Kaixin-San improves Aß-induced synaptic plasticity inhibition by affecting the expression of regulation proteins associated with postsynaptic AMPAR expression.

Objective: To explore the mechanism underlying Kaixin-San (KXS) regulation of postsynaptic AMPA receptor (AMPAR) expression to mitigate toxic effects of the amyloid-ß protein (Aß). Methods: An animal model was established via intracerebroventricular injection of Aß1-42. The Morris water maze test was conducted to evaluate learning and memory, while electrophysiological recording was conducted to assess the hippocampal long-term potentiation (LTP). Western blotting was used to detect expression levels of the hippocampal postsynaptic AMPAR and its accessory proteins. Results: The time spent to find the platform was significantly prolonged, the number of mice crossing the target site was significantly reduced, and the maintenance of LTP was inhibited in the Aß group than in the control group. In the Aß/KXS group, the time taken to find the platform was significantly shortened and the number of mice crossing the target site was significantly increased than in the Aß group; furthermore, the inhibition of LTP induced by Aß was reversed. The expression of GluR1, GluR2, ABP, GRIP1, NSF, and pGluR1-Ser845 was upregulated, while that of pGluR2-Ser880 and PKC δ was downregulated in the Aß/KXS group. Conclusion: The increased expression of ABP, GRIP1, NSF, and pGluR1-Ser845 and the decreased expression of pGluR2-Ser880 and PKC δ under the influence of KXS, followed by the upregulation of postsynaptic GluR1 and GluR2, alleviated the inhibition of LTP induced by Aß. Ultimately, the memory function of model animals was improved by KXS. Our study provides novel insights into the mechanism underlying KXS mitigation of Aß-induced synaptic plasticity inhibition and memory impairment by altering the levels of accessory proteins associated with AMPAR expression.

[Anti-depression targets and mechanism study of Kaixin San].

This study identified the anti-depression targets of Kaixin San(KXS) in the brain tissue with "target fishing" strategy, and explored the target-associated pharmacological signaling pathways to reveal the anti-depression molecular mechanism of KXS. The Balb/c mouse model of depression was established by chronic unpredictable mild stress(CUMS) and the anti-depression effect of KXS was evaluated by forced swimming test and sucrose preference test. KXS active components were bonded to the benzophenone-modified magnetic nanoparticles by photocrosslinking reaction for capturing target proteins from cortex, thalamus and hippocampus of depressive mice. The target proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS). The enrichment analysis on signaling pathways was performed by Cytoscape. The potential biological functions of targets were verified by immunohistochemistry and Western blot assay. The results showed that KXS significantly improved the behavioral indexes. There were 64, 91, and 44 potential targets of KXS identified in cortex, thalamus, and hippocampus, respectively, according to the target identification experiment. The functions of these targets were mainly associated with vasopressin-regulated water reabsorption, salmonella infection, thyroid hormone synthesis, and other signaling pathways. Besides, the results of immunohistochemistry and Western blot showed that KXS up-regulated the expressions of argipressine(AVP) in the cortex, heat shock protein 60(HSP60), cytochrome C oxidase 4(COX4), and thyrotropin-releasing hormone(TRH) in the thalamus, and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and nuclear factor kappa B(NF-κB) p65 in the thalamus. Therefore, KXS may exert anti-depression effect through regulating vasopressin signaling pathway in the cortex and inflammation, energy metabolism, and thyroid hormone signaling pathways in the thalamus, and the effect of KXS on hippocampus is not significant.

Chinese traditional formula Kaixin San suppressed ferroptosis of hippocampal neurons and cardiomyocytes in mice with paradoxical sleep deprivation.

