Effects of electroacupuncture on the microvascular structure and related protein expression in the hippocampus of vascular dementia rats. / ç”µé’ˆå¯¹è¡€ç®¡æ€§ç—´å‘†å¤§é¼ æµ·é©¬å¾®è¡€ç®¡ç»“æž„å’Œç›¸å ³è›‹ç™½è¡¨è¾¾çš„å½±å“.

OBJECTIVES: To explore the effect of electroacupuncture (EA) at "Baihui" (GV20) and "Shenting" (GV24) on the microvascular structure and related protein expression in the hippocampus of vascular dementia (VD) rat model, and to investigate the mechanism of EA in the treatment of VD. METHODS: A total of 24 SD rats were randomly divided into sham operation, model, EA, and oxiracetam groups, with 6 rats in each group. Multiple cerebral infarction method was used to establish VD model. In the EA group, EA was applied to GV20 and GV24 for 30 min, once daily for 14 days. Rats in the oxiracetam group were treated with oxiracetam (50 mg/kg) by intraperitoneal injection, and the course of treatment was the same as that in the EA group. Learning and memory ability were evaluated by using Morris water maze test and new object recognition experiment. The cerebral blood flow was detected by laser doppler. The microvascular structure in the hippocampus was observed by transmission electron microscopy. The expression of vascular structure related proteins of platelet-derived growth factor receptor (PDGFR)-ß, platelet endothelial cell adhesion molecule-1(CD31), neural cadherin N-Cadherin, Zonula occludens protein-1(ZO-1) in the hippocampus were measured by Western blot. RESULTS: Compared with the sham operation group, the rats in the model group had a significant increase in time of first crossing the platform, a significant decrease in the number of crossing platform and the new object preference index (P<0.05), a significant decrease in cerebral blood flow (P<0.05), and a significant increase in the brain weight (P<0.05). The structure boundary of pericyte and endothelial cells in the microvessels of the hippocampal CA1 area of model group was blurred, accompanied by obvious edema around the vessels and the reduction of tight junctions. The protein expression levels of PDGFR-ß, CD31, N-Cadherin, ZO-1 were significantly decreased in the model group compared with those in the sham operation group (P<0.05). Compared with the model group, the time of first crossing the platform of rats in the EA and oxiracetam group was shortened, the number of crossing platform were increased (P<0.05), the cerebral blood flow was increased (P<0.05), the brain weight was decreased (P<0.05), the morphology and structure of pericyte and endothelial cells in the microvessels of hippocampal CA1 area were intact, accompanied by the increase of tight junctions. Additionally, Compared with the model group, the EA group had a significant increase in the new object preference index (P<0.05), the protein expression levels of PDGFR-ß, CD31, ZO-1 in the EA group were increased (P<0.05), and the expression of PDGFR-ß, N-Cadherin, ZO-1 in the oxiracetam group were increased (P<0.05). CONCLUSIONS: EA at GV20 and GV24 can improve the learning and memory ability of VD rats, and the mechanism may be related to the repair of microvascular structures and improvement of cerebral blood flow.

Habitual glucosamine use, APOE genotypes, and risk of incident cause-specific dementia in the older population.

BACKGROUND: The relationship of glucosamine use with incident dementia in the older population remains uncertain. We aimed to evaluate the longitudinal association between habitual glucosamine supplement and the risk of cause-specific dementia and examine the possible effect modifiers on this association. METHODS: The study included 214,945 participants over the age of 60 who had available information on glucosamine use and did not have dementia at baseline in the UK Biobank. The APOE genotypes were determined by a combination variant of rs429358 and rs7412. The primary outcome was incident vascular dementia, incident Alzheimer's disease, and incident frontotemporal dementia, respectively. RESULTS: Over a median follow-up duration of 12 years, 1039, 1774, and 122 participants developed vascular dementia, Alzheimer's disease, and frontotemporal dementia, respectively. Overall, habitual glucosamine use was significantly associated with a lower risk of incident vascular dementia (adjusted HR, 0.82; 95%CI, 0.70-0.96), but not significantly associated with incident Alzheimer's disease (adjusted HR, 1.02; 95%CI, 0.92-1.14) and incident frontotemporal dementia (adjusted HR, 0.95; 95%CI, 0.63-1.43). Moreover, the inverse association between habitual glucosamine use and incident vascular dementia was more pronounced in participants with concomitant supplement of calcium (P-interaction = 0.011), and those without concomitant supplement of zinc (P-interaction = 0.018). However, APOE ε4 dosage and baseline cognitive function did not significantly modify the relationships of glucosamine use with incident vascular dementia or Alzheimer's disease (All P-interactions > 0.05). CONCLUSIONS: Regardless of APOE genotypes and baseline cognitive function, habitual glucosamine use was significantly inversely associated with incident vascular dementia in the older population.

[Effect of moxibustion on inositol requiring enzyme 1 / X-box binding protein 1 pathway in hippocampal CA1 region of rats with vascular dementia].

