Danggui Shaoyaosan Inhibits Neuroinflammation in AD Rats by Regulating NLRP3/Caspase-1 Pathway / 中国实验方剂学杂志

Objective:To investigate the neuroprotective effect of Danggui Shaoyaosan （DSS） in a rat model of amyloid-<italic>β</italic>-peptide_1-42 （A<italic>β</italic>_1-42）-induced Alzheimer's disease （AD） as well as its regulatory effect on NOD-like receptor protein 3 （NLRP3）/cysteinyl aspartate-specific protease-1 （Caspase-1） signaling pathway. Method:The AD animal model was established via intracerebral injection of A<italic>β</italic>_1-42 and treated with different concentrations of DSS after the division of rats into the sham operation group， model group， as well as the high-， medium-， and low-dose DSS groups. Morris water maze test was conducted to determine the learning and memory abilities of rats. The morphology and function of neurons were detected by hematoxylin-eosin （HE） staining and Golgi staining， followed by immunofluorescence co-localization of NLRP3 inflammasome activation. The mRNA expression levels of interleukin （IL）-1<italic>β</italic> and IL-18 were measured by Real-time polymerase chain reaction （Real-time PCR）， and the protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β </italic>were assayed by Western blot. Result:Compared with the sham operation group， the model group exhibited significantly decreased learning and memory abilities （<italic>P</italic><0.01）， impaired neuronal morphology and function， up-regulated IL-1<italic>β</italic> and IL-18 mRNA expression， enhanced NLRP3 inflammasome activation， and elevated NLRP3， Caspase-1， and IL-1<italic>β</italic> protein expression （<italic>P</italic><0.01）. Compared with the model group， DSS at both medium and high doses remarkably improved the learning and memory abilities of AD rats （<italic>P</italic><0.05， <italic>P</italic><0.01）， restored neuronal morphology and function， down-regulated the mRNA expression levels of inflammatory factors IL-1<italic>β</italic> and IL-18， reduced the activation of NLRP3 inflammasomes， and lowered the protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> （<italic>P</italic><0.01）. Conclusion:DSS inhibits inflammasome activation and neuroinflammatory response possibly by regulating the NLRP3/Caspase-1 signaling pathway， thus exerting the neuroprotective effect.

Mechanism of puerarin reversing SH-SY5Y cell injury induced by Aβ_(1-42) based on proteomics / 中国中药杂志

Puerarin has the anti-Alzheimer's disease (AD) activity,which can reverse nerve injury induced by Aβand inhibit neuronal apoptosis.However,its potential pharmacodynamic mechanism still needs to be further researched.The occurrence and development of AD is due to the change of multiple metabolic links in the body,which leads to the destruction of balance.Puerarin may act on multiple targets and multiple metabolic processes to achieve therapeutic purposes.Quantitative proteomic analysis provides a new choice to understand the mechanism as completely as possible.This research adopted SH-SY5Y cells induced by Aβ_(1-42)to establish AD cell model,and Aβimmunofluorescence detection showed that Aβdecreased significantly after puerarin intervention.The mechanism of puerarin reversing SH-SY5Y cell injured by Aβ_(1-42)was further explored by using label-free non-labeled quantitative technology and Western blot detection based on bioinformatics analysis result.The results showed that most of the differential proteins were related to biological processes such as cellular component organization or biogenesis,cellular component organization and cellular component biogenesis,and they mainly participated in the top ten pathways of P value such as pathogenic Escherichia coli infection,m TOR signaling pathway,regulation of autophagy,regulation of actin cytoskeleton,spliceosome,hepatocellular carcinoma,tight junction,non-small cell lung cancer,apoptosis and gap junction.Annexin V/PI flow cytometry and TUNEL were used to detect apoptosis,and the results showed that Aβdecreased significantly and the rate of apoptosis decreased significantly after puerarin intervention.Western blot analysis found that the protein expression level of autophagy related protein LC3Ⅱwas up-regulated after Aβinduction,and the degree of this up-regulation was further enhanced in puerarin intervention group.The trend of the ratio of LC3Ⅱ/LC3Ⅰamong groups was the same as the protein expression level of LC3Ⅱ，the protein expression level of p62 in the control group,AD model group and puerarin intervention group decreased successively.Protein interaction network analysis showed that CAP1 was correlated with TUBA1B,HSP90AB2P,DNM1L,TUBA1A and ERK1/2,and the correlation between CAP1 and ERK1/2 was the highest among them.Western blot showed that the expressions of p-ERK1/2,Bax and CAP1 were significantly down-regulated and the protein expression level of Bcl-2 was significantly up-regulated after puerarin intervention.Therefore,puerarin might improve the SH-SY5Y cells injured by Aβ_(1-42)through the interaction of multiple biological processes and pathways in cells multiple locations,and CAP1 might play an important role among them.

