| Backgroud:Methamphetamine(METH),an amphetamine-type stimulant,is a substance derived from the chemical structure of ephedrine.It is also known as deoxyephedrine because it looks pure white crystals and is commonly known as "ice".".In 1919,the Japanese chemist A.Ogata first synthesized crystal-like methamphetamine to make it possible for large-scale production;in 1945,it was determined that methamphetamine could be addictive;in 1951,Japan began to regulate methamphetamine..At the end of the 20th century,new types of drugs represented by methamphetamine were relatively simple to produce and process,and the use of drugs was cheaper.They have gradually replaced traditional drugs that use poppy as their main raw material and rapidly spread throughout the world.According to the "National Drug Abuse Monitoring Annual Report(2016)" issued by the China Food and Drug Administration(CDFA),276,980 drug abuse monitoring cases(copies)in 2016,and heroin abuse monitoring data accounted for 45.0%of all monitoring data,which was comparable to that of 2015.The rate of decline was 3.6 percentage points,and continued to decline in the past five years.The abuse of methamphetamine-containing drugs("ice-toxin" and "Ma-gu pills")accounted for 55.1%of the total,an increase of 3.2 percentage points from the same period in 2015,which was the main cause of the prevalence of abuse in China.Synthetic drugs,indicating that the rapid spread of traditional drugs represented by heroin has been further curbed,while new methamphetamine-based synthetic drugs The abuse of personnel has grown rapidly,and the drug situation in our country is still not optimistic.The long-term abuse of methamphetamine can cause all kinds of criminal offenses and bring great harm to the public security.Therefore,the study on the toxicity of METH and its prevention and control has become a major topic and research hotspot in the world.METH has a molecular weight of 149.233 and a molecular formula of C10H15N.Its structural formula isIts chemical structure is very similar to that of ephedrine and catecholamine transmitters.Therefore,its pharmacological properties include central nervous system excitability and hallucinogenic effects.At the same time,it also releases a large amount of catecholamines through the central and peripheral nerve endings,causing vasoconstriction and heartbeat.Accelerate and increase blood pressure and other sympathomimetic effects.According to a large number of clinical data summarized from epidemiological surveys,METH can cause damage to important parenchymal organs in the heart,brain,lungs,liver,kidneys,etc.;in the damage to many organs,the most serious damage to the central nervous system is The most in-depth study;and in recent years,more and more research shows that METH also has a major impact on the cardiovascular system,and even fatal damage.In 2017,there were 100 cases of methamphetamine poisoning-related deaths studied by Iranian pathologists.In all cases,forensic medicine determined the toxicity of methamphetamine as the direct cause of death,and 68%of the study cases found cardiovascular pathological changes.In the cardiovascular injury caused by METH,the incidence of aneurysm is increasing year by year,the condition is dangerous,and the disability rate and sudden death rate are high.The risk factors of aneurysm include hypertension,old age,atherosclerosis,and cardiovascular surgery.,Marfan syndrome,Ehlers-Danlos syndrome,etc.The sympathomimetic effect caused by METH,with the increase of systole and diastolic beat,and the increase of vascular wall pressure,the ascending aorta,aortic arch,and descending aorta are the sites most affected by blood flow,and the vascular elastic fibers are vulnerable to injury.It is easy to cause damage to the aortic wall,trauma,tear and tear in the media,and when the blood flows into the vascular wall space,it forms a dissection hematoma and an aortic aneurysm.MicroRNA(miRNA or miR),a non-coding