OBJECTIVE: To observe the effect of moxibustion at Governor Vessel acupoints on inositol requiring enzyme 1 （IRE1） / X-box binding protein 1 （XBP1） pathway in hippocampal CA1 region of rats with vascular dementia （VD）, so as to explore its mechanisms in the treatment of VD. METHODS: Male SD rats were randomly divided into normal, sham operation, model, moxibustion （Moxi） and medication groups （n=12）. The VD model was established by permanent ligation of bilateral common carotid arteries. For rats of the Moxi group, mild moxibustion was given to "Baihui" （GV20）, "Dazhui" （GV14） and "Fengfu" （GV16） for 20 min each point, once a day for consecutive 6 days per week, for a total of 4 weeks. For rats of the medication group, intragastric perfusion of nimodipine was given 3 times each day with total dose of 2 mg•kg-1•d-1 for 4 weeks. Morris water maze test was used to detect the learning and memory ability of rats before and after modeling as well as after intervention. The apoptosis rate of nerve cells in hippocampal CA1 region was detected by TUNEL staining. The proteins and mRNA expression levels of IRE1, XBP1, B-cell lymphoma/leukemia-2 （Bcl-2）, Bcl-2 associated X protein （Bax） in hippocampal CA1 region were detected by Western blot and real-time quantitative PCR, respectively. RESULTS: Compared with the sham operation group, the average escape latency was significantly prolonged （P<0.01）, the number of times crossing the original platform was significantly reduced （P<0.01）, the apoptosis rate of nerve cells in hippocampal CA1 region was significantly increased （P<0.01）, the proteins and mRNA expression levels of IRE1, XBP1 and Bax were significantly increased （P<0.01）, and the expression levels of Bcl-2 protein and mRNA were significantly decreased （P<0.01） in rats of the model group. After treatment, compared with the model group, the average escape latency was significantly shortened （P<0.01）, the number of times crossing the original platform was increased （P<0.05）, the apoptosis rate of nerve cells in hippocampal CA1 region was significantly decreased （P<0.01）, the protein and mRNA expression levels of IRE1, XBP1 and Bax were decreased （P<0.05, P<0.01）, and the expression levels of Bcl-2 protein and mRNA were increased （P<0.05, P<0.01） in rats of the Moxi group and medication group. There was no significant difference in the above indexes between the Moxi group and the medication group. CONCLUSION: Moxibustion at the acupoints of Governor Vessel can improve the cognitive function of VD rats, and its mechanism may be related to regulating IRE1/XBP1 pathway, inhibiting the release of pro-apoptotic protein Bax, increasing the expression of anti-apoptotic protein Bcl-2, and thus inhibiting the apoptosis of hippocampal nerve cells.

["Huayu Tongluo" moxibustion improves learning-memory ability and promotes myelin regeneration by regulating Sonic Hedgehog signaling pathway in vascular dementia rats].

OBJECTIVE: To investigate the effect of "Huayu Tongluo" (resolving blood stagnation to dredge meridian-collaterals) moxibustion on remyelination and Sonic Hedgehog (Shh) signaling pathway in the corpus callosum of vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD. METHODS: Male Wistar rats were randomized into sham-operation, model, medication and moxibustion groups, with 12 rats in each group.The VD model was established by bilateral common carotid artery occlusion. Moxibustion was applied to "Shenting"(GV24), "Baihui"(GV20) and "Dazhui"(GV14) for 20 min once a day, 7 d as a treatment course, for 3 courses, with one day's rest between every two courses. Rats of the medication group were treated by gavage of 10 mg/kg of chloromastine solution once a day, and the course of treatment was the same as that of the moxibustion group. The rat's learning-memory ability was assessed by Morris water maze test (escape latency). The neurological deficits were evaluated by using Longa's scale.The mRNA and protein expressions of Shh and Gli1 in the corpus callosum were measured by quantitative real-time fluorescence PCR and Western blot, separately. The ultrastructure of the myelin sheath and myelinated axons was observed under transmission electron microscopy (TCM). RESULTS: Compared with the sham-operation group, the neurologic score and escape latency were significantly increased and prolonged (P<0.01), and the mRNA and protein expression levels of Shh and Gli1 and the number of myelinated axons were obviously decreased in the model group (P<0.01). In comparison with the model group, the escape latency was apparently shortened (P<0.05), while the mRNA and protein expression levels of Shh and Gli1 as well as the number of myelinated axons were strikingly increased in both moxibustion and medication groups (P<0.01). Results of TCM showed that in the model group, the arrangement of myelin coil structures was sparse and fuzzy, and some structures were bulged and disbanded. The oligodendrocytes were irregular, and the number of myelin sheath was rare. These situations were relatively milder in both moxibustion and medication groups. CONCLUSION: "Huayu Tongluo" moxibustion can promote the differentiation and maturation of oligodendrocyte precursor cells after cerebral ischemia by regulating the expressions of Shh and Gli1 in Shh signaling pathway, thus promoting the regeneration of cerebral white matter myelin sheaths in VD rats, which may contribute to improving learning-memory ability.

Association of regular glucosamine use with incident dementia: evidence from a longitudinal cohort and Mendelian randomization study.

BACKGROUND: Emerging data suggests the neuroprotective and anti-neuroinflammatory effects of glucosamine. We aimed to examine the association between regular glucosamine use and risk of incident dementia, including dementia subtypes. METHODS: We conducted large-scale observational and two-sample Mendelian randomization (MR) analyses. Participants in UK Biobank having accessible data for dementia incidence and who did not have dementia at baseline were included in the prospective cohort. Through the Cox proportional hazard model, we examined the risks of incident all-cause dementia, Alzheimer's disease (AD), and vascular dementia among glucosamine users and non-users. To further test the causal association between glucosamine use and dementia, we conducted a 2-sample MR utilizing summary statistics from genome-wide association studies (GWAS). The GWAS data were obtained from observational cohort participants of mostly European ancestry. RESULTS: During a median follow-up of 8.9 years, there were 2458 cases of all-cause dementia, 924 cases of AD, and 491 cases of vascular dementia. In multivariable analysis, the hazard ratios (HR) of glucosamine users for all-cause dementia, AD, and vascular dementia were 0.84 (95% CI 0.75-0.93), 0.83 (95% CI 0.71-0.98), and 0.74 (95% CI 0.58-0.95), respectively. The inverse associations between glucosamine use and AD appeared to be stronger among participants aged below 60 years than those aged above 60 years (p = 0.04 for interaction). The APOE genotype did not modify this association (p > 0.05 for interaction). Single-variable MR suggested a causal relationship between glucosamine use and lower dementia risk. Multivariable MR showed that taking glucosamine continued to protect against dementia after controlling for vitamin, chondroitin supplement use and osteoarthritis (all-cause dementia HR 0.88, 95% CI 0.81-0.95; AD HR 0.78, 95% CI 0.72-0.85; vascular dementia HR 0.73, 95% CI 0.57-0.94). Single and multivariable inverse variance weighted (MV-IVW) and MR-Egger sensitivity analyses produced similar results for these estimations. CONCLUSIONS: The findings of this large-scale cohort and MR analysis provide evidence for potential causal associations between the glucosamine use and lower risk for dementia. These findings require further validation through randomized controlled trials.

