| Research background:Methamphetamine(methamphetamine,METH)is a kind of new synthetic drugs,easily soluble in water and alcohol,and with benzene ring structure,which is very similar to catecholamine neurotransmitter structure.Moreover,due to the appearance of like ice,commonly known as "ice",is the most common synthetic drugs.Over the past few years,the number of methamphetamine seizures accounted for the largest share of annual global amphetamine seizures,because of its quick effect,long duration of excitement,simple synthesis technology,which has become the world's second most widely used illegal drugs for the moment.At present,the quantity of METH abusers is estimated to be 33 million in the whole world,and since the 90s of last century,the abuse of METH has become more and more serious in China,it showing characteristics like gradual expansion of the region,sharp increase in the rate of abuse,age structure of younger and so on.What's more,the abuse of methamphetamine not only damage their own health,but also caused a series of social cases,have a serious negative impact on society.In the daily practice of forensic medicine,also encountered a lot of sudden death cases caused by METH.Therefore,the study of METH induced toxicity has become a hot research topic in the world.After methamphetamine brings brief mental and physical pleasure and pleasure,the direct side effect is brought great damage to the body and health.Research shows that methamphetamine can produce many adverse reactions,including neurotoxicity,neuropsychological deficits and cardiovascular toxicity.The great effects on the nervous and cardiovascular system is caused by the release of monoamine neurotransmitters,including dopamine,norepinephrine and 5-hydroxy tryptamine.With methamphetamine abuse intensifies,the reports of cardiovascular injury are increasing,the long-term abuse of methamphetamine,in addition to human body of the nervous system have serious adverse consequences,the cardiovascular system will also appear serious pathological damage,leading to the occurrence of many diseases.For example,acute methamphetamine intoxication can activate the sympathetic nervous system,causing arrhythmia and hypertension,and suck methamphetamine for long time can cause myocardial infarction,coronary heart disease,myocardial fibrosis and aortic dissection etc.Therefore,more and more researchers focus on it.Clinical observation and autopsy results showed that METH abusers cause serious cardiovascular diseases,such as arrhythmia,myocardial ischemia,aortic dissection,hypertension,or hypertrophic cardiomyopathy,acute coronary syndrome,congestive heart failure and sudden death is often seen.Research shows that after the treatment of methamphetamine can lead to catecholamines such as norepinephrine release,its release can directly stimulate cardiac a receptor,which can enhance the expression of collagen fibers,leading to fibrosis,secondly,the excessive release of catecholamine can lead to vasospasm,ischemia,reactive oxygen species and mitochondrial damage and so on.It is reported that after methamphetamine function in myocardial cells,the first occurrence is mitochondrial dysfunction(Morphological study of acute myocardial lesions experimentally induced by methamphetamine).Based on the above research,and on the basis of the previous research,this project is based on the existing model of treatment methods,explore to establish an animal model of methamphetamine induced myocardial fibrosis,and at the same time,from animal experiments and cell experiments to further explore the molecular mechanism of methamphetamine induced myocardial apoptosis.Objective:This project through the establishment of animal models in vivo and cell model in vitro of methamphetamine poisoning,to preliminary study on mechanism of methamphetamine induced myocardial fibrosis and apoptosis,explore the establishment of animal model of myocardial fibrosis induced by methamphetamine,and through the iTRAQ technology to find the differential expressed genes,lay the foundation for the follow-up study of the mechanism.On the one hand,explore the action mechanism of PUMA on myocardial apoptosis induced by methamphetamine on existing animal models and verify on the primary myocardial cells.On the other hand,detection of the expression and apoptosis of related proteins through proteomics and immunofluorescence technique.Finally,complete the establishment of fibrosis model induced by methamphetamine and elucidate the role of PUMA in myocardial apoptosis,and provide a theoretical basis for the study on the mechanism of METH induced cardiotoxicity and gene intervention therapy.Method:The first part:the establishment of the model of myocardial fibrosis induced by methamphetamine.1.Determination of the concentration and time of administration of isoproterenolSelected 180 g-200 g SD male