Roles And Mechanism Of C/EBPβ In Methamphetamine-induced Aortic Aneurysm And Dissection

Research background:Methamphetamine(methamphetamine,METH)is a new type of drug,with the appearance same as ice,commonly called "ice poison" and also known as Metamfetamine Sulfate,soluble in water and alcohol.Methamphetamine has a benzene ring,its structure and catecholamine neurotransmitter structure is very similar to amphetamine-typed neurostimulator(Amphetamine-Typed Stimulant,ATS).The abuse of methamphetamine is a global problem due to its simple synthetic chemistry and strong central excitatory effects,with the increasing abuse of methamphetamine and low age of the abusers.It can cause health damage,but also trigger a series of cases which have a serious negative impact.Methamphetamine has multiple organ toxicity,and its main role is nervous system toxicity,but the cases,due to excessive consumption,caused by sudden death of myocardial ischemia are not uncommon in forensic work and clinical work,studies have shown that cardiovascular complications(hypertension,Aortic dissection,acute coronary syndrome,pulmonary hypertension,methamphetamine-related cardiomyopathy,etc)has become the second leading cause of death for methamphetamine abusers.Aortic aneurysm(AA)and aortic dissection(AD)is a rare but high mortality disease,with acute onset,occult symptoms and poor prognosis.In recent years,the incidence of aortic aneurysm and aorta dissection showed an upward trend.Aortic aneurysm and aortic dissection occurred with a variety of factors,hypertension is the main risk factors for aortic aneurysm and aortic dissection,while age,dyslipidemia,smoking,the use of cocaine,connective tissue disease(such as Marfan synthesis Levy,Ehlers syndrome),vascular inflammation(for instance giant cell arteritis,arteritis,cardiovascular syphilis),trauma,etc.are also considered risk factors for aortic aneurysm and aortic dissection.In recent years,more and more studies have shown that there is a strong and significant association between abuse or dependence of amphetamine and aortic dissection,and amphetamine been the second risk factor for the occurrence of an interarterial dissection following hypertension.The association between Methamphetamine and aortic aneurysm or aortic dissection have many clinical reports,but its mechanism is not clear,there is no methamphetamine induced aneurysm and arterial dissection animal models and related molecular mechanisms reported.With the increasing abuse of methamphetamine,explorimg the relationship between methamphetamine and aortic aneurysm and aortic dissection,and the molecular mechanism have a great significance.Objectives:Our study plan to establish an animal model of aortic aneurysm and aortic dissection induced by methamphetamine and a cell model treated with methamphetamine.The animal model was examined by histological staining.By examining the expressions of extracellular matrix metalloproteinase(MMP2,MMP9),apoptosis maker genes(Caspase-3 and PARP),inflammation maker gene(IL-1β)in samples of aortic aneurysm and aortic dissection,we explored the role of methamphetamine in the development of aortic aneurysm and aortic dissection.To study the correlation between C/EBPβ and TGFβ pathway,we used siRNA interference technology,immunofluorescence staining,Western blotting,inhibition and other techniques in animal models and cell models to explore the role and the molecular mechanism of methamphetamine in aortic aneurysm and aortic dissection.The C/EBPβ and other factors were verified by Western blotting and immunofluorescence staining on human specimens,Which laid a good foundation for further study of the role and mechanism of methamphetamine in aortic aneurysm and aortic dissection.Methods:Part Ⅰ:Establishment animal models of aortic aneurysm and aortic dissection induced by methamphetamine1.To determine the time and concentration of Methamphetamine and 3-aminopropionitrile fumarate(β-aminopropionitrile monofumarate,BAPN)We choosed three-week-old male rats induced by combined treatment of BAPN(lg/Kg/day,i.g,for 4 weeks)and METH(5mg/Kg,6h×2,day,i.p,for 4 weeks),to establish aortic aneurysm and aortic dissection model.2.Through HE staining,VG staining examine animal model and count tumor formation rate.Three-week-old male SD rats were randomly divided into control group,BAPN-treated group,METH-treated group and BAPN combined with METH group.The experimental group was treated with BAPN(1g/Kg/day,i.g)at 9:00 am for 4 weeks and METH(5mg/kg,6h × 2,day,i.p)at 13:00 and 19:00 for 4 weeks.Control group was given equal amount of saline treatment.After the last administration for 24 h,anesthesia was performed.We fixed the aorta