small RNA of approximately 20-23 nucleotides in length;it is presumed to be involved in the regulation of gene expression in nearly one-third of the body;mainly through the interaction with target genes.Incomplete complementary pairing negatively regulates the expression of the target gene at the transcriptional and post-transcriptional levels,resulting in degradation of the mRNA or inhibition of translation.Objectives:Elucidate the role of microRNA-mediated apoptosis and autophagy in vascular smooth muscle cells(VSMCs)in METH-induced rat aortic aneurysm/dissection and possible regulatory mechanismsMethod:The first part of 1.0mmol/L METH treatment VSMCs 24h after chip screening and in vitro and in vivo QPCR validation(1)Isolated and cultured aortic smooth muscle cells of about 150-200 g of male SD rats;(2)Experimental group:control group,METH treatment group(1.0mmol/LMETH effect 24h)After Trizol treatment,cells were collected and sent to Shanghai Bohao Biotechnology Co.,Ltd.for gene chip screening;(3)Select the microRNAs to be verified according to P<0.05 and Fold change>2 or<0.5;(4)The expression of miR-351-5p,miR-20b-5p and miR-708-5p in VSMCs was detected by QPCR.(5)Aortic aneurysm model group:control group,BAPN+METH-group,BAPN-METH+ group,BAPN+METH+ group;(6)QPCR detection of miR-351-5p,miR-20b-5p,miR-708-5p expression changes in aortic aneurysm model;(7)According to the in vitro and in vivo validation results,select the study purpose microRNAs.Part Ⅱ The role of miR-351-5p in METH-induced VSMCs autophagy,target and its role in SD rat aortic aneurysm model(1)Experimental groups:NC group,miR-mimics group,NC+METH group,and miR-mimics+METH group;(2)QPCR detection of miR-351-5p mimics AGGCUCAAAGGGCUCCUC AGGGAUU overexpression efficiency;(3)Western-blot was used to detect the effect of miR-351-5p on autophagy induced by METH in VSMCs.(4)The effect of miR-351-5p overexpression on VSMCs autophagy induced by METH was detected by transfection with GFP-LC3;(5)The effect of overexpression of miR-351-5p on autophagy of VSMCs induced by METH was detected by transmission electron microscope;(6)Bioinformatics software predicts miR-351-5p target of action;(7)Western-blot was used to detect the effect of miR-351-5p overexpression on UVRA expression in VSMCs induced by METH.(8)Experimental grouping:NC group,siUVRAG#1 group,siUVRAG#2 group,siUVRAG#3 group siUVRAG#4 group,METH group,siUVRAG#1+METH group,siUVRAG#2+METH group,siUVRAG#3+METH Group,siUVRAG#4+METH group;#1 GGAACAUUGCUGCUCGGAATTUUCCGAGCAGCAAUGUUCCTT#2 GGAGUGAAGUGAUUAAGAATTUUCUUAAUCACUUCACUCCTT#3 GCUGUUGAUAGAGUGGAAATTUUUCCACUCUAUCAACAGCTT#4 GCAACAAAUGGCUCUACAATTUUGUAGACCCAUUUGUUGCTT(9)The effect of silencing UVRAG gene on autophagy of VSMCs induced by METH was detected by Western-blot.(10)Western-blot was used to detect the effect of miR-351-5p overexpression on STAT3 expression in VSMCs induced by METH.(11)Western-blot was used to detect the effect of miR-351-5p on the expression of Tyr-STAT3,Ser-STAT3 and LCN2 in VSMCs induced by METH.(12)Experimental groups:Control group,METH group,Stattic lum,3um,5um,7um,lOum group;(13)The effect of Tyr-STAT3 on the expression of LCN2 and autophagy induced by METH was detected by Western-blot.(14)Experimental grouping:NC group,siLCN2 group,NC+METH group,siLCN2+METH group;SiLCN2:CCCAGGACUCAACUCAGAATTUUCUGAGUUGAGUCCUGGGTT(15)The effect of silencing LCN2 on METH-induced autophagy was detected by Western-blot.(16)Western-blot was used to detect the expression of UVRAG and STAT3 in aortic aneurysm tissue.(17)Western-blot detection of autophagy expression in aneurysm tissue.Part Ⅲ Role,target of miR-20b-5p in autophagy induced by METH in VSMCs and its role in SD rat aortic aneurysm model(1)Experimental groups:NC group,miR-mimics group,NC+METH group,and miR-mimics+METH group;(2)QPCR detection of miR-20b-5p mimics ACCUGCACUAUGAGCACU UUGUU overexpression efficiency;(3)Western-blot was used to detect the effect of miR-20b-5p overexpression on the apoptosis of VSMCs induced by METH.