| BackgroundHeart failure(HF)is a common and frequent disease in the clinic,which is the final consequence of many cardiovascular diseases.Ventricular remodeling is the important pathological basis in the course of HF disease.,It has remained a hot and thorny topic to reverse ventricular remodeling and improve cardiac functioning.Autophagy is an important course of cell metabolism,while improving ventricular remodeling by regulating and controlling autophagy is also a hot research topic currently.Phosphoinositide-3-kinase,Protein kinase B,Mammalian target of rapamycin(PI3K,Akt,mTOR)target spots are the classical autophagy pathway,which play an important role in the course of HF.However,there is limited research and report about its role in traditional Chinese Medicine.Experiment 1:Research on Huangqibaoxin Decoction improveing the ventricular remodeling in rats with HF after myocardial infarction.Objective:To observe the effect of Huangqibaoxin Decoction on regulating inflammatory factors Interleukin-6(IL-6),Tumor necrosis factor-a(TNF-a),Matrix Metalloproteinase-2(MMP-2)and Matrix Metalloproteinase-9(MMP-9),improving ventricular remodeling and cardiac function.Methods:120 rats were divided into sham operation group(20 rats)and model group(100 rats).The sham operation group only threaded without ligaturing anterior descending branch,while the model group ligature anterior descending branch.After feeding 4 weeks,there were 14 rats left in the sham operation group and 65 rats succeed in the model group.Then they were randomly divided into groups:control group(the same volume of distilled water),captopril group(6.75 mg/kg of captopril),low-dose group with Huangqibaoxin Decoction(5.67 g raw herbs/kg/d),middle-dose group with Huangqibaoxin Decoction(11.34g raw herbs/kg/d),high-dose group with Huangqibaoxin Decoction(22.68 g raw herbs/kg/d),and 13 rats in each group.After 4 weeks,the general condition of these rats were observed in terms of the cardiac structure and functioning by echocardiography,haemodynamic indexes by intubation through carotid arteries,the results of N-terminal B-type natriuretic peptide(NT-proBNP),ST2,IL-6,TNF-?,MMP-2 and MMP-9 by ELISA,and the changes on myocardial tissue morphology by HE.Results:Compared with the sham operation group,the points of general condition in rats of the model group decreased(P<0.05),the LVEDD and LVESD increased,EF,FS,SV and CO decreased(P<0.01),the number of MAP,LVDP,+dp/dmax,-dp/dtma and LVSP(P<0.01),the number of LVEDP increased(P<0.01),the number of NT-proBNP,ST2,IL-6,TNF-?,MMP-2,MMP-9 increased(P<0.01),the myocardial cells were arranged in disorder and there were a lot of fibrous tissue hyperplasia.Compared with the model group,the low,middle and high dose group of Huangqibaoxin Decoction could increase the points of general condition to varying degrees(P<0.01),decrease the LVEDD and LVESD(P<0.05,P<0.01),increase the EF,FS,SV and CO(P<0.05,P<0.01),increase MAP,LVDP,+dp/dmax,-dp/dtma and LVSP(P<0.05,P<0.01),decrease LVEDP(P<0.05,P<0.01)and decrease NT-proBNP,ST2,IL-6,TNF-?,MMP-2,MMP-9(P<0.05,P<0.01).In addition,the myocardial cells were neatly arranged,the hyperplasia of fibrous tissue improved(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.Conclusion:Huangqibaoxin decoction regulate to some extent the inflammatory factors Interleukin-6(IL-6),Tumor necrosis factor-?(TNF-?),Matrix Metalloproteinase-2(MMP-2)and Matrix Metalloproteinase-9(MMP-9)in rats with HF after myocardial infarction and improve myocardial tissue morphology,ventricular remodeling and cardiac function.Experiment 2:Research on the influence of Huangqibaoxin Decoction on autophagy expression and the expression of related protein autophagy pathways of PI3K,Akt and mTOR in rats with HF after myocardial infarction.Objective:To verify the influence of Huangqibaoxin Decoction on autophagy expression and the expression of related protein autophagy pathways of PI3K,Akt and mTOR in rats with HF after myocardial infarction.Methods:The samples preserved in experiment 1 were used in order to observe the autophagy expression condition of bi-layers vesiculation structure in heart tissues under electron microscopy.The expression condition of LC3 was tested through immumofluorescence and the expression of LC3-II/LC3-I,protein p62,Akt and mTOR and its phosphorylation level.Results:Compared with the sham operation group,the autophagosome of bi-layers vesiculation structure in heart tissues in rats of the model group increased(P<0.01),the ration of LC3-?