tissue and calculated the tumor formation rate..HE and VG staining were performed then.3.To explore the role of methamphetamine in aortic aneurysm and aortic dissection model through molecular biology techniquesAfter administration,aortic vascular tissue was taken out and the expression of extracellular matrix metalloproteinase MMP2 and MMP9,apoptotic maker genes Caspase-3 and PARP,inflammation maker gene IL-1β was detected by Western blot,immunofluorescence and immunohistochemical staining and TUNEL staining.,Part Ⅱ:The Role of C/EBPβ in Methamphetamine-Induced Aortic Aneurysm and Aortic Dissection1.Establish a methamphetamine toxic vascular smooth muscle cells model and animal model testing(1)primary culture of vascular smooth muscle cells:we took about 100g SD rats,with neck severed,rapid extraction of aortic tissue,blunt dissection of the intima and adventitia,then took the vascular intima.The membrane was cut into pieces in DMEM medium added 20%FBS.Then,we used a sterile pasteurized pipette taking up tissue pieces into the cell culture flask and planked well-distributed.The culture flask stood upright at 3 7℃ and 5%CO2 in a cell incubator,gently flatten after 2h incubation and let stand absolutely for one week in incubator and change the fluid every three days.After passage of primary cultured vascular smooth muscle cells to the third generation,the cells were inoculated into 6-well plates and used for experiments when the cell density reached 75%.(2)Establishment of vascular smooth muscle cell methamphetamine toxic model:According to the results of pre-experiment,METH(1.0mmol/L)was used to set the time gradient to deal with primary culture of vascular smooth muscle cells after three generations.The cells were cultured in 20%FBS medium for METH(1.0mmol/L)induced 0h,2h,4h,8h,12h,24h respectively to establish the cell toxic model.The total cellular protein was extracted and the expression of C/EBPβ and TGFβ were detected by Western Blot.(3)Take the vascular tissue of METH induced aortic aneurysm and aortic dissection animal model,detected the expression changes of C/EBPβ,TGFβ by immunohistochemical staining and Western Blot.2.The role of C/EBPβ in METH toxic cell model(1)Protein of METH toxic cell was used to detect the expression of MMP2,MMP9,Caspase-3,PARP and IL-1β by Western Blot.(2)We designed the C/EBPβ siRNA interference fragment targeting rat vascular smooth muscle,and lipo3000 liposomes were used to transfect the siRNA into vascular smooth muscle cells.Western Blot was used to detect the expression of C/EBPβ and the interference efficiency of the interference sequences,thus to select out Effective interference fragments.(3)The vascular smooth muscle cells were divided into 4 groups:control group(Ctrl siRNA),METH-treated group(METH + Ctrl siRNA),METH-treated + small interfering group 1(METH + siC/EBPβ#1),METH-treated + small interfering group 2(METH + siC/EBPβ#2).The expression of MMP2,MMP9,Caspase-3,PARP and IL-1β were detected by Western Blot and immunofluorescence staining after treatment for 8 hours.3.C/EBPβ mediated METH-induced extracellular matrix degradation through the TGFβ pathway(1)After extracting protein of METH toxic and C/EBPβ siRNA treatmentt cell model,the expression of TGFβ,P-SMAD3 and P-ERK were detected by Western Blot.(2)The P-SAMD3 inhibitor SIS3(10umol)was introduced to divide vascular smooth muscle cells into four groups:control(Ctrl)group,Ctrl + SIS3 group,METH group,METH + SIS3 Group.After 8 hours of treatment,the total cellular protein was collected.The expression levels of P-SMAD3,MMP2 and MMP9 were detected by Western Blot.The P-ERK inhibitor U01263(20umol)was introduced to divide vascular smooth muscle cells into four groups:control group(Ctrl),Ctrl +U0126 group,METH + U0126 group.After 8 hours of treatment,the total cellular protein was collected.The expression of P-ERK,MMP2 and MMP9 was detected by Western Blot.(3)Take vascular tissue of METH-induced aortic aneurysm and aortic dissection animal model,the expression of P-SMAD3,P-ERK were detected by Western Blot.Part Ⅲ:The role of C/EBPβ in human aortic aneurysms and aortic dissectionsSpecimens of human aortic aneurysms and aortic dissection were collected during the prosecution work of the department and taken fresh tissue getting rid of epicardium to extract proteins.Western Blot was used to detect the expression of C/EBPβ,MMP2,MMP9,Caspase-3,PARP and IL-1β.Results:Part Ⅰ:Establishment animal models of aortic aneurysm and aortic dissection induced by methamphetamine1.METH and BAPN administration time and drug concentration determinationBased on the preliminary experimental results,we selected 3-week-old male rats pre-treated with