| Research background:Aortic dissection,which has been thought to have a rare incidence but high mortality rate,is a serious macrovascular disease,which is characterized by acute onset and poor prognosis.There are no other medical treatments outside the surgery(aortic prosthetic vascular replacement or endovascular isolation).In recent years,there are more and more cases of aortic dissection in our country,the age of onset is 40 years old and 50 years old,and the male is the main one.Most aortic aneurysms are usually asymptomatic and are often diagnosed by chance when diagnosing other diseases in the chest and abdomen,but,if the damaging factors persist,the aortic aneurysm will progress into an acute aortic dissection,leading to pericardial tamponade,stroke,acute aortic regurgitation,hemothorax,paraplegia and other life-threatening complications.Therefore,the high mortality rate and increasing incidence of aortic aneurysm/dissection lead to prevention and prediction becoming very active research in this field,and it is crucial to control the risk factors and to study some pathogenic target genes.In the past,the risk factors of the disease were hypertension,atherosclerosis,vascular connective tissue disease,vascular inflammation,dyslipidemia,positive family history,age,sex,smoking,trauma,and so on.Extracellular matrix degradation,apoptosis and so on.Among the risk factors,hypertension ranked first among the risk factors of aortic aneurysm and aortic dissection,but in recent years,more and more clinical cases have reported that methamphetamine(meth)is also related to the formation of aortic dissection.In the United States,the abuse of METH has been identified as the most common risk factor for fatal acute aortic dissection after hypertension.It is recommended that all patients under the age of 50 with aortic dissection should receive regular urine screening for methamphetamine.In 2010,The University of Texas in the United States conducted a population-based epidemiological study to examine the relationship between amphetamine abuse/dependence and aortic dissection.This study shows a cross-sectional study at 18-49 years old.In the population,the hospitalization rate of the aortic dissection increased,while the hospitalization rate of patients aged 50 years and older did not increase.Thus,in young adults,there is a strong and significant association between amphetamine abuse and aortic dissection(even after adjustment of known risk factors)while controlling known risk factors.However,experimental studies on the induction of aortic dissection by METH have not been reported.Based on the above analysis,we have successfully induced aortic aneurysm/dissection by using BAPN and METH in our previous study,and found that C/EBPβwas up-regulated in our aneurysm/dissection model.In vitro experiments showed that C/EBPβ could regulate the expression of MMP 2/MMP9,which could affect the stability of vascular media,and C/EBPβ could also regulate the apoptosis of vascular smooth muscle cells(VSMC),however,the specific regulation mechanism is not clear.The degradation of vascular media matrix and apoptosis of VSMC are frequently observed in human aortic dissection tissues,which are the main pathological changes of aortic aneurysm/dissection.So,whether METH-induced media matrix degradation and smooth muscle cell apoptosis ultimately lead to aortic aneurysm/dissection,whether C/EBPβ plays an important role in those processes,and how does it regulate these two biological processes?This is the core problem to be solved in this study.This study is to explore the molecular mechanism of METH-induced aortic aneurysm/dissection,and to provide an important molecular target for the prevention and treatment of aortic aneurysm/dissection.Objective:In this study,it has been proved that C/EBPβ can regulate the expression of MMP2/MMP9,and further verify that C/EBPβ can regulate the expression of MMP2/MMP9 and lead to the degradation of media matrix then aneurysm/dissection is promoted at the whole level of animals.We intend to establish animal model by injecting synthetic C/EBPβ adenovirus(AD-shC/EBPp)or negative control(AD-shGFP)into tail vein,and then combining BAPN and METH to establish animal model.HE and VG(hematoxylin-eosin and