| Coronary heart disease(CHD)is one of the common and frequently-occurring diseases of cardiovascular system,which seriously threatens human health.Coronary artery calcification(CAC)plays an important role in the development of CHD.CAC can change the mechanical properties of coronary artery,increase the risk of rupture of plaque and blood vessels and cause thrombosis,myocardial ischemia and infarction.Relevant studies have shown that calcification can occur in the early stage of atherosclerosis,and the severity of CAC is closely related to serious adverse cardiovascular events in patients with CHD such as acute myocardial infarction,sudden cardiac death and so on.Meanwhile,CAC is also a difficult disease to be treated by cardiovascular interventional therapy,which greatly increases the difficulty and risk of interventional operation.At present,effective prevention and treatment of CAC is a hot spot in cardiovascular disease research.Recent studies have shown that CAC may be an active regulatory process similar to bone ossification,including abnormal proliferation and migration of Vascular smooth muscle cells(VSMCs)and phenotypic transformation into osteogenic phenotypes.Epithelial-mesenchymal transitions(EMT)is a process in which epithelioid cells lose apical-basal polarity and intercellular adhesion and transform into aggressive stromal like cells.EMT plays an important role in the occurrence and development of a variety of diseases such as tumor,fibrosis,cardiovascular disease and so on by regulating phenotypic transformation and abnormal migration of cells.As the main member of the Transforming growth factor(TGF)family,TGF-?1 is considered to be one of the most important factors to induce EMT in organ tissue fibrosis,tumor and other pathological states.Related studies have shown that TGF-?1,as a key regulator of extracellular matrix deposition,can induce phenotypic conversion of VSMCs in high phosphorus environment.Apigenin(Api)is a kind of biological flavonoid compound extracted from celery,which has a variety of pharmacological activities.Current studies have shown that Api can play the role of scavenging oxygen free radicals,anti-tumor,anti-oxidation and anti-atherosclerosis by regulating a variety of signaling pathways and affecting the expression of effector molecules.However,its effect on coronary artery calcification has not been reported.On the basis of the existing literature textual research,in this study,we will use RNA knockdown and adenovirus mediated gene overexpression to clarify the mode of action and specific mechanism of TGF-?1 mediated EMT marker genes affecting cell calcification through regulating the migration of calcified VSMCs in ?-GP induced calcified VSMCs models.Moreover,the role of TGF-?1 and EMT marker genes in CAC will be studied at the clinical tissue level and animal model level.At the same time,this study will also explore whether Api can affect the migration of VSMCs by regulating TGF-?1 mediated EMT,and then delay the process of CAC.This study will further reveal the mechanism of action of Api against CAC,and provide theoretical basis and new ideas for targeted treatment of CAC with integrated traditional Chinese and western medicine.The specific content includes the following three chapters:Chapter ? Differential expression of TGF-?1 and EMT in CACObjective:To investigate the differential expression of TGF-?1 and EMT marker genes in calcification models of VSMCs,and to verify them by clinical specimens of CAC and CAC model rats.Methods:Firstly,we used CCK-8 kit to detect the effect of ?-GP concentration on the vitality of VSMCs,and determined the ?-GP working concentration to establish the cell calcification model and then treated VSMCs to construct the calcified model.Alizarin red staining was used to detect the cell calcification degree in calcified VSMCS model.Calcium content and alkaline phosphatase(ALP)activity were determined by calcium assay and ALP kit.The expression of calcification associated protein RUNX2 and smooth muscle marker protein ?-SMA in cells was tested by Western Blot.The above experiments were used to verify whether the cell calcification model was successfully constructed.On the basis of the successful construction of the cell calcification model,we used RNA-seq to conduct transcriptome detection and analysis of calcified VSMCs and normal VSMCs to identify the differentially expressed genes in calcified VSMCs.Secondly,through clinical case screening,we collected peripheral blood from hospitalized patients with CAC confirmed and CAC excluded by coronary angiography during 2018-2020.Expression of TGF-?1 and EMT marker genes N-cadherin(N-CAD),E-cadherin(E-CAD),?