[Effects of electroacupuncture on the blood-brain-barrier and proinflammatory cytokine IL-1ß and IL-18 in the hippocampus of rats with vascular dementia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on blood-brain barrier (BBB) permeability and proinflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) in the hippocampus of vascular dementia (VD) rats, so as to explore the mechanism of EA on treatment of VD. METHODS: SD male rats were randomly divided into sham operation, model, and EA groups, with 15 rats in each group. The VD rat model was established by permanently occlusion of the bilateral middle cerebral artery. Rats of the EA group received EA at "Baihui" (GV20), "Dazhui" (GV14), and bilateral "Shenshu"(BL23) for 30 min, 6 days a week for a total of 4 weeks. Morris water maze test was used to assess the cognitive function of rats. Evans blue staining was used to detect the BBB permeability, transmission electron microscopy and ELISA were used to detect the ultrastructure of BBB and the contents of hippocampal IL-1ß and IL-18, respectively. RESULTS: Following modeling, compared with the sham operation group, the mean escape latency of model group was significantly prolonged (P<0.01), the times of crossing the platform were significantly decreased (P<0.01), the content of Evans blue, and the contents of IL-1ß and IL-18 in hippocampus were increased (P<0.01). After the intervention, comparison between the model and EA groups showed that the average escape latency of rats in EA group was significantly shortened (P<0.01), the times of crossing the platform were increased (P<0.05), the content of Evans blue, and the contents of IL-1ß and IL-18 in hippocampus were significantly decreased (P<0.01). The ultrastructure of BBB was moderately damaged in the model group, which was evidenced by blurred endothelial cell membrane structure, obviously dropsical astrocyte foot process, and decreased tight junctions. The ultrastructure of BBB was slightly damaged and astrocyte foot had no obvious edema in the EA group. CONCLUSION: EA can significantly improve the learning and memory ability of VD rats and improve the BBB permeability, which may be related to its effect in inhibiting the expression of IL-1ß and IL-18 in the hippocampus.

[Effects of electroacupuncture on ROS-NLRP3 inflammatory pathway and autophagy related proteins in hippocampus of vascular dementia rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on learning-memory ability, ultrastructural changes of hippocampal CA1 neurons, reactive oxygen species (ROS) level, Nod-like receptor protein 3 (NLRP3) and auto-phagy-related proteins expression in the hippocampus of vascular dementia (VD) rats, so as to reveal its partial mechanisms in treating VD. METHODS: Male SD rats were randomly divided into sham operation, model, and EA groups (n=10 rats in each group). The VD model was established by permanent ligation of bilateral common carotid arteries. Rats of the EA group were treated with EA at "Baihui" (GV20), "Dazhui" (GV14) and bilateral "Shenshu" (BL23) for 30 min, once a day for 4 weeks. Morris water maze was used to evaluate the learning and memory ability of rats before modeling, 4 weeks after modeling and after intervention. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of hippocampal CA1 neurons. The level of ROS in hippocampus was detected by DCFH-DA fluorescence probe. The expressions of NLRP3, autophagy-related protein Beclin1 and microtubule-associated protein 1 light chain 3 (LC3) were measured by Western blot. RESULTS: In comparison with the sham operation group, the average escape latency of rats in the model group was prolonged (P<0.01), and the times of crossing the original platform were reduced (P<0.05), the level of ROS, the expression levels of LC3-Ⅱ/LC3-Ⅰ ratio, Beclin1 and NLRP3 proteins in hippocampus were increased (P<0.01, P<0.05) in the model group. After EA intervention, the average escape latency of rats was significantly shortened (P<0.01), and the times of crossing the original platform were increased (P<0.05), the level of ROS, the expression levels of LC3-Ⅱ/LC3-Ⅰ ratio, Beclin1 and NLRP3 proteins in hippocampus were decreased (P<0.01, P<0.05) in the EA group compared with those of the model group. Outcomes of TEM showed that CA1 neurons in the hippocampus were damaged, chromatin aggregation, mitochondria pyknosis, cristae structure disorder, rough endoplasmic reticulum expanded and degranulated, the number of free ribosomes decreased, and autophagy could be seen in the model group, which were milder in the EA group. CONCLUSION: EA at GV20, GV14 and BL23 can improve the learning and memory abilities of VD rats, alleviate the ultrastructural damage of neurons in hippocampal CA1 area, and repair the damaged neurons. The mechanism may be related to the reduction of ROS level, LC3-Ⅱ/LC3-Ⅰ ratio, NLRP3 and Beclin1 protein expression, the decrease of neuronal autophagy, inhibition of activation of NLRP3 inflammasome and alleviation of central inflammatory response.