verhoeff-van gieson staining)were used to observe the changes of aortic structure in rats,and the formation rate of aortic aneurysms/dissection was counted,the mechanism of C/EBPβ regulating MMP2/MMP9 expression and smooth muscle cell apoptosis was also discussed at the whole level of animals.At the same time,the cell model of METH poisoning was established in vitro,by using siRNA interference technology,western blotting,immunohistochemical staining,immunofluorescence staining,TUNEL and other techniques to detect the expression levels of apoptosis maker gene(cleaved Caspase 3 and cleaved PARP),C/EBPβ,IGFBP5,P53,and PUMA in aortic tissue of animal models and the METH-infected VSMCs model,to explore the molecular mechanism of C/EBPβ regulating apoptosis in aortic dissection/aneurysm induced by METH.The C/EBPβ,IGFBP5 and apoptosis markers were verified by wstern blotting on human samples to provide a new theoretical basis for the study of the pathogenetic target and preventive treatment of aortic aneurysm/dissection.Methods:Part I:Effect of silencing C/EBPβ on METH+BAPN-induced Aortic Aneurysm/dissection1.Effect of adenovirus AD-shC/EBPβ silencing C/EBPβ on METH-induced Aneurysm/dissection.In the early stage,we used BAPN and METH to construct a rat aortic aneurysm/dissection animal model and detected the up-regulation of C/EBPβexpression.To further study the role of C/EBPβ in METH-induced aortic aneurysm/dissection,we constructed an adenovirus that silenced the expression of C/EBPβ and injected it into the rat via tail vein,then observed the effect of C/EBPβ silence on the formation of METH-induced aortic aneurysm/dissection.Three-week-old male SD rats(about 40g)were randomly divided into four groups:AD-shGFP group,AD-shC/EBPβ group,AD-shGFP+METH+BAPN group,AD-shC/EBPβ+METH+BAPN group.Four groups of rats were injected intravenously with 1.6×10 10 PFU AD-shC/EBP β or AD-shGFP at 9:00 on day 1,day 12,day 23 and day 34 during the course of the experiment.And the AD-shGFP+METH+BAPN group and the AD-shC/EBPβ+METH+BAPN group were treated with BAPN(1 g/kg/day,i.g)on the first day,f or 4 weeks.In the third week,METH(5mg/kg,twice/day,i.p)was administered for 4 weeks,and AD-shGFP group and AD-shC/EBP β group were treated with the same amount of saline.Establishment of animal model of aortic aneurysm/dissection in the above way.2.Animal models were detected by HE staining and VG staining to observe the change of rat aortic structure,and the aortic aneurysm formation rate was calculated.24 hours after the last administration of METH,the anaimal’s aortic tissue were taken after left ventricular perfusion under anesthesia and the aneurysm formation rate was counted by naked eyes,then the part of the aortic arch was fixed in paraformaldehyde solution,paraffin sections were made for subsequent HE and VG staining.3.Exploring the effect of silencing C/EBPβ on the expression of MMP2/MMP9 and its regulatory mechanism at animal level by molecular biological techniques.At the end of the administration,some tissues of aortic arch were taken from each group and the protein was extracted.The expression of C/EBPβ and MMP2,MMP9,p-Smad3,p-ERK in each group were detected by Western blot and analyzed.Part Ⅱ:C/EBPβ mediates methamphetamine-induced aortic aneurysm/dissection by regulating apoptosis of smooth muscle cells.1.Study on the regulation mechanism of C/EBPβ on apoptosis of VSMC induced by METH at cell level.(1)Primary culture of aortic vascular smooth muscle cells(VSMC):about 100 g of SD rats were sacrificed and the spinal cord was sacrificed then the vascular middle layer was quickly extracted in the ultra-clean platform.The removed medium membrane was cut,and then the tissue block was uniformly inoculated into a cell culture flask containing 20%FBS in DMEM culture solution by a sterile bagel suction pipe,the culture bottle is vertically placed in a cell culture box containing 37℃ 5%CO2,and the culture bottle is gently put into a flat after 2 to 3h,then stand still in the cell incubator for one week,and then change the culture solution once every three days.After the passage of the above-mentioned primary cultured vascular smooth muscle cells to the third generation,the cells were seeded into a 6-well plate and used for experiments when the cell density reached 70%to 80%.