Experimental Study On The Active Components Of Xionggui Prescription Regulating The Polarized Phenotype Of Microglia

Background:The pathological mechanism after the onset of ischemic stroke involves oxidative stress,immune response,inflammatory response,mitochondrial energy metabolism,etc.A full understanding of the pathological link is conducive to clinical targeted diagnosis and treatment.High-throughput genomics,proteomics,and bioinformatics provide a large amount of gene expression data for the modern study of diseases,and also provide new research methods for researchers to discover disease genes,underlying mechanisms,molecular diagnosis,drug screening,and so on.Microglia cells are the permanent immune cells in the brain involved in the immune response and the phenotypic changes associated with the functional changes are often closely related to the prognosis of stroke.The phagocytosis embodied by the M1 phenotype can effectively remove necrotic cells and tissues,while the inflammatory cascade caused by the overactivation of the M1 phenotype can expand tissue damage.The M2 phenotype can effectively inhibit inflammation and promote tissue repair,but it is only briefly activated in the early stages of stroke.Therefore,it is important to control the balance of microglia phenotype in the treatment of ischemic stroke.Chuanxiong-Angelica is a common clinical drug pair for ischemic stroke.Chuanxiong-Angelica is a common clinical drug pair for ischemic stroke.The combination of Chuan-xiong and Dang-gui has the function of nourishing blood,promoting blood circulation,removing blood stasis and clearing collaterals.The team previously confirmed that its active components,ligustrazine and ferulic acid,can reduce cell apoptosis and promote the growth of neuronal axons.Therefore,whether its active ingredients can participate in the regulation of microglia phenotype and the specific mechanism behind it is worthy of further study.Objective:Bioinformatics data show that the differentially expressed genes were screened out and their related biological functions and molecular mechanisms were explored through the analysis of data sets related to Acute ischemic stroke(AIS)in the Gene Expression Omnibus(GEO)database.By using a network pharmacological approach,we screened and predicted the target genes of the active ingredients of Xiong-gui fang,and explored the biological function process that it may participate in and the potential molecular mechanism.Based on the team’s preliminary work,in vitro model was used to study the effect and mechanism of active ingredients of Xiong-gui fang on microglial phenotype changes.Methods:1.Through the GEO database retrieval data associated with ischemic stroke,screening of AIS data sets,using R language,DAVID online database,KEGG database,STRING,Cytoscape,database Coremine Medical online database for differences in gene screening,GO function enrichment,KEGG pathway enrichment,to explain the pathogenesis of ischemic stroke from the molecular level.2.Target collection and prediction of active ingredients TMP and FA of Xiongguifang were carried out through CTD and Genemania online platform,and the results were mapped to Web Gestalt online platform for GO analysis and KEGG analysis.Cytoscape 3.8.2 was used to construct an "active ingredient-target-pathway" network,so as to explore the possible mechanisms of TMP and FA in interfering with biological processes.3.LPS was used to induce BV2 cells to establish an in vitro model of inflammation,and the active ingredients TMP and FA of Xionggui Formula were used for drug intervention.They were divided into: normal control group,LPS group,LPS + drug group(FA: 25/50/100μM,TMP: 25 /50/100μM).CCK8 method toxicity test to screen the safe concentration of TMP and FA;cell morphology parameters(cell area to axon length ratio)to screen the effective concentration of TMP and FA;q RT-PCR method to detect the effect of TMP and FA on the microglia M1 markers(i NOS,CD86),M2 markers(Arg-1,CD206)mRNA;immunofluorescence staining to observe the changes in the protein levels of i NOS and CD206.4.q RT-PCR and Western Blot methods were used to detect the changes of TMP and FA on M1 type microglia NFAT5 mRNA and protein levels;immunofluorescence staining was used to observe the nuclear translocation of NFAT5 and NF-κB protein;q RT-PCR method to detect TMP and FA pairs The influence of M1 type microglial pro-inflammatory factors(TNF-α,IL-6,IL-1β)mRNA,ELISA method was used to detect the content of pro-inflammatory factors(TNF-α,IL-6)in BV2 culture supernatant.Results:1.Download the mouse data set GSE23160 from the GEO gene