ETHNOPHARMACOLOGICAL RELEVANCE: Kaixin San (KXS) is one of the most famous traditional Chinese formulas prescribed by Sun Simiao in 652 Christian era. It is composed of Panax ginseng C.A.Mey, Polygala tenuifolia, Poria cocos and Acorus calamus var. angustatus Besser. KXS is widely used for the treatment of emotion-thought disease, such as settling fright, quieting the spirit and nourishing the heart. However, whether KXS benefits hippocampal neurons and myocardial cells of mice impaired by paradoxical sleep deprivation (PSD) and its mechanism remains unclear. AIM OF THE STUDY: This study was aimed to investigate the effect of KXS on hippocampal neuron and cardiac ferroptosis in rapid-eye-movement (REM) sleep deprived mice and clarify its potential mechanism. MATERIALS AND METHODS: PSD was induced by a modified multi-platform method. Morris water maze (MWM) was used to detect the ability of learning and memory. Cardiac morphological changes were assessed by hematoxylin and eosin (HE) staining. Heart rate was detected by a PowerLab multichannel physiological recorder. Serum levels of atrial natriuretic peptide (ANP) and lactate dehydrogenase (LDH) were measured with biochemical kits. Transmission electron microscopy (TEM), immunofluorescent, and Western blotting analysis were used to observe the process and pathway of ferrotosis in hippocampus tissue and heart tissue of PSD mice. RESULTS: KXS administration improved the impaired learning and memory of PSD mice. It prevented the damage of mitochondria in the hippocampus and heart of PSD mice. KXS also alleviated the myocardial injury, such as morphological damage, abnormal heart rate, serum ANP, and serum LDH induced by PSD. Further study disclosed that KXS reversed the expressions of proteins involved in ferroptosis such as TFRC, SLC7A11/xCT, GPX-4, ACSL4, and FTH1 in hippocampus and heart tissues. CONCLUSIONS: KXS improved learning and memory of mice with REM sleep deprivation, which was closely associated with suppressed ferroptosis in hippocampal neurons and myocardiocytes.

Analysis on mechanism of Kaixin San in the treatment of Alzheimer disease based on integrated pharmacology and GEO chip technology / 国际中医中药杂志

Objective:To analyze the mechanism of Kaixin San in treating Alzheimer disease (AD) based on the TCM integrated pharmacology platform combined with GEO chip differential gene analysis method.Methods:By searching TCMIP and Drugbank database, the active components and related molecular targets of Kaixin San were obtained. GSE4757 chip data was obtained through GEO database, and its differential genes were obtained using R language to draw heat map and volcano map. Molecular target map of differentially expressed genes between Kaixin San and AD was constructed through Cytoscape 3.7.2. Bisogenet and CytoNCA were used to draw the target topological network, and GO enrichment analysis and KEGG enrichment analysis of Kaixin San and AD gene were carried out.Results:86 active components of Kaixin San were obtained to treat AD, and 29 differential genes shared with GEO were obtained. PPI topological network was constructed. 6 core candidate genes were screened, and were merged with KEGG pathway enriched genes to obtain important genes for disease treatment, such as CHRM1, CHRM2, ACHE, CHRM3, CASP8, PTGS2, DRD1, CACN1S, ADRB1. 375 GO entries were obtained, mainly involving biological processes such as vasoconstriction, postsynaptic membrane plasticity, neurotransmitter transmission, etc. KEGG enrichment analysis mainly involved cholinergic synaptic signal pathway, cAMP signal pathway, calcium signal pathway, nerve ligand receptor interaction signal pathway, etc.Conclusions:Kaixin San shows the features of multi-component, multi-target and multi-channel in treating AD. It can play a role in the treatment of AD by inhibiting inflammatory reaction, reducing the activity of acetylcholinesterase and regulating the concentration of calciumion.

Anti-depression targets and mechanism study of Kaixin San / 中国中药杂志

This study identified the anti-depression targets of Kaixin San(KXS) in the brain tissue with "target fishing" strategy, and explored the target-associated pharmacological signaling pathways to reveal the anti-depression molecular mechanism of KXS. The Balb/c mouse model of depression was established by chronic unpredictable mild stress(CUMS) and the anti-depression effect of KXS was evaluated by forced swimming test and sucrose preference test. KXS active components were bonded to the benzophenone-modified magnetic nanoparticles by photocrosslinking reaction for capturing target proteins from cortex, thalamus and hippocampus of depressive mice. The target proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS). The enrichment analysis on signaling pathways was performed by Cytoscape. The potential biological functions of targets were verified by immunohistochemistry and Western blot assay. The results showed that KXS significantly improved the behavioral indexes. There were 64, 91, and 44 potential targets of KXS identified in cortex, thalamus, and hippocampus, respectively, according to the target identification experiment. The functions of these targets were mainly associated with vasopressin-regulated water reabsorption, salmonella infection, thyroid hormone synthesis, and other signaling pathways. Besides, the results of immunohistochemistry and Western blot showed that KXS up-regulated the expressions of argipressine(AVP) in the cortex, heat shock protein 60(HSP60), cytochrome C oxidase 4(COX4), and thyrotropin-releasing hormone(TRH) in the thalamus, and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and nuclear factor kappa B(NF-κB) p65 in the thalamus. Therefore, KXS may exert anti-depression effect through regulating vasopressin signaling pathway in the cortex and inflammation, energy metabolism, and thyroid hormone signaling pathways in the thalamus, and the effect of KXS on hippocampus is not significant.

