Mechanism Of Panax Notoginseng Saponins On Macrophage M1 Polarization Based On Notch Pathway

Objective: To revel the molecular mechanism of Panax notoginseng Saponins(PNS)to promote early healing of Diabetic foot ulcers(DFU)through inhibition of hyperglycemia induced M1 polarization of RAW264.7 macrophages in mice by PNS.Methods: This experiment is divided into the three parts.Part Ⅰ: Prediction of relevant targets for PNS by Swiss Target Prediction and Pharm Mapper databases;prediction of relevant targets for DFU by Gene Cards,Drug Bank and Dis Ge NET databases.PPI network construction and core target screening of common targets were performed using Cytoscape 3.9.1 software and STRING database,and GO function and KEGG pathway enrichment analysis were performed using DAVID database.Part Ⅱ: 1.NO(Nitric Oxide)content was determined by NO kit to determine the high sugar concentration of mouse RAW264.7 macrophages polarized to M1;2.Different concentrations of PNS(37.5 μg/m L,75 μg/m L,150 μg/m L,300 μg/m L,600 μ g/m L)on the proliferation of RAW264.7 macrophages in mice;3.In vitro models of cellular inflammation induced by high sugar in mice RAW264.7 macrophages with M1 polarization were established and divided into control group,high sugar group and high sugar + PNS.The optimal concentration of PNS was screened by CCK-8 assay.4.Protein immunoblotting(Western Blot,WB)was used to detect the expression of Cleaved-Notch1 and Hes1 proteins;5.Real-time fluorescence quantitative PCR assay was performed to detect the expression of Notch1,TNF-α,IL-6 and IL-1β mRNA;6.Immunofluorescence assay to detect Cleaved-Notch1 nuclear translocation.Part Ⅲ: 1.The effects of different concentrations of Notch pathway activator Jagged-1(10 μM,20 μM,30 μM,40 μM,50 μM,100 μM)on the proliferation of mouse RAW264.7macrophages were examined by CCK-8 assay;2.In vitro cellular inflammation was established in a high sugar-induced M1 polarization of mouse RAW264.7 macrophage The model was divided into control group,high glucose group,high glucose+PNS group,and high glucose+PNS+Jagged-1 group,and the optimal concentration of Jagged-1 was screened by CCK-8 assay.3.WB was used to detect the expression of Cleaved-Notch1 and Hes1 protein;4.Real-time fluorescence quantitative PCR assay was performed to detect the expression of Notch1,TNF-α,IL-6 and IL-1β mRNA;5.Immunofluorescence assay was performed to detect Cleaved-Notch1 nuclear translocation.Results:Part Ⅰ:The results showed that 260 potential targets of PNS,1307 disease targets,and88 common targets were screened out.According to the PPInetwork,the core targets were GAPDH、PPARG、ADRB2、MTOR,etc.The common targets were mainly involved in the GO terms,such as negative regulation of the protein modification process,DNA repair,and other biological process.KEGG showed that PNS was correlated with the Notch signaling pathway significantly.Part Ⅱ:1.The 33.3 m M glucose induced RAW264.7 cells polarized to the M1 subtype.2.Compared with the control group,PNS at 37.5-300 μg/m L did not significantly affect the growth rates of RAW264.7 cells(p > 0.05),however PNS at 600 μg/m L could inhibit RAW264.7 cells proliferation effectively(p < 0.001).3.Compared with the 33.3 m M group,PNS at 150 μg/m L promited the activity of RAW264.7 cells(p<0.001),and the survival rate of RAW264.7 cells was(76.6±2.3)%.The final concentration of PNS was 150 μg/m L.4.Compered with the control group,the 33.3 m M glucose group significantly increased the protein expression of Cleaved Notch1 and Hes1(P<0.01 或 P<0.001);Compered with the 33.3 m M glucose group,33.3 m M glucose+PNS group significantly decreased the protein expression of Cleaved Notch1 and Hes1(P<0.01).5.Compered with the control group,the 33.3 m M glucose group significantly increased the mRNA expression of IL-6、TNF-α、IL-1β and Notch1(P<0.001);Compered with the 33.3 m M glucose group,33.3 m M glucose+PNS group significantly decreased the mRNA expression of IL-6、TNF-α、IL-1β and Notch1(P<0.001).6.Compered with the control group,33.3 m M glucose group Cleaved-Notch1 protein migrated from cytoplasm to nucleus;Compered with the 33.3 m M glucose group,33.3 m M glucose+PNS group Cleaved-Notch1 protein migrated from nucleus to cytoplasm.Part Ⅲ:1.Compared with the control group,Jagged-1 at 10-30 μM did not significantly affect the growth rates of RAW264.7 cells(p>0.05),however Jagged-1 at 40-100 μM could inhibit RAW264.7 cells proliferation effectively(p < 0.01 or p < 0.001).2.Compared with the 33.3 m M glucose+PNS group,Jagged-1 at 10 μM promited the activity of RAW264.7 cells(p < 0.001),the survival rate of RAW264.7 cells was(53.8±6.7)%.The final concentration of Jagged-1 was 10 μM.3.Compered with the 33.3 m M glucose+PNS group,33.3 m M glucose+PNS+Jagged-1 group increased the protein expression of Cleaved Notch1 and Hes1(P<0.05).4.Compered with the 33.3 m M glucose+PNS group,33.3 m M glucose+PNS+Jagged-1 group significantly increased the mRNA expression of IL-6 、TNF-α、IL-1β and Notch1(P<0.001 or P<0.01).5.Compered with the 33.3 m M glucose+PNS group,the 33.3 m M glucose+PNS+Jagged-1 group Cleaved-Notch1 protein migrated from the cytoplasm to the nucleusConclusion: PNS plays a role in promoting DFU wound repair by inhibiting M1-type polarization of macrophages and reducing the expression levels of Notch1 mRNA,Cleaved-Notch1,and Hes1 proteins in macrophages,thereby inhibiting Notch pathway activation and reducing inflammatory damage.