| BackgroundAdriamycin (ADM), a member of the anthracycline-based antibiotics family, has great advantages such as strong anti-tumor effects and wide antineoplastic spectrum. Adriamycin, as one of the most effective anti-tumor drug at present, is widely used in clinical treatment for leukemia and many other kinds of solid tumors. However, since Lefrak first found and reported the cardiac toxicity of adriamycin in 1973, a growing number of studies have found that ADM can lead to cumulative, dose dependent and irreversible myocardial damage, and eventually can lead to dilated cardiomyopathy (DCM) or congestive heart failure (CHF), etc. People have shown great concerns to the cardiac toxicity of adriamycin and thus its application to clinical treatment has been greatly limited. However, people are still ignorant of the exact cause that leads to cardiac toxicity of adriamycin. At the present time, it is generally believed that the cardiac toxicity is caused by several factors which can influence each other and finally trigger the cardiac toxicity in joint force. It is widely accepted that the mechanism of adriamycin myocardial damage mainly includes the following aspects:free radical damage, abnormal mitochondria damage, energy metabolism, calcium overload, apoptosis, cell shrinkage, etc. In the future, further discussions and verifications of these basic mechanisms are still required in order to provide more luminous ideas and laboratorial data for preventing and treating adriamycin cardiomyopathy, which is an important subject to be solved in applying adriamycin to clinical treatment.A great deal of evidence has shown that inflammation can play a role in the process of myocardial injury. For example, it can affect the myocardial contraction and hypertrophy, promote cell apoptosis and accelerate myocardial remodeling. In addition, many existing researches have confirmed that statins also display a number of pleiotropic properties besides lowering blood lipid, such as anti-inflammatory, antioxidant, anti-free radicals. Through inhibiting hydroxymethyl glutaric acid single acyl coenzyme A (HMG CoA) reductase and then reducing mevalonic acid, statins can help reduce blood fat and resist atherosclerosis so that they are always used as second-level prevention drug for coronary artery diseases. Statins can not only reduce the generation of mevalonic acid, but also can reduce the generation of intermediates in metabolic process at the same time. The reduction of these intermediates, which are important neurotransmitters of intercellular signal transduction, can affect the cell proliferation, differentiation and cell function. These are probably related to satins’ effects beyond lipid-lowering. They can help reduce the level of angiotensin Ⅱ, inhibit the generation of matrix metalloproteinases and cell apoptosis, elevate NO synthesis in blood vessels and endothelium, reduce endothelial injury and improve endothelial function so that they can delay the progression of heart failure, improve the left ventricular function and increase physical endurance of DCM patients. Statins have shed some light for patients who are eaten up with cardiomyopathy and heart failure. Statins can also reduce the NT-proBNP level, and they are effective in curing antiarrhythmia, restoring balance of autonomic nervous system and protecting renal function and blood pressure. Previous studies have shown that statins can reduce oxidative stress of myocardial and blood vessels by inhibiting the Rac induced NADPH oxidase activity and reduce the reactive oxygen species (ROS) of endothelial cells through the TRX S-nitroso, so as to improve endothelial function. In addition, DCM patients are exposed in risks of thrombosis and embolism, while statins can inhibit the formation of fibrinolytic enzyme activation inhibitor-1 and strengthen the effect of tissue plasminogen activator. Statins, therefore, play an important role in promoting fibrinolytic activity and antithrombotic effects.Compared with other existing statins, Rosuvastatin has several advantages:it has a stronger effect, a higher affinity with human enzyme and a reversible inhabitation of enzyme; it only competes with the substrate HMG-CoA rather than common substrates nicotinamide adenine-two nucleotide phosphate (NADPH), so that there is less interaction between different drugs; after using rosuvastatin, liver selectivity becomes better and hepatic metabolism becomes less; it takes a longer time to eliminate half-life so that it can be delivered only once per day; age and gender have no effect on pharmacokinetics, etc.Heart gap junction (GJ), an important way of chemical coupling and electric coupling between myocardial cells, has a part to play in cell growth, proliferation and differentiation. It is also involved in protecting local mechanism of the whole body, maintaining the stability of internal environment, coordinating the synchronization of systole and diastole, and participating excitation-contraction coupling indirectly, etc. Apart from physiological functions, gap junction is also related to many human cardiovascular diseases such as atherosclerosis, myocardial infarction, high blood pressure and cardiomyopathy. GJ is composed of connexins (Cx), among which Cx37 and Cx43 are two