| Objective1.To explore the understanding of the etiology and pathogenesis of atherosclerosis(AS)in Traditional Chinese Medicine,the connotation of the theory of "spleen-ying-vessel correlation" and its association with the pathogenesis of AS.2.To clarify the effectiveness of Jian-Pi-Tong-Mai formula(JPTMF)in the prevention and treatment of AS,reveal its mechanism of regulating the phenotypic transformation of vascular smooth muscle cells(VSMCs)to inhibit the occurrence and development of AS,and verify the scientificity and practicability of the theory of "spleen-ying-vessel correlation",and provide theoretical and experimental basis for clinical treatment of atherosclerotic related diseases from the spleen.Methods By combining theoretical research with experimental research,theoretical research is used to guide and design experiments,and experimental research is used to verify and support the theory.1.Theoretical study:Analysis of etiology and pathogenesis of AS in traditional Chinese medicine by consulting and summarizing ancient and modern literature;the etiology and pathogenesis of AS were explored from the perspective of modern medicine,and then the two were combined.The phenotypic transformation of VSMC in AS was taken as the breakthrough point to clarify the theory of "spleen-ying-vessel correlation".The internal relationship between this theory and VSMC,and the rationality of this theory in the treatment of early AS were discussed.Under the guidance of this theory,the preventive measures and therapeutic principles of early AS were discussed.2.Experimental study:ApoE-/-mice were fed with high-fat diet to establish AS model to verify the mechanism of JPTMF in preventing and treating AS.On this basis,the main effective components of JPTMF-containing serum were detected by liquid chromatography-mass spectrometry(LC-MS).The effects of JPTMF on the proliferation,migration,invasion and phenotypic transformation of VSMC were verified in vitro by MTT,scratch repair,Transwell,phalloidin,RT-PCR,Western-Blot and immunofluorescence,and the molecular mechanism and key targets of JPTMF inhibiting the phenotypic transformation of VSMC intervening AS were further explored by lentivirus transfection technology.Results1.Based on the theory of "spleen-ying-vessel correlation" the evolution of Traditional Chinese Medicine pathogenesis of AS was partially clarified,VSMC was found to be an important physiological basis of the theory,and it was confirmed that the treatment from the spleen is particularly important in the early stage of AS.2.The results of small animal ultrasound showed that JPTMF improved the formation of aortic plaque in AS mice.RT-PCR and Western-Blot results showed that JPTMF inhibited the expression of IKKα,IKKβ,p-IKK,p-IκBα and p-p65 related indicators of IKK/IKBα/NF-κB signaling pathway.3.MTT,wound healing and Transwell experiments showed that serum containing JPTMF could inhibit the proliferation,migration and invasion of VSMC in vitro.philloidin experiment results showed that JPTMF medicated serum can improve the skeleton morphology of VSMC in vitro.RT-PCR and Western-Blot results showed that serum containing JPTMF could inhibit the expression of PCNA,MMP-2,MMP-9 and OPN in vitro,and up-regulate the expression of PPARγ,SM22α,α-SM-actin and SM-MHC.4.Western-Blot results showed that serum containing JPTMF inhibited the expression of IKK/IKBα/NF-κB signaling pathway related proteins IKKα,IKKβ,p-IKK,p-κBα and p-p65 in vitro.The results of Western-Blot and immunofluorescence showed that serum containing JPTMF inhibited the nuclear translocation of NF-κB p65 in vitro.5.MTT,wound healing and Transwell results showed that BMS-345541 and PDTC could inhibit the proliferation,migration and invasion of VSMC in vitro.The results of philloidin experiment showed that BMS-345541 and PDTC could improve the skeleton morphology of VSMC in vitro.RT-PCR and Western-Blot results showed that BMS-345541 and PDTC could inhibit the expression of PCNA,MMP-2,MMP-9 and OPN in vitro,and up-regulate the expression of PPARy,SM22α,α-SM-actin and SM-MHC.Western-Blot results showed that BMS-345541 and PDTC could inhibit the expression of IKKα,IKKβ,p-IKK,p-IκBα and p-p65 related to IKK/IKBα/NF-κB signaling pathway in vitro.Western-Blot and immunofluorescence results showed that BMS-345541 and PDTC could inhibit the nuclear translocation of NF-κB p65 in vitro.6.MTT,wound healing and Transwell results showed that after silencing IKKα or IKKβ,the effect of JPTMF on inhibiting the proliferation,migration and invasion of VSMC in vitro decreased.The results of philloidin experiment showed that after silencing IKKα or IKKβ,the effect of JPTMF on improving the cytoskeleton morphology of VSMC in vitro decreased.RT-PCR and Western-Blot results showed that after silencing IKKα or IKKβ,JPTMF inhibited the expression of PCNA,MMP-2,MMP-9 and OPN in vitro,and up-regulated the expression of PPARγ,SM22α,α-SM-actin and SM-MHC.The results of Western-Blot experiment showed that after silencing IKKα or IKKβ,the effect of JPTMF on inhibiting the expression of IKKα,IKKβ,p-IKK,p-IκBα and p-p65 related proteins in IKK/IKBα/NF-κB signaling pathway in vitro decreased.The results of Western-blot and immunofluorescence showed that after silencing IKKα or IKKβ,the inhibitory effect of JPTMF on NF-κB P65 nuclear translocation decreased in vitro.Conclusion1.Based on the theory that the spleen and pulse are tongqi at nutrient qi and defensive qi,combined with the study of the characteristics of AS,the theory of "spleen-ying-vessel correlation" was put forward.The key pathogenesis of AS is to clarify that spleen qi deficiency,ying blood deficiency and adverse pulse channel are the key pathogenesis,and invigorating spleen and replenishing qi and removing blood stasis and dredging pulse are the important therapeutic principles and methods for early prevention and treatment of AS.2.JPTMF can improve aortic plaque formation in AS mice and interfere with AS progression by inhibiting IKK/IKBα/NF-κB signaling pathway.3.JPTMF can inhibit the proliferation,migration and invasion of VSMC by mediating IKK/IKBα/NF-κB signaling pathway,and regulate the phenotypic transformation of VSMC,thus playing an effective role in the intervention of AS.IKK may be one of the key targets of JPTMF in its anti-AS effect. |
PDF Full Text Request