-catenin(?-CAT)and Vimentin(VIM)was detected by ELISA method,and the correlation between TGF-?1 and EMT marker genes expression was analyzed.Finally,the CAC rat model was established by intraperitoneal injection of vitamin D3 in rats.The calcification degree and TGF-?1 expression in the coronary artery tissues of CAC model rats were detected by alizarin red staining and immunohistochemistry.Total RNA and protein were extracted from calcified coronary artery tissues of CAC model rats.The mRNA expression level of EMT marker genes was detected by RT-qPCR assay,and protein expression level of RUNX2,?-SMA,TGF-?1 and EMT marker genes was detected by Western Blot assay.Results:CCK-8 cell viability assay showed that compared with normal control group(NC group),different concentration of ?-GP treatment of VSMCs had a dose-dependent effect on cell viability.And 15 mmol/l ?-GP group showed statistical difference compared with NC group,indicated that ?-GP at this concentration had cytotoxic effect on VSMCs.Therefore,10 mmol/l ?-GP was selected as the working concentration for cell calcification modeling.After ?-GP treatment of VSMCs for 10 days,Alizarin red staining showed that there were more orange calcium nodules and cytoplasmic red staining in the calcified VSMCs model group(?-GP group)compared with the NC group.Calcium detection results showed that the calcium content and ALP activity were significantly increased in ?-GP group compared with the NC group.Western Blot results showed that the expression of RUNX2 was significantly increased and the expression of ?-SMA was significantly decreased in ?-GP group compared with the NC group.The results indicated that the calcification model of VSMCs was successfully established.The results of RNA-seq assay and analysis showed that compared with the NC group,the expression of 1767 genes in ?-GP group was up-regulated and 1395 genes down-regulated.Differential genes expression analysis showed that the expression of TGF-?1 and EMT marker genes(E-CAD,N-CAD and ?-CAT)was altered in ?-GP group.The expression of TGF-?1 and EMT marker genes in peripheral blood of CAC patients and CAC patients excluded(the control group)by concurrent angiography was detected by ELISA.The results showed that the expression of TGF-?1,N-CAD in serum of CAC patients was increased,and the expression of E-CAD and ?-CAT was decreased compared with the control group.Gene correlation analysis showed that the expression of N-CAD and VIM in serum of CAC patients was significantly positively correlated with the expression of TGF-?1,while the expression of E-CAD and ?-CAT were significantly negatively correlated with the expression of TGF-?1.At the same time,we used vitamin D3 to establish CAC rat model.Alizarin red staining showed that compared with the control group rats(Ctrl group),the elasticity of the coronary artery tissue in the CAC model rats(VC group)was reduced,the media was disordered,and many red/orange granules appeared between the elastic fibers of the media.Western Blot results showed that compared with the Ctrl group,the expression of RUNX2 was increased and the expression of ?-SMA was decreased in the VC group.The results indicated that the CAC model was successfully constructed in rats.The CAC model rats were used as the research object,and the expression of TGF-?1 was detected by immunohistochemistry and Western Blot.The results showed that compared with the Ctrl group,the staining degree of TGF-?1 and the protein expression level of TGF-?1 were significantly increased in the VC group.The results of RT-qPCR showed that compared with the Ctrl group,the mRNA expression of N-CAD was increased and the mRNA expression of E-CAD was decreased in the VC group,while the expression of VIM and ?-CAT was not significantly different.The results of Western Blot showed that compared with the Ctrl group,the protein expression of N-CAD was increased and the protein expression of E-CAD and ?