Research Progress of Buyang Huanwu Tang and Its Components in Treatment of Alzheimer's Disease / 中国实验方剂学杂志

Alzheimer' s disease (AD) is a neurodegenerative disease with insidious onset and complex etiology and pathogenesis. The main pathological changes are the damage of cholinergic neurons and the loss of synapses. Because of advantages of multi-pathway and multi-target intervention, traditional Chinese medicine(TCM) compound prescriptions have a significant effect in the prevention and treatment of AD. Buyang Huanwu Tang, which is the representative prescription for benefiting Qi and activating blood circulation, has been widely used in cerebrovascular diseases, with significant effects in protecting neurons, repairing blood-brain barrier, reducing permeability, resisting cerebral edema and vascular endothelial cell injury and promoting new angiogenesis and maturation. Due to its powerful therapeutic effect the brain, a large number of scholars have found that Buyang Huanwu Tang has a significant effect in improving cognitive impairment, and different components can improve the therapeutic effect of cognitive impairment through different mechanisms. However, different studies focus on a relatively single mechanism of action, which is not conducive to a comprehensive understanding of its mechanisms of action and intervention targets. This article summarizes relevant literatures in recent years for the effect of Buyang Huanwu Tang and its component in reducing beta amyloid precursor protein (APP) expression and beta amyloid protein deposition, inhibiting the central nervous system inflammatory signaling pathways in reducing inflammatory cytokines release factor expression protect neurons, repair, neuron apoptosis blood brain barrier, preventing harmful substances from the central nervous system, improving the low-density lipoprotein receptor (LRP)-1 content, lowering ages receptor (RAGE) beta amyloid protein expression, and increasing peripheral clearance of β amyloid protein, and elaborated the mechanisms in protecting neurons and alleviating learning and cognitive dysfunction, in order to provide strong literature support for the treatment of Alzheimer's disease with Buyang Huanwu Tang.

Moxibustion at acpoints of governor vessel on regulating PI3K/Akt/mTOR signaling pathway and enhancing autophagy process in APP/PS1 double-transgenic Alzheimer's disease mice / 中国针灸

