Effects Of Corilagin Against Atherosclerosis And Its Mechanisms:Based On CD40-CD40L/TRAF-2/NF-κB Signaling Pathway

Background:It is of great significance to find new anti-atherosclerosis drugs from natural medicinal plants for the treatment of atherosclerosis.The previous studies showed that Corilagin,an active ingredient from Yunnan characteristic medicinal plant phyllanthus,has a potential anti-atherosclerosis effect and deserves further development.In this study,we intend to take minipigs as animal experimental model and ox-LDL induced macrophages as cell experimental model to study the anti-atherosclerotic effect of Corilagin,explore which key targets Corilagin intervenes and their relationship using the method of network pharmacology,and explore the effect of Corilagin on key factors of CD40-CD40L signaling pathway.The effects of Corilagin on atherosclerosis were systematically studied in vitro and in vivo,and the mechanism was explored.Objective:To study the effects of Corilagin on experimental atherosclerosis and explore the molecular mechanism.Methods:1 To study the anti-atherosclerotic effect of Corilagin in vivo1.1 Atherosclerosis model was established by feeding minipigs a high-fat diet combined with balloon injury.1.2 The level of TG,TC,LDL-C,HDL-C,ApoB,ApoE and Lp(a)in serum were determined.1.3 Oil red O staining,HE staining and Masson staining were used for pathological observation of plaques,and the degree of lipid deposition,collagen hyperplasia and severity of lesions were evaluated.1.4 The expression of MMP-1,-2,and-9 in plaques was detected by immunohistochemistry.1.5 The expression of TNF-α,NF-κB,MMP-1,-2,and-9 in plaques was detected by western blotting and RT-qPCR.2 To study the effect of Corilagin on macrophage proliferation and foaming in vitro2.1 Murine RAW264.7 macrophages were cultured and induced with ox-LDL.2.2 Effects of Corilagin on macrophage proliferation were detected by MTT method.2.3 The degree of macrophage foaming was evaluated after oil red O staining.2.4 Lipid level in macrophages was measured by ELISA.2.5 The degree of NF-κB nuclear translocation was detected by immunohistochemistry.2.6 The expression of TNF-α,NF-κB,MMP-1,-2,and-9 in macrophages was detected by western blotting and RT-qPCR.3 Network pharmacology research3.1 The potential targets of Corilagin were predicted by Swiss and PharmMapper database,and atherosclerosis-related genes were screened by Disgenet and GeneCards database.3.2 Cytoscape 3.9.0 software was used to establish and construct the component-target-disease network visualization network.3.3 The protein interaction network was constructed using String data platform,and the GO and KEGG pathways were analyzed using David database.4 Effect of Corilagin on CD40-CD40L/TRAF-2/NF-κB signaling pathway4.1 The expression of CD40,CD40L,TRAF-2 and NF-κB proteins in plaques was detected by western blotting.4.2 The degree of macrophage foaming was evaluated after oil red O staining.4.3 The expression of CD40 and CD40L in macrophages was detected by immunofluorescence.4.4 The expression of CD40,CD40L,TRAF-2 and NF-κB in macrophages was detected by western blotting and RT-qPCR.Results:1 Corilagin has obvious therapeutic effect on experimental atherosclerosis in minipigs1.1 Corilagin can significantly reduce the serum level of TG,TC,LDL-C,ApoB,Lp(a),and increase the level of ApoE and HDL-C.1.2 The results of oil-red O staining,HE staining and Masson staining showed that Corilagin could significantly reduce the lipid deposition of plaques,alleviate the lesion degree,and inhibit the collagen hyperplasia.1.3 Immunohistochemical staining results showed that Corilagin could significantly down-regulate the expression of MMP-1,-2 and-9 in plaques.1.4 Western blotting and RT-qPCR results showed that Corilagin could significantly down-regulate the protein and mRNA expression of TNF-α,NF-κB,MMP-1,-2 and-9 in plaques.2 Corilagin can inhibit proliferation and foaming of macrophages2.1 MTT assay showed that Corilagin could significantly inhibit the activity of macrophages.2.2 Oil red O staining results showed that Corilagin could significantly reduce the degree of macrophage foaming.2.3 The results of ELISA showed that Corilagin could significantly reduce TC and CE level in macrophages,and reduce CE/TC value.2.4 Immunohistochemical results showed that Corilagin could significantly inhibit the nuclear translocation of NF-κB in macrophages.2.5 Western blotting and RT-qPCR results showed that Corilagin could significantly down-regulate the protein and mRNA expression of TNF-α,NF-κB,MMP-1,-2 and-9 in macrophages.3 Network pharmacology prediction related molecular targets3.1 There are 116 core targets of Corilagin in the treatment of atherosclerosis,mainly including MMP-2 and MMP-9.3.2 Corilagin treats AS by regulating CD40-CD40L/TRAF-2/NF-κB signaling pathway and other biological processes involving cell proliferation,inflammatory response,oxidative stress and apoptosis through key targets.4 Effect of Corilagin on CD40-CD40L/TRAF-2/NF-κB signaling pathway4.1 Western blotting results showed that Corilagin could significantly down-regulate the expression of CD40,CD40L,TRAF-2 and NF-κB proteins in plaques.4.2 Oil red O staining results showed that Corilagin could significantly reduce the foaming degree of macrophages.4.3 Immunofluorescence results showed that Corilagin could significantly down-regulate the protein expression of CD40 and CD40L.4.4 Western blotting and RT-qPCR results showed that Corilagin could significantly down-regulate the protein and mRNA expression of CD40,CD40L,TRAF-2 and NF-κB in macrophages.Conclusion:Corilagin has obvious therapeutic effect on atherosclerosis.Its mechanism is closely related to down-regulating the expression of TNF-α,NF-κB,MMP-1,-2,and-9,inhibiting macrophage proliferation and foaming,and regulating CD40-CD40L/TRAF-2/NF-κB signaling pathway.