| Object ive1.To clarify whether Nuanxin Capsule improves the myocardial energy expenditure in heart failure patients with preserved ejection fraction.2.To clarify whether Nuanxin Capsule improves the cardiac dysfunction with preservation of ejection fraction induced by oxidative stress.3.To clarify whether Nuanxin Capsule improves the myocardial energy metabolism disorder induced by oxidative stress by activating AMPK/PGC-1?signaling pathway to resist heart failure with preserved ejection fraction.4.To clarify whether Nuanxin Capsule inhibits the mitochondrial apoptosis pathway induced by oxidative stress by activating the AMPK/JNK signaling pathway to resist heart failure with preserved ejection fraction.MethodsThe first part is clinical observation:Nuanxin Capsule improves myocardial energy consumption of heart failure patients with preserved ejection fraction35 patients with heart failure with preserved ejection fraction were initially collected because of limited mobility during the epidemic period,and through the strict case screening process,20 cases of clinical observation were completed.They were randomly divided into treatment group(n=10)and control group(n=10).Both groups were given standardized western medicine treatment.On the basis of the control group,the treatment group was additionally treated with Nuanxin Capsule(NX,composed of red ginseng,cooked aconite,coix seed and Pummelo Peel,prepared by the Drug Manufacturing Room of Guangdong Hospital of traditional Chinese medicine,Guangdong medicine preparation word Z20080138,0.5g per capsule,3 capsules each time,3 times a day,oral)for 4 weeks.The changes of MEE,cess,LVM,LVMI,NT proBNP,LVEF,TCM syndrome score and safety index were observed before and after treatment.The second part is the basic experiment:the study of Nuanxin Capsule on improving the cardiac dysfunction induced by oxidative stress1.Eight week old male C57BL/6J mice were selected to establish HFpEF model by TAC.Three days after operation,the mice were randomly divided into four groups:? Sham group,?TAC group,?TAC+ NXL group,and? TAC+NXH group.In TAC+NXL group and TAC+NXH group,the dosage of NX was 0.64g/kg/d and 1.72g/kg/d respectively and the remaining two groups were given the same volume of normal saline by gavage.After 4 weeks of continuous administration,the functional indexes of mice were detected.2.H9c2 cardiomyocytes from rat embryos were selected to establish oxidative stress model by stimulating cells with 3%H2O2.The cells were incubated with different concentrations of NX for 4 h,and then exposed to different concentrations and different times of 3%H2O2 for inducing oxidative stress.According to the cell viability test,the best concentration and time of NX and 3%H3O2 on cardiomyocytes were screened out.3.Echocardiography was used to evaluate the cardiac function of mice,including LVIDd,LVIDs,EF,FS;serum BNP level was detected to evaluate the degree of HFpEF.4.MTT assay was used to detect the viability of cardiomyocytes and LDH assay was used to detect cell damage.5.The level of ROS in cardiomyocytes was evaluated by flow cytometry with DCFH-DA probe.6.Seahorse XFe24 analyzer was used to evaluate mitochondrial respiratory function of H9c2 cardiomyocytes by OCR value.The third part is the basic experiment:the research of Nuanxin Capsule in improving the myocardial energy metabolism disorder induced by oxidative stress1.Eight week old male C57BL/6J mice were selected to establish HFpEF model by TAC.Three days after operation,the mice were randomly divided into four groups:? Sham group,? TAC group,?TAC+NX group,and? TAC+NXH+Compound C(Comp C)group.TAC+NX group was given 0.64g/kg/d of NX by gavage,TAC+NX+Comp C group was given compound C(a selective ATP competitive AMPK inhibitor)by gavage,and the remaining two groups were given the same volume of normal saline by gavage.After 4 weeks of continuous administration,the functional indexes of mice were detected.2.H9c2 cardiomyocytes from rat embryos were selected to establish oxidative stress injury model by stimulating H9c2 cardiomyocytes with 3%H2O2.The cardiomyocytes were first incubated with 4 mg/ml NX for 4 h,then incubated with 5?M Compound C for 2 h,and finally exposed to 600 ?M 3%H2O2 for 1.5 h to induce oxidative stress.Cell experiment was divided into four groups:? Control(Con)group,? H2O2 group,?H2O2+NX group,and? H2O2+NX+Comp C group.3.The method of detecting by echocardiography and serum BNP level was the same as that in the second part of the basic experiment.4.The detection of MTT and LDH was the same as the second part of basic