main myocardial connexins that differ from each other in expression, distribution and function in the heart. In recent years, there are several studies found that the change of content or distribution of Cx43 may suppress the whole function of heart, causing all sorts of heart diseases. While the change of Cx37 expression and it’s gene Single Nucleotide Polymorphism (SNP) have close relation with some cardiovascular diseases such as atherosclerosis and coronary heart disease.In recent years, many animal experiments and clinical studies have confirmed that statins can improve various ischemic or non-ischemic cardiomyopathy, and protect myocardial injury so as to increase the survival rate. In addition, several studies have found that abnormal expression, re-distribution and change of phosphorylation in connexins, such as Cx37, Cx40 and Cx43, are found in heart failure, arrhythmia, myocarditis and other heart diseases. While statins can repair connexin’s abnormal expression, protect connexins away from hyperphosphorylation, restore its distribution so as to protect myocardial cells and maintain the integrity of intercellular gap junction. More and more studies have shown that statins have a variety of effects besides lipid-lowering and at least a part of its protective effect on heart is related to its ability to adjust connexin expression and distribution. For all this, it is still not clear that how does adriamycin cause myocardial injury. Besides, at the time being, there is no report about rosuvastatin’s protective effects on the damage or its relationship with Cx43, whether at home or abroad.Objectives This study adopted the ADM tail intravenous injection method, established adriamycin DCM and chronic congestive heart failure model, and used Rosuvastatin to intervene the ADM-DCM rats. The experiment was aimed at detecting different indexes in ADM-DCM rats, evaluating whether doxorubicin would cause changes in Cx43 expression or abnormal distribution in rat myocardial cells and discussing rosuvastatin’s protective effects on myocardial injury caused by adriamycin and its possible influence on myocardial Cx43. The objectives were to explore the key roles Cx43 and the gap junction played in cardiomyopathy triggered by adriamycin, to discuss the possible mechanisms of rosuvastatin’s protective effects on myocardial injury, and to provide basic research data which can help to find effective drugs to prevent and control adriamycin-induced cardiomyop thy and heart failure.Methods Twenty-four healthy male six-week-old SD rats, all weighted 220～290g for the experiment. All rats were randomly divided into three groups:1) the normal Control group (Control, n=8):the rats were injected with the same amount of saline (tail vein injection), and were lavaged with double steaming water instead of drug therapy.2) the adriamycin group (ADM, n=8):the rats were injected with adriamycin (1.67mg/kg/day, dosing every other day at 8:00 am, single tail intravenous injection) for 3 weeks. At the same time, they also underwent lavage with double steaming water instead of drug therapy.3) the rosuvastatin group (RSV, n=8):the rats were injected with adriamycin (1.67mg/kg/day, dosing every other day, single tail intravenous injection) for 3 weeks. At the same time, they accepted rosuvastatin gavage therapy (11.54 mg/kg/day, dosing every other day) for 21 days.The weight of the rats and the change of some general situations were recorded every day. All rats were executed on the 21st day of the experiment to collect specimens for further studies and analysis.In the third week after the rats were injected, they were narcotized with 10% chloral hydrate (0.2 ml for each one) and fixed for transthoracic echocardiography examination. The cardiac structure and cardiac function change of the rats were observed. Take left ventricular long axis view and left papillary muscle level of M type ultrasonic mode to record LVEDd, LVEDs, and then measure the LVEF%, LVFS%, IVS and LVPW. The above measurements should be the average value of three consecutive and complete cardiac cycles. After the echocardiography examination, the rats’blood was collected from their abdominal aorta by a microcapillary tube (5ml) and then was pumped into a 5ml centrifuge tube for 30 minutes. Next, separate serum by centrifugation at 3000 rpm for 15 minutes and then store the serum in a centrifuge tube at a 4℃ refrigerator. The serum was used for detecting biochemical parameters, which include ALT, AST, LDH, CK, BNP, TC, TG, LDL-C, VLDL-c, HDL-C and TC/HDL and LDL-C/HDL-C. The rats were then killed and their hearts, livers and left ventricles were removed, flushed with prepared cool saline and then weighed after the surface saline was absorbed by a filter paper. Calculate the whole heart hypertrophy index, left ventricular hypertrophy index and liver coefficient. Whole heart hypertrophy index=(the whole heart weight/body weight, HW/BW)×100%. Left ventricular hypertrophy index=(left ventricular weight/body weight, LVW/BW)×100%. After being weighed, the hearts and livers were fixed with 10 times 4% paraformaldehyde for a whole night, and then they were dealt with tissue cutting, paraffin embedding, routine sectioning, HE staining, gradient alcohol dehydration, transparentization