Study On Anti-myocardial Apoptosis Effect Of ShenJu Decoction After Myocardial Infarction Through The LOX-1 Pathway

BackgroundVentricular remodeling after myocardial infarction(Ventricular Remodeling,VR)refers to the emergence of Hemodynamic changes and activation of neurohumoral regulatory mechanism induce the change of complex molecular signals and the re-expression of embryonic gene and protein in the heart when the heart is under damaged and overloaded condition after an acute myocardial infarction.This result in myocardial cells' maladaptation of hypertrophy,myocardial apoptosis and myocardial extracellular matrix fibrosis,clinically manifested as ventricular hypertrophy,increased ventricular volume and ventricular structural changes.The further development of ventricular remodeling after myocardial infarction can lead to heart failure,sudden death.It is one of the most important risk factors for future cardiogenic death.Apoptosis plays an extremely important role in the Ventricular remodeling process.Apoptosis is a spontaneous death process of the body's normal cells when suffered physiological and pathological stimulus,which is an active,highly ordered,gene controlled and a series of enzymes involved process.Apoptosis plays a key role in the healthy survival process.The new cells are produced while the aging and mutant cells are cleared through apoptosis mechanism,making the organs and tissues mutant normal development and metabolism.Excessive apoptosis leads to a reduced ability of macrophage phagocytosis of apoptotic cells,causing abnormal metabolism of normal cells around apoptotic cells.The enhanced expression of matrix metalloproteinase results in abnormal extracellular matrix degradation,thereby causing myocardial fibrosis and Ventricular remodeling.Lectin-like oxidized low-density lipoprotein receptor(LOX-1),an new ox-LDL receptors,which expressed in the vascular,endothelial tissue and vascular organs,such as placenta,brain and lung under physiological conditions.LOX-1 has a high degree of ligand specificity,it can mediate the cellular uptake and metabolism of ox-LDL and ox-LDL damage effect.Studies have shown that LOX-1-mediated myocardial ischemia and reperfusion,thrombosis,atherosclerosis formation and hypertensive left ventricular remodeling.LOX-1 may be a new target for cardiovascular disease prevention.ERK1/2,JNK,P38MAPK are the family members of MAPK in vivo.MAPK,the major transmembrane signal transduction pathway existed in most cells.MAPK can regulate a wide range of cell activity,ischemia,hypoxia,hormones,growth factors,and cytokines and other extracellular stimuli-induced gene expression,cell proliferation,nuclear reactions common pathway or a focal point.once activated,it can regulated the cell growth,proliferation,differentiation and apoptosis.It Has been proven to be an important signaling molecule in the process of ventricular remodeling,the expression of these molecules can be activated by gene targeting ventricular remodeling.NF-?B is a nucleoprotein factor having the effect of multi-directional transcriptional regulation,which exists in a variety of cells and play a role in physiological and pathological processes such as immunity,inflammation,oxidative stress,cell proliferation,apoptosis,etc.Recent studies suggest that NF-?B and apoptosis is closely related to each other,NF-?B participated in the transcriptional regulation of a variety of apoptosis-related genes,played dual role of inhibit apoptosis and pro-apoptotic,these depended on the different stimulus cell types,and the type and quantity of the activation of NF-?B subunit.P65(RelA)was significantly increased when apoptosis.Caspase 3 is the main terminal cutting enzyme in the process of cell apoptosis,it is in orderly apoptotic cascade downstream,it is one of the most important Caspase,it is a critical executioner of apoptosis,it is the major effect factors in the process of cell apoptosis,it is the focal point of the signal transmission which induced by a variety of apoptotic stimulus,its activation is the flag of irreversible stage of apoptosis.AMI belongs to XiongBi,XinTong,ZhenXinTong in Traditional Chinese Medicine(TCM).The pathogenesis is that the deficiency of Xiong Yang,blockade of QiJi,YuXue and TanZhuo lead to the contracture and blockade of XinMai.The principle of treatment no more