BAPN(1g/Kg/day,ig)for 2 weeks and later BAPN co-treated with METH(5mg/Kg,6h × 2,day,ip)for 2 weeks,and finally treated with METH alone for 2 weeks to establish an animal model.2.Tumor formation rate statistics and HE staining,VG staining results(1)The control group did not develop tumor,the constituent tumor rate of METH +BAPN treatment group and the aneurysm rupture mortality rate were higher than that of METH and BAPN treatment groups.A total of 20 rats in METH+BAPN groups,11 of them observed aneurysm or severely damaged vascular tissues,4 died of tumor rupture,a total of 15/20 tumor formation rates.In METH treated groups,no tumors were observed among these 6 rats,the tumorigenesis rate was 0/6.In the BAPN treated group,there were 6 rats,and one tumor was seen with naked eyes,and the tumor formation rate was 1/6.(2)The results of staining showed that there was no tissue injury in the control group,and the tissue structures in the BAPN group and the METH group were disordered.The BAPN + METH group showed obvious cleavage of the elastic fibers and formation of the dissection.3.Related factor detected in METH induced aortic aneurysm and aortic dissection animal modelThe results of Western blot showed that the expressions of MMP2,MMP9,Caspase-3,PARP and IL-1β in METH + BAPN group were higher than those in the control group,BAPN group and METH group.Immunohistochemistry,immunofluorescence and TUNEL staining are consistent with Western blot results.Part Ⅱ:The Role of C/EBPβ in Methamphetamine-Induced Aortic Aneurysm and Aortic Dissection1.Establish a methamphetamine toxic vascular smooth muscle cells model and animal model testing(1)After respectively treated with METH(1.0mmol/L)for 0h,2h,4h,8h,12h,24h,the results of Western Blot showed that the expression of C/EBPβ and TGFβgradually increased with the prolongation of METH,The expression began to rise at2 hours and peaked at 8 hours.(2)The Western blot results of METH induced aortic aneurysm and aortic dissection animal model showed that the expression of C/EBPp and TGFp in BAPN+METH group was significantly higher than that in BAPN group,METH group and control group;Chemical staining results are consistent with Western blot.2.The role of C/EBPβ in METH toxic cell model(1)The expression of MMP2,MMP9,PARP,Caspase3 and IL-1β were increased with the prolongation of treatment time and bagan increasing at 2h and peaked at 8h orl2h.(2)After C/EBPβ was silenced by siRNA,the expression of C/EBPβ in METH +siC/EBPβ#1 group was decreased and the expression of C/EBPβ in METH +siC/EBPβ#2 group was significantly decreased;The expression of IL-1β,MMP2,MMP9,PARP and Caspase3 decreased with the silence of C/EBPβ.3.C/EBPβ mediated METH-induced extracellular matrix degradation through the TGFβ pathway(1)In METH toxic cell model,the expression of P-SMAD3 and P-ERK increased with the prolongation of METH treatment.The expression of P-SMAD3 and P-ERK increased at 2h and peaked at 8h.TGF-P,P-SMAD3 and P-ERK expression in METH+ siC/EBPβ#2 group were significantly decreased after C/EBPβ was silenced by siRNA.(2)After SIS3 inhibited the expression of P-SMAD3,the expression of P-SMAD3,MMP9 and MMP2 in Ctrl+SIS3 group was lower than that in Ctrl group and METH+SIS3 group was lower than that in METH group.After U0126 was used to inhibit the expression of P-ERK,the expression of P-ERK and MMP9 in Ctrl+U0126 group was lower than that in Ctrl group and METH+ U0126 group was lower than that in METH group,but the expression of MMP2 was not decreased.(3)The Western blot results of METH-induced aortic aneurysm and aortic dissection model animals showed that the expression of P-SMAD3 and P-ERK in BAPN + METH group was significantly higher than that in BAPN group,METH group and control group.Part Ⅲ:The role of C/EBPβ in human aortic aneurysms and aortic dissectionsThe results showed that the expression of C/EBPβ in human aortic aneurysms and aortic dissection specimens was higher than that in the normal control group.Compared with the controls,MMP9,Caspase-3,PARP,IL-1β expression level was also significantly increased,but the expression of MMP2 did not increase.Conclusion:1.METH exposure can induce the occurrence of aortic aneurysm and aortic dissection,and C/EBPβ plays an important role in METH-induced aortic aneurysm.2.C/EBPβ may lead to the development of aortic aneurysm by regulating the apoptosis of vascular smooth muscle cells and regulating the expression of matrix metalloproteinase MMP2 and MMP9,at the same time,C/EBPβ can promote the production of inflammatory cytokines,Thus inducing the occurrence of aortic aneurysm.3.C/EBPβ plays a role in the development of human aortic aneurysm and aortic dissection.