/LC3-? increased,p62 decreased(P<0.01),the ration of p-Akt/Akt and p-mTOR/mTOR both decreased(P<0.01).Compared with the model group,the autophagosome in different dose groups of Huangqibaoxin Decoction decreased(P<0.05,P<0.01),the ration of LC3-?/LC3-?decreased,p62 increased(P<0.05,P<0.01),the ration of p-Akt/Akt and p-mTOR/mTOR both increased(P<0.01).There was a dose-effect relationship between the groups with different doses towards the improvement on the above indexes.Conclusion:Huangqibaoxin decoction can reduce the autophagy expression level in rats with HF after myocardial infarction,decrease the ration of LC3-?/LC3-?,increase the function of protein p62,and decrease the Akt,mTOR phosphorylation level in the PI3K,Akt and mTOR autophagy pathways.Experiment 3:Drug serum with Huangqibaoxin decoction relieves the damage of myocardial anoxia and reoxygenation by inhibiting the autophagy pathways of PI3K,Akt and mTOR to reduce the autophagy level.Objective:To verify the regulation of drug serum with Huangqibaoxin decoction to autophagy pathways of PI3K,Akt and mTOR,reduce the autophagy level and relieve the damage of myocardial anoxia and reoxygenation.1.Cultivation and identification of primary neonatal rat cardiac myocytes,cardiac myocytes of primary neonatal rats of about 1-2 days to primarily cultivate them outside the body.Inoculating on the basis of the density as the petri dish about 5×105cells/60mm after normal digestion and separation.The liquid was changed normally,and model was established after 4 days' cultivation.2.Establish anoxia and reoxygenation model.After cultivation,the primary cardiac myocytes was placed in the hypoxic box for 12 hours,and then followed by reoxygenation for 4 hours.3.The preparation for drug serum with Huangqibaoxin decoction and the determination for the best drug concentration and time.60 SD rats were divided into 2 groups randomly with 30 rats in each group.The Chinese medicine group was offered intragastric administration with Huangqibaoxin decoction while the control group was given intragastric administration with distilled water,for the first 4 days fed as normal,then fasting(no fodder and water only)on the 5th day,given intragastric administration twice before taking blood,and the interval time was 1 hour between the two intragastric administration,taking blood 1 hour later after the second intragastric administration.Optimal drug concentration and time of drug serum were ensured for myocardial cells by the method of CCK-8 and LDH.4.Experimental grouping and administration method:?Control group(Con):adherence for 48 hours of cardiac myocytes,then hatching normally for 17 hours;?Anoxia and reoxygenation(A/R)group:adherence for 48 hours of cardiac myocytes,then hatching normally for 1 hours,followed by anoxia for 12 hours and reoxygenation for 4 hours.?Drug serum(DS)+A/R group:adherence for 48 hours of cardiac myocytes,hatching normally for 0.5 hours,then preprocessing for 0.5 hours after mixing the best drug concentration of drug serum,followed by anoxia for 12 hours and reoxygenation for 4 hours.?Rapamycin(Ra)+A/R group:adherence for 48 hours of cardiac myocytes,hatching normally for 0.5 hours,then preprocessing for 0.5 hours after mixing Ra,and followed by geting anoxia for 12 hours and reoxygenation for 4 hours.?Rapamycin(Ra)+DS+A/R group:adherence for 48 hours of cardiac myocytes,hatching normally for 0.5 hours,then preprocessing for 0.5 hours after mixing Ra,processing 0.5 hours after mixing DS,followed by get anoxia for 12 hours and reoxygenation for 4 hours.? Akt signal inhibitor(API-2)+A/R group:adherence for 48 hours of cardiac myocytes,hatching normally for 0.5 hours,then preprocessing for 0.5 hours after mixing API-2,followed by get anoxia for 12 hours and reoxygenation for 4 hours.? Akt signal inhibitor+DS++A/R group:adherence for 48 hours of cardiac myocytes,then preprocessing for 0.5 hours after mixing API-2,processing 0.5 hours after mixing DS,followed by anoxia for 12 hours and reoxygenation for 4 hours.5.Test indexUsing Try pan blue to test cell viability,CCK-8,LDH to test the optimal drug concentration and time of Huangqibaoxin decoction,transmission electron microscope to test the number and form of autophagosome,immunofluorescence to test LC3.Western blot was adopted to test the expression of protein LC3-?