradually deosis and DNA damage occurred at 24 h and 72 h, respectively. Conclusion: Our results show that SM significantly inhibited T lymphocyte proliferation as well as induced CD3+CD4+ and CD3+CD8+ upregulation. SM intoxication also significantly increased the levels of pro-inflammatory cytokines(IL-1β, IL-6 and TNF-α) and inhibited the level of anti-inflammatory cytokine IL-10. Our results may partly be due to the significant SM induced significant apoptosis and necrosis of lymphocytes as well as DNA damage of bone marrow cells. The results provided a favorable evaluation of SM immune toxicity in an animal model.

Improvements in SOD mimic AEOL-10150, a potent broad-spectrum antioxidant

AEOL-10150 is a broad-spectrum metalloporphyrin superoxidase dismutase(SOD) mimic specifically designed to neutralize reactive oxygen and nitrogen species. Research has shown that AEOL-10150 is a potent medical countermeasure against national security threats including sulfur mustard(SM), nerve agent exposure and radiation pneumonitis following a radiological/nuclear incident sufficient to cause acute radiation syndrome(ARS). AEOL-10150 performed well in animal safety studies, and two completed phase 1 safety studies in patients demonstrated that the drug was safe and well tolerated, indicating that AEOL-10150 has potential as a new catalytic antioxidant drug. In this article, we review improvements in AEOL-10150 in preclinical pharmacodynamic studies, especially regarding anti-SM,chlorine gas and radiation exposure studies.

Effect and mechanism of LW-AFC on chronic unpredictable mild stress-induced mood and cognition impairment of mice

OBJECTIVE To explore the effect and mechanisms of LW-AFC,a new formula derived fromLiuwei Dihuang decoction,on chronic unpredictable mild stress(CUMS)-induced mood and cogni.tion impairment in mice.METHODS C57 BL/6 J mice were randomly placed into seven groups(n=10):normal control group,CUMS group,Fluoxetine(10 mg·kg^(-1),once per day) group,Liuwei Dihuang decoction group(LW,10 g·kg^(-1),once per day),and LW-AFC(0.8 g·kg^(-1),1.6 g·kg^(-1),3.2 g·kg^(-1),once per day) group.The stressed group was given CUMS for 4 weeks to set up a chronic multiple-stressed model.LW and LW-AFC was oral administered a week prior to CUMS and until the end of the study(a total of 35 d),while fluoxetine was administrated orally for 4 weeks.The anxiety behavior was analyzed using the open field test(OFT) and elevated plus maze test(EPM).The depression behavior was ana.lyzed using the sucrose preference test(SPT) and forced swimming test(FST).Spatial cognition was evaluated using Morris water maze(MWM) test and working memory was evaluated using new object recognition test(NORT).RESULTS CUMS for 28 d increased depressive-and anxiety-like behaviors.LW-AFC(1.6 g·kg^(-1)) significantly increased the numbers of entries into the open arm and time in the open arm of CUMS mice(P<0.05).LW-AFC(3.2 g·kg^(-1)) increased sucrose consumption and de.creased the immobility time of FST(P<0.01) of CUMS mice.The MWM test showed that spatial learning andmemory in CUMS mice were remarkably affected relative to controls,whereas LW-AFC(3.2 g·kg^(-1)) im.proves cognitive functions(P<0.05).CONCLUSION The mood and theability of learning and memory of thestressed group can be affected after exposure to CUS.Oral administration of LW-AFC significant.ly improved CUMS-induced impairments of mood and cognition in mice.