rats,then set up 0.1mg/kg,0.5mg/kg,1.0mg/kg three concentration gradient,and divided into three days group and five days group,finally,MASSON staining was used to determine the optimal concentration of isoproterenol.2.The degree of fibrosis was detected by MASSON stainingSelected 180g-200g SD male rats,divided into four groups,namely control group,isoproterenol group,methamphetamine group,isoproterenol plus methamphetamine group.We treated the control group with commensurable saline injection,at the same time,the isoproterenol plus methamphetamine group was treated with morning 10:00 injection of isoproterenol(0.1mg/kg),14:00 PM and 19:00 intraperitoneal injection of methamphetamine(5mg/kg),continuous injection of 30 days.Then,the heart tissue was fixed and stained with MASSON.3.Detection of differentially expressed proteins by iTRAQAt the end of the drug's administration,the rats were sacrificed at the same time in the second day,and the fresh apical tissues were taken immediately and placed at-80?,each group was given 9 apical tissues,randomly divided into 3 groups,then we detected differential proteins by iTRAQ technique.The second part:the role of PUMA in METH induced cardiac apoptosis1.Establishment of primary myocardial cells and animal models of METH poisoning(1)Culture of primary myocardial cell.Taked 1?3 days new birth of SD suckling mice,rapidly extracted the myocardial tissue,and 0.2%trypsin incubated overnight.The second day,cells were digested with collagenase type two and recovered the suspension,then added with high glucose DMEM after centrifugation and the samples were inoculated in the 10cm culture dish after mixed.After 90 minutes,the culture medium was collected and counted and inoculated at the pore of6 holes.The inoculation density was 4?6×105 and incubated at 37?and 5%C02.When the cell density reached 70%,respectively,with the use of 0 mmol/L,0.6mmol/L,0.9mmol/L,1.2 mmol/L,1.5 mmol/L METH culture medium of 10%FBS for 24 hours,then extracted the total protein and used western blot to detect the expression of PUMA protein in cell,and then the appropriate concentration was selected to establish the model of cell poisoning.(2)Through the above experiments,selected the appropriate METH concentration,with the concentration of METH solution respectively treated with 0 hours,2 hours,4 hours,8 hours,16 hours,24 hours to establish the cell model of poisoning.After the end of treatment,the total proteins were extracted.The expression of PUMA protein in cell was detected by Western blot,selected the appropriate METH action time according to the result.(3)The animal models of METH poisoning were established by referring to the literature and the previous modeling methods,we selected 200g-250gSD male rats,established subacute poisoning model in rats(15mg/kg,Injection 1 times every 12 hours,,a total of 2 injections,continuous injection of 4 days)and chronic poisoning model(gradient increased to 5mg/kg,continuous injection of 14 days),the animals were sacrificed at the end of the last injection,and the myocardial tissue protein was extracted.The expression of PUMA and mitochondrial apoptosis pathway related marker protein(Bcl2 and Bax)was detected by Western blot.2.The role of PUMA in METH induced cardiomyocyte apoptosis(1)PUMA siRNA interference fragments were designed for myocardial cells,and the cells were treated with Lipo 3000 liposome transfection method,the expression of PUMA protein in cells was detected by Western Blot,then selected the effective interference fragments by the above methods.(2)On the basis of the above experiments,the myocardial cells were divided into 6 groups,respectively named blank control group(Ctrl),control+small interfering group 1(siPUMA#1))control+small interfering group2(siPUMA#2),medication administration group(MEH),medication administration+small interfering group 1(siPUMA#1),medication administration+small interfering group 2(siPUMA#2).After 24 hours,collected the cell protein to detect the expression of PUMA,Caspase3,PARP.(3)Detected cell apoptosis in each group by using TUNEL staining.3.PUMA mediates METH induced cardiomyocyte apoptosis via mitochondrial pathway(1)Detected the expression of Bcl-2 and Bax in these groups,calculated and compared the ratio of Bax to Bcl-2.