(4)bioinformatics software predict miR-20b-5p target;(5)The effect of miR-20b-5p on the expression of PTEN in VSMCs induced by METH induced by Western-blot(6)Experimental groups:NC group,SiPTEN#1 group,SiPTEN#2 group,METH group,SiPTEN#1+METH group,SiPTEN#2+METH group;(7)Western-blot was used to detect the effect of silencing PTEN gene on the apoptosis of VSMCs induced by METH.(8)The expression of PTEN in aortic aneurysm was detected by Western-blot.(9)Western-blot detection of apoptosis in rat aneurysm tissue.Results:The first part of 1.Ommol/L METH treatment VSMCs 24h after chip screening and in vitro and in vivo QPCR validation(1)1.0mmol METH treatment VSMCs 24h after chip screening,the results showed a total of 57 microRNAs expression changes,which METH group compared to Control group miR-351-5p,miR-20b-5p decreased significantly,miR-708-5p rose significantly.(2)In vitro experiments confirmed that the miR-351-5p and miR-20b-5p were significantly decreased and the miR-708-5p was significantly increased.(3)In vivo experiments showed that miR-351-5p and miR-20b-5p were decreased in BAPN-METH+ group compared with control group,and miR-351-5p and miR-20b-5p in control group were significantly lower in BAPN+METH+ group;MTR and BAPN,METH combined treatment group miR-708-5p increased to a certain extent.(4)The miR-351-5p and miR-20b-5p were selected for the purpose of microRNAs.Part Ⅱ The role of miR-351-5p in METH-induced VSMCs autophagy,target and its role in SD rat aortic aneurysm model(1)The synthesized miR-351-5p mimics can be effectively over-expressed in the cells:1.0mmol/L METH treated VSMCs 24h after the NC+METH group was significantly lower than the NC group,overexpression of miR-351-5p after NC The group rose rapidly and the increase was statistically significant.(2)Overexpression of miR-351-5p protects VSMCs from autophagy induced byMETH:1.Western-blot detection of autophagy marker gene:Compared with NC group,autophagy was more obvious in NC+METH group,marker gene beclinl and LC3-II were significantly expressed,and after overexpression of miR-351-5p,the expression of Beclin-1 and LC3-II was increased.With the decline,the trend is obviously statistically different;2.The degree of LC3-II aggregation was detected by transfecting GFP-LC3:the number of green and bright spots in NC+METH group was significantly higher than that in NC group,and LC3-II was heavily accumulated.After over-expression of miR-351-5p,the number of green spots rapidly decreased.The significant decrease in fluorescence intensity was statistically significant.3.The number of autophagosomes was detected by transmission electron microscopy:The number of autophagosomes in the NC+METH group was significantly higher than that in the NC group,and the autophagy was evident.After overexpression of miR-351-5p,the amount of autophagosomes rapidly decreased.The statistically significant difference in the number of autophagosomes was significantly reduced.(3)Targctscan bioinformatics software predicts that UVRAG and STAT3 are targets of miR-351-5p;(4)UVRAG was significantly negatively regulated by miR-351-5p:the expression of UVRAG in NC+METH group was significantly higher than that in NC group,and UVRAG expression was decreased after overexpression of miR-351-5p,with a significant difference.(5)The synthesized small interference siUVRAG can effectively silence the expression of UVRAG in cells:The expression of UVRAG in the NC+METH group was significantly higher than that in the NC group.After transfection with siUVRAG,the expression of UVRAG in the cells was effectively reduced.The difference was statistically significant.