Immunopotentiating activities and TLR4/MAPK related molecular mechanisms research of Lycium barbarum polysaccharide

OBJECTIVE To observe the effect and molecular mechanisms of Lycium barbarum polysaccharide(LBP) and glycopeptides on T,B lymphocytes and macrophages.METHODS ~3H-TdR incorporation method was used to compare the effects of LBP and glycopeptides on the proliferation lymphocytes.Peritoneal macrophages induced by sodium thioglycolate were used to compare the effects of LBP and glycopeptides.T and B lymphocytes were purified by immunomagnetic beads method.Using antibody blocking methods screening polysaccharide activity related receptors.C3H/HeJ mice were further used to observe the activity of LBP.Biolayer interference method was used to observe the binding kinetics of LBP with TLR4 in vitro.TLR4 level was tested by flow cytometry.Western blotting was used to observe the phosphorylation of p-38,SAPK/JNK and ERK.RESULTS The monosaccharide compo.sition of LBP is rhamnose,arabinose and galactose,and does not contain amino acids.The mixed lymphocyte proliferation experiment showed that LBP had more obvious effect on the proliferation of B cells,and glycosides induced T cells proliferation was more obvious.On the purify lymphocytes,it was found that LBP-induced B cells proliferation requires the involvement of macrophages.Further research found that anti-TLR4 antibody had significant inhibitory effect on LBP-induced macrophage release of TNF-α and IL-1β but not the anti-CR3 treatment.C3 H/HeJ mice related results further demonstrated that TLR4 is necessary for LBP activity.Although biolayer interference showed no obvious binding of TLR4/MD2 with LBP,flow cytometry confirmed that LBP could increase TLR4 expression.Western Blot experiments showed that the effect of LBP on macrophage was related to its activation of p-38/MAPK pathway and inhibition of ERK/MAPK and JNK/MAPK pathways.CONCLUSION TLR4 is the activity related receptors of LBP.LBP cannot directly bind to TLR4/MD2 complex in vitro,but can increase TLR4 expression and activate macrophage p-38/MAPK signaling pathway,inhibiting ERK-MAPK and JNK-MAPK signaling pathways.

LW-AFC,a new formula derived from Liuwei Dihuang decoction,ameliorates behavioral and pathological deterioration via modulating the neuroendocrine-immune abnormalities in PrP-hAβPPswe/PS1^(ΔE9) transgenic mice

OBJECTIVE To investigate the effect of LW-AFC,a new formula of the main active components extracted fromLiuwei Dihuang decoction,on treatment of Alzheimer disease(AD) in mouse models.METHODS After treatment LW-AFC,mice were cognitively evaluated in behavioral experiments.Neuron loss,amyloid-β(Αβ) deposition,and Αβ level were analyzed using Nissl staining,immunofluorescence,and an Alpha LISA assay,respectively.Multiplex bead analysis,a radioimmunoassay,immunochemiluminometry,and an ELISA were used to measure cytokine and hormone levels.Lymphocyte subsets were detected using flow cytometry.RESULTS LW-AFC ameliorated the cognitive impairment observed in APP/PS1 mice,including the impairment of object recognition memory,spatial learning and memory,and active and passive avoidance.In addition,LW-AFC alleviated the neuron loss in the hippocampus,suppressed Αβ deposition in the brain,and reduced the concentration of Aβ1-42 in the hippocampus and plasma of APP/PS1 mice.LW-AFC treatment also significantly decreased the secretion of corticotropin-releasing hormone and gonadotropin-releasing hormone in the hypothalamus,and adrenocorticotropic hormone,luteinizing hormone,and follicle-stimulating hormone in the pituitary.Moreover,LW-AFC increased CD8+CD28+T cells,and reduced CD4^+CD25^+Foxp3^+T cells in the spleen lymphocytes,down-regulated interleukin(IL)-1β,IL-2,IL-6,IL-23,granulocyte-macrophage colony stimulating factor,and tumor necrosis factor-α and-β,and up-regulated IL-4 and granulocyte colony stimulating factor in the plasma of APP/PS1 mice.CONCLUSION LW-AFC ameliorated the behavioral and pathological deterioration of APP/PS1 transgenic micevia the restoration of the NIM network to a greater extent than either memantineor donepezil,which supports the use of LW-AFC as a potential agent for AD therapy.