(2)Repectively extracted the cytosolic and mitochondrial protein in each group,then detected the changes of Cytc expression in cytoplasm and mitochondria.Results:Part I:Establishment of methamphetamine-induced myocardial fibrosis models1.Determination of isoproterenol administration concentration and timeIsoproterenol in the 0.1 mg/kg three-day group has appeared fibrogenic changes,0.1 mg/kg five days showed significant changes in fibrous proliferation.the higher concentration and the longer time,the more severe of the degree of fiber changes.By comparison,we finally determined that isoproterenol was administered at a dose of 0.1 mg/kg for three days.2.The result of MASSON staining in each groupIn the four groups,the fibrosis of methamphetamine and isoproterenol group was the most severe,and the fibrogenic hyperplasia was observed in the isoproterenol group and methamphetamine group in the cardiomyocyte gap,but the degree of fibrosis is not serious,the saline group did not see fibrosis changes.The result of iTRAQ protein analysisThe results of iTRAQ differential protein analysis showed that the expression of Collagen3al and Fibnection were up-regulated in the methamphetamine group.Part II:The role of PUMA in methane-induced cardiac apoptosis1.Establishment of METH-induced primary cardiomyocyte model and animal model(1)primary cardiomyocytes were treated with 0 mmol/L,0.3 mmol/L,0.6 mmol/L,0.9 mmol/L,1.2 mmol/L and 1.5 mmol/L METH solution,respectively.Western Blot showed that the expression of PUMA increased with the increase of the concentration of METH,the expression level of PUMA increased the most significant difference compared 0 mmol/L group with 0.9 mmol/L group.(2)Primary cardiac myocyte were treated with 0.9 mmol/L METH solution for 0 hours,2 hours,4 hours,8 hours,16 hours,24 hours.,Western Blot showed that the expression of PUMA increased gradually with the prolong of METH treatment time,and the expression of PUMA began to increase at 2 hours and peaked at 16 hours.2.The role of PUMA in methane-induced cardiomyocyte apoptosis(1)The expression of PUMA protein decreased after the specific siRNA interference fragment relative to the use of the METH group alone.The expression of PUMA was significant lower than the METH group after Using of METH + siPUMA#1 and METH + siPUMA#2.The expression of Caspase3 and PARP protein in the METH group was increased compared with the control group.The expression of Caspase3 and PARP protein in the METH + siRNA group was decrease compared with the METH group.(2)TUNEL method was used to detect the apoptosis of each group.The results showed that there was no significant difference between siPUMA#1 and siPUMA#2 groups compared with the control group.While the number of positive apoptotic cells in the METH group was significantly increased.METH + siRNA group compared with METH group,the cells of both groups were significantly decreased.3.PUMA mediates METH-induced apoptosis of cardiomyocytes through mitochondrial pathway(1)The expression of Bcl-2 and Bax protein was detected by Western Blot after the administration of the specific siPUMA interference fragment,and the expression of Bax was increased and Bcl-2 was decreased in the METH group compared with the control group after 24 hours of administration.And the ratio of Bax/Bcl-2 increased,while that of METH + siRNA group was reversed compared with METH group,Bax expression was decreased,Bcl-2 expression was increased and Bax/Bcl-2 ratio decreased.(2)After the specific siPUMA interference fragment was used,the cytoplasmic protein and mitochondrial protein were extracted after 24 hours of administration.The cytochrome C protein was detected by Western Blot.It was found that the expression of cytochrome C in the mitochondria was decreased and the expression of cytochrome C in the cytoplasm was increased compared with the control group,while that of METH + siRNA group was reversed compared with METH group,the expression of cytochrome C in the mitochondria increased,while the cytochrome C expression in the cytoplasm decreased.Conclusion:Part I:Establishment of methamphetamine-induced myocardial fibrosis model1.Methamphetamine and isoproterenol combined,using method of isopropyl adrenaline is 0.1 mg/kg for three days.2.Isoprenaline can induce cardiac fibrosis,combined with methamphetamine has synergistic effect to induced myocardial fibrosis.3.The results of iTRAQ differential protein analysis showed that the expression of Collagen3al and Fibnection was up-regulated in the methamphetamine group.Part II:The role of PUMA in methamphetamine-induced cardiac apoptosis1.PUMA expression was increase in METH-treated cells and animal models.2.After treatment with MEH,the apoptosis of primary myocardial cells and myocardium tissue was increased,the apoptosis decreased after knockdown of PUMA In the cell model.3.The expression of Bcl-2 and Bcl-2 were decreased after the treatment of primary myocardial cells with METH,and the expression of Bcl-2 was decreased after knockdown of PUMA.4.After treatment of primary cardiomyocytes with Methamphetamine,cytochrome c from the mitochondrial outflow cytoplasm increased;The cytochrome C from the mitochondria out of the cytoplasm was blocked after knock down the PUMA.5.The expression of Caspase3 and PARP was decreased after treatment of primary cardiomyocytes with METH,and the expression of Caspase3 and PARP decreased after knockdown of PUMA. |
PDF Full Text Request