than "Tong and Bu",that is to increase XinQi for the restoration of blood running.ShenJu Decoction is an efficacious prescription for the treatment of VR after AMI.The prescription which take into account QiXu,XueYu,TanZhuo and composed of SenMaiSan,ShiXiaoSan,YueJuWan,had achieved good curative effect in clinic.Previous research results show that the PKC,ERK1/2,NF-?B,as the target molecules of ShenJu Decoction,play an important role in the LOX-1 signaling pathway.In this study,we observed the effects of ShenJu Decoction on VR after AMI from whole-cell-protein level,studied the intervention effect and mechanism of ShenJu Decoction on the cardiomyocyte apoptosis which involved in the process of VR,using a variety of modern experimental methods.ObjectiveWe choose the Myocardial infarction rats and myocardial cell H9C2 as the research object,to investage the effect of ShenJu Decoction on Ventricular Remodeling and its probable mechanism and provided a scientific basis for the further widespread application of ShenJu Decoction.Method1.The making and grouping of rat myocardial infarction modelSelect 36 adult male Wistar rats SPF level,weighing between 150g-200g,ligating the left coronary artery and resulting in the myocardial infarction.Divide them into 2 groups Randomly:Model control group,ShenJu Decoction group;with additional Sham operation group which only hanged line without ligation of coronary artery after thoracotomy.The rats fed with medicine the postoperative day.ShenJu Decoction group:5.5g/kg·d herb medicine decoction administered orally;Sham operation group and Model control groups:distilled water administered orally for 12 weeks.2.Observation of the rats' General life status and heart shape.We observat the rats,General life status,including food intake,hair condition,activity,then execute the rats after 12 weeks,cut out the heart and observed the overall shape,measur the size of heart with a ruler.3.Myocardial connective tissue stainingWe carry out Pathological observation using HE staining technic and TUNEL staining technic,calculate transverse area of myocardial cells through the medical pathology image system.4.Detection of Caspase 3Probe solution can be diluted to the required concentration in fresh broth,the cell culture fluid is replaced by this dyeing liquid.Dihydrogen b organism concentration can be chosen in the range of 1?mol to 100?mol and incubated at 37 ? or room temperature,away from light.After the incubation,cells are cleaned with fresh solution,observed under a fluorescence microscope.Cells that activated by different fluorescence present different color.5.Dectecting the expression of related proteins through Western blottingForty micrograms of protein extracts is mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)sample buffer.They are boiled for five minites,and undergo electrophoresis on a 10%SDS polyacrylamide gel.They are transferred onto PVDF membranes.The membranes are blocked with 5%non-fat dry milk in TBST buffer and then incubated overnight with primary rabbit antibody against LOX-1,BCL-2,BAX,P-P38,P-ERK1/2,P65 at 4?.They are then washed 3 times in TBST(10 min for each wash)befor incubating the secondary antibody for 1 hour.The results are analyzed by KODAK Image Station 4000MM exposure imaging system and quantified using Image tool V3.0 software.6.The cultivation and grouping of H9C2 CellH9C2 rat myocardial cell lines are cultivate for use,then randomly divided into six groups,the normal group(DMEM,10%FBS),the Model control group(DMEM,10%FBS,100?mmol/L H2O2),the ShenJu Decoction with low dosage group(DMEM,10%FBS,100?mmol/L H2O2,0.5mg/ml ShenJu Decoction decoction),the ShenJu Decoction with medium dosage group(DMEM,10%FBS,100?mmol/L H2O2,lmg/ml ShenJu Decoction),the ShenJu Decoction with high dosage group(DMEM,10%FBS,100?mmol/L H2O2,2mg/ml ShenJu Decoction decoction),?-Car group(DMEM,10%FBS,100?mmol/L H2O2,250mg/L?