[Effect of electroacupuncture on gut microbiota and serum IL-1ß and IL-18 in rats with vascular dementia based on principle of "curing brain disorders by treating intestines"].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Baihui"(GV20), "Dazhui"(GV14), "Shenshu" (BL23)and "Zusanli"(ST36) on the intestinal flora and serum interleukin (IL)-1ß and IL-18 contents in vascular dementia (VD) rats. METHODS: SD rats were randomized into sham operation, VD model, GV20+GV14+BL23 (EA-basic acupoints), and EA-basic acupoints+ST36 and EA-basic acupoints+probiotics groups (n=10 in each group). EA (10 Hz/50 Hz) was conducted for 30 min, once daily for 4 consecutive weeks. Rats of the EA-basic acupoints+probiotics received gavage of probiotics (2 mL/d containing 2.0×109 CFU of live bifidobacterium), once a day for 4 weeks, and those of the EA-basic acupoints and EA-basic acupoints+ST36 groups received gavage of the same dose of normal saline. The Morris water maze test was used to evalua-te the rats' lear-ning and memory ability before and after the treatment. The serum IL-1ß and IL-18 levels were determined by ELISA, and the histopathological changes of the intestinal mucosa were observed by H.E. staining. The ultrastructural changes of hippocampal neurons were observed by using transmission electron microscopy and 16S rDNA sequencing technique was used to analyze the composition of intestinal microbiome. RESULTS: Compared with the sham operation group, the escape latency, serum levels of IL-1ß and IL-18, as well as the relative abundance of harmful bacteria (including Catabacter, obinsoniella and Desulfovibrio) in the intestine were significantly increased (P<0.01). In comparison with the model group, the escape latency, serum levels of IL-1ß and IL-18 in the three treatment groups, and the relative abundance of harmful bacteria (such as the Catabacter, Robinsoniella and Desulfovibrio) in the EA-basic acupoints+ST36 group were down-regulated obviously(P<0.05,P<0.01), and the relative abundance of Clostridiales-unclassified in both EA-basic acupoints+probiotics and EA-basic acupoints+ST36 groups was significantly up-regulated (P<0.05). The effects of EA-basic acupoints+ST36 and EA-ba-sic acupoints+probiotics were significantly superior to that of EA-basic acupoints in down-regulating IL-18 content (P<0.05). H.E. staining showed atrophy of the whole mucosal layer, loss of goblet cells, destruction of glands, infiltration of a large number of inflammatory cells, and transmission microscope displayed fuzziness of the nucleus membrane boundary, cystic dilation of the rough endoplasmic reticulum with unclear structure swelling of the mitochondria, and disordered arrangement or dissolution of the inner cristae in the model group, which was relatively milder in the EA-basic acupoints+ST36 and EA-basic acupoints+probiotics groups. CONCLUSION: EA of GV20+GV14+BL23+ ST36 can improve the cognitive dysfunction of VD model rats, which may be related to its function in regulating the imbalance of intestinal microbiota, thereby inhibiting the peripheral inflammatory factor.

Network pharmacology and in vitro studies reveal the pharmacological effects and molecular mechanisms of Shenzhi Jiannao prescription against vascular dementia.

BACKGROUND: Shenzhi Jiannao (SZJN) prescription is a type of herbal formula adopted in the management of cognitive impairment and related disorders. However, its effects and related regulatory mechanisms on vascular dementia (VD) are elusive. Herein, network pharmacology prediction was employed to explore the pharmacological effects and molecular mechanisms of SZJN prescription on VD using network pharmacology prediction, and validated the results through in vitro experiments. METHODS: Through a search in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database, chemical composition and targets for SZJN prescription were retrieved. The potential targets for VD were then obtained from the GeneCards and DisGeNET databases. The network was constructed that depicted the interactions between putative SZJN prescription and known therapeutic targets for VD using Cytoscape 3.7.1. Analysis of protein-protein interaction was achieved via STRING 11.0 software, followed by Gene Ontology (GO) functional enrichment and Kyoto Gene and Genome Encyclopedia (KEGG) pathway analyses. To validate the computer-predicted results, in vitro experiments based on an excitotoxic injury model were designed using glutamate-exposed PC12 cells, and treated with varying concentrations (low, 0.05; medium, 0.1 and high, 0.2 mg/mL) of SZJN prescription. Cell viability and cell death were detected using the IncuCyte imaging system. Moreover, the expression profiles of Caspase-3 were analyzed through qRT-PCR. RESULTS: Twenty-eight potentially active ingredients for SZJN prescription, including stigmasterol, beta-sitosterol, and kaempferol, plus 21 therapeutic targets for VD, including PTGS2, PTGS1, and PGR were revealed. The protein-protein interaction network was employed for the analysis of 20 target proteins, including CASP3, JUN, and AChE. The enrichment analysis demonstrated candidate targets of SZJN prescription were more frequently involved in neuroactive ligand-receptor interaction, calcium, apoptosis, and cholinergic synaptic signaling pathways. In vitro experiments revealed that SZJN prescription could significantly reverse glutamate-induced cell viability loss and cell death, and lower the levels of Caspase-3 mRNA in glutamate-induced PC12 cells. CONCLUSIONS: Collectively, this study demonstrated that SZJN prescription exerted the effect of treating VD by regulating multi-targets and multi-channels with multi-components through the method of network pharmacology. Furthermore, in vitro results confirmed that SZJN prescription attenuated glutamate-induced neurotoxicity.

Tongluo Huatan capsule improves cognitive function by regulating the endocytosis of N-methyl-D-aspartic acid receptors mediated by clathrin in a rat model of vascular dementia.

