| BackgroundDilated cardiomyopathy(DCM)is the most common type of the primary cardiomyopathies.Besides,it has a poor prognosis and high mortality.The incidence of DCM increases year by year and becomes a serious threat to human health.Ventricular remodeling(VR)is the main pathological basis of heart failure with DCM,while inhibiting or reversing ventricular remodeling are key to treat DCM.The mechanism of ventricular remodeling is complex,and it is closely related to the neurohumoral regulation,apoptosis and inflammatory factors.Apoptosis is one of the most important mechanisms of cardiac remodeling.It is closely related to the occurrence and development of heart failure,and reduces cardiac remodeling by inhibiting myocardial apoptosis,then improves the cardiac function.In recent years,endoplasmic reticulum stress(ERS)pathway was found to be a new signal path of apoptosis,except of the mitochondrial pathway and death receptor pathway.Researchers believe that inhibiting ERS reaction and reducing myocardial cell apoptosis can improve and reverse ventricular remodeling.Traditional Chinese Medicine(TCM)has rich experience in theory research and treatment with DCM,which is multi-channel,multi-link,multi-target,safe and effective.It provides a new direction for the modern treatment of DCM.Based on the basic theory of Chinese medicine,Professor Zhang Peiying,who summarized his many years of clinical experience,invented the clinical prescription,Huangqibaoxin Decoction.And the clinical efficacy is significant after years of clinical practice.The early clinical studies have shown that Huangqibaoxin Decoction can improve the cardiac function and prognosis in patients with DCM,inhibit their myocardial fibrosis and reverse the ventricular remodeling.Besides,the anti-myocardial fibrosis were verified by preliminary experiments.Based on the early clinical and basic work,we assumed that Huangqibaoxin Decoction could inhibit the activation of neuroendocrine system,reverse the occurrence of DCM ventricular remodeling and its molecular mechanism might be related to the function that it inhibited myocardial apoptosis mediated by ERS.In this study,we used Adriamycin to induce DCM rats' model.Then we explored the effects that Huangqibaoxin Decoction inhibits neuroendocrine factors and improves the ventricular remodeling in DCM rats.We also discussed the molecular mechanism that Huangqibaoxin Decoction improves ventricular remodeling in DCM rats through the apoptosis mediated by ERS.Meanwhile,we take these rats as donors of drug-containing serum by the serum pharmacological methods.And we use the myocardial cells of the original generation training rats as the objects of study,then establish the model of newborn rats with myocardial cell apoptosis induced by angiotensin.After this,we need to further study the influence that Huangqibaoxin Decoction drug-containing serum has on myocardial cell apoptosis induced by Ang? and ERS apoptosis related proteins.Then we explore the molecular mechanism that Huangqibaoxin Decoction inhibits ventricular remodeling through inhibiting ERS-mediated cell apoptosis from cellular level.The study is divided into four parts:Experiment 1:Influence of Huangqibaoxin Decoction on ventricular remodeling in Rats with dilated cardiomyopathyObjective:To observe the effect of Huangqibaoxin Decoction on inhibiting RAAS activation and improving ventricular remodeling in DCM rats.Methods:The 90 SD rats were given intraperitoneal injection of Adriamycin to establish the DCM model.And 55 rats succeed.Then divided them into 5 groups randomly(11 rats in each group):model group(the same volume of physiological saline solution),low-dose group with Huangqibaoxin Decoction(5.67 g raw herbs/kg),middle-dose group with Huangqibaoxin Decoction(11.34 g raw herbs/kg),high-dose group with Huangqibaoxin Decoction(22.68 g raw herbs/kg),and captopril group(6.75 mg/kg of captopril).The rats were administrated the drugs once a day according to the above methods.We also set a normal control group with 10 rats(the same volume of physiological saline solution).4 weeks later,we monitor the rats in every group by their echocardiography to identify the construction and function of the left ventricular,the intubation through carotid arteries to monitor