/LC3-??p62 and p-Akt/Akt?p-mTOR/mTOR.Results:1.Try pan blue to test:Compared with Control group,cell viability in A/R group decreased(P<0.01).Compared with A/R group,preprocessing of drug concentration can reduce the damage of cell viability,indicating that drug concentration can protect myocardium in A/R group.2.Transmission electron microscope:Compared with Control group,the number of autophagosome in A/R group increased(P<0.01).Compared with A/R group,the number of autophagosome decreased(P<0.01).3.Immunofluorescence:Compared with Control group,the number of autophagosome in A/R group increased(P<0.01).Compared with A/R group,the number of autophagosome decreased(P<0.01).4.Westren blot:4.1 The influence of Ra on the expression of P13K/Akt/mTOR autophagy pathways of myocardial cells.?Compared with the Control group,the ration of LC3?/? in A/R group increased(P<0.01);the expression of protein p62 decreased(P<0.01).Compared with Control group,LC3-? in DS+A/R group increased,the ration of LC3-?/LC3-? decreased(P<0.01),the expression of protein p62 increased(P<0.01).Compared with A/R group,the autophagy level in Ra+A/R group increased(P<0.01),the ration of LC3-?/LC3-? increased(P<0.01),the decrease of the expression of protein p62 was not statistically significant(P>0.05).Compared with DS+A/R group,the ration of LC3-?/LC3-? in Ra+A/R group increased dramatically(P<0.01),the expression of protein p62 decreased(P<0.01).Compared with Ra+A/R group,the change of LC3?/LC3-? and p62showed no statistical significance.? Compared with the Control group,the ratio of p-mTOR/mTOR in A/R group decreased significantly(P<0.01).Compared with A/R group,the ration of p-mTOR/mTOR in DS+A/R group increased(P<0.01).Compared with A/R group,p-mTOR/mTOR in Ra+A/R group decreased(P<0.01).Compared with DS+A/R group,the ration of p-mTOR/mTOR in Ra+DS+A/R group decreased(P<0.01);Compared with Ra+A/R group,the ratio of p-mTOR/mTOR in Ra+DS+A/R group showed no statistical significance(P>0.05).? Compared with Control group,the ratio of p-Akt/Akt in A/R group decreased significantly(P<0.01).Compared with A/R group,the ration of p-Akt/Akt in DS+A/R group increased(P<0.01).Compared with A/R group,the ration of p-Akt/Akt in Ra+A/R group showed no statistical difference(P>0.05).Compared with Ra+A/R group,the ratio of p-Akt/Akt in Ra+DS+A/R group increased significantly(P<0.01).4.2 The influence of API-2 on the expression of PI3K/Akt/mTOR autophagy pathways of myocardial cells.? Compared with the Control group,the ratio of LC3?/? in A/R group increased significantly(P<0.01)while the expression of protein p62 decreased(P<0.01).Compared with A/R group,LC3-? in DS+A/R groupincreased significantly(P<0.01),the ratio of LC3?/?decreased and the expression of protein p62 increased(P<0.01).Compared with A/R group,the autophagy level in API-2 group increased,the ratio of LC3?/? increased(P<0.01)and the expression of protein p62 decreased(P<0.01).Compared with DS group,the ratio of LC3?/? in API-2+DS+A/R group increased obviously(P<0.01)and the expression of protein p62 decreased(P<0.01).Compared with API-2+A/R group,there was no significant statistical difference between the two groups(P>0.05).?Compared with the Control group,the ratio of p-mTOR/mTOR in A/R group decreased significantly(P<0.01).Compared with A/R group,the ratio of p-mTOR/mTOR in DS+A/R group increased(P<0.01).Compared with A/R group,the ratio of p-mTOR/mTOR in API-2+A/R group decreased significantly(P<0.01).Compared with DS+A/R group,the ratio of p-mTOR/mTOR in API-2+DS+A/R group decreased obviously(P<0.01).Compared with API-2 group,there was no significant statistical difference between API-2+DS+A/R group in terms of p-mTOR/mTOR(P>0.05).? Compared with the Control group,the ratio of p-Akt/Akt in A/R group decreased significantly(P<0.01).Compared with A/R group,the ratio of p-Akt/Akt in DS+A/R group increased(P<0.01).Compared with A/R group,the ratio of p-Akt/Akt in API-2+A/R group decreased significantly(P<0.01).Compared with DS+A/R group,the ratio of p-Akt/Akt in API-2+DS+A/R group decreased significantly(P<0.01).There was no statistical significance in terms of the difference between API-2+A/R group and API-2+DS+A/R group(P>0.05).Conclusions:1.Preprocessing of Drug serum with Huangqibaoxin decoction could relieve the damage of myocardial anoxia and reoxygenation by inhibiting the formation of autophagosome.2.Huangqibaoxin decoction can induce the autophagy inhibitory effect of myocardial anoxia and reoxygenation process through the autophagy pathways of PI3K/Akt/mTOR. |
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