Effects of Liuwei Dihuang decoction and its compatible prescriptions on the function of hypothalamus-pituitary-adrenal axis in senescence-accelerated mouse

OBJECTIVE To investigate age-related functional change of hypothalamus-pituitary-adrenal(HPA)axis in senescence accelerated mouse(SAM)and the effects of Liuwei Dihuang decoction(LW)and its San-bu(three tonics)and San-xie(three eliminators)components on the function of HPA axis.METHODS Male SAM-resistance/1(SAMR1)and SAM-prone/8(SAMP8)at the ages of 6and 12 months old were used.SAMP8 were orally administered with LW,three tonics and three eliminators at the doses of 10,6.4and 3.6g·kg-1·d-1,respectively,for consecutive 60 d.Serum level of CORT was assayed with ELISA method.The levels of hypothalamic CRH and pituitary ACTH was determined with radioimmunoassay.RESULTS The levels of hypothalamic CRH,pituitary ACTH and serous CORT were much higher in 6 and 12 months old SAMP8 than those in age-matched SAMR1,which suggested the abnormal function of HPA axis in SAMP8.Oral administration of LW and three tonics significantly decreased the level of hypothalamic CRH of 6 and 12 months old SAMP8,and reduced the levels of pituitary ACTH and serous CORT of 6 month old SAMP8.Three eliminators significantly decreased the level of hypothalamic CRH of 6 months old SAMP8.The results indicated that oral administration of LW,three tonics and three eliminators improved the function of HPA axis of SAMP8.CONCLUSION The results showed the hyperactivity of HPA axis of SAMP8,and LW improved the hyperactivity of 6 month old SAMP8.Three tonics and three eliminators had similar effects as LW,which had better effect after compatibility.

Effects of LW-AFC,a new formula derived from Liuwei Dihuang decoction,on intestinal microbiome in senescence-accelerated mouse prone 8 strain,a mouse model of Alzheimer disease

OBJECTIVE To investigate the effects of LW-AFC,a new formula derived fromLiuwei Dihuang decoction,on gut microbiota and the behavior of learning and memory of SAMP8 mice,a mouse model of Alzheimer Disease(AD),and identify the specific intestinal microbiota correlating with cognitive ability.METHODS Morris-water maze test,novel object recognition test and shuttle-box test were conducted to observe the ability of learning and memory.16S rRNA amplicon sequencing(Illumina,San Diego,CA,USA)was employed to investigate gut microbiota.RESULTS The treatment of LW-AFC improved cognitive impairments of SAMP8 mice,including spatial learning and memory ability,active avoidance response,and object recognition memory capability.Our data indicated that there were significantly 8 increased and 12 decreased operational taxonomic units(OTUs)in the gut microbiota of SAMP8 mice compared with senescence accelerated mouse resistant 1(SAMR1) strains,the control of SAMP8 mice.The treatment of LW-AFC altered 22(16 increased and 6 decreased)OTUs in SAMP8 mice and among them,15 OTUs could be reversed by LW-AFC treatment resulting in a microbial composition similar to that of SAMR1 mice.We further showed that there were7(3 negative and 4 positive correlation)OTUs significantly correlated with all the three types of cognitive abilities,at the order level,including Bacteroidales,Clostridiales,Desulfovibrionales,CW040,and two unclassified orders.LW-AFC had influences on bacterial taxa correlated with the abilities of learning and memory in SAMP8 mice and restored them to SAMR1 mice.CONCLUSION The effects of LW-AFC on improving cognitive impairments of SAMP8 mice might be via modulating intestinal microbiome and LW-AFC could be used as a potential anti-AD agent.