hemodynamic indexes,observe the general form of heart and get the cardiac weight index.Besides,we also need to know the changes on myocardial tissue morphology by HE and Masoon,the results of NT-proBNP,ST2,Ang?,ALD,and Renin by ELISA,and the activity of SOD,MDA,and LDH by Coomassie brilliant blue method.Results:Compared with the normal group,the LVEDD and LVESD increased in rats of the model group,EF,FS and SV decreased(P<0.01),the LVDP,ądp/dtmax decreased,the LVEDP increased(P<0.01),the heart weight index increased(P<0.01),the myocardial cells are arranged in disorder and there were a lot of fibrous tissue hyperplasia.The NT-proBNP,ST2,Renin,Ang? and ALD increased(P<0.01).The level of SOD decreased,while the content of LDH and MDA increased(P<0.01).Compared with the model group,the low,middle and high dose group of Huangqibaoxin Decoction could reduce the LVEDD and LVESD to varying degrees,increase the EF,FS and SV(P<0.05,P<0.01),increase LVDP and ądp/dtmax,decrease LVEDP(P<0.05,P<0.01),decrease HWI and LVMI(P<0.05,P<0.01).And the myocardial cells were neatly arranged,the hyperplasia of fibrous tissue were improved.Besides,the levels of NT-proBNP.ST2,Renin,Ang? and ALD were decreased(P<0.05,P<0.01),LDH and MDA decreased,SOD increased(P<0.05,P<0.01).There was a dose-effect relationship between the groups with different doses towards the improvement on the above indexes,while the high-dose group was the best,and there was no significant differences between the high-dose group and the positive drug group(P>0.05).Conclusion:Huangqibaoxin decoction can improve the cardiac hypertrophy and fibrosis in rats of dilated cardiomyopathy.It also improves the cardiac function and inhibits ventricular remodeling by regulating the oxidation factors and inhibiting the activation of RAAS neuroendocrine factors.Experiment 2:Research on the mechanism of Huangqibaoxin Decoction inhibiting the apoptosis of endoplasmic reticulum stress cells in DCM ratsObjective:To verify the mechanism of Huangqibaoxin Decoction inhibiting the apoptosis of endoplasmic reticulum stress cells in DCM rats.Methods:We use the tissue samples preserved in experiment 1 in order to get the cell apoptosis level by TUNEL,levels of protein related to the apoptosis of Bcl-2,Bax,Caspase-3 by immunohistochemistry and Western blotting and the protein expression of GRP78,CHOP and Caspase-12 by Western blotting.Results:Compared with the normal group.the level of myocardial cell apoptosis in model groups was significantly increased(P<0.01).The protein expression of Bax and Caspase-3 were increased,while the anti-apoptotic factor Bcl-2 was decreased(P<0.05).The protein expression of GRP78,Caspase-12 and CHOP were increased in the myocardial tissue(P<0.05).Compared with the model group,the low,middle and high dose group of Huangqibaoxin Decoction could reduce the level of myocardial cell apoptosis to varying degrees(P<0.05,P<0.01).However,the low dose group of Huangqibaoxin Decoction can't improve the expression of pro-apoptotic factors Bcl-2.Bax and Caspase-3(P>0.05),while the other two groups can decrease the expression of pro-apoptotic factors Bax and Caspase-3 and increase the expression level of anti-apoptotic factor Bcl-2(P<0.05).The expressions of GRP78 and Caspase-12 and CHOP protein in the low dose group have not obviously change,while in the other two groups,those expressions decrease to different degrees(P<0.05).There was a dose-effect relationship between the groups with different doses towards the improvement on the above indexes,while the high-dose group was the best,and there was no significant differences between the high-dose group and the positive drug group(P>0.05).Conclusion:DCM rats induced by Adriamycin appear the apoptosis mediated by ERS.While Huangqibaoxin Decoction could inhibit the expression of ERS,decrease the protein expression of GRP78,Caspase-12 and CHOP apoptosis,increase the expression of Bcl-2,decrease the expression of Bax and Caspase-3 proteins,so as to inhibit the myocardial cell apoptosis.Experiment 3:Influence of Huangqibaoxin Decoction serum on the apoptosis of endoplasmic reticulum cardiomyocytes induced by Ang ?Objective:To verify the Huangqibaoxin Decoction serum could inhibit the apoptosis of endoplasmic reticulum cardiomyocytes induced by Ang ?.Methods:1.After cultivating the neonatal rats' cardiomyocytes,we detect them by Try pan blue,morphological and ?