OBJECTIVE: To explore the neuroprotective mechanisms of Tongluo Huatan capsule (THC) in a rat model of vascular dementia (VD). METHODS: A rat model of VD was established by repeated clamping of bilateral common carotid arteries with the intraperitoneal injection of sodium nitroprusside solution. VD rats were administered THC, memantine hydrochloride, or distilled water daily for 14 d after operation. Learning and memory abilities were assessed using the step-down passive avoidance test, novel object recognition (NOR) test, and Morris water maze (MWM) test. Pathological changes in the hippocampus were observed through hematoxylin and eosin and Nissl staining. The expression levels of clathrin, RAB5B, and N-methyl-D-aspartic acid receptor 1 (NMDAR1) were measured by immunohistochemistry staining, real-time quantitative polymerase chain reaction and Western blot. RESULTS: Rats in VD group showed impaired learning and memory abilities (step-down passive avoidance, NOR, and MWM) and abnormalities in neuronal morphology (light microscopy) in the hippocampus. The mRNA or protein expression levels of clathrin and RAB5B were decreased, and NMDAR1 was increased in hippocampal tissues (P < 0.05). Administration of THC promoted the learning and memory abilities and the morphological structure of hippocampal neurons in VD rats. Besides, THC enhanced mRNA or protein expression levels of clathrin and RAB5B, and decreased NMDAR1 (P < 0.05). CONCLUSION: THC may improve cognitive functions by regulating the endocytosis of NMDA receptors mediated by clathrin.

[Acupuncture improves cognitive function of vascular dementia rats by regulating PI3K/Akt/mTOR pathway].

OBJECTIVE: To explore the mechanism of acupuncture in improving cognitive ability by regulating hippocampal phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in vascular dementia (VD) rats. METHODS: A total of 80 male SD rats were randomized into sham operation, model, non-acupoint and acupoint groups (n=18 per group). The VD model was established by ligation of the bilateral common carotid arteries. For rats of the acupoint group, "Baihui" (GV20) and bilateral "Zusanli "(ST36) were needled and stimulated by twirling the needles with reinforcing method, and for rats of the non-acupoint group, the bilateral subcostal spots (about 10 mm superior to the iliac cresta) were needled and stimulated by twirling the needles with uniform reinforcing and reducing method. The treatment was conducted once daily, 6 times a week for two weeks, beginning 3 days after successful modeling. Rats of the sham operation group and model group received grasps as those in the acupoint groups. Morris water maze test was used to detect the abilities of learning and spatial memory. The contents of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and the activities of acetylcholinesterase (AChE) and acetylcholine transferase (ChAT) in the hippocampus tissue were detected by using ELISA, changes of hippocampal mitochondrial membrane potential (MMP) detected using JC-1 fluorescence probe, and the expression levels of hippocampal phosphorylated (p)-PI3K, p-Akt and mTOR proteins measured using Western blot. RESULTS: Compared with the sham operation group, the escape latency, contents of ROS and MDA, and AChE activity were significantly increased (P<0.05), and the spatial memory ability, SOD activity, ChAT activity, MMP, p-PI3K, p-Akt and mTOR expression levels were significantly decreased in the model group (P<0.05). Compared with the model group, carotid artery ligature-induced increase of the escape latency, contents of ROS and MDA, and AChE activity, and decrease of the spatial memory ability, SOD activity, ChAT activity, MMP, p-PI3K, p-Akt and mTOR expression levels were significantly reversed in the acupuncture group (P< 0.05), but not in the non-acupoint group (P>0.05). The therapeutic effects of acupoint needling were obviously superior to those of non-acupoint needling in decreasing the escape latency, contents of ROS and MDA, and AChE activity (P<0.05), and in increasing the spatial memory ability, SOD activity, ChAT activity, MMP, p-PI3K, p-Akt and mTOR expre-ssion levels (P<0.05). CONCLUSION: Acupuncture can improve cognitive function of VD rats, which may be related with its functions in easing oxidative stress and MMP reduction by activating PI3K/Akt/mTOR signaling pathway in the hippocampus.

Exploring the regulatory mechanism of Nao Tai Fang on vascular Dementia's biological network based on cheminformatics and transcriptomics strategy.

ETHNOPHARMACOLOGICAL RELEVANCE: Nao Tai Fang (NTF) is modified from Buyang Huanwu Decoction. Modern pharmacological research showed that NTF has a good anti-cerebral ischemic effect and can improve the learning and memory ability of cerebrovascular disease. AIM: The purpose of this study is to explore the regulation mechanism of NTF on the regulation mechanism of vascular dementia (VD)'s biological network based on chemoinformatics and transcriptomics strategies. METHOD: First, the bilateral common carotid artery ligation method was used to create a rat VD model. After NTF intervention for 30 days, the treatment effect was evaluated by HE staining and water maze experiment. Then, the Agilent mRNA expression profiling chip was used to obtain mRNA expression data of hippocampal tissues of VD model rats before and after NTF intervention, and microarray analysis was used to screen for genes with significant differential expression. The BATMAN database was utilized to obtain the potential targets of NTF and the Genecards and OMIM were utilized to collect the VD potential genes. The cytoscape was utilized to construct and analyze the networks. RESULT: The animal experiments showed that NTF can improve VD. A total of 180 up-regulated proteins and 289 down-regulated proteins were identified. The top 20 up- and down-regulated differentially expressed genes were utilized to construct differentially expressed gene's protein-protein interaction (PPI) network. A total of 677 NTF potential targets and 550 VD genes were obtained and were utilized to construct NTF-VD PPI network. The cheminformatics analysis showed that NTF can regulate a lot of biological processes and signaling pathways (such as inflammation modules, vasodilation and contraction modules, hypoxia modules, angiogenesis, coagulation, neurovascular unit modules, Neuroactive ligand-receptor interaction, Calcium signaling pathway, Serotonergic synapse). CONCLUSION: NTF may play a role in the treatment of VD through the targets, signaling pathways and biological processes discovered in this study.

Exploring the Oxidative Stress Mechanism of Buyang Huanwu Decoction in Intervention of Vascular Dementia Based on Systems Biology Strategy.