(2)Establishing VSMCs model of METH poisoning:based on the results of the previous study,VSMCs in 6-well plates after primary culture for 3 generations were infected with 1.0 mmol/L METH and a time gradient of 0-24 h was set to establish a model of cell poisoning.At the end of the experiment,the total protein was extracted and the expression of C/EBPβ and cleaved Capase3,cleaved PARP was detected by Western blot.(3)Regulation of C/EBPβ on apoptosis of VSMCs poisoned by METH:According to the results of previous studies,VSMCs were randomly divided into 4 groups by using two effective interference fragments of C/EBPβ siRNA against rats:blank control group(Ctrl siRNA),administration group(METH+ Ctrl siRNA),administration +small Interfering fragment group 1(METH + siC/EBPβ 1#),administration + small interference fragment 2 group(METH +siC/EBPβ 2#),firstly transfecting small interference fragments into VSMCs using lipo3000 liposome,transfected for 24h,then 1.0mmol/L METH was used for 8 hours in the experimental group.After the treatment,C/EBP β,cleaved Caspase3 and cleaved PARP were detected by Western Blot and TUNEL staining.(4)C/EBPβ mediates METH-induced apoptosis of VSMCs via the IGFBP5-P53-PUMA pathway:VSMCs model protein poisoned by METH and cell protein treated with C/EBPβ siRNA interference fragment were taken,and the expression level of IGFBP5,P53,PUMA was detected by Western Blot.(5)IGFBP5 siRNA interference fragment targeting rat vascular smooth muscle was designed,the VSMCs were divided into four groups:control group(Ctrl),administration group(METH+ Ctrl siRNA),administration + small interference fragment Group 1(METH + siIGFBP5 1#),drug administration+small interference fragment 2 group(METH H+siIGFBP5 2#).VSMCs were transfected with lipo3000 liposome for 24 hours,then the experimental group was infected with 1.0mmol/L METH for 8 hours,and then the expression level of IGFBP5,P53,PUMA,cleaved Caspase3,cleaved PARP was detected by immunofluorescence staining and Western Blot.2.Confirm that C/EBPβ regulates apoptosis of smooth muscle cells and its specific mechanism at animal level(1)The vascular tissues of aortic aneurysm/dissection model induced by synthetic C/EBPβ adenovirus +METH+BAPN were collected and the expression of C/EBPβ、IGFBP5、P53、PUMA and cleaved Capase3,cleaved PARP in each group was detected by Western Blot.(2)The aortic tissues of aortic aneurysm/dissection model induced by METH+BAPN were obtained.And the expression levels of C/EBPβ,IGFBP5,P53,PUMA,cleaved Caspase3 and cleaved PARP were detected by Western Blot,immunohistochemistry,immunofluorescence staining and TUNELPart Ⅲ:C/EBPβ is involved in the apoptosis of human aortic aneurysm/dissection specimens induced by non-methamphetamineThe specimens of aortic aneurysm/dissection were collected from the forensic identification center of our university.The fresh tissue was removed from the outer membrane and the protein was extracted then the expression levels of C/EBPβ,IGFBP5,P53,PUMA,cleaved Caspase3 and cleaved PARP were detected by Western Blot.Results:Part I:Effect of silencing C/EBPβ on METH+BAPN-induced Aortic Aneurysm/dissection1.Statistics of aneurysm formation rate,and the results of HE staining and VG staining of animal model.According to the macrosomal view of aorta and the staining results of HE and VG:there was no aneurysm or obvious rupture of elastic fiber and dissection formation was found in AD-shGFP group and AD-shC/EBPβ group.In the AD-shGFP+BAPN+METH group,there were 4 rats with obvious aneurysm and severe elastic fiber disruption and rupture of aortic tissue.In the AD-shC/EBPβ+BAPN+METH group,only one rat had elastic fiber disorder and rupture in aortic tissue,and the degree of rupture was mild,but no obvious aneurysm was found.2.Exploring the effect of silencing C/EBPβ on the expression of MMP2/MMP9 and its regulatory mechanism at animal level by molecular biological techniquesWestern blot analysis showed that the expression level of C/EBPβ in AD-shGFP+METH+BAPN group was higher than that in AD-shC/EBPβ+ METH+BAPN group.The expression level of C/EBP[in AD-shGFP group was also higher than AD-shC/EBPβ group but lower than the AD-shGFP+METH+BAPN group.The expression of P-Smad3,p-ERK,MMP2 and MMP9 decreased with the silence of C/EBPβ.Part Ⅱ:C/EBPβ mediates methamphetamine-induced aortic aneurysm/dissection by regulating apoptosis of smooth muscle cells.1.Study on the regulation mechanism of C/EBPβ on apoptosis of VSMC induced by METH at cell level.