and then were sealed so that they can be observed by the optical microscope later. Use immunohistochemical method to detect the expression and distribution of Cx43 and waiting for testing myocardial cell apoptosis in rats by TUNEL examination method and observing myocardial ultrastructure with transmission electron microscope.Results1. General situationsGeneral situations of the rats were observed and recorded every day. In the first four days, rats of each group all behaved normally and their hair were soft and glossy. However, since the sixth day, rats of the ADM group began to show a low spirit and they ate much less than before and lost gloss of their hair. In the last phase of the experiment, the ADM rats even caught hyperaemia in their eyes, diarrhea and some other symptoms. On the 14th day, a rat died and later two rats died respectively on the 17th and 19th day. The mortality rate of the ADM group was 37.5%. The rats in the RSV group showed similar symptoms with the ADM group, but the time when they began to show the symptoms was much later, and the symptoms were less serious with only one rat died at the 20th day, which means the mortality rate was 12.5%. It is probable that it was left heart failure that led to the death of the only rat in the RSV group. The rats in the Control group showed well general situations with none of the rats died. The mortality rate of the ADM group and the Control group showed a rather big difference while there was no significant difference between the RSV group and the ADM group in terms of mortality rate.2. The weight of rats, the weight of hearts and heart weight coefficientThe change of weight of rats in each group was recorded every other day. The rats in the Control group gained weight while the rats in the ADM group apparently lost weight. There were significant differences between these two groups. The weight of rats in the RSV group declined when compared with the Control group, but there was no statistical difference between these two groups. Compared with the Control group, hearts of rats in the ADM group gained weight, which made their whole heart hypertrophy index become higher than that of the Control group. Compared with the ADM group, hearts of those rats that had accepted rosuvastatin therapy lost weight. Compared with the Control group, the weight of the left ventricle of the rats in the ADM group increased, while that of rats in the RSV group significantly decreased. But there were no significant differences of whole heart hypertrophy index and whole left heart hypertrophy index between these two groups and the Control group. Compared with the Control group, the hepatic index of rats in the ADM group decreased and that in the RSV group increased. But there was no statistical difference of liver index between these two groups and the Control group.3. The effects of rosuvastatin on cardiac function influenced by adriamycin-induced myocardial damage.The serum indexes have shown that indexes such as ALT, AST, LDH, CK and BNP significantly increased in the ADM group compared with the Control group. The differences between these two groups were significant. However, the indexes mentioned above of the RSV group decreased significantly compared with the ADM group, which confirmed that rosuvastatin have alleviated the myocardial injury induced by adriamycin and have improved the cardiac function that had been injured by adriamycin.4. The effects of rosuvastatin on blood lipid influenced by adriamycin-induced myocardial damage.TG, TC, LDL-C, VLDL-c and atherogenic indexes (TC/HDL and LDL-C/HDL-C) in the ADM group increased when compared with those in the Control group. The indexes mentioned above in the RSV group decreased compared with the ADM group. However, there was no significant change in terms of TG, TC, LDL-C, VLDL-c levels and atherogenic indexs (TC/HDL and LDL-C/HDL-C) between each groups (P>0.05). The HDL-C level in the ADM group decreased compared with the Control group, while there was also no significant change of HDL-C level in the RSV group compared with the Control group. All these have revealed that the dose of rosuvastatin did not affect the blood lipid level distinctly.5. The change of cardiac structure and cardiac functionThe results of transthoracic echocardiograph examination showed that LVEDd and LVEDs of rats in the ADM group increased significantly, while LVEF% and LVFS% reduced significantly compared with the Control group. The differences were statistically significant. IVS and LVPW increased in the ADM group but there was no statistically significant difference between the ADM group and the Control group. Compared with the Control group, hearts of rats in the ADM group rats showed several symptoms:cavity expansion and remodeling (especially in the left ventricular), non-significant hypertrophy of the ventricular wall and significant reduction of cardiac systolic function. All the symptoms confirmed to DCM pathological changes. LVEF% and LVFS% level of rats in the RSV group distinctly increased, while LVEDd and LVEDs significantly reduced compared with the ADM group. IVS and LVPW level also fell but the difference between the two groups was not significant. It is shown that the RSV group significantly improved the ventricular