The Effect Of Doxorubicin On Cardiovascular System In APOE^-/-Mice And The Underlying Cellular Mechanism

Objective: Doxorubicin(DOX),an anthracycline antitumor drug,which is a broad spectrum and high efficacy anti-tumor agent,is mainly used for the treatment of hematological tumors and various solid tumors.However,the clinical application of anthracyclines is often forced to stop because of its serious toxicity.Among them,cardiovascular toxicity is the main reason for its forced withdrawal,and the mechanism of doxorubicin-induced cardiovascular toxicity is not fully understood.In recent years,clinical studies have found that children with leukemia who have been treated with doxorubicin for more than 10 years have significantly impaired vascular endothelial function and are associated with abnormal blood lipid levels,while the heart function has no significant change.Therefore,the detrimental effect of DOX on vascular endothelium may contribute to its cardiotoxicity.this study intends to use APOE-/-mice to observe whether doxorubicin can aggravate the cardiovascular damage induced by high-fat diet in mice,and use human umbilical vein endothelial cells(HUVECs)cultured in vitro to observe its endothelial toxicity by using doxorucbicin alone or in combination with oleic acid or palmitic acid with MTT method.The possible toxicity mechanisms are analysed.This study can explain the cardiotoxicity mechanism of doxorubicin in a new perspective.Method: 1 Whole animal experimentTwenty male APOE-/-mice aged 7-8 weeks,weighing 27±2 g,were randomly divided into 5 groups and 4 per group.They were normal diet group,high fat diet,high fat diet+DOX 2.5 mg/kg group,5 mg/kg group and 10 mg/kg group.One week after the animals were given a high-fat diet,DOX treatment groups were given DOX 2.5,5,and 10 mg/kg respectively.Thenormal diet group and the high-fat diet group were given the same amount of normal saline,and each group was administered by intraperitoneal injection.The administration volume was 1.0 ml.The daily food intake of each group of animals was observed and recorded.The mice were weighed once a week or so,and the serum total cholesterol(TC)level was measured every 2 weeks.At 8 weeks after DOX administration,the animal's cardiac ejection fraction(EF),shortening score(FS),left ventricular isovolumic contraction time(IVCT)and left ventricular isovolumic relaxation time(IVRT)were measured using a small animal ultrasound analyzer.The mice were sacrificed after the last weighing on the 9th weekend.The heart and the abdominal aorta were separated and extracted,and fixed with 4% paraformaldehyde overnight.The abdominal aorta was stained with oil red O to observe the formation of plaques in the aorta,and the heart tissue was dehydrated by sucrose gradient(15% sucrose 24 h,30% sucrose 24 h)48 h,atrial tissue(part of the heart outflow tract)was embedded with OCT,frozen in liquid nitrogen,sliced with a cryostat.The slice thickness was 9 ?m.Oil red O staining was used to observe the formation of lipid deposits in the cardiac outflow tract.The ventricular tissue was examined by H&E staining to observe the lesions of the ventricular muscle.2 In-vitro cell culture experiments2.1 Effects of DOX,OA and PA on the survival rate of HUVECsHUVECs were cultured in vitro,and different concentrations of DOX,oleic acid(OA,unsaturated fatty acid),palmitic acid(PA,saturated fatty acid),and DOX and OA/PA were administered for 24 h and 48 h,respectively.The effects of DOX or OA/PA on the survival rate of HUVECs at 24 h and 48 h were observed,and the time and concentration of the above drugs in achieving 50%-80% survival rate of HUVECs were determined.The survival rate of HUVECs were further determined in the precence of DOX in combination with OA/PA.2.2 Effects of DOX alone or in combination with OA or PA on lipid deposition in HUVECsHUVECs in logarithmic growth phase were randomly divided into 7 groups:(1)Control group(abbreviation: Con group),(2)DMSO group,(3)DOX group,(4)OA group,(5)PA group,(6)DOX+OA group,(7)DOX+PA group.The concentrations of DOX,OA and PA used in this experiment were 1 ?M,150 ?M and 100 ?M respectively.The treatment