experiment.5.The detection of ROS in cardiomyocytes is the same as the second part of basic experiment.6.The ATP concentration in mice and cardiomyocytes was detected by chemiluminescence.7.Seahorse XFe24 analyzer was used to evaluate mitochondrial respiratory function of H9c2 cardiomyocytes by OCR value;glucose,fatty acid and glutamine pathway inhibitors were used to evaluate energy metabolism substrates of H9c2 cardiomyocytes by OCR value.8.The protein levels in mice and cardiomyocytes of p-AMPK,AMPK and PGC-1? were detected by Western blot.The fourth part is the basic experiment:the study of Nuanxin Capsule on inhibiting mitochondrial apoptosis induced by oxidative stress1.The experimental objects and grouping of animals and cells are the same as the third part of basic experiments.2.Annexin V-FITC/PI staining was used to evaluate the apoptosis of cardiomyocytes by flow cytometry.3.JC-1 staining was used to evaluate the mitochondrial membrane potential of cardiomyocytes by flow cytometry.4.The protein levels of in mice and cardiomyocytes Bcl-2,BAX,cytochrome c,cleaved caspase-3,p-AMPK,AMPK,p-JNK and JNK were detected by Western blot.ResultsThe first part is clinical observation:Nuanxin Capsule improves myocardial energy consumption of heart failure patients with preserved ejection fraction1.After treatment,the value of MEE in the treatment group decreased compared with that before treatment(P>0.05),the value of MEE in the control group increased compared with that before treatment(P<0.05),and the value of MEE in the treatment group was significantly lower than that in the control group after treatment(P<0.05).2.After treatment,there was no significant change in the value of cESS in the treatment group(P>0.05),but the value of cESS in the control group increased(P>0.05),and the value of cESS in the treatment group was significantly lower than that in the control group(P<0.05).3.After treatment,the LVM and LVMI values of the treatment group decreased compared with those before treatment(P<0.05),and the LVM and LVMI values of the control group did not change compared with those before treatment(P>0.05).The LVM and LVMI values of the treatment group after treatment were lower than those of the control group after treatment(P>0.05).4.After treatment,the value of NT-proBNP in the treatment group was significantly lower than that before treatment(P<0.05),the value of NT-proBNP in the control group was significantly higher than that.before treatment(P>0.05),and the value of NT-proBNP in the treatment group was lower than that in the control group after treatment(P>0.05).5.After treatment,the value of LVEF in the treatment group was slightly higher than that before treatment(P>0.05),the value of LVEF in the control group after treatment had no change(P>0.05),and the value of LVEF in the treatment group after treatment was higher than that in the control group after treatment(P<0.05).6.After treatment,the value of TCM syndrome integral in the treatment group decrea.sed compared with that before treatment(P<0.05),and there was no significant change in the value of TCM syndrome integral in the control group after treatment(P>0.05).The value of TCM syndrome integral in the treatment group after treatment was significantly lower than that in the control group after treatment(P>0.05).7.There were no significant differences in blood routine,electrolyte,liver function and renal function indexes between the treatment group and the control group before and after treatment(P>0.05),and there were no allergic reactions,adverse reactions related to the test drug and deterioration of the disease in the treatment group and the control group during the treatment.The second part is the basic experiment:the study of Nuanxin Capsule on improving the cardiac dysfunction induced by oxidative stress1.Four weeks after TAC,compared with Sham group,the LVIDd of HFpEF mice in TAC group increased slightly(P>0.05),LVIDs increased significantly(P<0.05),and the ventricles expanded significantly;compared with TAC group,the LVIDd of NXL group decreased significantly(P<0.05),the LVIDd of NXH group decreased(P>0.05),and the LVIDs of NXL and NXH groups decreased significantly(P<0.05).Compared with Sham group,the EF and FS of HFpEF mice in TAC group were significantly decreased(P<0.05);compared with TAC group,the EF and FS of NXL and NXH groups were significantly increased(P<0.05).2.After 4 weeks of TAC,compared with Sham group,the serum BNP level of TAC group was significantly increased(P<0.05);compared with TAC,the serum BNP levels of NXL group and NXH group