Research On Cardiac Myocytes Apoptosis Induced By Methamphetamine In Rats

[Background]Methamphetamine (MA), the subtype of amphetamine, was abused widely in the world. As the situation of drug dependence and abuse become more and more serious, it trends to be the most harmful.drug in the 21st century instead of morphine and cannabis sativa. Hence, methamphetamine abuse is considered as a global social problem. Numerous experiments and clinical data indicate that methamphetamine can lead to the lesions in cardiovascular, digestive, endocrine, urinary and genital systems, not just causing mentation and neurotoxicity. Of all these systemic dysfunctions, cardiovascular disease is the most important and fatal lesion, and most causes of deaths of MA abusers were demonstrated to be the angiocardiopathy and complications. For that reason, researches of the cardiovascular-toxicity of methamphetamine become hot issue for related areas gradually.Methamphetamine can lead to various cardiovascular diseases, and the cardiotoxicity of MA which caused sudden death was found by autopsy and pathological examinations. Recent studies about the cardiotoxicity of MA focus on the mitochondria damages of cardiac myocytes induced by MA, such as inner mitochondrial membrane swelling and cristae disappearing. Moreover, related experiments have found that Cytochrome C derived from mitochondrial membrane space was released into cytoplasm and caused a cascade of Caspase reactions which resulted in cell death finally.Therefore, it is widely believed that myocardial damage induced by MA is in connection with mitochondria death pathway. But there are still many unclear aspects on research of cardiotoxicity, for example:1. although it is known to all MA may lead to cardiac myocytes damages, cardiac myocytes apoptosis induced by MA that cannot be demonstrated for lack of evidence by far; 2. Caspase is the regulatory protein for cell apoptosis which locates in the downstream sequence. Myocardial damage caused by MA is assumed to be related to the Caspase cascade reactions. However, effects of the gene which is in upstream sequence of Caspase and related to apoptosis have not been discussed in studies of cardiotoxicity induced by MA. The effects and mechanism of methamphetamine in cardiotoxicity and myocardial damage have significant research values.[Objective]To demonstrate that MA could cause myocardial damage by observing cardiac myocyte apoptosis after exposure to MA.To study on the change of expressions of Fas, FasL, Bax and Bcl-2 in cardiac myocytes.To find out the mechanism of myocardial damages induced by MA.[Method]1. Establish the rats MA poisoning models:According to different administration time, all rats of experimental group were divided into three sub-groups, including acute group, subacute group and chronic group. Dose:3mg/kg. Form of injection:int raperitoneal injection once a day. The acute group was keep in giving drug for 3 days, the subacute group for 2 weeks and the chronic group for 8 weeks, while equal volume of saline was injected in control group.2. Detecting cardiac myocytes apoptosis in MA poisoning rats:The apoptotic cell was situ labeled by TUNEL method, and then compared the changes of apoptotic cardiac myocytes induced by MA in three groups.3. The expressions of Fas, FasL, Bax and Bcl-2 were detected by immunohistochemistry in the cardiac myocytes of MA poisoning rats. Analyze the changes in three experimental groups.4. Detecting the mRNA of Fas, FasL, Bax and Bcl-2 by RT-PCR method in the cardiac myocytes of MA poisoning rats. Compare the changes of the expressions of mRNA of Fas, FasL, Bax and Bcl-2 in three experimental groups. [Result]1.TUNEL assayOnly very few apoptotic cells could be found in control group, while they appeared or increased in the acute, subacute and chronic groups, which were diffused distribution and had no distinctive features. In acute, subacute and chronic groups, apoptotic positive indexes increased gradually as administrate time extended. No statistic difference was between the acute group and the subacute group, but compared with the chronic group, there was significant difference between it and the other two groups.2. Immunohistochemistry results of Fas,FasL, Bax and Bcl-2Compared with control group, Fas and FasL increased significantly in each experimental group(P<0.05). The highest expressions of Fas and FasL were found in the chronic group but lower in acute and subacute groups by analysis of group comparison in three experimental groups. Also, there was no significant difference between acute group and subacute group (P>0.05).From another point of view, Bax