OBJECTIVE: To explore the oxidative stress mechanism of modified Buyang Huanwu decoction (MBHD) in intervention of vascular dementia (VD) based on systems biology strategy. METHODS: In this study, through the reverse virtual target prediction technology and transcriptomics integration strategy, the active ingredients and potential targets of MBHD treatment of VD were analyzed, and the drug-disease protein-protein interaction (PPI) network was constructed. Then, bioinformatics analysis methods are used for Gene Ontology (GO) enrichment analysis and pathway enrichment analysis, and finally find the core biological process. After that, in animal models, low-throughput technology is used to detect gene expression and protein expression of key molecular targets in oxidative stress-mediated inflammation and apoptosis signaling pathways to verify the mechanism of MBHD treatment of VD rats. Finally, the potential interaction relationship between MBHD and VD-related molecules is further explored through molecular docking technology. RESULTS: There are a total of 54 MBHD components, 252 potential targets, and 360 VD genes. The results of GO enrichment analysis and pathway enrichment analysis showed that MBHD may regulate neuronal apoptosis, nitric oxide synthesis and metabolism, platelet activation, NF-κB signaling pathway-mediated inflammation, oxidative stress, angiogenesis, etc. Among them, SIRT1, NF-κB, BAX, BCL-2, CASP3, and APP may be important targets for MBHD to treat VD. Low-throughput technology (qRT-PCR/WB/immunohistochemical technology) detects oxidative stress-mediated inflammation and apoptosis-related signaling pathway molecules. The molecular docking results showed that 64474-51-7, cycloartenol, ferulic acid, formononetin, kaempferol, liquiritigenin, senkyunone, wallichilide, xanthinin, and other molecules can directly interact with NF-κB p65, BAX, BCL-2, and CASP3. CONCLUSION: The active compounds of MBHD interact with multiple targets and multiple pathways in a synergistic manner, and have important therapeutic effects on VD mainly by balancing oxidative stress/anti-inflammatory and antiapoptotic, enhancing metabolism, and enhancing the immune system.

Acupuncture Attenuates Inflammation in Microglia of Vascular Dementia Rats by Inhibiting miR-93-Mediated TLR4/MyD88/NF-κB Signaling Pathway.

BACKGROUND: It is widely accepted that inflammation may contribute to cognitive impairment in patients with vascular dementia (VD). Our prior clinical researches have reported that acupuncture can alleviate cognitive function in VD, but the underlying mechanisms are still unclear. The purpose of this research was to explore whether acupuncture alleviates cognitive impairment by suppressing the microRNA-93- (miR-93-) mediated Toll-like receptor (TLR) signaling pathway, which triggers inflammatory responses in the central nervous system. METHODS: VD was established by permanent bilateral common carotid artery occlusion in male Wistar rats. Three days after operation, the rats began daily treatment with acupuncture for two weeks. The levels of miR-93, Toll-like receptors (TLR2 and TLR4), intracellular signaling molecules (myeloid differentiation factor 88 (MyD88) and nuclear factor-kappa B (NF-κB)), and inflammatory cytokines were subsequently detected. TLR4 colocalized with neurons, microglia, and astrocytes in the hippocampus was evaluated. Neuroinflammation and cognitive function were determined after intracerebroventricular injection of TLR4 antagonist TAK-242 or agonist lipopolysaccharide (LPS) with or without acupuncture. RESULTS: We found that acupuncture notably repressed the expression of inflammatory cytokines in the hippocampus and plasma of VD rats. The expression of TLR4, but not TLR2, was markedly downregulated by acupuncture, accompanied by a decrease in miR-93 and MyD88/NF-κB signaling pathway activation. The overexpression of TLR4 in microglia, but not in astrocytes and neurons, was reversed by acupuncture. Furthermore, intracerebroventricular injection of TAK-242 had similar effects to acupuncture on inflammation and cognitive function, while LPS injection abolished the beneficial effects of acupuncture. CONCLUSIONS: Taken together, these findings provide evidence that acupuncture attenuates cognitive impairment associated with inflammation through inhibition of the miR-93-mediated TLR4/MyD88/NF-κB signaling pathway in experimental VD. Acupuncture serves as a promising alternative therapy and may be an underlying TLR4 inhibitor for the treatment of VD.

[Effect of moxibustion on learning-memory ability and expression of hippocampal inflammatory factors and microtubule associated proteins in vascular dementia rats].

