| Objective: To observe the protective effect of Chaihu Sanshen Capsule on doxorubicin-induced cardiotoxicity in neonatal rat model and heart failure model in mice,by studying the PI3K/AKT and P38 MAPK pathways and some of the involved effect indicators,we explored the underlying mechanisms of the protective effect of Chaihu Sanshen Capsule.Methods: Neonatal rat ventricular myocytes(NRVMs)were isolated from 1~3 day old Sprague Dawley rats via combined trypsin and collagenase type II digestion.After culturing NRVM with different concentrations of doxorubicin(0,0.1,0.5,1,3,5,10,20 and30umol/L)for 24 h,the cells were divided into phosphate buffered saline solution without fetal bovine serum DMEM control group(NT),doxorubicin-treated group(Model),and adriamycin-treated group after pretreatment with Chaihu Sanshen capsules(CHSSC).Sixty healthy adult male SPF C57BL/6 mice were randomly divided into normal control group(No Treatment,NT),model group(Model),and Chaihu Sanshen capsule pretreatment group(CHSSC),with 20 mice in each group.No Treatment(NT)mice orally received an equivalent volume of saline,doxorubicin-treated mice(Model)were injected with a single dose of doxorubicin dissolved in normal saline(15 mg/kg i.p.)and received an orally equivalent volume of saline.doxorubicin plus CHSSC treatment mice(CHSSC)were pretreated with Chaihu Sanshen capsule solution(0.3ml/20g)by gavage for 3days and administered Chaihu Sanshen capsule for 3 additional days after the injection of the same dose doxorubicin as the Model group.Detection indicators: 1.Efficacy evaluation indicators:(1)Regularly observe the general condition and body weight changes of mice during gavage;(2)Color echocardiography to detect cardiac function indexes after modeling;(3)CCK8 method and Ed U method to detect cell viability;(4)The kit detects the concentration of ROS,CK and LDH;(5)HE staining was used to observe the pathological changes of mouse ventricle.2.The mechanism indicators:(1)The kit detect the concentration of SOD,MDA,GSH and GPX;(2)Immunofluorescence assay on frozen sections to detect tissue reactive oxygen species;(3)TUNEL staining and flow cytometry to detect cell apoptosis;(4)Western Blot detect the expression of Caspase3,c-Caspase3,p-P38 MAPK,p-PI3 K,p-AKT,Bcl2,Bax protein.Results:1.Body weight and animal model evaluation: During the feeding period,there was no statistical difference in the body weight of mice in each group(P>0.05).Mice were sacrificed the next day after the last administration,and echocardiography was performed before sacrifice.The EF decreased significantly(P<0.01),and the LVIDs,LVIDd,LVESV and LVEDV increased(P<0.05).Cardiac function was impaired,suggesting that the heart failure model was successfully established.2.Evaluation of cell model: After culturing with different concentrations of doxorubicin for 24 hours,the viability of NRVM cells was significantly decreased(P<0.01),the level of reactive oxygen species was increased(P<0.01),and the concentrations of CK and LDH were increased(P>0.05),It suggested that the myocardial cell injury model in vitro was successfully established.3.Cardiac color ultrasound results:(1)Compared with NT group,EF in Model group decreased significantly(P<0.01),while LVIDs,LVIDd,LVESV and EDV increased(P<0.05);(2)Compared with the Model group,the ultrasound indexes of mice in CHSSC group improved,EF increased(P<0.05),LVESV(P<0.05),LVIDs,LVIDd and LVEDV were significantly decreased(P<0.01). 4.Determination of serum CK and LDH levels:(1)Results in NRVM:(1)Compared with NT group,CK and LDH in Model group showed an increasing trend,but there was no statistical difference(P>0.05);(2)Compared with Model group,CK and LDH in CHSSC group decreased,but the difference between groups was not statistically significant(P>0.05).(2)Results in mouse serum:(1)Compared with NT group,the levels of CK and LDH in Model group were significantly increased(P<0.01);(2)Compared with Model group,CK(P<0.05)and LDH in CHSSC group were different degree decreased(P<0.01).5.Cell viability results :(1)Results in NRVM:(1)Compared with NT group,the cell viability of Model group was significantly decreased in a dose-dependent manner.The higher the concentration of doxorubicin,the lower the cell viability(P<0.05).When the concentration of doxorubicin was 1umol/L,the cell viability decreased significantly(P<0.01);(2)After treatment with different concentrations of drug-containing plasma,the cell viability at 5%CHSSC concentration was the highest(P<0.01);(3)NRVM was pretreated with CHSSC at different times,and with the treatment time prolonged cell viability increased,but decreased again after 24hours(P<0.01).(2)Results in mouse myocardial tissue:(1)Compared with NT group,the fluorescence of myocardial tissue stained cells in Model group was weakened(P<0.05);(2)Compared with Model group,the proliferation of cells in CHSSC group was improved,and the fluorescence of myocardial stained cells was enhanced(P<0.05).6.Active oxygen test results:(1)Results in NRVM:(1)Compared with the NT group,the fluorescence intensity in the Model group was significantly enhanced(P<0.01);(2)Compared with the Model group,the fluorescence intensity in the CHSSC group was significantly reduced(P<0.05).