LW-AFC and its active components ameliorate corticosterone-induced long-term potentiation impairment in mice

OBJECTIVE LW-AFC is extracted from the classical traditional Chinese medicinal prescription-Liuwei Dihuang Decoction.Previous studies have showed that LW-AFC could improve learning&memory ability in amny animal models.In this study,we focused on evaluating the effect of several main active components fromLW-AFC(B-B;loganin,LOG;morroniside,MOR;paeoniflorin,PF and stachyose,STA)on LTP.METHODS In vivo recording of LTP was used in this study to evaluate the effects of LW-AFC and it′s active components on coticorsterone(Cort)induced LTP impairment.RESULTS The results showed that LW-AFC could ameliorate Cort-induced LTP impairment.The effect of LW-AFC was abolished when the immune function was inhibited.Single administration(ig,ip,icv)of any of the components had no effect on Cort-induced LTP impairment.Consecutively intragastric administration or intraperitoneal injections(chronic administration)of B-B,LOG,MOR or PF for 7 d showed protective effect on Cort-induced LTP impairment.Intragastric administration of STA for 7 d protected LTP from impairment induced by Cort,while there was little improving effect when STA was administrated via intraperitoneal injection.In addition,when the intestinal microbiota was disrupted by applying the antibiotic cocktail,STA showed little protective effect against Cort.CONCLUSION In conclusion,LW-AFC and it′s components showed positive effects against cort induced LTP impairment,it seems that all displayed protective effects via indirectly,immune modulation might be the common pathway for all components;the exact pathways are different in each component,B-B,LOG,MOR and PF could be absorbed into the bloods tream and then modulate the peripheral immune function,while STA could not be absorbed and modulates the immune function via modulating intestinal microbiota.Further studies are needed to invesgate the underlying mechanisms and the synergetic effects of all components.

SOD mimicAEOL-10150 improve lifespan and delay brain aging via inhibition of senescence-associated secretory phenotype in SAMR1 strain

OBJECTIVE To observe the anti-aging effects of SOD mimic AEOL^(-1)0150 in antisenescence accelerated mouse resistant 1(SAMR1)strain.METHODS The lifespan of SAMR1 mice were observed by subcutaneous injection AEOL^(-1)0150 2 mg·kg-1once a week.Morris water maze,new object recognition,nesting and forced swimming were used to observe the behavioral changes of animals.Lymphocyte subgroups and ROS were measured by Flow cytometry.The cytokines levels were determined by Luminex method.The number of DCX+neurons in brain tissue was observed by immunofluorescence.RESULTS The results showed that AEOL^(-1)0150 could prolong the mean lifespan of SAMR1 mice,but it had no obvious effect on maximal lifespan.What′s more,AEOL^(-1)0150 could significantly improve the spatial learning memory of aged mice,but it could not increase the number of DCX+neurons in the hypothalamic MBH and hippocampal DG regions.Then,we observed the effects of AEOL^(-1)0150 on peripheral blood lymphocyte subgroups and cytokines.We found that AEOL^(-1)0150significantly modulated the lymphocyte subgroups and cytokine release.Especially,AEOL^(-1)0150 can dose-dependently inhibit plasma levels of SASP related inflammatory cytokines TNF-αand IL^(-1)7.CONCLUSION The results indicate that AEOL^(-1)0150 has anti-aging effects,and the effects are closely related to modulating immunity and inhibiting SASP production.

Neuroprotective effect of Liu Wei Di Huang Tang on adult hippocampal neurogenesis in mice