[Modified Kaixin San improves memory and synaptic damage of mice with Alzheimer's disease by modulating αCaMKâ ¡-PSD95 protein binding through inhibition of neuroinflammation:a study of mechanism].

To investigated the mechanisms underlying the effects of modified Kaixin San(MKXS) on improving memory and synaptic damage of Alzheimer's disease(AD) mouse model with conditional presenilin 1/2 conditional double knockout(PS cDKO). Specifically, 60 PS cDKO mice(3-3.5 months old) and their age-matched wild-type(WT) littermates were randomized into three groups: WT group(n=20), PS cDKO group(n=20), and PS cDKO+MKXS group(n=20). Mice in WT and PS cDKO groups were fed with standard chow and those in PS cDKO+MKXS group were given chow containing MKXS(at 2.55 g·kg~(-1)) for 60 days. Novel object reco-gnition task was employed to detect the recognition memory of mice, and Western blot to detect the protein levels of synapse-associated proteins in the hippocampus(HPC) of mice, such as NR1, NR2 A, NR2 B, p-αCaMKⅡ, tau, and p-tau. Microglial morphology in the HPC CA1 of mice was observed based on immunohistochemistry. Quantitative real time-PCR(qRT-PCR) was employed to detect the mRNA levels of the pro-inflammatory factors and synapse-associated proteins in the HPC of mice, including COX-2, iNOS, IL-1ß, IL-6, TNF-α, PSD95, NR1, NR2 A, NR2 B, and MAP2. The protein levels of IL-1ß, TNF-α, and IL-6 were tested by enzyme-linked immunosorbent assay(ELISA). The interaction between PSD95 and αCaMKⅡ and between PSD95 and p-αCaMKⅡ was tested by co-immunoprecipitation(Co-IP). The results showed that PS cDKO+MKXS demonstrated significantly higher preference index and recognition index of the new objects, lower protein level of p-tau(ser 396/404) and mRNA levels of COX-2, iNOS, TNF-α, IL-1ß, and IL-6 in HPC, higher protein levels of NR1, NR2 A, NR2 B, and p-αCaMKⅡ and mRNA levels of NR1, NR2 A, NR2 B, PSD95, and MAP2, and stronger interaction of αCaMKⅡ with PSD95 and interaction of p-αCaMKⅡ with PSD95 than the PS cDKO group. Immunohistoche-mical staining showed that MKXS inhibited the activation of microglia. In conclusion, MKXS improves memory and synaptic damage in mice with AD by modulating αCaMKⅡ-PSD95 protein binding through inhibition of neuroinflammation.

[UHPLC-Q-Exactive Orbitrap MS/MS-based rapid identification of chemical components in substance benchmark of Kaixin San].

Ultra-performance liquid chromatography-quadrupole-electrostatic field Orbitrap mass spectrometry(UHPLC-Q-Exactive Orbitrap MS/MS) was used for rapid identification of the chemical components in Kaixin San substance benchmark. The gradient elution was performed through a Waters ACQUITY~(TM) BEH C_(18) column(2.1 mm×150 mm, 1.7 µm) with water-acetonitrile as mobile phase, a column temperature of 30 ℃, a flow rate of 0.3 mL·min~(-1), and a sample size of 1 µL. The scanning was performed in the negative ion mode. The complex component groups in Kaixin San substance benchmark were quickly and accurately identified and clearly assigned based on the comparison of the retention time and MS data with those of the reference substance as well as the relative molecular weight of the same or similar components in the mass spectrum database and literature. A total of 77 compounds were identified, including 26 saponins, 13 triterpenoid acids, 20 oligosaccharide esters, 5 xanthones, and 13 other compounds. The qualitative method established in this study can systematically, accurately, and quickly identify the chemical components in Kaixin San substance benchmark, which can provide a basis for the further analysis of its active components in vivo and the establishment of its quality control system.