-CAT was decreased in the VC group,while the expression of VIM with not significantly different.Conclusion:TGF-?1 and EMT marker genes are differentially expressed in coronary artery calcification.TGF-?1 and EMT marker genes may be involved in the occurrence and development of coronary artery calcification.Chapter ? The mechanism of TGF-?1 mediated EMT marker genes affecting CACObjective:To establish VSMCs models with N-CAD knockdown and E-CAD overexpression,and to investigate the specific mechanism of TGF-?1 mediated EMT regulating coronary artery calcification.Methods:We verified the RNA-seq result by RT-qPCR and Western Blot.Transwell cell migration assay,cell scratch assay and immunofluorescence staining F-actin assay were used to detect the effect of cell calcification on the migration ability of VSMCs.RT-qPCR assay was used to detect the mRNA expression level of EMT marker genes.Western Blot assay was used to detect the protein expression level of TGF-?1 and EMT marker genes.The enrichment of N-CAD and E-CAD promoter sequences in calcified VSMCs by TGF-?1 antibody was detected by ChIP assay combined with qPCR.We constructed VSMCs models with N-CAD knockdown and E-CAD overexpression.After the VSMCs models successfully constructed,We used ?-GP to induce calcification and examined the effect of N-CAD knockdown and E-CAD overexpression on calcium content and ALP activity in calcified cell models.The effect of N-CAD and E-CAD on the migration ability of calcified cells was investigated by cell scratch assay and Transwell cell migration assay.Western Blot assay was used to detect the effect of N-CAD and E-CAD on the expression of calcification associated protein RUNX2 and smooth muscle marker protein ?-SMA in calcified cell model.Results:Transwell cell migration assay and cell scratch assay showed that compared with NC group,the number of cell migration and cell scratch healing rate of ?-GP group was significantly increased.Immunofluorescence staining of F-actin showed that ?-GP group had less tension fibers,loose arrangement between cells,abundant F-actin at cell margins,and more filamentous and lamellar pseudopods than NC group.The results of RT-qPCR and Western Blot showed that the mRNA and protein level of N-CAD was increased in?-GP group,while the mRNA and protein level of E-CAD and ?-CAT was decreased in?-GP group compared with NC group.We used anti-TGF-?1 antibody for ChIP which detected by ChIP-qPCR.The results showed that calcified VSMCs could increase enrichment of N-CAD promoter sequence and decrease enrichment of E-CAD promoter sequence.At the same time,?-GP calcification treatment was used to construct successful N-CAD knockdown and E-CAD overexpression cell models.Through calcium corresponding index detection,it was found that N-CAD knockdown and E-CAD overexpression significantly reduced the calcium contentand and ALP activity of calcified VSMCs.Western Blot analysis showed that N-CAD knockdown and E-CAD overexpression down-regulated the expression of RUNX2,and up-regulated the expression of ?-SMA in calcified VSMCs.Transwell cell migration assay and cell scratch assay showed that N-CAD knockdown and E-CAD overexpression could reduce the migration ability of calcified VSMCs,and partially reverse the promotion effects of calcified on the migration ability of VSMCs.Conclusion:TGF-?1 mediated EMT marker genes N-CAD,E-CAD and ?-CAT affect cell calcification by regulating the migration of VSMCs.Chapter ? Apigenin affects CAC by regulating TGF-?1 mediated EMT marker genes expressionObjective:To investigate whether Apigenin affects CAC by regulating TGF-?1 mediated EMT marker genes expression.Methods:Firstly,calcified VSMCs were treated with Apigenin(Api)at different concentrations.The effects of Api on VSMCs cell viability,proliferation,cycle and apoptosis were detected by CCK-8 cell viability assay,EdU cell proliferation assay and flow cytometry.At the same time,the effects of Api and N-CAD knockdown on calcification related indicators in calcified VSMCs were detected by the calcification related indicators detection experiments(alizarin red staining,calcium content and ALP activity,and the expression of RUNX2 and ?