OBJECTIVE@#To observe the eliminating effects of moxibustion at "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) on amyloid β-peptide (Aβ) in brain of the amyloid precursor protein/presenili1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD) by regulating the phosphoinositide 3-kinases/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway.@*METHODS@#A total of 60 APP/PS1 double-transgenic mice with AD were randomly divided into a model group, a moxibustion group, a rapamycin group and a combination group (treated with moxibustion and inhibitor), 15 mice in each group, another 15 male C57BL/6J mice with same age and background were selected as the control group. In the moxibustion group, pressing moxibustion was applied at "Baihui" (GV 20) while the mild moxibustion was applied at "Fengfu" (GV 16) and "Dazhui" (GV 14). The treatment was manipulated for 20 min each time, once a day for 2 weeks. In the rapamycin group, rapamycin (2 mg/kg) was given by intraperitoneal injection once a day for 2 weeks. On the basis of the treatment in the moxibustion group, 3-methyladenine (1.5 mg/kg) was given by intraperitoneal injection once a day for 2 weeks. The mice in the control and the model group received normal diet and no intervention was given for 2 weeks. Immunohistochemica method was used to measure the levels of Aβ in the cerebral cortex and hippocampal, transmission electron microscopy was used to observe the formation of autophagosome in hippocampus, and Western blot method was used to observe the levels of PI3K, Akt, p-Akt, mTOR and p-mTOR in hippocampus.@*RESULTS@#Compared with the control group, the levels of Aβ in the cerebral cortex and hippocampal were increased in the model group (0.05）. Compared with the rapamycin group, the levels of Aβ in the cerebral cortex and hippocampal were increased in the combination group (<0.01). In the model group, the cytoplasmic utophagic vacuoles and organelles of neuron were reduced. In the moxibustion group, the utophagic vacuoles were increased, and the organelles showed deformation and atrophy. In the rapamycin group, the utophagic vacuoles were widely disturbed and few deformed organelles were found. In the combination group, few utophagic vacuoles were found and additional organelles showed deformation and atrophy. Compared with the control group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the model group (all <0.01). Compared with the model group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were reduced in the moxibustion group, the rapamycin group and the combination group (all <0.01). Compared with the moxibustion group, the levels of PI3K、Akt、and p-mTOR were increased in the rapamycin group and the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the combination group (all <0.01). Compared with the rapamycin group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the combination group (<0.01).@*CONCLUSION@#Moxibustion at acupoints of governor vessel can enhance the autophagy process on Aβ in brain of the APP/PS1 double-transgenic AD mice, which may be associated with its effects on inhibiting the abnormal activation of PI3K/Akt/mTOR signaling pathway.

Effect of isoflurane anesthesia on hippocampus neuroapoptosis in zinc-deficient APP/PS1 transgenic mice of Alzheimer's disease models / 吉林大学学报(医学版)

Objective: To study the effect of isoflurane anesthesia on the hippocampal neuroapoptosis in the zincdeficient APP/PS1 transgenic mice, and to clarify its related mechanisms. Methods: The eight-month or ninemonth old APP/PS1 transgenic mice were randomly assigned to four groups (n=24): zinc adequate (ZA) group, the mice were given a standard diet for 2 months; zinc adequate + isoflurane (ZA+Iso) group, the mice were given a standard diet for 2 months then exposed to 1. 4% isoflurane for 2 h; zinc-deficient (ZD) group, the mice were given zinc deficient diet for 1 month; zinc deficient with isoflurane (ZD+Iso) group, the mice were given zinc deficient diet for 1 month and then exposed to 1. 4% isoflurane for 2 h. The hippocampus tissue of the mice were obtained 24 h after anesthesia. The immunofluorescence double staining was performed to measure the apoptotic rates of hippocampal neurons. The Western blotting method was used to detect the expression levels of Aβ and Cleaved caspase-3 and the ratio of Bax/Bcl-2. Results: Compared with ZA group, the apoptotic rate of neurons, the ratio of Bax/Bcl-2 and the expression level of Cleaved caspase-3 of the mice in ZA+Iso group were increased 6 h after isoflurane exposure (P0. 05). Compared with ZA group, the apoptotic rates of neurons, the ratios of Bax/Bcl-2, the expression levels of Aβ42 and Cleaved caspase-3 of the mice in ZD group and ZD+Iso group were increased 6 and 24 h after isoflurane exposure (P<0. 05 or P<0. 01). Compared with ZD group, the apoptotic rate of neurons, the expression levels of Aβ42, Bax/Bcl-2 and Cleaved caspase-3 and the ratio of Bax/Bcl-2 of the mice in ZD+Iso group were elevated significantly 6 and 24 h after isoflurane exposure (P<0. 05 or P<0. 01). Conclusion: 1.4% isoflurane exposure for 2 h can induce the transient elevation of hippocampal neuroapoptosis in the ten-month old APP/PS1 transgenic mice. 1.4% isoflurane exposure for 2 h can significantly aggravate the hippocampal neuroapoptosis in the zinc-deficient APP/PS1 transgenic mice, it is probably associated with the hippocampal Aβ aggregation, activation of Bax and Caspase-3 and inhibition of Bcl-2.

