| Purpose:To investigate the potential mechanism of action of the Chinese medicine compound Yixiangkang,including the drug pair "Astragalus-Red Ginseng",in the prevention and treatment of diabetic cerebral infarction,and to predict the potential targets,pathways and modes of action by applying the network pharmacology method and molecular docking technology to combine the compounds with the core targets.Further experiments were conducted to verify that the neuroprotective effect of the Chinese herbal formula Yixiangkang on diabetic cerebral infarction rats may be related to its promotion of microglia conversion from M1 to M2 type and alleviation of their neuroinflammatory response.It provides a new reference and direction for clinical treatment as well as future experimental studies.Methods:Network pharmacology sectionWe collected compounds and active targets of Astragalus-Red Ginseng pair by TCMSP pharmacology analysis platform;collected diabetes and cerebral infarction related targets from four common disease databases,TTD,OMIM,DRUGBANK and Dis Ge NET,took the intersection of the three and plotted the Venn diagram;used Cytoscape software to construct drug-active ingredient-key target We used Cytoscape software to construct the drug-active ingredient-key target network;obtained the intersection target protein interaction(PPI)network from String database;performed gene function GO analysis and KEGG pathway enrichment analysis;used Py MOL and Auto Dock to perform molecular docking.Animal experiment partExperimental validation of the Model of Middle cerebral artery occlusion(MCAO)in rats with type 2 diabetes and cerebral ischemia,observation of neurological function,behavioral tests,observation and calculation of cerebral infarct volume changes and brain water content in rats,Western blot method to detect The expression levels of i NOS,TNF-?,ARG-1,TGF-? and the expression of Iba1,CD206,CD86 by immunofluorescence method were detected.Results:Network pharmacology section1.24 active compounds were obtained,215 drug targets,a total of 2633 targets for type 2diabetes disease,823 targets for cerebral infarction disease,and 64 intersecting targets.2.The key targets of PPI network include IL6,AKT1,VEGFA,CCL2,TP53,etc.3.GO functional analysis is mainly related to the response of small molecules(including lipopolysaccharide,molecules of bacterial origin,metal ions,etc.),regulation of response to external stimuli(including biological stimuli,nutrient levels,oxygen levels,external stimuli,oxidative stress,drugs,etc.),regulation of apoptotic signaling pathways,development of reproductive structures,development of reproductive system,regulation of reactive oxygen species metabolism,leukocyte migration,etc.4.KEGG pathway enrichment analysis mainly focused on diabetes complications AGE-RAGE signaling pathway and Fluid shear stress and etherosclerosis signaling pathway,inflammation-related signaling pathway(including TNF,IL-17,HIF-1 signaling pathway,etc.),pertussis,kallikrein,etc.Pertussis,Kaposi's sarcoma-associated herpesvirus infection,Chagas disease,EGFR tyrosine kinase inhibitor resistance,proteoglycans in cancer,human cytomegalovirus infection,and endocrine resistance were also ranked high.5.Molecular docking results showed that the core compound components interacted with the core targets mainly with amino acid residues such as methionine at 522,serine at 49 and548,histidine at 39,asparagine at 382,and threonine at 206 by hydrogen bonding,hydrophobic,and van der Waals forces,with binding energies below-5.6 kcal/mol.Animal experiment part1.The Chinese medicine compound Yixiangkang can improve the neurological deficits in diabetic cerebral infarct rats.2.The Chinese medicine compound Yixiangkang can reduce the volume of brain infarction in diabetic cerebral infarction rats.3.The Chinese herbal formula Yixiangkang can reduce the brain water content of diabetic cerebral infarct rats.4.By observing the expression levels of M1 and M2 polarization markers i NOS,TNF-?,ARG-1 and TGF-? in microglia of three groups of rats,compared with the control group,the expression of M1 polarization markers i NOS,TNF-?,M2 polarization markers ARG-1 and TGF-? in microglia of the diabetic + MCAO model group was increased after surgery;compared with the diabetic +MCAO model group,the expression of M1-type polarization markers i NOS and TNF-?,M2-type polarization markers ARG-1 and TGF-? were decreased in microglia in the diabetes+MCAO model+Chinese medicine group,and the expression of M2-type polarization markers ARG-1 and TGF-? were increased in the diabetes+MCAO model+Chinese medicine group.5.Through the observation of immunofluorescence staining results of M1 type and M2 type polarization markers of microglia in the three groups,compared with the control group,the expression of Iba1 and CD86 increased and CD206 decreased in the diabetes+MCAO model group;compared with the diabetes+MCAO model group,the expression of Iba1 and CD86 decreased and CD206 decreased in the diabetes+MCAO model+Chinese medicine group.increased.Conclusion:1.Through network pharmacological analysis,the core targets of "Astragalus-Red Ginseng" drug in the Chinese medicine compound Yixiangkang with preventive and curative effects on diabetic cerebral infarction are PTGS2,PTGS1,F2,NOS2 and DPP4.2.According to the network pharmacological analysis,the biological functions of "Astragalus-Red Ginseng" that exert protective functions against diabetic cerebral infarction mainly involve responses to small molecules(including lipopolysaccharide,molecules of bacterial origin,metal ions,etc.),regulation of responses to external stimuli(including biological stimuli,nutrient levels,oxygen levels,external stimuli,oxidative stress,drugs,etc.),and regulation of responses to external stimuli.The KEGG pathway is used to regulate apoptotic signaling pathways,development of reproductive structures,development of the reproductive system,regulation of reactive oxygen species metabolism,leukocyte migration,etc.The results showed that AGE-RAGE signaling pathway was significantly associated with Fluid shear stress and etherosclerosis signaling pathway.The results showed that the significance and enrichment number of AGE-RAGE signaling pathway and fluid shear stress and etherosclerosis signaling pathway were high,and most of the genes in the key targets were enriched in this pathway,indicating that these two signaling pathways are most likely the key signaling pathways of "Astragalus-Red Ginseng" for the treatment of diabetic cerebral infarction.4.The Chinese medicine compound Yixiangkang can improve the neurological function and behavior of rats,reduce the volume of cerebral infarction and the water content of brain tissue.5.The traditional Chinese medicine compound Yitangkang has neuroprotective effects on rats with diabetic cerebral infarction.Its mechanism may be through inhibiting the activation of microglia,inhibiting the polarization of microglia to M1 type,and promoting the polarization to M2 to alleviate the neuroinflammatory response.. |
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