and Bcl-2 increased in acute, subacute and chronic groups, which tended to increase with time elapsing. And there was significant difference between experimental groups and control group (P<0.05). Besides, the level of Bax/Bcl-2 expressions in subacute group was lower than in acute group but there was no significant difference.3. RT-PCR findingsThe expressions of Fas/FasL mRNA increased significantly in both acute and subacute groups compared with control group (P<0.05). And the expression of FasL mRNA in subacute group was lower than in acute group without significant difference (P>0.05).The expressions of Bax/Bcl-2 mRNA in acute and subacute groups were significant higher than control group (P<0.05). The expression of Bcl-2 showed gradually increasing trend and it was higher in subacute group than in acute group. Bax and the ratio between Bax and Bcl-2 were found increased in acute group but decreased in subacute group which even lower than it was in control group.[Conclusion]1.methamphetamine and cardiac myocyte apoptosisMethamphetamine can lead to the apoptosis in cardiac myocytes, which may be the major pathway to cause myocardial damage. Our studies indicated that there was slightly difference between 3 days of injection with MA and 2 weeks, but serious myocardial damage could be found after long-term using MA.2. The relationship between cardiac myocytes apoptosis induced by MA and the expressions of Fas/FasL and Bax/Bcl-2(1) The effect of MA on Fas/FasL and Bax/Bcl-2Fas/FasL and Bax/Bcl-2 showed different changes in acute, subacute and chronic groups. It indicated that MA could cause apoptosis of cardiac myocytes by regulating the expressions of Fas/FasL and Bax/Bcl-2. We believe that Fas/FasL and Bax/Bcl-2 play an important part in cardiac myocytes apoptosis induced by MA.(2) Immunohistochemistry findingsWith the time of giving MA extending, the expression of Bcl-2 increased gradually. However, Fas/FasL in subacute group was slightly lower than in acute group and there was no significant difference between them by the method of TUNEL. So the expression of Fas/FasL in cardiac myocytes apoptosis induced by MA was negatively regulated by Bcl-2. After 8 weeks, apoptosis of cardiac myocytes in chronic group increased significantly compared with subacute group, and so did Fas/FasL. Meanwhile, Bcl-2 increased slightly and the ratio between Bax and Bcl-2 decreased markedly. It suggested that the negative regulation of Bcl-2 was diminished with time of exposure to MA increasing.(3) RT-PCR resultsBy analyzing the expressions of all target genes and comparing them with immunohistochemistry results mentioned above, we found several distinctions. The expressions of Bcl-2 mRNA were the same as immunohistochemistry results, which showed that the positive findings were higher in subacute group than in acute group and much higher than in control group. In contrast, Bax mRNA was observed increasing at beginning and then decreasing with the time extending, but all the results of experimental groups were higher than control group. Immunohistochemistry finding of Bax suggested that level of Bax expression increased a bit in subacute group compared with acute group. We believed that Bax revealed slow-growth and even reduction after 2 weeks exposure to MA, and it resulted in the significant decline of the ratio between Bax and Bcl-2,even lower than the ratio of control group.TUNEL method was used to detect cardiac myocytes apoptosis induced by MA. We found that the longer exposure to MA was, the more apoptosis cell could be observed. But no remarkable difference was showed between 3 days and 2 weeks. Fas/FasL and Bax/Bcl-2 gene expressions showed up-regulated or down-regulated rather than simplex increase or decrease in both 3 days(acute) and 2 weeks(subacute) groups by the means of immunohistochemistry and RT-PCR detecting. It suggested that drug resistance and self-regulation of body developed gradually over time, especially the anti-apoptosis gene Bcl-2 presented difference in these two groups with different detection methods. Bcl-2 is presumed to play an important role in self-regulation and self-healing of body, also to be the significant factor of cardiac myocytes apoptosis induced by MA.In chronic group, apoptosis of cardiac myocytes increased dramatically due to long-term exposure to MA (8 weeks). The immunohistochemistry results showed that the related genes of apoptosis, Fas/FasL and Bax/Bcl-2, significantly increased again. When the time of exposure to MA exceeds a certain point, myocardial damages become irreversible and apoptosis cell and related genes increased markedly once again.