UHPLC-Q-Exactive Orbitrap MS/MS-based rapid identification of chemical components in substance benchmark of Kaixin San / 中国中药杂志

Ultra-performance liquid chromatography-quadrupole-electrostatic field Orbitrap mass spectrometry(UHPLC-Q-Exactive Orbitrap MS/MS) was used for rapid identification of the chemical components in Kaixin San substance benchmark. The gradient elution was performed through a Waters ACQUITY~(TM) BEH C_(18) column(2.1 mm×150 mm, 1.7 μm) with water-acetonitrile as mobile phase, a column temperature of 30 ℃, a flow rate of 0.3 mL·min~(-1), and a sample size of 1 μL. The scanning was performed in the negative ion mode. The complex component groups in Kaixin San substance benchmark were quickly and accurately identified and clearly assigned based on the comparison of the retention time and MS data with those of the reference substance as well as the relative molecular weight of the same or similar components in the mass spectrum database and literature. A total of 77 compounds were identified, including 26 saponins, 13 triterpenoid acids, 20 oligosaccharide esters, 5 xanthones, and 13 other compounds. The qualitative method established in this study can systematically, accurately, and quickly identify the chemical components in Kaixin San substance benchmark, which can provide a basis for the further analysis of its active components in vivo and the establishment of its quality control system.

Rapid discovery of quality-markers from Kaixin San using chinmedomics analysis approach.

BACKGROUND: Alzheimer's disease (AD), a progressive neurodegenerative disease, is more common disease of dementia among the elderly by multiple factors and presents enormous challenges in terms of diagnosis and treatment. Kaixin San (KXS), is a classic prescription for the treatment of memory decline and applied for AD nowadays. However, the quality-markers of KXS for the treatment of AD remain unclear. PURPOSE: To investigate the effects and potential quality-markers of KXS against an APP/PS1 transgenic mouse model of AD. METHODS: Two month old APP/PS1 transgenic model mice of AD were orally given KXS for 10 month to intervene. Through the novel object recognition (NOR), the classic Morris water maze (MWM), immunohistochemistry detection of Aß1-42, Hematoxylin-eosin staining (HE), blood metabolic profiling evaluated the therapeutic effect of KXS on AD. PCMS software was applied to analysis correlations between biomarkers and serum constituents and became a powerful tool for excavating effective material basis. Behavior, histopathology and Chinmedomics were applied for assessing the efficacy and discovering potential quality-markers. RESULTS: The result of MWM showed oral KXS could shorten the escape latency and increased the times of crossing the platform. The result of NOR showed oral KXS increased discrimination index (DI). Though the histopathology, KXS reduced the necrosis of neuron in brain tissue and the deposition of Aß1-42. Chinmedomics strategy was used to analyze the biomarkers and blood components. KXS called back 20 biomarkers of AD. The effective material basis of KXS was ginsenoside Rf, ginsenoside F1, 20-O-glucopyranosyl ginsenoside Rf, dehydropachymic acid and E-3, 4, 5-trimethoxycinnamic acid. CONCLUSION: This study demonstrate that KXS significantly improved cognitive function of transgenic mice of AD, repaired the damage caused by Aß, regulated amino acid metabolism and lipid metabolism abnormalities and determined the effective material basis of KXS treating AD. Clarifying the quality-markers of KXS can establish scientific quality standard to reflect the safety and effectiveness of Traditional Chinese Medicine (TCM).

[Effect of six class of Kaixin San formulas on pharmacological and preliminary mechanism of Alzheimer's disease mice].