(1)The results of Western blot showed that the expression of C/EBP β and cleaved Capase3,cleaved PARP began to increase at 2 h and reached the peak at 8 h or 12 h after VSMCs was treated with 1.Ommol/L METH at 0 h,2 h,4 h,8 h,12 h and 24 h,respectively.(2)After silencing C/EBPβ with siRNA:Western blot results showed that the expression of C/EBPβ in METH+ Ctrl siRNA group was significantly increased,which was consistent with the protein expression of METH-infected VSMCs model,and the expression level of this group is significantly different from the expression of other groups.The expression of C/EBPβ was decreased in the METH + siC/EBPβ 1#group and the METH+siC/EBPβ 2#group;however,the decrease in C/EBPβ was more significant in the METH+siC/EBPβ 2#group.The expression levels of cleaved Caspase3 and cleaved PARP decreased with the decrease of C/EBPβ.The results of TUNEL staining of siC/EBPβ 2#transfected VSMCs were consistent with result of Western blot.(3)C/EBPβ mediates METH-induced apoptosis of VSMCs via the IGFBP5-P53-PUMA pathway:Western Blot results of METH-infected VSMCs model protein showed that the expression levels of IGFBP5,P53 and PUMA in METH-infected VSMCs model protein were similar to C/EBPβ,and the expression levels increased at 2 h after treatment,and peaked at 8h or 12h.The results of cell protein detection after C/EBPβ siRNA interference fragment treatment showed that the expression levels of IGFBP5,P53 and PUMA decreased with the silence of C/EBPβ.(4)After silencing IGFBP5 with siRNA,Western blot results showed that the expression of IGFBP5 in METH+ Ctrl siRNA group was significantly increased,which was consistent with the protein expression of METH-infected VSMCs model,and the expression level of this group was significantly different from that of other groups.The expression levels of IGFBP5 in METH+ siIGFBP5 1#group and METH+siIGFBP52#group were all decreased;however,the decrease of IGFBP5 in METH+siIGFBP51#group was significant.The expression levels of P53,PUMA,cleaved Caspase3,and cleaved PARP all decreased with the silencing of IGFBP5.Immunofluorescence staining results of siIGFBP5 1#transfected VSMCs were consistent with Western blot results.2.Confirm that C/EBPβ regulates apoptosis of smooth muscle cells and its specific mechanism at animal level(1)The results of Western Blot in aortic tissue of animal model treated with adenovirus+METH+BAPN showed that the expression levels of IGFBP5,P53,PUMA,cleaved Capase3,cleaved PARP and C/EBPβ were the highest in the AD-shGFP+METH+BAPN group,and other factors decreased with C/EBPβ silencing.(2)Western Blot results of aortic tissue of aortic aneurysm/dissection animal model induced by METH+BAPN in the early stage of our group showed:The expression level of C/EBPβ,IGFBP5,P53,PUMA,apoptotic factor(cleaved Caspase3 and cleaved PARP)in BAPN+METH treated group was significantly higher than that in the BAPN group,the METH group,and the control group.The immunohistochemical results of C/EBPβ and IGFBP5 and the TUNEL staining results of apoptotic factors were consistent with the results of Western blotting.Part Ⅲ:C/EBPβ is involved in the apoptosis of human aortic aneurysm/dissection specimens induced by non-methamphetamineThe results of human specimens showed that the expression levels of C/EBPβ,IGFBP5,P53,PUMA,cleaved Caspase3 and cleaved PARP were higher in the aortic aneurysm and aortic dissection than in the normal control group.Conclusion:1.C/EBPβ regulates the expression of MMP2/MMP9,leading to the degradation of the mesangial matrix,which promotes the occurrence of aneurysm/dissection.Silencing C/EBPβ will reduce the occurrence of METH-induced aortic aneurysm/dissection.2.C/EBPβ regulates apoptosis of smooth muscle cells via IGFBP5-P53-PUMA pathway mediates methamphetamine-induced aortic aneurysm/dissection3.The C/EBPβ-IGFBP5-P53-PUMA pathway is also involved in the apoptosis of human aortic aneurysm and aortic dissection which not induced by METH. |
PDF Full Text Request