-SMA proteins),and the working concentration of Api was screened.Secondly,cell scratch assay and Transwell cell migration assay were used to detect and compare the effects of Api and N-CAD knockdown on the migration ability of calcified VSMCs.The effects of Api on TGF-?1 and EMT marker genes in calcified VSMCs were determined by RT-qPCR and Western Blot assay.Finally,CAC model rats were treated with Api and Atorvastatin(Ats)respectively.The coronary artery tissues of rats in each group were stained with Aligarin red.The expression of TGF-?1 was detected by immunohistochemistry,the expression of mRNA level of EMT marker genes was detected by RT-qPCR,and the expression of protein level of TGF-?1,RUNX2,?-SMA and EMT marker genes was detected by Western Blot.Results:CCK-8 cell viability assay showed that Api inhibited the viability of VSMCs in a concentration-dependent way.Compared with NC group,when the concentration of Api increased to 80 ?mol/l,the viability of VSMCs began to decrease significantly.Based on this,we set the concentration gradient of Api(low concentration group:10?mol/l,medium concentration group:20?mol/l,high concentration group:40?mol/l).The effects of the above concentrations of Api on the proliferation,cell cycle and apoptosis of VSMCs were detected by EdU cell proliferation assay,flow cytometry assay and apoptosis assayrespectively.The results showed that the above drug concentrations had no significant effects on cell proliferation,cell cycle and cell apoptosis of VSMCs.When the concentration of Api reached 40 ?mol/l,the effects began to appear on cell cycle and cell apoptosis of VSMCs.At the same time,calcified VSMCs were treated with low,medium and high concentrations of Api,and the effects of Api on cell calcification were detected by alizarin red staining,calcium assay and ALP activity assay,and Western Blot to detect the expression of RUNX2 and ?-SMA proteins.The results showed that compared with?-GP group,Api in each concentration group could inhibit cell calcification,and the medium concentration group and high concentration group were more significant,with statistical difference.The inhibition effects of medium concentration Api treatment group(Api-M)and high concentration Api treatment group(Api-H)on cell calcification were more similar to that of N-CAD knockdown group(KD N-CAD).Considering the cytotoxicity and inhibitory effect of Api on cell calcification,20?mol/l of Api was selected as the working concentration for subsequent experiments.We investigated the effects of Api on transcriptome in calcification model cell of VSMCs by RNA-seq assay.The results showed that compared with ?-GP group,115 genes were significantly up-regulated and 187 genes down-regulated in Api treatment group.Among them,the expression of TGF-?1 and EMT marker genes(E-CAD,N-CAD and ?-CAT)in Api treatment group was significantly altered.Then we examined the effects of Api and KD N-CAD on the cell migration of calcified VSMCs by cell scar and Transwell cell migration assay.Compared with ?-GP group,both Api and KD N-CAD could inhibit the cell migration of calcified VSMCs,especially the inhibition effects of KD N-CAD were more significant.Immunofluorescence staining for F-actin showed that compared with?-GP group,there were more tension fibers,relatively tightly arrangement between cells,and the cell margins of F-actin,filopodia and lamellar pseudopodia were significantly reduced in Api treatment group and KD N-CAD group.RT-qPCR and Western Blot results showed that compared with ?-GP group,the expression of E-CAD and ?-CAT was increased in Api treatment group and KD N-CAD group,while the expressions of TGF-?1,N-CAD and VIM were decreased.At the same time,we treated CAC model rats with Api and Ats,and the results of angializarin red staining of coronary artery showed that compared with the VC group,the coronary artery tissue in the Api treatment group and Ats treatment group had better elasticity,less damage to the media structure,and the red/orange particles between the media elastic fibers were significantly reduced.Western Blot results showed that Api and Ats significantly down-regulated the expression of calcification associated protein RUNX2 and up-regulated the expression of smooth muscle marker protein ?-SMA compared with VC group.Meanwhile,Api can up-regulate TGF-?1,E-CAD and ?-CAT,and down-regulate N-CAD,while Ats has no significant effect on TGF-?1 and EMT marker genes expression in coronary artery tissues of CAC model rats.Conclusion:Api can inhibit the migration of calcified VSMCs and delay the process of CAC by regulating TGF-?1 mediated EMT marker genes expression. |
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