Protective Effect Of Prosulinin A On C5b-9-mediated Podocyte Injury And Its Proteomics

OBJECTIVE Complement system-mediated cell damage is one of the important mechanisms of glomerular disease development.Caesalpinia sappan L has many functions such as anti-oxidation,anti-tumor,anti-inflammatory,anti-bacterial and immunosuppression.Proteomics is a omics technology developed after tandem mass spectrometry after genomics.Our previous study found that rosulin A has protective effects on C5b-9-mediated podocyte injury,but its molecular mechanism is still unclear.In this study,we constructed a C5b-9 attack podocyte injury in vitro model,evaluated the protective effect and underline mechanism of prosulinin A on C5b-9-mediated podocyte injury based on proteomics platform.METHODS The C5b-9 membrane attack complex podocyte injury model was constructed.The effects of prostaglandin A on podocyte proliferation were detected by CCK-8 method.The original hematoxylin A was used to detect podocyte marker protein by immunofluorescence assay.The effect of Nephrin expression and cell localization,flow detection of the effect of prosulinin A on podocyte apoptosis,iTRAQ proteomics method to detect proteomic changes after prostaglandin A,screening differentially expressed proteins,and performing GO functional enrichment analysis and KEGG Pathway enrichment analysis,and PPI interaction network analysis of differentially expressed proteins revealed the potential molecular mechanism of the protective effect of prosulinin A on podocyte injury,and the expression of related differential proteins was confirmed by Western blotting assay.RESULTS(1)Proliferation of podocytes in the 20,40,80,and 160 ?mol/L pro hematoxylin treated groups was obvious in 0-48 h,while the proliferation of podocytes in the C5b-9 group and 10 ?mol/L pro-hematoxylin group was inhibited,and the concentration was80 ?mol/ When L,the podocyte proliferation is best;(2)Under the C5b-9 attack,the podocyte density decreased and the expression of nephrin decreased significantly.After the original hematoxylin A,the expression of nephrin was significantly higher than that of the model group.(3)The apoptosis rate of the blank group was the lowest,and the apoptosis rate of the model group was the highest.Compared with the model group,the apoptosis rate of podocytes in the 20,40 and 80?mol/L groups was significantly lower than that in the model group(P<0.05).(4)A total of 3367 proteins were identified in the iTRAQ proteomics,and134 differentially expressed differential proteins and 185 up-regulated differential proteins were screened for a total of 319 differential proteins;(5)GO functional enrichment analysisresults,biological processes are mainly enriched in transcription,ribosomal RNA processes,cytoplasmic translation,ribosomal small subunit biosynthesis,ribosomal large subunit biosynthesis,apoptosis Negative regulation,cell proliferation,etc.;cell composition is mainly concentrated in ribosomes,ribosomal subunits,focal adhesions,extracellular bodies,nucleus,cytoplasm and extracellular matrix;molecular functions are mainly enriched in ribosome structure,RNA binding,cadherin binds to cell-cell adhesion,protein complex binding,protein kinase binding,and actin binding;(6)Pathways enriched by differential proteins include: Ribosome,Focal adhesion,ECM-receptor interaction,Metabolic pathways,Oxidative Related signaling pathways such as phosphorylation,Complement and coagulation cascades,and Fatty acid metabolism;(7)PPI network map results show that the protective effect of prosulinin A on podocyte injury may involve biological processes such as ribosome function,cell proliferation and cell cycle regulation,complement pathway and podocyte protein;(8)Compared with the blank control group,the expression levels of Cav1,Nestin and Carm1 were significantly down-regulated in the C5b-9 model group,and the expression levels of Bax were significantly up-regulated.Compared with the model group,the original hematoxylin A group Cav1,Nestin and Carm1 Protein expression levels were significantly up-regulated,and Bax protein expression levels were significantly down-regulated,consistent with the results of the iTRAQ proteome assay.CONCLUSION(1)The original hematoxylin A can promote podocyte proliferation,inhibit podocyte apoptosis,up-regulate the expression level of nephrin protein in podocytes,and have significant protective effect on C5b-9-mediated podocyte injury;(2)protosumin A on foot The protective effects of cells may involve ribosome function,cell proliferation,regulation of cycle and apoptosis,biological processes such as the complement pathway,and functions of podocyte-associated proteins.(3)The original hematoxylin A can up-regulate the expression of Cav1,Nestin and Carm1 protein in podocytes,and down-regulate the expression of Bax protein,which may be related to the inhibition of podocyte apoptosis.The specific molecular mechanism needs further investigation.