Study On The Effects And Mechanism Of Asiatic Acid Promoting Positive Vascular Remodeling Of Aortic Dissection By Regulating Macrophage Polarization

Background:Aortic dissection(AD)is one of the most serious and dangerous vascular diseases,which can cause aortic rupture and lead to death in a short time,with poor prognosis.Thoracic endovascular aortic repair(TEVAR)promotes false lumen thrombosis by isolating the proximal intimal entry tear,stabilizing endothelial cells,vascular smooth muscle cells(VSMC)and extracellular matrix in the aortic wall,and promoting the positive aortic remodeling.However,there are still some adverse events,such as proximal retrograde progression,distal dilatation,and recurrent rupture in some patients after operation,which seriously affect the prognosis of the patients.Studies have shown that the inflammation is an important factor in mediating negative vascular remodeling for AD,which is closely related to aortic adverse events.As the inflammatory cells with the highest proportion of infiltration in the pathological aortic wall,macrophages play an important role in the aortic remodeling.There are two polarization phenotypes of macrophages: M1 phenotypic macrophages could promote inflammatory response and aggravate the destruction of diseased tissue;on the contrary,M2 phenotypic macrophages could inhibit inflammatory response,stabilize aortic wall structure,and promote local tissue repair.Therefore,the search for potential drugs that could effectively regulate the polarization of macrophages to M2 phenotype is of great significance to prevent the occurrence and progression of dissection.Asiatic acid(AA)is a pentacyclic triterpene organic compound,which has many biological activities,such as anti-inflammation,anti-oxidant stress,anti-apoptotic,immune regulation and so on,especially in improving the progression of cardiovascular diseases.However,there is a lack of reports on the biological effects and molecular mechanism of AA in the occurrence,development,and prognosis of AD.Therefore,the purpose of this study is to explore the molecular mechanism of AA promoting positive aortic remodeling in mouse AD model by regulating macrophage polarization,provide new interventional targets for AD therapy,and provide theoretical and scientific basis for the treatment of AD patients with AA in the future.Objective:Part one: to explore whether AA could regulate the polarization of macrophages and then affect the biological effect of VSMC phenotypic transformation.Part two: to explore whether AA could alleviate negative aortic remodeling in mouse AD model.Part three: to explore and verify the molecular mechanism of AA in promoting positive aortic remodeling in AD.Method:Part one: 100ng/m L lipopolysaccharide(LPS)used was to stimulate mouse RAW264.7 cells and induce its polarization to M1 pro-inflammatory phenotype.The main results were as follows:(1)different doses of AA were intervened to explore the biological effects of AA on LPS-induced polarization of M1 pro-inflammatory phenotype.(2)the mouse RAW264.7 cells and VSMC coculture system in vitro was established to explore the effect of AA on affecting the phenotypic transformation of VSMC by regulating macrophage polarization.Part two: 3-week-old C57BL/6J male mice were fed with 0.25%β-aminopropionitrile(BAPN)for 4W to establish AD model,during which normal saline(Saline),low dose(10mg/kg/d)and high dose AA(30mg/kg/d)were treated by oral gavage daily:(1)to observe the effects of AA on negative aortic remodeling such as dilation and rupture of aorta,inflammatory cell infiltration,extracellular matrix degradation and the survival rate of mice in mouse AD model.(2)the levels of inflammatory cytokines and related proteins were detected by serum ELISA and Western blot(WB)to explore the effect of AA on macrophage polarization and the secretion of systemic and local inflammatory factors in mouse AD model.Part three: the aortic tissues of mice in AD model group and AD model plus high dose AA intervention group were separated and sequenced.The main results are as follows:(1)the differentially expressed genes and signal pathways of AA promoting forward aortic remodeling in mouse AD model were screened by bioinformatics analysis,and verified at gene and protein level.