lasted for 24 h,then oil red O dye was used to observe the lipid deposition in each group.2.3 Effects of OA or PA alone or in combination with DOX or FABP4 on ROS contens in HUVECsCultured HUVECs were grouped and treated as mentioned above.Cells were stained with reactive oxygen species(ROS)-specific fluorescent probe(2,7-dichlorofluorescenin diacetate,DCFH-DA)1 ?M for 30 min.The fluorescence intensity reflecting the content of ROS of the cells was determined by laser confocal microscopy.Another group of HUVECs were randomly divided into 5 groups,namely(1)Control group(Abbreviated into: Con group),(2)DMSO group,(3)OA group,(4)PA group,(5)FABP4+OA group,(6)FABP4+PA group.The concentrations of FABP4,OA and PA were 100 ng/ml,150 ?M and 100 ?M,respectively.ROS levels were detected by laser confocal microscopy.2.4 Effects of DOX alone or in combination with OA or PA on MDA content in HUVECsHUVECs were cultured,grouped and administered as before,the lysates of the cells were taken,protein concentration was determined by ultraviolet spectrophotometer,and MDA content was determined according to the MDA kit instructions.2.5 Effects of DOX alone or in combination with OA or PA on mRNA expression levels of FABP4 and Mn-SOD in HUVECsHUVECs were cultured,grouped and treated as before.The mR NA expression levels of FABP4 and Mn-SOD in the presence or absence of DOX alone or in combination with OA or PA were analysed by RT-PCR.Result:1 In vivo animal experiment:1.1 General conditions of animalsIn the normal diet group and the high-fat diet group,the general activities,food intake and body weight were not abnormal throughout the observation period.DOX 2.5 mg/kg group,5.0 mg/kg group and 10.0 mg/kg group compared with the high-fat diet group,the mice showed reduced activity,rough hair and vertical hair.After 36 days,the body weight was significantly reduced(P < 0.05 or 0.01)and continued until the end of the whole observation period in DOX 5.0 mg/kg group and 10.0 mg/kg group.There was no significant difference in food intake among the groups.1.2 Effect of DOX on cardiac function in APOE-/-miceEchocardiographic results showed that there were no significant differences in EF,FS,VICT,and VIRT in the APOE-/-mice with high-fat diet compared with the normal diet group.Compared with the high-fat diet group,the values of EF and FS in DOX 5.0 mg/kg and DOX 10.0 mg/kg group were lower significantly than those in high-fat diet group(P < 0.01),but there was no significant change in VICT and VIRT.The high-fat diet + DOX 2.5 mg/kg group had no significant effects on the above parameters.1.3 Effect of DOX on plasma TC levels in APOE-/-miceThere was no significant difference in plasma TC levels in each group before administration.On the 33 rd,47th and 60 th day after administration,compared with the normal diet group,the plasma TC was significantly increased in the high-fat diet group(P < 0.01).There was no significant change in plasma TC in DOX 2.5 mg/kg group and DOX 5 mg/kg group compared to the high-fat diet group.The plasma TC level in DOX 10.0 mg/kg was significantly increased(P < 0.05).1.4 Effect of DOX on the formation of aortic plaque in APOE-/-miceThere was only a small number of plaques in the aortic vessels in the normal diet group;the number and area of plaques in the high-fat diet group were larger than those in the normal diet group;the number and area of plaques in the high-fat diet plus DOX group were higher than those in the high-fat diet group.The trend became more obvious with the dose increasing,accompanied by arterial wall hardening and smaller lumen.1.5 Effect of DOX on lipid deposition in cardiac outflow tract of APOE-/-miceThere was no lipid deposition in the heart outflow tract of the normal diet group;compared with the normal diet group,the lipid outgrowth was found in the cardiac outflow tract in the high-fat diet group;compared with the high-fat diet group,parts of the cardiac outflow tract and the tricuspid valve were covered with more lipid deposits in DOX-administered group.1.6 Pathological effects of DOX on heart tissue in APOE-/-miceHE staining of ventricular myocytes showed that there was no