| Background and purpose:Along with the improvement of therapeutics, the number of long-term survivors of malignant hematology diseases and solid tumors is increasing. It has been taken attention that how to lessen side effects caused by chemical therapy and then promote life quality of patients. Chemotherapy is the main method, and anthrocycline is the most important one among those drugs. They have been used in curing leukemia, lymphoma and a lot of solid tumors. However their cardiotoxicity limits their use. It will increases dosage and than promote healing rate to protect heart from cardiotoxicity. Daunorubicin (DNR), one of anthrocyclin drugs, is important in therapeutic schedule of children leukemia. It is applied in almost all proposals of acute leukemia. We developed this experiment for following purpose (1) to observe ultrastructure alter of myocardial cells, moniter the change of serum enzymology, and clarity the detail mechanism of daunorubicin cardiotoxicity, find a useful way by wich we can monitor cardio lesion in clinical. (2) to investigate the possible protective role and mechanism of nuerugulin, the gland of ErbB2 a member of endothelium growth factor, in that to find a new way of myocardium protecting. (3) to make sure the effect and its protect mechanism of zinc sulphate in cardiomyopathy, and study whether zinc sulphate interfere cytotoxicity of daunorubicin in vivo.Method:Twenty wistar rats (4 weeks old, 120—150g) were individually housed in Macrolon cages at a constant temperature of 26-27 °C with 12h light-dark cycle for 1 week prior to starting the experiment. They were randomized into two groups. Rats in group DNR were injected s.c. with DNR 3.5 mg/ kg once a week for four weeks. Another ten rats were injected s.c. with physiological saline served as control groups.The extent of cardiac muscle cell lesions was rated under a light microscope employing Billingham scale. Apoptosis was tested by TdT-mediated dUTP nick end labelling(TUNEL) so to get apoptosis indexes. Cardiac function representing by ejection fraction (EF) of rats were tested using ultrasonic cardiography. Glutathione peroxidase (GSH-PX), maleic dialdehyde (MDA), CuZn superoxide dismutase (CuZn-SOD) and reaction oxygen species(ROS) were tested in rat cardiac muscle. Cardiac troponin T (cTnT) was tested in serum of rats by enzyme-linked immunosorbant assay (ELISA). Expression of ErbB2mRNA in myocardial cells by reverse transcription polymerase chain reaction (RT-PCR).Wistar rats (4 weeks old, 120-150g) were randomized into three groups. Rats were injected s.c. with 3.5 mg DNR/ kg once a week for four weeks as group DNR. Another ten rats injected s.c. with 3.5mg DNR/ kg once a week for four weeks and injected by caudal vine with neuregulin 10 u g/kg. b.w everyday for 5 days from first injection of DNR as N group. Rats were injected with physiological saline served as control groups. Maleic dialdehyde and superoxide dismutase were tested in rat cardiac muscle. Apoptosis was tested by TUNEL assay and counted apoptosis index. Cardiac function represented by ejection fraction was tested using ultrasonic cardiography. The expression of ErbB2 mRNA was tested by RT-PCR.Thirty wistar rats (4 weeks old, 120-150g) were individually housed in Macrolon cages at a constant temperature of 26-27 °C with 12 h light-dark cycle for 1 week prior to starting the experiment. They were randomized into three groups.Rats in group DNR were injected s.c. with 3.5 mg DNR/ kg once a week for four weeks. Ten rats in group zinc sulphate were injected s.c. with 3.5 mg DNR/ kg once a week for four weeks, and pour ZnSCU into stomach with 8mg/kg. b.w everyday until last injection. 10 rats were injected with physiological saline served as control groups. Glutathione peroxidase, maleic dialdehyde and superoxide dismutase(SOD) were tested in rat cardiac muscle. Extent of cardiac muscle cell lesions was rated under a light microscope employing Billingham scale. Cardiac functions of rats were tested using ultrasonic cardiography. The expression of metallothionion (MT-1) mRNA was detected by RT-PCR.Seven concentrates(0.25~16ug/ml) Zinc sulphate were added in 96 wells plate with human acute T-lymphocyte leukemia cell line (CEM) as group ZnSC>4. 7 concentrations (0.001~0.512ug/ml) DNR were added in 96 wells plate with CEM. 7 concentrations (0.001~0.512ug/ml) DNR and zinc sulphate (8ug/ml) were added in 96 wells plate with CEM as a group DNR+ZnSC>4. All plates mentioned above were incubated in incubator under 37 °C -. 