| Purpose:To predict and analyze the main active ingredients of the traditional Chinese medicine compound Yitangkang and its core target,potential mechanism and pathway for treating DCI by means of network pharmacology,and to evaluate and verify the efficacy in various aspects through in vitro and in vivo experiments,aiming to deeply explore the efficacy and potential mechanism of the traditional Chinese medicine compound Yitangkang in the prevention and treatment of DCI.Material and method:(1)The effective chemical components of "Compound Yitangkang" were screened by TCMSP database,and the targets of DCI diseases were obtained by OMIM,TTD,Genecards and Drugbank databases.Then,the PPI network of drug target-disease is constructed by using String database and Cytoscape 3.9.0 software,and the relevant network topology analysis is realized through Cyto NCA plug-in in the software,so as to screen and obtain the core target.GO and KEGG pathway enrichment analysis was carried out on the selected core targets through Metascape database.(2)Sixty SPF male and 9-week-old male db/db mice were randomly divided into model group(db/db group),positive control group(LIRA group),high-dose group of Yitangkang(YTKH group),middle-dose group of Yitangkang(YTKM group)and low-dose group of Yitangkang(YTKL group),with 12 mice in each group.Another 12 SPF male and 9-week-old male db/m mice were selected as blank control group(db/m group).After a week of adaptive feeding,the experimental animals were given liraglutide at 200 μ g/kg d,YTK high,medium and low dose groups were given 60 g/kg d,30 g/kg d and 15 g/kg d by gavage respectively,and db/db group and db/m group were given the same volume of distilled water by gavage for 6 weeks.During the experiment,the blood sugar and weight of mice were recorded,and Morris water maze experiment was conducted in the last week of the experiment to judge the cognitive function of mice.After the behavioral experiment,the mice were killed,and the serum levels of FINS,IL-1β,IL-6,IL-18 and TNF-α were detected by ELISA,and the HOMA-IR index was calculated to evaluate the insulin secretion,resistance and inflammation level of mice in each group.The oxidative stress index SOD inhibition rate,CAT and MDA contents of mice in each group were detected to evaluate the oxidative stress level of mice in each group.Meanwhile,HE staining and Nissl staining were used to observe the pathology of mice in each group to judge the changes of the morphology and quantity of neurons in mice.Western blot was used to detect the expression of synapse-related proteins SYN,BDNF and PSD95 in mice.(3)Based on the results of the first and second papers,Western blot was used to detect the expression of the autophagy-related proteins Beclin-1,LC3 and p62 in each group of mice to determine whether the autophagy flow was complete;At the same time,the distribution and expression of LC3 protein in mouse hippocampus were detected by immunofluorescence method.Western blot method was used to detect the inflammation related proteins NLRP3,ASC,Caspase-1 and IL-1 of mice in each group β、 IL-18 expression to determine the activation of inflammatory bodies;At the same time,the distribution and expression of NLRP3 protein in mouse hippocampus were detected by immunofluorescence method.Western blot was used to detect the expression of AMPK/mTOR signal pathway protein in mice of each group.Finally,the above research results were summarized to analyze the effect of Yitangkang on autophagy,inflammation and expression of AMPK/mTOR signal pathway in db/db mice.(4)The model of DCI in vitro was established by the injury of HT22 neurons induced by high glucose,and was divided into six groups: blank group,model group,Yitangkang group,AMPK inhibitor group,autophagy inhibitor group,and NLRP3 inhibitor group.The corresponding drug-containing serum and inhibitor were given for intervention.The morphological changes of cells in each group were observed by inverted microscope,and the cell viability and glucose consumption of HT22 neurons in each group were measured by CCK8 method.Western blot method was used to detect the expression level of autophagy-related proteins Beclin-1,LC3,p62,inflammation-related proteins NLRP3,ASC,Caspase-1 and AMPK/mTOR signal pathway related proteins of HT22 cells in each group.Results:1.According to the research of network pharmacology,the main active components of the traditional Chinese medicine compound Yitangkang include quercetin,β-sitosterol,kaempferol,formononetin,luteolin,etc.Its core targets for treating DCI include AKT1、mTOR、IL-6、NLRP3、TNF 、PTGS2、STAT3、TP53、FOS、VEGFA、ESR1、MYC,etc.Core pathways include AMPK signaling pathway,mTOR signaling pathway,PI3K-AKT signaling pathway,MAPK signaling pathway,TOLL signaling pathway,inflammatory signaling pathway,AGE-RAGE signaling pathway,insulin signaling pathway,cholinergic signaling pathway,etc.The potential mechanism may be related to autophagy and inflammation.2.Yitangkang can significantly reduce the fasting blood glucose level of db/db mice,correct hyperinsulinemia in mice,improve insulin resistance and improve the memory and learning ability of db/db mice.In addition,Yitangkang can significantly improve the oxidative stress and inflammatory reaction in db/db mice,and has obvious protective effects on hippocampus and neuronal synapses in db/db mice.3.Yitangkang can significantly up-regulate the expression of Beclin-1 and LC3 protein and down-regulate the expression of p62 protein,suggesting that it can effectively improve the autophagy level in db/db mice;Yitangkang can significantly reduce the expression of IL-1β and IL-18 in NLRP3/ASC/Caspase-1 signaling pathway and its downstream inflammation,suggesting that it can effectively inhibit the activation of inflammatory corpuscles and the release of inflammatory factors caused by it.Yitangkang can significantly up-regulate AMPK protein expression and down-regulate mTOR protein expression,suggesting that it can effectively intervene AMPK/mTOR expression.4.Yitangkang can protect HT22 neurons induced by high glucose.The potential mechanism of Yitangkang’s neuroprotection may be achieved by activating AMPK/mTOR signaling pathway and then regulating autophagy-mediated activation of NLRP3/ASC/Caspase-1 inflammatory body pathway.Conclusion:1.The research of network pharmacology shows that Yitangkang,a traditional Chinese medicine compound,has the characteristics of multi-components,multi-targets and multi-channels,which provides a solid basis for its application in the prevention and treatment of DCI.2.Yitangkang can improve the blood sugar level of db/db mice,reduce the oxidative stress and inflammatory reaction,reduce the degree of cognitive impairment,and delay the related pathological changes in hippocampus.3.Yitangkang,a traditional Chinese medicine compound,may play an anti-DCI role by regulating AMPK/mTOR signaling pathway,autophagy and NLRP3/ASC/Caspase-1 inflammatory body pathway.4.Yitangkang may protect HT22 neurons induced by high glucose by regulating AMPK/mTOR signaling pathway and regulating autophagy-mediated activation of NLRP3/ASC/Caspase-1 inflammatory corpuscles.。... |
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