(6)Silencing UVRAG had a protective effect on METH-induced VSMCs autophagy:NC+METH group had significantly higher levels of autophagy marker genes Beclin-1 and LC3-Ⅱ than NC group.After effective UVARG silencing,Beclin-1 and LC3-Ⅱ The decrease was statistically significant;(7)STAT3 was significantly negatively regulated by miRA-351-5p:the expression of S TAT3 in NC+METH group was significantly higher than that in NC group,and STAT3 was decreased after over-expression of miR-3 51-5p,with a statistically significant difference.Tyr-The changes of STA3 and Ser-STAT3 expression were consistent with the STAT3 trend.(8)Stattic can effectively inhibit the expression of Tyr-STAT3 in cells:Compared with the control group,the expression of Tyr-STAT3 is significantly increased in the METH group,but there is no significant change in the expression of the Ttyr-STAT3 in the Stattic cells,and 5-10 um acts on the cells.With effective inhibition,the expression of Tyr-STAT3 was significantly decreased,and the decrease was statistically significant.(9)Inhibition of tyr-STAT3 has a protective effect on METH-induced VSMCs autophagy:NC+METH group had significantly higher expression of autophagy marker genes Beclin-1 and LC3-Ⅱ than NC group.After effectively inhibiting Tyr-STAT3,LCN2 There was a statistically significant difference in the decline of Beclin-1 and LC3-Ⅱ.(10)Silencing LCN2 had a protective effect on METH-induced VSMCs autophagy:NC+METH group had significantly higher levels of autophagy marker gene Beclin-1 and LC3-Ⅱ than NC group.After effective silencing of LCN2,Beclin-1 and LC3-ⅡThe decrease was statistically significant;(11)Expression of miR-351-5p target in aortic aneurysm tissue:Expression of UARAG and STAT3 in BAPN-METH+ group increased to some extent,and UVRAG and STAT3 expression in BAPN+METH+ group increased significantly;(12)Expression of autophagy in aortic aneurysm tissue:Expression of Beclin-1 and LC3-Ⅱ in BAPN-METH+ group increased to some extent,and expression in BAPN+METH+ group increased significantly.Part Ⅲ Role,target of miR-20b-5p in autophagy induced by METH in VSMCs and its role in SD rat aortic aneurysm model(1)The synthesized miR-20b-5p mimics can be effectively over-expressed in cells:1.0 mmol/L METH treated VSMCs 24 h later,NC+METH group decreased significantly compared with NC group,over-expression miR-20b-5p compared to NC The group rose rapidly,and the increase was significantly different;(2)Overexpression of miR-20b-5p has a protective effect on apoptosis of VSMCs induced by METH:NC+METH group has more obvious apoptosis than NC group,marker gene Cleaved-PARP and Cleaved-Caspase3 are significantly higher,overexpression of miR-After 20b-5p,Cleaved-PARP and Cleaved-Caspse3 decreased,and the drop was significantly different.(3)Targct scan bioinformatics software predicts that PTEN is the miR-20b-5p target;(4)PTEN was significantly negatively regulated by miR-20b-5p:NC+METH group was lower than NC group PTENThe expression of PTEN was significantly increased after overexpression of miR-20b-5p,and the expression of PTEN was significantly decreased.CHOP,as a downstream molecule of PTEN,was consistent with PTEN trend.(5)The synthesized small-interfering siPTEN could effectively silence the expression of PTEN in the cells:NC+METH group had significantly higher PTEN than NC group.After transfected with siPTEN,the amount of PTEN in the cells decreased with a significant decrease.(6)Silencing PTEN had a protective effect on the apoptosis of VSMCs induced by METH:NC+METH group had significantly higher apoptosis marker genes Cleaved-PARP and Cleaved-Caspse3 than NC group.After silencing PTEN,Cleaved-Caspase3 and Cleaved-PARP were expressed.With the decrease,the drop has obvious statistical difference;(7)Expression of miR-20b-5p target in aortic aneurysm tissue:Expression of PTEN in BAPN-METH+ group increased to a certain extent,and PTEN expression in BAPN+METH+ group increased significantly;(8)Expression of autophagy in aortic aneurysm tissue:The expression of apoptosis markers Cleaved-PARP and Cleaved-Caspase3 in BAPN-METH+ group increased to a certain extent,and BAPN+METH+ group expressed significantly increased. |
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