-actin staining.2.Establishing the apoptosis of cardiomyocytes induced by Ang ?,cultivating the cardiomyocytes in vitro,giving them different concentration of Ang II and different time.The highest percentage of positive cells by TUNEL was the best concentration and the best time for the experiment.3.The preparation of Huangqibaoxin Decoction serum and the best amount of blank serum.The normal SD rats were given Huangqibaoxin Decoction twice a day for one week.The serum was separated under aseptic conditions and cryopreserved after filtration.The MTT assay was used to determine the best amount of blank serum.Using HPLC method to determine the best time for blood sampling.After intragastric administration for SD rats,divided them into groups according to the time intervals after the last administration.The HPLC chromatograms were recorded at different time points.The total area of it was recorded by the normalization method.The optimal blood collection time was determined by comparing the total area of the chromatographic peaks at different time.4.Preparation of serum containing Huangqibaoxin Decoction and grouping.Thirty-two male SD rats were divided into 4 groups randomly,and 8 rats in each group.They were divided into blank serum group,low-dose serum group(28.25 g/kg),medium-dose serum group(56.5 g/kg)and high-dose serum group(113 g/kg).Each group was given the corresponding dose of drugs.The blank control group was given the corresponding volume of sterile water twice a day for 7 days.60 min after the last administration,we take inferior vena cava blood,and make sterile preparation for blank serum and Huangqibaoxin Decoction of low,medium and high dose group serum.5.The cardiomyocytes cultivated and identified were divided into 7 groups--normal control group,blank serum group.Ang 11 group of low,medium and high dose medicine serum group and captopril group.The Ang ?-induced apoptosis of cardiomyocytes was induced into the latter 5 groups.Apoptotic cells were detected by Annexin V-FITC/PI after 24 hours cultivation in the 7 groups,while the protein expressions of Bcl-2,Bax,Caspase-3,GRP78,Caspase-12 and CHOP were detected by Western blotting.Results:1.Cardiomyocytes were successfully cultivated.The cell viability measured by try pan blue staining was above 90%.a-actin staining showed that the positive rate of myocardial cells was above 95%.The optimal concentration of Ang ? was 10-7 mol/L and the optimal time was 24 hours.TUNEL showed the highest percentage of apoptotic cells under this condition.2.The optimal ratio of the serum containing was 20%.Under this condition,the serum containing has no significant effect on the normal activity of neonatal rat cardiomyocytes.HPLC method,which was determined by the clinical human equivalent dose(5 times),was giving the administration twice daily for 7 days.The peak area of the serum appears after 60 minutes at the last administration.3.The levels of Bcl-2,Bax.Caspase-3,GRP78,Caspase-12 and CHOP in myocardium of blank serum group had no significant difference compared with the normal control group(P>0.05).While the apoptosis of cardiomyoctes significantly increased in Ang ? group(P<0.01),the expression of Bax and Caspase-3 increased and Bcl-2 decreased(P<0.05),the expressions of GRP78,Caspase-12 and CHOP protein expression increased(P<0.05).Compared with Ang II group,groups with different doses can decrease the level of apoptosis to varying degrees(P<0.01).Actually,the low dose group cannot change the expression of Bcl-2,Bax.Caspase-3,GRP78,Caspase-12 and CHOP obviously(P>0.05),while the other two groups can decrease the expression of Bax,Caspase-3,GRP78,Caspase-12 and CHOP(P<0.05),and increase Bcl-2(P<0.05).These effects were in a dose-dependency.There was no significant difference between the high-dose group and the captopril group.(P>0.05)Conclusion:Ang II could induce ERS-mediated apoptosis,while Huangqibaoxin decoction could inhibit Ang ?-induced apoptosis of cardiomyocytes.The mechanism works probably by inhibiting ERS and decrease GRP78,Caspase-12,CHOP,and the expression of Bax,Caspase-3,then regulate Bcl-2. |
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