Effect of microenvironment on β-site amyloid precursor protein cleaving enzyme 1 expression in PC12 Cells / 上海交通大学学报（医学版）

Objective • To investigate the effect of microenvironment on the expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) in PC12 cells. Methods • The PC12 cells were respectively treated with thapsigargin (Tg), lipopolysaccharide (LPS), H2O2, hypoxia incubator and hypoglycemic medium to produce mild endoplasmic reticulum stress (ERS), inflammatory environment, mild oxidative stress, hypoxia and low glucose conditions. Western blotting and fluorescence quantitative PCR were used to detect the expression of BACE1 protein and BACE1 mRNA. Results • After PC12 cells were treated with Tg (0.13 μmol/L) for 12 h or LPS (0.01 mg/mL) for 36 h, BACE1 protein and mRNA levels were significantly up-regulated (P0.05). Conclusion • Mild ERS and inflammatory condition can up-regulate BACE1 mRNA and protein expression in PC12 cells.

Effect of isoflurane anesthesia on hippocampus neuroapoptosis in zinc-deficient APP/PS1 transgenic mice of Alzheimer's disease models / 吉林大学学报(医学版)

Objective:To study the effect of isoflurane anesthesia on the hippocampal neuroapoptosis in the zincdeficient APP/PS1 transgenic mice,and to clarify its related mechanisms.Methods:The eight-month or ninemonth old APP/PS1 transgenic mice were randomly assigned to four groups (n=24):zinc adequate (ZA) group,the mice were given a standard diet for 2 months;zinc adequate+ isoflurane (ZA+ Iso) group,the mice were given a standard diet for 2 months then exposed to 1.4％ isoflurane for 2 h;zinc-deficient (ZD) group,the mice were given zinc deficient diet for 1 month;zinc deficient with isoflurane (ZD+Iso) group,the mice were given zinc deficient diet for 1 month and then exposed to 1.4％ isoflurane for 2 h.The hippocampus tissue of the mice were obtained 24 h after anesthesia.The immunofluorescence double staining was performed to measure the apoptotic rates of hippocampal neurons.The Western blotting method was used to detect the expression levels of Aβ and Cleaved caspase-3 and the ratio of Bax/Bcl-2.Results:Compared with ZA group,the apoptotic rate of neurons,the ratio of Bax/Bcl-2 and the expression level of Cleaved caspase-3 of the mice in ZA+Iso group were increased 6 h after isoflurane exposure (P＜0.05 or P＜0.01);but no differences were found in the apoptotic rate of neurons and the expression levels of total Aβ,Aβ40 and Aβ42 24 h after isoflurane exposure (P＞0.05).Compared with ZA group,the apoptotic rates of neurons,the ratios of Bax/Bcl-2,the expression levels of Aβ42 and Cleaved caspase-3 of the mice in ZD group and ZD+ Iso group were increased 6 and 24 h after isoflurane exposure (P＜0.05or P＜0.01).Compared with ZD group,the apoptotic rate of neurons,the expression levels of Aβ42,Bax/Bcl-2and Cleaved caspase-3 and the ratio of Bax/Bcl-2 of the mice in ZD+Iso group were elevated significantly 6 and 24 h after isoflurane exposure (P＜0.05 or P＜0.01).Conclusion:1.4％ isoflurane exposure for 2 h can induce the transient elevation of hippocampal neuroapoptosis in the ten-month old APP/PS1 transgenic mice.1.4 ％ isoflurane exposure for 2 h can significantly aggravate the hippocampal neuroapoptosis in the zinc-deficient APP/PS1 transgenic mice,it is probably associated with the hippocampal Aβ aggregation,activation of Bax and Caspase-3 and inhibition of Bcl-2.