remodeling and prevented cardiac malfunction caused by adriamycin.6. The change of myocardial tissue pathologyHearts of rats in the ADM group obviously enlarged which can be seen by visual inspection. Grey lesion areas on the surface of the hearts, heart dilatation, ventricular cavity expansion and muscle relaxation could also be visible. In the RSV group, the hearts were smaller with less surface lesion area and the expansion of four heart cavities was less obvious than that of the ADM group. It could be observed through the HE staining microscope that in the ADM group, myocardial cells bleeding, myocardial fibers hypertrophy, muscle fibers arrangement disorder, muscle fiber fracture, nucleus pycnosis and deformation could be observed. There were also vacuoles in cytoplasm. Heart tissue structure was basically normal in the RSV group, meaning most of the hearts did not show myocardial cell bleeding. The myocardial muscle fiber structure was clear with fine color and intact nucleus. Pathological results showed that rosuvstatin could reduce the morphological changes of myocardial tissue induced by adriamycin in rats. In addition, there were severe liver injuries shown in rats of the ADM group. For example, the livers have shown large areas of diffuse hemorrhage; the hepatic lobule structure was obscure; hepatic cells showed to dissolve putrescence and the lymphocytes tended to aggregate. Rats in the RSV group also showed severe liver cell injuries such as diffuse hemorrhage, liver cell swelling, nuclei cytolysis and cell necrosis. All these indicated that rosuvstatin could alleviate myocardial injuries induced by adriamycin, but it could not alleviate liver injuries.7. The analysis of Rosuvstatin’s effects on the expression and distribution of Cx43 in myocardial tissue by using immunohistochemical methodIn the Control group, Cx43 positive particles distributed regularly in alignment in the intercalated disc areas and connected with each other end-to-end. Myocardial cells were neatly arranged with integral structures. There was no degeneration necrosis in myocardial cells and the nucleus structure was clear. Compared with the Control group, Cx43 positive particles in the ADM group obviously increased and were in disorder. Most of the particles were located in the side of myocardial cells or inside of the cells. The myocardial tissue boundary was obscure and was seriously damaged, which manifested that the Cx43 myocardial gap junction remodeling had take place in this group. Compared with the ADM group, Cx43 reduced in the RSV group and the Cx43 positive particles regularly distributed in the intercalated disc areas, which was similar to the Control group. The results suggested that rosuvastatin could inhibit abnormal expression of Cx43 in left ventricular myocardium of rats caused by adriamycin, and improve the Cx43 gap junction remodeling.8. The analysis of Rosuvastatin’s effects on myocardial cell apoptosis injured by adriamycin in rats by using the TUNEL methodRosuvastatin’s protective effects on cardiomyocytes of rats injured by adriamycin were observed by adopting the TUNEL technique. The results showed that the colored nuclei were positive cells, while there were no or rare apoptosis myocardial cells in rats of the Control group. However, in the ADM group, apoptosis of cardiomyocytes could be observed in a wide distribution range. Myocardial cell apoptosis in the RSV group significantly decreased when compared with the ADM group.9. The changes of the myocardial ultrastructureThe myocardial structure of intercalated disc was disorder in rats of ADM group, partial interruption, gap junction distribution in disorder, which is far away from the intercalated disc, forming side to side connection between myocardial cells. Intercalated disc structure in RSV group was basicly normal, gap junctions distribution were by rules and were located in the ruffle of intercalated disc mostly.Conclusions1. Indexes such as the general situations, the whole heart hypertrophy index, left ventricular hypertrophy index, echocardiography, myocardial enzymology, serum BNP level and pathological cell morphology have shown that Adriamycin could induce dilated cardiomyopathy in rats.2. Compared with the Control group, adriamycin could induce more abnormal expression and disorganized arrangements of Cx43 in rats, most of which located at the side of ventricular muscle cells or inside of the cells. It indicated that Cx43 myocardial gap junction remodeling had taken place in myocardium of rats injured by adriamycin.3. Apoptosis of cardiomyocytes could be observed in a wide distribution range in the ADM group by using TUNEL technique. It suggested that apoptosis of cardiomyocytes significantly increased because of adriamycin.4. Rosuvastatin could inhibit abnormal expression and distribution of Cx43 in left ventricular myocardium and improve the Cx43 gap junction remodeling.5. Rosuvastatin could inhibit myocardial cell apoptosis caused by Adriamycin.6. There was no huge difference among the three groups in terms of blood lipid indexes.The effects of rosuvastatin on Cx43 expression and myocardial cell apoptosis may be caused by its other functions other than lowering blood lipid. |
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