OBJECTIVE To explore the effect and mechanisms of LW-AFC,a new formula derived fromLiu Wei Di Huang Tang,on irradiation-induced reduction of mice adult hippocampal neurogenesis.METHODS C57 BL/6 J mice were randomly divided into seven groups(n=10):control group,LW-AFC group(1.6 g·kg^(-1)),Liu Wei Di Huang Tang(LW) group(10 g·kg^(-1)),brain derived neurotrophic factor(BDNF) group,irradiation group,irradiation + LW group,and irradiation + LW-AFC group.Reduction of mice adult hippocampal neurogenesis was induced by cranial irradiation.LW-AFC was administered by oral gavage for 30 d after cranial irradiation treatment.Immunofluorescence and Nissl′s staining were performed for histological morphology assessment.Bromodeoxyuridine(BrdU) staining was used in the detection of proliferation cells.The peripheral blood and hippocampal homogenate were collected to measure the content of tumor necrosis factor-α(TNF-α),interferon-gamma(INF-γ),interleukin-1β(IL-1β),IL-4 and IL-10.The hippocampal homogenate was used for Western blot to detect the BDNFTrkB signal pathway,including extracellular regulated protein kinases1/2(ERK1/2) and BDNF target protein.Morris water maze and new object recognition test were performed to examine the cognitive function of mice.The mice forced swimming and tail suspension test were used to assess alteration in depressive behavior.Long term potentiation was used to examine the synaptic plasticity change of mice.RESULTS Adult hippocampal neurogenesis was significantly reduced after irradiation of 20 Gray dose(10 Gray per day,total 2 d).LW-AFC treatment increased the BrdU number of irradiated mice(P<0.05).In Morris water maze test,LW-AFC group showed decreased escape latency in the learning period(P<0.05),while increased the number of crossing the platform in the memory period.LW-AFC can also reduce the immobility time of mice in the tail suspension test(P<0.01).CONCLU.SION LW-AFC modulates adult neurogenesis to ameliorate cognitive impairment and reduce depres.sive behavior in radiation injury mice.

Metabolism alternation of schisandra chinensis in carbon tetrachloride-intoxicated rats and patients with advanced hepatocellular carcinoma

OBJECTIVE To study the pharmacokinetics change of schisandra chinensis under the pathological condition of liver dysfunction for safe and rational use of herbal medicines.METHODS The metabolism of four effective lignans from schisandra chinensis(SC),schisandrin,schisantherin A,deoxyshisandrin and γ-schisandrin was studied using microsomes from patients with advanced hepato.cellular carcinoma.In situ intestinal and hepatic perfusions were conducted to clarify the contributions from impairments of gut and liver on the pharmacokinetics of the four schisandra lignans in CCl4-intoxi.cated rats.The metabolism in rat and human liver microsomes and transport in Caco-2 monolayer cell model were studied to reveal the key factors for the in vivo disposition of the four lignans.RESULTS When SC alcoholic extract was orally administrated to CCl4-intoxicated rat for a short term(4 d),the pharmacokinetics of four active SC lignans was significantly changed while its hepatoprotective effect was not obviously observed.The plasma concentrations of the four schisandra lignans were dramatical.ly elevated compared with the control.The Cmax,AUC and MRT were all increased or prolonged signif.icantly while parameter CLz/F was obviously reduced in rat pretreated with CCl4.In hepatic perfusion study and liver microsomes incubation,it was found that the hepatic metabolism of the four lignans was markedly decreased mainly due to the activity reduction of multiple CYP450 isoenzymes involved the metabolism,which,eventually,might lead to the alternation of their pharmacokinetic profiles in CCl4-intoxicated rats or patients with advanced hepatocellular carcinoma.CONCLUSION The pharmacoki.netic studies of SC components in pathological situation of liver dysfunction are expected to provide useful data for rational and safe application of SC preparations in clinic or further pharmacological and toxicological research.

LW-AFC,a new formula derived from Liuwei Dihuang decoction,ameliorates behavioral and pathological deterioration via modulating the neuroendocrineimmune system in Alzheimer disease mouse models