expression comprehensive database.This data set contains 8 control samples and 24 acute ischemic stroke mouse brains obtained within 24 hours after middle cerebral artery occlusion(MCAO).Tissue sample.In the brain tissue of mice in the acute phase,the expression of 37 genes increased,and the expression of 1 gene decreased.The result of GO enrichment of these 38 differential genes is 80,and the results indicate that they are mainly involved in immune-related processes,such as:inflammation,immune response,neutrophil chemotaxis,cell response to tumor necrosis factor,neutral Chemotaxis of granulocytes,etc.The result of KEGG enrichment is 20 pathways.The signal pathways include Toll-like receptor signaling pathway,TNF signaling pathway,NF-κB signaling pathway,MAPK signaling pathway,etc.,all of which are related to inflammation.2.There were 19 common targets of ligustrazine and ferulic acid collected and predicted from CTD database.The GO functional enrichment analysis showed that the active components of Xiong-guifang were mainly reflected in biological processes,including biological regulation,metabolic process,response to stimulation,multicellular biological process,cell communication,etc.The in-depth analysis of "response to stimulation" found that the active ingredients of Xiong-guifang were involved in regulating the biological processes related to hypoxia response and inflammatory stimulation.A total of 111 signaling pathways were obtained by KEGG enrichment analysis of potential targets.Among them,TNF signaling pathway,IL-17 signaling pathway,Toll-like receptor signaling pathway,T cell receptor signaling pathway,B cell receptor signaling pathway,NF-κB signaling pathway,TGF-βsignaling pathway and other important pathways are closely related to inflammation.3.After LPS stimulation,the cell membrane was observed to retract from a round or spindle shape to an "amoeba" like appearance,and the cell area was increased.At the same time,slender branches were observed,and the ratio of long axis to short axis was also increased,indicating that the microglia cells presented an activated morphology.Compared with model group,TMP(1-5)μm and FA(1-5)μm had no significant effect on the morphology of microglia after LPS stimulation.TMP(25-100)μm and FA(25-100)μm could restore the activated microglia to the "resting" state to some extent,and the ratio of cell area to axon length was improved.The quantitative results of q RT-PCR and immunofluorescence staining showed that the levels of i NOS and CD86 genes of M1 classical phenotype markers were up-regulated after the stimulation of BV2 microglia cells by LPS,which proved that LPS could induce the polarization of microglia cells into M1 phenotype.Meanwhile,the levels of M2 substitution phenotypic markers Arg-1 and CD206 were down-regulated.TMP and FA could dosedependentially decrease the mRNA levels of i NOS and CD86.However,TMP and FA had no significant effect on the mRNA levels of Arg-1 and CD206,indicating that TMP and FA could not promote the protein expression of M2-substituted phenotypic markers and achieve the phenotypic polarization of microglia from M1 to M2.4.The expression of NFAT5 gene and protein increased after LPS stimulation of BV2 microglia,same with the mRNA expression trend of M1 phenotypic marker.TMP and FA had no significant effects on the mRNA and total protein levels of NFAT5.Immunofluorescence results showed that LPS stimulation increased the nuclear translocation of NFAT5 and NF-κB.TMP and FA had no effect on the nuclear translocation of NFAT5.However,the two can inhibit the nuclear translocation of NF-κB.Conclusion:1.Analysis of the GSE23160 dataset showed that functional enrichment and pathway enrichment of differential genes in ischemic stroke were associated with inflammatory response.2.Target prediction,GO analysis and KEGG analysis of active ingredients TMP and FA of Xiongguigui Formula showed that TMP and FA could participate in the regulation of biological processes and pathways related to inflammation.3.TMP and FA can inhibit the expression of M1 phenotype,but cannot increase the M2 phenotype,nor can it complete the conversion of M1 to M2.4.Under the M1 phenotype of BV2 cells,the expression of NFAT5 mRNA and total protein increased,and TMP and FA had no effect on NFAT5 mRNA and total protein;nuclear translocation of NFAT5 and NF-κB increased,and TMP and FA could inhibit nuclear translocation of NF-κB,But it cannot inhibit the nuclear translocation of NFAT5;the expression and secretion of inflammatory factor mRNA increase,and TMP and FA can inhibit the expression and secretion of inflammatory factor mRNA.