OBJECTIVE: To observe the effect of moxibustion on learning-memory ability and expression of hippocampal inflammatory factors and microtubule-associated protein doublecortin (DCX, a marker of neuronal regeneration) in vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD. METHODS: SD rats were randomly divided into normal control, sham operation, VD model, moxibustion and medication groups (n=15 rats in each group). The VD model was established by repeated occlusion of the bilateral common carotid arteries and reperfusion. Moxibustion was applied to "Guanyuan" (CV4), "Mingmen" (GV4) and "Dazhui"(GV14) for 15 min, once a day, 6 days a week for 4 weeks. Rats of the medication group were treated by gavage of Nimodipine (2mg·kg-1·d-1) 3 times daily for 4 weeks. Morris water maze test was used to detect the average escape latency of location navigation tasks for assessing the rats' learning-memory ability. H.E. staining was used to detect histopathological changes of the hippocampus tissue. The number of DCX-positive neurons (DCX/NeuN co-expression) in the dentate gyrus (DG) region of hippocampus was counted under microscope after immunofluorescence double staining, the immunoactivity of hippocampal DCX detected by using immunohistochemistry stain and the expression of DCX, TNF-α, IL-1ß, MPO, NF-κB p65 and IL-6 proteins in the hippocampus tissue detected using Western blot. RESULTS: Following modeling, the average escape latency was significantly longer in the model group than in the normal control and sham operation groups (P<0.01), and notably shorter in both the moxibustion and medication groups than in the model group after the treatment (P<0.01, P<0.05). The number of DCX-positive neurons, and the expression levels of DCX, TNF-α, IL-1ß, MPO, NF-κB p65 and IL-6 proteins in the hippocampus were significantly increased in the model group in comparison with the normal control and sham operation groups (P<0.01, P<0.05). After the interventions and in comparison with the model group, the number of DCX-positive neurons and the expression level of DCX were further up-regulated in both moxibustion and medication groups (P<0.01), while the expression levels of hippocampal TNF-α, IL-1ß, MPO, NF-κB p65 and IL-6 proteins were considerably down-regulated in the moxibustion and medication groups (P<0.01). The effect of moxibustion was weaker than that of medication in down-regulating the expression of TNF-α,MPO, NF-κB p65, IL-6 and IL-1ß, and in up-regulating DCX-positive neuron number and DCX expression (P<0.05, P<0.01). H.E. staining showed loose arrangement of neurons (with vague neuronal membrane in some cells), uneven organelle chromatin, disappearance of partial nucleolus, necrocytosis, and infiltration of small number of lymphocytes after modeling, which was relatively milder in both moxibustion and medication groups. CONCLUSION: Moxibustion can improve learning-memory ability in VD rats, which may be related to its effect in down-regulating the expression of inflammatory factors and up-regulating the expression of DCX to promote neuronal repair and regeneration.

Wuzang Wenyang Huayu decoction regulates differentially expressed transcripts in the rats' hippocampus after cerebral hypoperfusion.

The modified Wenyang Huayu decoction has been widely used to treat vascular dementia in China for thousands of years. We have previously proved that a modified version, Wuzang Wenyang Huayu decoction has the potential to be a more effective clinical treatment that can attenuate cerebral ischaemic injury. However, the global transcript profile and signalling conduction pathways regulated by this recipe remains unclear. This study established a two-vessel occlusion rat model by bilateral common carotid artery occlusion. Two groups of rats were intragastrically treated Wuzang Wenyang Huayu 2.5 g/kg vs or Piracetam 0.15 g/kg for 2 weeks. Learning and memory abilities were measured with Morris water maze. Neuronal plasticity was observed by HE staining. Differentially expressed transcripts of rat hippocampus were analysed by transcriptomics with Illumina HiSeq2500 platform. Results showed that Wuzang Wenyang Huayu decoction significantly alleviated learning, memory deficits, coordination dysfunction and prevented hippocampus cellular injury; Results further revealed the increased gene expression in KEGG metabolic pathways (MT-ND2. MT-ND3, MT-ND4, MT-ND4L, MT-ND5 and MT-ATP8) and genes involved in signal transduction, carcinogenesis, immune system, endocrine system, nervous system etc (Results further revealed differential expression of genes involved in various systems, including MT-ND2) Our discovery is likely to provide new insights to molecular mechanisms of Wuzang Wenyang Huayu regarding hippocampal transcripts in a murine vascular dementia model.

Ling-Yang-Gou-Teng-decoction prevents vascular dementia through inhibiting oxidative stress induced neurovascular coupling dysfunction.

ETHNOPHARMACOLOGICAL RELEVANCE: Vascular dementia (VaD) is the common cognitive disorder derived mainly from lacunar stroke (LS). The oxidative stress induced neurovascular coupling (NVC) dysfunction involves in the pathogenesis of VaD. Currently, there is no specific drug for VaD. Ling-Yang-Gou-Teng -Decoction (LG), a well-known traditional Chinese formula, has been used for preventing VaD in clinic. AIM OF THE STUDY: In this study, we aimed to investigate the underlying mechanism of LG on VaD in rats. MATERIALS AND METHOD: VaD was replicated with autologous micro-thrombi against the background of hypercholesterolemia induced with high fatty diet. PTX (68.90 mg/kg/day), LG with three dosages (2.58, 8.14, 25.80 g/kg/day) was orally administrated to VaD rats, respectively. The NVC sensitivity was defined as the ratio of the microcirculative cerebral blood velocity (CBV) to the electroencephalograph (EEG) before and after penicillin stimulation. Behavioral performance, pathological changes of brain and oxidation related molecules were detected to assess the effects of LG on VaD. RESULTS: LG exhibited beneficial effects on the VaD, which was demonstrated as improved exploratory, learning and memory abilities, relieved vascular or neural pathological changes in cerebral cortex or hippocampus. LG maintained NVC sensitivity, which was confirmed as significantly increased ΔCBV and the elevated ratio of ΔCBV/ΔqEEG. The underlying mechanisms of LG was associated with antioxidant effects, which was confirmed as significantly decreased nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression, and increased superoxide dismutase 3 (SOD3) expression. LG also reduced iNOS, increased nNOS and eNOS expression to restore NO bioavailability. CONCLUSIONS: The results suggested that LG prevented VaD may associate with inhibiting oxidative stress, protecting NO bioavailability, and then maintaining NVC sensitivity.

Puerarin Up-regulates Methyl-CpG Binding Protein 2 Phosphorylation in Hippocampus of Vascular Dementia Rats.