The efficacy of Chinese herbal formulas in treating Alzheimer has been proved in many studies. In this study, six different Kaixin San formulas were compared to investigate their effects on learning and memory decline, brain-derived neurotrophic factor (BDNF) in the hippocampus, tau protein, acetylcholinesterase (AChE) and N-terminal pro-brain natriuretic peptide (NT-proBNP). Kunming mice were selected and established a mouse model of Alzheimer's by intraperitoneal injection of D-galactose and sodium nitrite, continued intragastric 4 weeks, using the ability of learning and memory in Morris water maze test to evaluate the animals in each group; the content of BDNF in the hippocampus of mice with Western blotting detected; ELISA method for the detection of each group of mice hippocampal tau protein,p-Tau protein, Aß,Ach,AchE and serum NT-proBNP levels. The results showed that, Kaixin San of Qianjin Yaofang three dose recorded significantly improved learning and memory ability of mice; increased the content of BDNF and Ach in the hippocampus; decreased the content of Aß, Tau protein, p-Tau protein in the hippocampus; high, middle dose significantly decreased the serum NT-proBNP and AchE in hippocampus, the effect is most significant. Part dose of Kaixin San of Yixin Fang, Kaixin Wan of Yimen Fang, Dingzhi Xiaowan of Beji Qianjin Yaofang and Dingzhi Wan of Guji Luyan could improve the learning and memory ability evaluation indicators, significantly increased BDNF and Ach in the hippocampus of AD model mice, reduced the Aß, Tau protein, p-Tau protein in hippocampus of AD model mice, decreased the NT-proBNP and AchE in serum of AD mice, the effect is more significant. Three does of Buxin Tang of Qianjin Yi had no effects of treatment in Alzheimer's disease. The results showed the treatment in AD of Kaixin San of Qianjin Yaofang is the most significant.

Effect of six class of Kaixin San formulas on pharmacological and preliminary mechanism of Alzheimer's disease mice / 中国中药杂志

The efficacy of Chinese herbal formulas in treating Alzheimer has been proved in many studies. In this study, six different Kaixin San formulas were compared to investigate their effects on learning and memory decline, brain-derived neurotrophic factor (BDNF) in the hippocampus, tau protein, acetylcholinesterase (AChE) and N-terminal pro-brain natriuretic peptide (NT-proBNP). Kunming mice were selected and established a mouse model of Alzheimer's by intraperitoneal injection of D-galactose and sodium nitrite, continued intragastric 4 weeks, using the ability of learning and memory in Morris water maze test to evaluate the animals in each group; the content of BDNF in the hippocampus of mice with Western blotting detected; ELISA method for the detection of each group of mice hippocampal tau protein,p-Tau protein, Aβ,Ach,AchE and serum NT-proBNP levels. The results showed that, Kaixin San of Qianjin Yaofang three dose recorded significantly improved learning and memory ability of mice; increased the content of BDNF and Ach in the hippocampus; decreased the content of Aβ, Tau protein, p-Tau protein in the hippocampus; high, middle dose significantly decreased the serum NT-proBNP and AchE in hippocampus, the effect is most significant. Part dose of Kaixin San of Yixin Fang, Kaixin Wan of Yimen Fang, Dingzhi Xiaowan of Beji Qianjin Yaofang and Dingzhi Wan of Guji Luyan could improve the learning and memory ability evaluation indicators, significantly increased BDNF and Ach in the hippocampus of AD model mice, reduced the Aβ, Tau protein, p-Tau protein in hippocampus of AD model mice, decreased the NT-proBNP and AchE in serum of AD mice, the effect is more significant. Three does of Buxin Tang of Qianjin Yi had no effects of treatment in Alzheimer's disease. The results showed the treatment in AD of Kaixin San of Qianjin Yaofang is the most significant.

Effect of Kaixin-San on depression behavior and hippocampal brain-derived neurotrophic factor in mice / 第二军医大学学报

Objective To investigate the effect of Kaixin-San (KXS) on behavior, brain monoamine transmitters, and hippocampal brain-derived neurotrophic factor (BDNF) in mice receiving tail suspension test (TST), so as to explore the correlation of the antidepressant effect of KXS and hippocampal BDNF level. Methods The effect of KXS on the immobility time of mice in the TST was observed. The levels of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) in the brain were determined by high-performance liquid chromatography (HPLC). Western blotting analysis was used to examine the expressions of cAMP-response element-binding protein (CREB), p-CREB, and BDNF in the hippocampus. BDNF mRNA expression was also examined by RT-PCR. Pearson's correlation analysis was used to evaluate relationship between anti-depression effect of Kaixin-San and hippocampal BDNF level. Results KXS reduced the immobility time of mice in the TST (P<0.05); it also increased the brain levels of monoamine transmitters (DA and 5-HT) and hippocampal levels of CREB, p-CREB and BDNF compared with the control mice(P<0.05). Moreover, the anti-depression effect of KXS was correlated with BDNF level (protein:r=-0.694, P<0.01; mRNA:r=-0.547, P<0.01) in the hippocampus. Conclusion The anti-depression effect of KXS is positively correlated with the hippocampal BDNF level, indicating KXS may exert anti-depression effect via increasing hippocampal BDNF.