Application of the animal model of intracerebral injection of amyloid-β oligomers to the study of Alzheimer's disease / 药学学报

Progressive accumulation of the amyloid-β peptide (Aβ) in the brain plays a central role in the pathogenesis of Alzheimer's disease (AD). The animal model of intracerebral injection of Aβ oligomers not only provides a method for further exploring the mechanism of Aβ in AD, but also can be used to screen drug candidates targeting Aβ oligomers. This animal model has been widely used in the study of anti-AD drugs and mechanism of AD. In this paper, we summarize the research progress in the animal model of intracerebral injection of soluble Aβ oligomers, including experimental animals, the types of Aβ, the preparation of Aβ oligomers in vitro, injection sites and doses, the duration of modeling, animal behavioral changes, and the pathological mechanisms relating to this animal model, which will contribute to the application of the animal model to various conditions.

A Natural Formulation Increases Brain Resistance to Oxidative Stress.

Aims: Oxidative stress is an imbalance in the pro-oxidant/antioxidant homeostasis, characterized by excess accumulation of reactive oxygen/nitrogen species (ROS/RNS) and free radicals that can be toxic for cells by initiating disruptive peroxidation reactions on cellular substrates such as proteins, lipids, and nucleic acids. Neurons have a high content of unsaturated fatty acids which are easily peroxidable by the elevated levels of ROS and RNS produced by brain oxygen metabolism, yielding isoprostanes among which 8-iso-PGF2α derived from arachidonic acid represents a stable marker of lipoperoxidation, in vivo. Numerous findings pointed to the protective role of natural products against oxidative stress in the brain. Methodology: In the present work we evaluated the effects of a natural formula containing bacopa extract, vitamin E, astaxanthin and phosphatidylserine on lipoperoxidation in rat brain cortex, both in vivo and in vitro. Results: The results demonstrate that the natural formula could reduce basal and hydrogen peroxide- and amyloid β peptide-induced oxidative stress, as evidenced by the reduction of 8-iso-PFG2α and ROS/RNS production in the rat brain. Conclusion: Results could account for a rational use of herbal products in the treatment of conditions characterized by increased burden of oxidative stress and defective antioxidant mechanisms, such as aging and neurodegenerative disorders.

The Protective Effects of Butylphthalide on Amyloid β Peptide_（25-35） Induced Apoptosis in PC-12 Cells / 中国医科大学学报

Objective To investigate the protective effects of dl-3-n-butylphthalide（NBP）on amyloid β peptide 25-35（Aβ25-35）-induced apoptosis in PC-12 cells.Methods Cultured PC12 cells were divided into 6 groups：normal group,Aβ25-35 treated model group,0.1,1.0,10,100 μmol/L NBP pretreatment groups.MTT assay was employed to analyze the PC12 cell viability.The ultrastructural changes of neuronal mitochondria were viewed under transmission electron microscope.In order to observe the effects of oxidative stress,MDA and SOD activities were detected by spectrophotometry.Results NBP pretreatment could significantly prevent the cell viability induced by Aβ25-35（P 0.05）.Pretreatment with 10 μmol/L NBP could significantly inhibit the viability decrease induced by Aβ25-35（P 0.05）.Compared with the cells in the model group,the number and morphology of neuronal mitochondria changed distinctly in the NBP pretreated cells.The activity of SOD in the NBP pretreated cells was obviously higher than that of the cell in the model group,while MDA activity had opposite result.Conclusion NBP could protect the mitochondria in Aβ25-35 induced apoptosis by inhibiting the MDA activity and activating the SOD activity.