5%CO2 for 48 hours. The rates of inhibition were tested by MTT assay.DNR (0.016ug/ml) was group DNR. DNR ( 0.016ug/ml ) and Zn sulphate(2ug/ml) was DNR+ ZnSC>4. The two groups with CEM were incubated for 24 hours. Apoptosis was tested by flow cytometer.DNR (0.016ug/ml) with CEM was added in 24 wells plate as group DNR. DNR (0.016ug/ml) and Zn sulphate (2ug/ml) were aded in 24 wells plate as group DNR+ ZnSC?4. These two groups were incubated for 24 hours. CEM without any drugs was control. Giemsa stain. Morphologic change was observed by light microscope and apoptosis cells were counted so we concluded a apoptosis index. ResultScale counted by Billingham assay is significantly high in group DNR comparing with control group (t=6.83, p<0.001) . Group N showed lower Billinghamscale than group DNR (t=2.97, p<0.01) , and higher than control (t=2.84, p<0.05) . Billingham scale in group zinc sulphate were lower than in group DNR (t=2.4, p<0.05) and higher than control (t=3.76, p<0.01) .There are obviously degenerative changes with a mitochondrion gather and mitochondrion vacuolization, myofibrillar rarefaction, Z line vague.The apoptosis index increased significantly in group DNR comparing with control group (t=15.99, pO.Ol ) . Group N appeared a lower apoptosis index than group DNR (t=14.75, p<0.01) and a same apoptosis index with control (t=1.06, P>0.05) .Ejection fraction was lower in group DNR than that in control (t=8.72, p<0.01 ) and associated with Billingham scale (r=-0.7733, PO.05) . Ejection fraction in group N was as same as control (t=1.182, P>0.05)and higher comparing with group DNR (t=5.33, p<0.01) . Rats in group zinc sulphate showed higher EF than group DNR (t=2.98,p<0.01) and lower EF than control (t=5.46,p<0.01) .Reaction oxygen species in rats of group DNR were significant higher than control (t=2.37, p<0.05)o .CuZn-SOD in group DNR decreased significantly (t=2.94, p<0.01) comparing with control. In contrast, in group zinc sulphate CuZn-SOD were significantly higher than in group DNR(t=3.02, p<0.01), and lower than control(t=2.73, PO.05). Group N showed a lower SOD level comparing with control (t=3.58, p<0.01) , and as same as group DNR (t=0.488, P>0.05) .GSH-PX were lower in group DNR than in control (t=4.42, p<0.01) . In group zinc sulfate GSH-PX were higher than group DNR (t=2.96, p<0.01) , and remain lower than control (t=2.34, PO.05) .MDA were higher in group DNR than control (t=5.93, PO.01) . Group zinc sulphate appeared a low level of MDA comparing with group DNR (t=5.93, PO.01), and as the same as control (t=1.69, P>0.05) . MDA showed significantly lower ingroup N than in group DNR(t=8.63, p<0.01)and interestedly, even lower than control (t=1.89,p>O.O5) .Troponin T were low or undetectable in all groups even in those rats with severe injury on hart, all under 0.006ng/ml.ErbB2mRNA had a low expression in group DNR(t=3.65, PO.01) comparing with control and over-expression in group N comparing with group DNR (t=9.69, PO.01).MT-lmRNA over-expression in group Zn than in group DNR(t=2.6, P<0.05) and control (t=2.61, PO.05) .DNR+ ZnSC>4 leads to apoptosis and smartly inhibit as well as daunorubicin alone. Conclusion:DNR induced a lesion in rats heart which showed a high Billingham scale, increasing apoptosis index and a low ejection fraction. The ultrastructure alter observed by electric microscope was in mitochondrion.In cardiac myocyte, DNR caused generation of reaction oxygen species which trigger lipid peroxidation and produce lipid peroxidation product, including MDA. The activitis of SOD and GSH-PX are inhibited, usually these enzymes clearing reaction oxygen species in cardiac myocyte, then more radicals wear generated in cardiac muscle cells. Reaction oxygen species and lipid peroxidation products attack membrane of cell and mitochondrion. This lead to disorder of mitochondrion bridge and form vacuolization. Expression of ErbB2mRNA was low in group DNR. The generation of reaction oxygen species and low-expression of ErbB2mRNA may be one of mechanisms by which DNR cause apoptosis. Apoptosis results in loss of cardiac muscle cells.The alter of heart function caused by DNR can be observed by ultrasonic cardiography. Ejection fraction is correlated with pathological degeneration.
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