| Objective:In this study,we used network pharmacology methods combined with previous laboratory studies to screen the pathways and key targets of Xiongma Drop Pills in the treatment of vascular cognitive impairment.We used the 2-VO method to establish the VCI model,and intervened with Xiongma Drop Pills.We detected the changes in key pathways related proteins in the brain,serum,and serum exosomes,explored the correlation between vascular cognitive impairment and serum exosomes and the correlation between changes in related proteins in the brain,serum,and serum exosomes,in order to provide preliminary theoretical and experimental basis for subsequent laboratory research on exosomes and VCI.To provide empirical evidence for rapid detection of vascular cognitive impairment in vitro.Methods:Using the method of network pharmacology combined with previous laboratory research,the key pathways and targets of Xiongma Dropping Pills in the treatment of VCI were screened.The rat VCI model was reproduced using a vascular occlusion tensiometer combined with the 2-VO method,and cerebral blood flow was measured using a laser Doppler cerebral blood flow meter after ligation surgery,on 7d and 14d after ligation.Morris water maze experiment and social experiment were conducted on 28d and 56d after the establishment of the model.After the 56d water maze,blood and brain tissue were collected.The content of acetylcholine(ACh)and the activity of acetylcholinesterase(AChE)in the brain and the content of AKT1,AChE and PKCβ1 in serum of rats in each group were detected by using ELISA kit.The expression of AKT1,AChE,and PKCβ1 in the brain of rats in each group was detected by Western blot.Serum exosomes were isolated by ultracentrifugation,and the morphological characteristics of the exosomes were observed by transmission electron microscopy.The particle size and concentration of the exosomes were analyzed by nanoflow cytometry.The marker proteins of the exosomes(CD9,CD63,and TSG101)and the expression of AKT1,AChE,and PKCβ1 in the serum exosomes of rats in each group was detected by Western blot.Results:The results of network pharmacology analysis indicated that Xiongma Dropping Pills may play a role in the treatment of VCI by regulating.cholinergic synaptic signaling pathways by acting on AKT1,AChE,and PKCB.The results of cerebral blood flow(CBF)measurement suggested that compared with the sham operation(SO)group,the CBF of the model group rats significantly decreased after surgery,on 7d and 14d after ligation(p<0.01).In contrast with the model group,on 7d and 14d after ligation,the CBF of rats in each treatment group significantly increased(p<0.05),while the difference in blood pressure before and after ligation was not statistically significant(p>0.05).The results of Morris water maze experiment showed that on 28d and 56d after ligation,compared with the SO group,the escape latency and total movement distance of the model group rats were significantly increased(p<0.05,p<0.01),and the number of times they crossed the platform was markedly decreased(p<0.01).In contrast with the model group,the escape latency and total exercise distance of rats in the high-dose group of Xiongma(XM-H)and positive drug group were substantially reduced(p<0.05,p<0.01),and the number of times of crossing the platform was substantially increased(p<0.05,p<0.01).The results of social experiment suggested that on 28d and 56d after ligation the contact time and contact times of the model group rats were significantly reduced(p<0.01,p<0.05);Compared with the model group,the continuous contact time of rats in each administration group significantly increased(p<0.01,p<0.05),and on 56d after ligation,the contact times of rats in each medication group significantly increased(p<0.01,p<0.05).The measurement results of biochemical indicators in the brain of rats in each group suggested that compared with the SO group,the model group was markedly decreased in ACh content(p<0.01),increased in AChE activity(p<0.01),and downregulated in the experssion of AKT1,AChE,and PKCβ1(p<0.01).In contrast with the model group,the ACh content was significantly increased(p<0.05),and the expression level of PKCβ1 was significantly upregulated(p<0.05,p<0.01)in the XM-H group and positive drug group in the brain of rats.The AChE activity was dramaticslly increased(p<0.05,p<0.01),and the expression of AKT1 and AChE was significantly upregulated(p<0.05,p<0.01)in the brain of rats in each administration group.The determination results of AKT1,AChE,and PKCβ1 content in the serum of rats in each group showed that compared with the SO group,the content of AKT1,AChE,and PKCβ1 were dramaticslly increased(p<0.01).In contrast with the model group,the serum AChE content of was dramaticslly reduced(p<0.05,p<0.01),while the serum AKT1 and PKC content of rats in the XM-H group and positive drug group were significantly reduced(p<0.05,p<0.01).The results of isolation and identification of serum exosomes showed that the serum exosomes were in a saucer or cup-shaped structure,with complete morphology and uniform shape,consistent with the basic characteristics of exosomes;Serum exosomes particle concentration was 1.47×1010 Particles/mL detected by nanoflow cytometry,the measured average particle size was 85.44 nm;Western blot detection showed that there were bands of CD9,CD63 and TSG101 in serum exosomes,which supported each other,indicating that the ultracentrifugation method was successful in extracting serum exosomes.The detection results of the expression levels of AKT1,AChE,and PKCβ1 in the serum exosomes of rats in each group showed that there was no significant difference in the expression of AKT1 in the serum exosomes of rats in each group(p>0.05).Compared with the SO group,the expression levels of AChE and PKCβ1 in the serum exosomes of rats in the model group were significantly decreased(p<0.05,p<0.01).In contrast with the model group,the expression levels of PKCβ1 and AChE in the serum exosomes of rats in each administration group were significantly upregulated(p<0.05,p<0.01).Conclusion:Based on the results of network pharmacology analysis and previous laboratory research,this experiment focused on cholinergic synaptic signaling pathways.The rat model of VCI was established using the 2-VO method combined with a blood vessel blocking tensile apparatus.After 28 days of ligation,the learning and memory abilities of VCI rats decreased,accompanied by social impairment,which lasted until 56 days.Xiongma Dropping Pills may regulate cholinergic synaptic signaling pathways by acting on AKT1,AChE,and PKCβ1,improving the learning and memory abilities of VCI rats;The changes in the expression of related proteins in the serum exosomes of VCI rats are basically consistent with the trend of changes in the brain,suggesting that vascular cognitive impairment may affect the expression of related proteins in the serum exosomes which mediates the transmission of disease information between the central nervous system and the periphery through the serum exosomes.Xiongma Dropping Pill upregulates the expression of AChE and PKCβ1 in serum exosomes,suggesting that the serum exosomes participate in and mediate the process of Xiongma Dropping Pills acting on related proteins and regulating cholinergic synaptic signaling pathways. |
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