(2)the mouse RAW264.7 cells and VSMC coculture system was constructed in vitro,and lentivirus was transfected into RAW264.7cells to explore the effect and mechanism of AA on VSMC phenotypic transformation by regulating macrophage polarization.(3)the molecular pathway of AA regulating macrophage polarization was explored by ELISA-based transcription factor assay and WB experiment,and verified by WB.Result:Part one: in the cell experiment,(1)through q-PCR,WB,and immunofluorescence techniques,we found that AA increased the expression of specific marker gene and protein arginase-1(Arg-1)and CD206 in M2 phenotypic macrophages,while decreased the expression of specific marker gene and protein inducible nitric oxide synthase(i NOS)in M1 phenotypic macrophages.(2)through the analysis of ELISA results in the supernatant,we found that AA could reduce the secretion of pro-inflammatory inflammatory factors and increase the secretion of anti-inflammatory inflammatory factors.(3)through the establishment of coculture model,we found that AA could promote the polarization of macrophages to M2 phenotype and then transform VSMC to contractile phenotype,that is,the proportion of contractile VSMC increased.Part two: in animal experiments,(1)through the analysis of the results of regular ultrasound in mice,we found that AA could delay the dilatation and rupture of blood vessels in mouse AD model induced by BAPN,and then improve the survival rate of mice.(2)through the analysis of histopathology(HE staining and EVG staining),we found that AA reduced inflammatory cell infiltration and elastic fiber breakage.(3)through the analysis of serological ELISA and WB results,we found that AA could promote the polarization of RAW264.7 cells to M2 phenotype in BAPN-induced mouse AD model,and reduce the systemic and local wall inflammation.(4)through histopathology,we found that AA had no organ damage in BAPN-induced AD model in mice.Part three: through m RNA sequencing analysis,we found that(1)AA could significantly reduce the expression of CX3C-chemokine ligand 1(CX3CL1)in aortic tissue,KEGG is enriched in the intercellular inflammatory signal pathway,and studies have shown that there is a corresponding receptor CX3CR1 on the membrane of VSMC that regulates the phenotypic transformation of cells,suggesting that AA may affect the phenotypic transformation of VSMC by inhibiting the secretion of CX3CL1 by macrophages.(2)the mouse RAW264.7 cells and VSMC coculture system was constructed,and different doses of AA were used to interfere with M1 phenotypic macrophages induced by LPS.Through the detection of gene and protein levels,we found that AA could reduce the expression of CX3CL1 gene and inhibit the activation of ERK signal pathway.Furthermore,RAW264.7 cells were infected with lentivirus overexpressing CX3CL1.The phenotypic transformation of VSMC was detected by immunofluorescence and WB technique.The results showed that the proportion of contractile VSMC decreased,while synthetic formation increased.(3)some literature studies have found that AA can exert its anti-inflammatory effect by inhibiting Toll-like receptor-4(TLR4)/Nuclear factor kappa-B(NF-κB)p65 signal pathway.We used AA to interfere with LPS-induced RAW264.7 cells,and through ELISA-based transcription factor assay,we found that AA could inhibit the phosphorylation level of p65 signal pathway.Furthermore,when RAW264.7 cells were infected with lentivirus overexpressing p65,it was found that the expression of CX3CL1 protein increased and the expression of specific marker protein i NOS in M1 phenotypic macrophages increased.Finally,we detected the protein phosphorylation level of p65 in cytoplasm and nucleus of LPS-induced RAW264.7 cells by WB.It was found that AA could promote M2 polarization of macrophages and reduce the expression of CX3CL1 by inhibiting p65 nuclear translocation.Conclusions:Based on the three experimental results above,we concluded that(1)AA could promote the polarization of macrophages to M2 phenotype and improve the phenotypic transformation of vascular smooth muscle cells by regulating macrophage polarization;(2)AA could delay the negative remodeling of vascular wall in mouse aortic dissection model and improve the survival rate of mice.(3)AA could regulate macrophage polarization and promote the positive aortic remodeling of AD by inhibiting NF-κB p65/CX3CL1 signal pathway,which is expected to provide new ideas and therapeutic targets for AD.