significant change in the APOE-/-mice with high-fat diet group compared with the normal diet group except for the increased myocardial space.However,compared with the high-fat diet group,the DOX administration group had disordered cell arrangement,thickened blood vessel wall,atrophy of cells,increased interstitial space,cytoplasmic lysis of myocardial cells,and extranuclear flow.With the increase of the dose,the injury was more serious;myocardial interstitial inflammation cell infiltration was also found in DOX 10.0 mg/kg group.2 In vitro cell experiment2.1 Cytotoxicity test resultsThe cell growth curve showed that the toxicity of DOX,OA and PA on HUVECs was time and concentration dependent.50~80% Survial ratio of HUVECs when cultured for 24 h was achieved in the presence of DOX 1 ?M,150 ?M OA and 100 ?M PA respectively.Considering the influence of cell growth and metabolism,the final administration time was 24 h.Therefore,in the next experiments,the effects of DOX alone or in combination with OA or PA on HUVEC cells were observed by using the concentrations seleted.The combination effects of DOX+OA or DOX+PA were larger than those of the single drug.2.2 Effect of DOX alone or in combination with OA or PA on lipid deposition in HUVECsOil red O staining showed that there were a few normal lipid deposits inthe cells of the Con and DMSO groups,and the lipid deposits in the DOX,OA,PA,DOX+OA,and DOX+PA groups were increased abnormally.More abnormal lipid deposits were found in DOX+OA and DOX+PA groups,accompanied by reduced cell number.2.3 Effect of DOX alone or in combination with OA or PA on ROS levels in HUVECsThe results of laser confocal microscopy showed that the HUVECs were swelled,and the fluorescence intensity(reflecting ROS content)was increased(P < 0.01)after treated with DOX for 24 h,while the fluorescence intensity of the cells in the presence of OA or PA had no obvious change.The fluorescence intensity was higher in the combination groups than that in DOX,OA and PA alone group(P < 0.01).2.4 Effect of DOX alone or in combination with OA or PA on MDA contents in HUVECs.Compared with the Con group and the DMSO group,MDA contents of HUVECs were increased in the presence of DOX,OA,PA,DOX+OA,or DOX+PA.The MDA contents in DOX+OA or DOX+PA groups were higher than those in DOX,OA or PA alone group(P < 0.01).2.5 Effect of DOX alone or in combination with OA or PA on the mRNA expression of Mn-SOD and FABP4 in HUVECsRT-PCR results showed that mRNA expressions of Mn-SOD in DOX,OA or PA group were decreased while no effect was observed compared with Con group or DMSO group(P < 0.01 respectively).After DOX combined with OA/PA,The mRNA expression of SOD was decreased even lower and FABP4 mRNA expression was increased in the combination of DOX with OA/PA(P < 0.05 or 0.01 respectively).2.6 Effect of DOX alone or in combination with OA or PA on the mRNA expression of Mn-SOD and FABP4 in HUVECsRT-PCR results showed that mR NA expressions of Mn-SOD in DOX group,OA group and PA group were decreased while no effect was observed compared with Con group and DMSO group(P < 0.01 respectively).AfterDOX combined with OA/PA,The mRNA expression of SOD decreased even lower and FABP4 mRNA expression increased in the combination of DOX with OA/PA(P < 0.05 or 0.01 respectively).2.7 Effect of FABP4 plus OA/PA on ROS content in HUVECsThe results of laser confocal microscopy showed that there were no differences in ROS content and fluorescence intensity between the OA/PA group and DMSO group,but the intracellular ROS contents were significantly increased after the combination of OA/PA with FABP4(P < 0.01).Conclusion:1.In vivo experimental results show that doxorubicin can increase the blood cholesterol level in APOE-/-mice feeded with high-fat diet,then causes the myocardial damage and decreased cardiac function by accelerating arterial intimal lipid deposition.2.In vitro cell experiments show that the combination of DOX with OA or PA can reduce the survival rate of HUVECs,which is related to its promotion of lipid deposition and aggravation of oxidative stress.FABP4-mediated signaling pathways may be involved in this process.