OBJECTIVE Alzheimer disease(AD),the most common cause of dementia among older people,could not be prevented,halted,or reversed up till now.A large body of pharmacological study has revealed that Liuwei Dihuang(LW) possesses potential therapeutic effects on AD.LW-AFC is key fractions fromLW.In the present study,we investigated the effect of LW-AFC on AD mouse models.METHODS PrP-hAβPPswe/PS1ΔE9(APP/PS1) mice and senescence-accelerated mouse prone 8 strain(SAMP8),classic AD animal models,were employed.After the treatment of LW-AFC,mice were cognitively evaluated in behavioral experiments.Neuron loss,amyloid-β(Αβ)deposition,and Αβ level were analyzed using Nissl staining,immunofluorescence,and an AlphaLISA assay,respectively.Multiplex bead analysis,a radioimmunoassay,immunochemiluminometry,and an ELISA were used to measure cytokine and hormone levels.Lymphocyte subsets were detected using fl ow cytometry.RESULTS LW-AFC ameliorated the cognitive impairment observed in APP/PS1 and SAMP8 mice,including the impairment of object recognition memory,spatial learning and memory,and active and passive avoidance.In addition,LW-AFC alleviated the neuron loss in the hippocampus,suppressed amyloid-β(Αβ) deposition in the brain,and reduced the concentration of Aβ1-42 in the hippo.campus and plasma of APP/PS1 mice.LW-AFC treatment also significantly restored the imbalance of hypothalamic-pituitary-adrenal(HPA) and hypothalamic-pituitary-gonadal(HPG) axis,enhanced the proliferation of splenocytes,corrected the disorder of lymphocyte subsets,and regulated the abnormal production of cytokine in APP/PS1 and SAMP8 mice.Effects of LW-AFC on pharmacodynamics and neuroendocrine immunomodulation network in APP/PS1 and SAMP8 mice were better than meman.tine and donepezil.CONCLUSION LW-AFC ameliorated the behavioral and pathological deterioration of AD mouse models via the restoration of the NIM network,which supports the use of LW-AFC as a potential agent for AD therapy.

Construction of NIM sub-network regulated by Liuwei Dihuang decoction and its characteristics

OBJECTIVE To construct the neuroendocrine immunomodulation(NIM) sub-network regulated by Liuwei Dihuang decoction(LW) and analyze its characteristics.METHODS We took the GSE57273 in GEO database and screened the differentially expressed genes(DEGs)(P<0.01) by the GEO2 R tool as gene expression signature of LW.The global PPI network was constructed in the context of whole PPI network through direct interaction algorithm and forest algorithm respectively.Then the enrichment and the topological characteristics of NIM signaling molecules were evaluated by permutation test.Finally,we abstracted the NIM sub-network by NIMNT,which combined the NIM molecular network and forest algorithm,and analyzed the topological characteristics of it by the Network Analyzer(release 2.7) plugin in Cytoscape v3.5.1.RESULTS We got 2468 DEGs in the gene expression signature of LW.After analyzing the global PPI network of LW got by two kinds of algorithms,we found that the NIM signaling molecules significantly enriched and located in important positions in the global PPI network.The NIM sub-network regulated by LW contained 1099 nodes and 1056 edges.We screened out 22 hub nodes(Degree>10).Among them,there were 19 NIM signaling molecules in which only ESR1 changed significantly and 3 non-DEGs(NFATC2,RARA,TP53).However,the down.stream of the hub nodes were significantly changes.CONCLUSION The results suggested that LW might mainly regulate the non-hub nodes to recovery of the network imbalance of the body in the state of disease.

Liuwei Dihuang Decoction improves cognitive impairments via regulating immune system in APP/PS1 transgenic mouse, a mouse model of Alzheimer disease

OBJECTIVE To investigate the effect and mechanisms of Liuwei Dihuang Decoction(LW) on cognition in PrP-hAβPPswe/PS1ΔE9(APP/PS1) transgenic mice.METHODS LW was adminis.trated with oral for 3 months.The locomotor activity test was performed to investigate the spontaneous motor activity of mice.The Morris water maze test and shuttle box test were performed to investigate the spatial learning and memory and active avoidance response respectively.The Αβ deposits and neuron loss in the hippocampus was detected by immunofluorescence staining and nissl staining respectively.The flow cytometry was employed to investigate the lymphocyte subsets of the mice.The 3 H-thymidine incorporation was performed to investigate the splenocytes proliferation.RESULTS The treatment of LW ameliorated the impairments of spatial learning and memory and active and passive avoidance in APP/PS1 mice.The administration of LW alleviated neuron loss in the brain,suppressed amyloid-β(Αβ) deposits in the hippocampus of APP/PS1 mice.The treatment of LW significantly increased ConAand LPS-induced proliferation of splenocytes,increased CD3+ T cells and CD19+ B cells in the spleen lymphocytes and reduced Gr1+ cells in APP/PS1 mice.CONCLUSION This data indicated the adminis.tration of LW ameliorated behavioral and pathological deterioration via regulating immune function.