(2)Immunofluorescence staining results of frozen sections of cardiac tissue suggest:(1)Compared withNT group,the fluorescence intensity in Model group was significantly enhanced(P<0.05);(2)Compared with the Model group,the fluorescence signal intensity in the CHSSC group was weakened(P<0.05).7.HE staining results of mouse ventricular tissue:(1)Compared with NT group,the arrangement of myocardial muscle fibers in Model group was disordered,some myocardial muscle fibers were coagulated and blurred,and myocardial fiber hyperplasia was occasionally seen;myocardial cells were partially dissolved;Lipid droplets and vacuoles appear,and cells appear without nuclei or punctate granular nuclei and pyknosis.(2)Compared with the Model group,the myocardial cells of the CHSSC group were not arranged neatly;mild myocardial cell lysis was seen;a small amount of lipid droplets or vacuoles were seen in some cells,and occasionally cells without nuclei or punctate granular nuclei were seen;no myocardial fibers were seen hyperplasia.8.SOD,MDA,GSH,GPX conc entration results :(1)Results in NRVM:(1)Compared with NT group,MDA in Model group had a tendency to increase,but there was no statistical difference(P>0.05),SOD decreased(P <0.05),GSH increased(P<0.01),GPX There was no statistical difference in GPX reduction(P>0.05);(2)Compared with the Model group,there was no significant difference in the decrease of MDA in CHSSC group(P>0.05),SOD and GPX increased,but no statistical difference(P>0.05),and GSH decreased(P<0.01).(2)Results in mouse serum:(1)Compared with NT group,MDA in Model group was significantly increased(P<0.01),SOD and GPX were decreased(P<0.01),GSH was increased(P<0.01);(2)Compared with Model group,in CHSSC group,MDA decreased(P<0.01),SOD and GPX increased significantly(P<0.01),GSH decreased(P<0.01).9.TUNEL apoptosis results :(1)Results in NRVM:(1)The number of apoptosis-positive staining cells increased after treatment with different concentrations of doxorubicin,and the higher the concentration of doxorubicin,the more positive staining cells(P<0.01);(2)Compared with the NT group,the number of apoptotic cardiomyocytes in the Model group was significantly increased(P<0.01);(3)Compared with the Model group,the positive staining cells of the cardiomyocytes in the CHSSC group decreased(P<0.01).(2)Results in mouse myocardial tissue:(1)Compared with NT group,the apoptotic cells of myocardial cells in Model group were significantly increased(P<0.01);(2)Compared with the Model group,the number of myocardial apoptosis-positive cells in the CHSSC group was significantly decreased(P<0.01).10.NRVM flow cytometry apoptosis results:(1)Compared with the NT group,the apoptotic cells in the Model group were significantly increased,and the early apoptosis was more obvious than the late apoptosis(P<0.05);(2)Compared with the Model group,the apoptotic cells in the CHSSC group decreased,and the late apoptotic cells decreased more significantly(P<0.01).11.Caspase3,c-Caspase3,p-P38 MAPK,p-PI3 K,p-AKT,Bcl-2,Bax protein expression :(1)Results in NRVM:(1)The expression of p-PI3 K,c-Caspase3,p-P38 MAPK and Bax increased after doxorubicin treatment,among which the phosphorylation of P38 MAPK increased most obviously,and it increased in a dose-dependent manner with doxorubicin(P<0.01),the expressions of p-AKT,Bcl-2,and Caspase3 decreased(P<0.01);(2)Compared with the NT group,the expressions of p-P38 MAPK,c-Caspase3,Bax,and p-PI3 K in the Model group were significantly increased(P<0.01),p-AKT and Bcl-2 decreased(P<0.01);(3)Compared with the Model group,the expressions of p-P38 MAPK,c-Caspase3,Bax and p-PI3 K in the CHSSC group were significantly decreased(P<0.01),p-AKT, Bcl-2 increased(P<0.01).(2)Results in myocardial tissue:(1)Compared with NT group,the expressions of c-Caspase3(P<0.05),p-P38 MAPK,Bax and p-PI3 K in Model group were increased(P<0.01),p-AKT,Bcl-2.Caspase3 decreased(P<0.01);(2)Compared with the Model group,the expressions of c-Caspase3(P<0.05),p-P38 MAPK,Bax,p-PI3K(P<0.01)in the CHSSC group were significantly decreased,while the expressions of p-AKT,Bcl-2 and Caspase3 were increased(P<0.01).Conclusion:1.Intraperitoneal injection of doxorubicin can build a mouse model of acute heart failure and cause abnormal cardiac function in the model mice.2.In vitro cardiomyocyte injury model can be established by inducing neonatal rat ventricular myocytes with doxorubicin.3.Doxorubicin promotes oxidative stress and apoptosis to induce cardiomyocyte injury,which may be related to the promote ROS generation,increased CK,LDH,MDA concentrations,decreased SOD,GPX,increased c-Caspase3,p-P38 MAPK and Bax,decreased p-AKT and Blc-2 were associated with decreased cell viability and increased peroxidative damage.4.Chaihu Sanshen Capsules can improve cardiac function in mice with acute heart failure,increase EF,decrease LVIDs,LVIDd,LVESV and LVEDV,and reduce myocardial steatosis and cell necrosis.5.Chaihu Sanshen Capsules can reduce doxorubicin-induced cardiotoxicity by regulating PI3K/AKT and p38 MAPK pathways,inhibiting oxidative stress and apoptosis. |
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