OBJECTIVE: To observe the effect of puerarin on methyl-CpG binding protein 2 (MeCP2) phosphorylation (pMeCP2) in the hippocampus of a rat model of vascular dementia (VD). METHODS: Thirty-six healthy Sprague-Dawley rats were randomly assigned to the sham-operated group, dementia group and puerarintreated group using a random number table (n=12 per group). The modifified permanent bilateral common carotid artery occlusion method was used to establish the VD model. The sham-operated and dementia groups were given 2 mL/d of saline, while the puerarin-treated group was given 100 mg/(kg•d) of puerarin for 17 days. The learning and memory abilities were evaluated by the Morris water maze test. Hematoxylin-eosin staining, immunohistochemical (IHC) staining and Western blot analysis were carried out to observe changes in neuron morphology and in level of pMeCP2 in the hippocampus, respectively. RESULTS: The morphologies of rat hippocampal neurons in the puerarintreated group were markedly improved compared with the dementia group. The escape latency of the dementia group was significantly longer than the sham-operated group (P<0.05), while the puerarin-treated group was obviously shorter than the dementia group (P<0.05). Cross-platform times of the dementia group were signifificantly decreased compared with the sham-operated group (P<0.05), while the puerarin-treated group was obviously increased compared with the dementia group (P<0.05). IHC staining showed no significant difference in the number of MeCP2 positive cells among 3 groups (P>0.05). The number of pMeCP2 positive cells in the CA1 region of hippocampus in the dementia group was signifificantly increased compared with the sham-operated group, and the puerarin-treated group was signifificantly increased compared with the dementia group (both P<0.05). Western blot analysis showed no signifificant difference of MeCP2 expression among 3 groups (P>0.05). The expression of pMeCP2 in the dementia group was signifificantly increased compared with the sham-operated group, while it in the puerarin-treated group was signifificantly increased compared with the dementia group (P<0.05). CONCLUSION: Puerarin could play a role in the protection of nerve cells through up-regulating pMeCP2 in the hippocampus, improving neuron morphologies, and enhancing learning and memory ablities in a rat model of VD.

Ligustrazine suppresses neuron apoptosis via the Bax/Bcl-2 and caspase-3 pathway in PC12 cells and in rats with vascular dementia.

The aim of this study was to examine the comprehensive neuroprotective mechanism of ligustrazine, which is extracted from Ligusticum Chuanxiong Hort., against vascular dementia (VD) in rats and apoptosis in oxygen and glucose deprivation (OGD) PC12 cells. Rats were subjected to bilateral common carotid artery occlusion (BCCAO) surgery and administered ligustrazine intragastrically for 6 weeks. At the end of the experiments, the hippocampal biomarkers brain-derived neurotrophic factor (BDNF), monocyte chemotactic protein 1 (MCP-1), and homocysteine (Hcy) were examined. In experiments in vitro, OGD PC12 cells were treated with ligustrazine for 0.5, 1, 3, 6, 12, or 24 h. The cell-released biomarkers BDNF, MCP-1, and Hcy were examined. Microscopy, acridine orange-ethidium bromide (AO/EB) staining, and flow cytometry assays were performed to investigate apoptosis. Cleaved caspase-3, Bcl-2 associated X protein (Bax), and B cell lymphoma 2 (Bcl-2) expression was examined using Western blot assays. The results showed that biomarkers, including MCP-1 and Hcy, were significantly increased in both the in vivo and in vitro models, while the BDNF level was significantly decreased compared with the sham or vehicle models. Microscopy, AO/EB staining, and flow cytometry analysis showed that severe cell damage occurred in OGD PC12 cells, and apoptosis played a major role in this environment. Further Western blot studies showed that the apoptosis-related Bax/Bcl-2 protein ratio and cleaved caspase-3 were significantly increased in the experiment. However, ligustrazine profoundly suppressed the imbalance of these biomarkers, reduced cell damage, decreased the Bax/Bcl-2, and downregulated cleaved caspase-3. Pro- and anti-apoptotic biomarkers of multiple pathways including BDNF, MCP-1, and Hcy played a joint role in triggering the activation of the mitochondria-related Bax/Bcl-2 and caspase-3 apoptosis pathway in VD. Ligustrazine attenuated VD by comprehensively regulating BDNF, MCP-1, and Hcy and inactivating the Bax/Bcl-2 and caspase-3 apoptosis pathway. Our data provide novel insight into ligustrazine, which is a promising neuroprotective agent for VD disease treatment strategies. © IUBMB Life, 70(1):60-70, 2018.

Protective effect of Shouwu Yizhi decoction against vascular dementia by promoting angiogenesis.

Shouwu is a traditional Chinese medicine (TCM) with neuroprotective effect. Shouwu Yizhi decoction (SYD) was designed based on TCM theory. However, little is known about the roles of SYD in Vascular dementia (VaD). The present study aimed to evaluate the potential effects of SYD on the vascular cognitive impairment and explore the underlying mechanism by establishing focal cerebral ischemia/reperfusion (I/R) rat model to induce VaD. SYD administration (54 mg·kg-1) for 40 days obviously improved the vascular cognitive impairment in the middle cerebral artery occlusion (MCAO) rats as evidenced by the declined neurological deficit score and shortened escape latency via neurological deficit assessment and Morris water maze test. Moreover, SYD decreased neuron damage-induced cell death and ameliorated the ultrastructure of endothelial cells in the MCAO rats, thereby alleviating VaD. Mechanistically, SYD caused increases in the expression of vascular endothelial growth factor (VEGF), CD34 and CD31, compared with the MCAO rats in coronal hippocampus. Simultaneously, the expression level of miR-210 was elevated significantly after SYD administration, compared with the vehicle rats (P < 0.01). The expression of Notch 4 at both mRNA and protein levels was upregulated remarkably along with the notably downregulated DLL4 expression under SYD administration compared with the vehicle rats (P < 0.05). Overall, the above results indicated that SYD promoted angiogenesis by upregulating VEGF-induced miR210 expression to activate Notch pathway, and further alleviated neuron damage and ameliorated the ultrastructure of endothelial cells in the MCAO rats, ultimately enhancing the cognition and memory of MCAO rats. Therefore, our findings preliminarily identified the effect and the mechanism of action for SYD on VaD in rats. SYD could be a potential candidate in treatment of VaD.