Neural stem cells derived from sporadic Alzheimer disease iPSCs exhibit excessive cell apoptosis and premature neuronal differentiation

OBJECTIVE To establish an in vitro cell model based on patient-specific human neural stem cells to study the pathomechanism of sporadic AD as well as screen candidate drugs.METHODS The peripheral blood cells from sporadic AD patients and cognitive normal controls were repro.grammed into inducedpluripotent stem cells(iPSCs),which were further induced into neural stem cells and neurons.The cell growth curve during the differentiation process was recorded by the IncuCyte ZOOM,and neural stem cells and neurons were identified by immunofluorescence.The apoptosis of neural stem cells and neurons was detected by Click-iT~Plus TUNEL Assay.RESULTS Neural stem cells derived from AD patients and cognitive normal controls can express neural stem cell markers Nes.tin,Sox1,Sox2 and Ki67.TUNEL assay results showed that the number of TUNEL-positive cells in neu.ral stem cells derived from AD patients was significantly higher than that of cognitive normal controls(P<0.01).When neural stem cells were differentiated into neurons,the percentage of MAP2 positive cells in the neural stem cell-derived culture dish of AD patients was significantly higher than the cogni.tive normal controls at day 16 of neuronal differentiation(P<0.01);the TUNEL assay showed that the number of TUNEL-positive cells in AD-derived neurons was significantly greater than that in cognitive normal controls(P<0.01) at day 16 of neuronal differentiation.CONCLUSION Our study revealed that AD-iPSC-derived neural stem cells exhibit premature neuronal differentiation and increased neural apoptosis,which might be relevant to the neuronal loss of AD,thus may provide valuable new tools to screen candidate drugs for the disease and to discover the mechanisms underlying AD pathogenesis.

Design and synthesis of cyclic acylguanidines as BACE1 inhibitors

Based on the lead compound 1 reported in literature, a series of novel BACE1 inhibitors were designed and synthesized, among which compound 11 exhibited a 14-fold improvement in potency over the lead compound 1. This represents a good lead for the discovery of more promising BACE1 inhibitors for the potential treatment of AD.

Design and synthesis of 3＇-(prop-2-yn-1-yloxy)-biphenyl substituted cyclic acylguanidine compounds as BACE1 inhibitors

Based on the lead compounds 1 and 2, a series of novel BACE1 inhibitors were designed and synthesized,among which compound 9h exhibited a 60 fold improvement in potency over the lead compound 1. This represents a good lead for the discovery of more promising BACE1 inhibitors for the potential treatment of AD. The result also showed that the prop-2-yn-1-yloxy is a suitable fragment for modification of cyclic acylguanidine BACE1 inhibitors.

Design and synthesis of 5-cyclopropyl substituted cyclic acylguanidine compounds as BACE1 inhibitors

By taking compound 1 as a lead, a series of 5-cyclopropyl substituted cyclic acylguanidine compounds were designed and synthesized as BACE1 inhibitors, compound 4d exhibited 84-fold improved inhibition efficiency than lead compound 1. The diphenyl fragment at the P3 position and the substituents at the second phenyl ring were essential for the compounds to achieve improved inhibition efficiency. This SAR studies provides new insights into the design and synthesis of more promising BACE1 inhibitors for the potential treatment of AD.