-Carrageenan).7.Hoechst33258 stainning analysisAdjusted the density of cardiomyocytes to 2×105/ml with 10%FBS containing DMEM medium,inoculated with the 6-well plates,each plant hole 2ml.After incubate 48h?72h.After 24h of serum starvatiocells with DMEM containing 2%FBS.The cells are fixed with 70%ethanol at room temperature for 30 minites,washed two times with PBS,then added PBS containing 10?g/mL hoechst 33342.Placed at room temperature for 30 minutes,and observed through fluorescence microscope.8.Annexin V-FITC/PIWe detecte the effect of ShenJu Decoction on the apoptosis induced by H22 through Annexin V-FITC/PI staining.We found that normal cells are not dyed but apoptotic cells shown red fluorescence by Annexin V-FITC staining and green fluorescence by PI staining.9.Western blottingAfter various treatments,myocytes are harvested and lysed in 150 ?l NP-40 lysis buffer.The protein concentration is determined by the BCA method.The metho of protein detection is the same as above.10.Statistical analysisResults are expressed as mean±SD of at least three independent experiments.One-way ANOVA is applied to test the level of significance for the expression among different doses using post-hoc analysis with Bonferroni correction.When the variance of arrhythmia,the use of robust estimation Welch,then the use of T3 compared methods.A value of P<0.05 was considered as significant.All the analyses are performed using SPSS 13.0.Result1.A total of 5 died in the process of modeling,divided the survival rats into two groups(The Model group has 16,the ShenJu Decoction group has 15).A total of 3 died in the process of feeding(The Model group has 1,the ShenJu Decoction group has 2)2.Compared with the Model control group,ShenJu Decoction group can improve the general life condition of AMI model rats Significantly.The heart of Sham group is smaller and more smooth than Model group.3.As the myocardial tissue staining shown,the myocardial tissue dyeing uniformity and neat striated muscle were observed in normal group,in Model control group,we can see that the fibers changed long,arranged disorder.The nuclei is large and hyperchromatic,and inflammatory cell infiltration around.After the treatment of ShenJu Decoction,the myocardial infarction area decreases significantly,hypertrophy cells and hyperplasia of fibre was observed in the infarct border zone,the degree of cellular degeneration is eased around the area of infarction.Myofibril arranged more tidy than model control group,Sarcoplasmic reticulum has a slight expansion.Myofilament arranged tidy,necrosis degree of ease,interstitial edema is not obvious,hyperplasia of collagen was reduced.Compaired to the model control group,the myocardial apoptosis rate decreased obviously in ShenJu Decoction group.4.As the Western blot result shown,the expression of LOX-1 protein in model control group are significantly higher than normal group.Compaired with the model control group,ShenJu Decoction can significantly reduce the expression of LOX-1,BAX,P-P38,P65,P-ERK1/2 in cardiac muscle tissue after myocardial infarction,meanwhile,increase the expression of BCL-2.5.As the Hoechst33358 staining result shown,high concentration of ShenJu Decoction had certain influence in the H9C2 cardiomyocytes activity.Under the light microscopy,we found that there are more apoptotic cells in the model control group than the normal group.We can saw that the degree of myocardial cell viability to varying to improve in all ShenJu Decoction groups,especially the ShenJu Decoction with high dosage group.This suggests that ShenJu Decoction can reduce the myocardial cell apoptosis induced by H2O2.6.As the Annexin V-FITC/PI staining shown,there are much more apoptotic cells in the H2O2 group than the control group,ShenJu Decoction can improve such phenomenon,as the concentration increased,the effect is more obvious,effect of high dosage group is the best,which is similar to the ?-Car group.7.The expression of BAX in model control group is higher than normal group,and the BCL-2/BAX ratio is lower than normal group.However,lower expression of BAX and higher expression of BCL-2 can be seen in the ShenJu Decoction groups.The best effect can be seen in the ShenJu Decoction with high dosage group as well as the ?-Car group.ConclusionShenJu Decoction can suppress the LOX-1 and the NF-?B pathway,reduce the expression of P38,ERK1/2 which are the downstream signal molecules of LOX-1,reduce the expression of P65 which is the subunit of NF-?B,reduce the expression of BAX,rise the expression of BCL-2.Therefore,we believed that ShenJu Decoction can inhibit the LOX-1 pathway,protect the Myocardial cells,Reduce the myocardial cell apoptosis,intervene in the pathological process of VR after AMI and slow the development of heart failure.