were significantly decreased(P<0.05).3.MTT assay showed that compared with 0 ? M H2O2,the cell viability gradually decreased with the gradual increase of H2O2 concentration(P<0.05),in which the cardiomyocyte viability of 600 ?M H2O2,was significantly decreased by 0.48±0.04(P<0.05);compared with 0 h H2O2,the cell viability gradually decreased with the gradual increase of H2O2 incubation time(P<0.05),in which the cardiomyocyte viability was 1.5 h compared with 0 mg/ml NX group,the cell viability of NX 4 mg/ml and NX 6 mg/ml increased significantly from 0.42 ± 0.01 to 0.73±0.02 and 0.68 ± 0.01(P<0.05),and the final protective concentration of NX was 4 mg/ml and 6 mg/ml,of which 4 mg/ml had the strongest.protective effect.4.MTT and LDH results showed that compared with Con group,cell viability in H2O2 group was significantly decreased(P<0.05),and cell damage was significantly increased(P<0.05);compared with H2O2 group,cell viability in NXL and NHX groups was significantly increased(P<0.05),and cell damage was significantly decreased(P<0.05).5.Compared with Con group,ROS production in H2O2 group increased significantly(P<0.05);compared with H2O2 group,ROS production in NXL and NHX groups decreased significantly(P<0.05).6.Seahorse XFe system results showed that compared with Con group,the OCR values of basal respiration,maximal respiration and ATP production in H2O2 group were significantly decreased(P<0.05);compared with H2O2 group,the OCR values of basal respiration and ATP production in NX group were significantly increased(P<0.05),and the OCR values of maximal respiration were significantly increased(P>0.05).The third part is the basic experiment:the research of Nuanxin Capsule in improving the myocardial energy metabolism disorder induced by oxidative stress1.Four weeks after TAC,compared with Sham group,LVIDd of HFpEF mice in TAC group increased slightly(P>0.05),LVIDs increased significantly(P<0.05),and ventricular dilation was obvious;compared with TAC group,LVIDd and LVIDs of NX group decreased significantly(P<0.05).Compared with Sham group,the EF and FS of HFpEF mice in TAC group were significantly decreased(P<0.05);compared with TAC group,the EF and FS of HFpEF mice in NX group were significantly increased(P<0.05).After Compound C pretreatment,LVIDd and LVIDs were significantly higher than those in NX group(P<0.05),EF and FS were significantly lower than those in NX group(P<0.05).2.Four weeks after TAC,compared with Sham group,the serum BNP level of TAC group was significantly increased(P<0.05);compared with TAC group,the serum BNP level of NX group was significantly decreased(P<0.05);Compound C pretreatment significantly increased the serum BNP level of NX group(P<0.05).3.Four weeks after TAC,the ATP concentration in TAC group was significantly lower than that in Sham group(P<0.05);the ATP concentration in NX group was significantly higher than that in TAC group(P<0.05);the ATP concentration in Compound C group was significantly lower than that in NX group(P<0.05).4.In H9c2 cells,compared with Con group,ATP concentration in H2O2 group was significantly lower(P<0.05);compared with H2O2 group,ATP concentration in NX group was significantly higher(P<0.05);ATP concentration after Compound C pretreatment was significantly lower than that in NX group(P<0.05).5.In H9c2 cells,compared with Con group,the cell viability of H2O2 group was significantly decreased(P<0.05),the cell damage was significantly increased(P<0.05),and the production of ROS was significantly increased(P<0.05);compared with H2O2 group,the cell viability of NX group was significantly increased(P<0.05),the cell damage was significantly decreased(P<0.05),and the production of ROS was significantly decreased(P<0.05)After pretreatment with Compound C,compared with NX group,cell viability was significantly decreased(P<0.05),cell injury was significantly increased(P<0.05),and ROS production was significantly increased(P<0.05).6.In H9c2 cells,compared with Con group,the basal respiration,maximal respiration,spare respiration and OCR value of ATP production in H202 group were significantly decreased(P<0.05);compared with H2O2 group,the basal respiration,maximal respiration,spare respiration and OCR value of ATP production in H2O2 group were significantly increased(P<0.05).After pretreatment with Compound C,the OCR values of basal respiration and ATP production decreased(P>0.05),and the OCR values of maximal respiration and reserve respiration decreased(P<0.05).7.In H9c2 cells,the OCR values of mitochondria in Media-con group,Etomixir-con group,Media-H2O2 group,Etomixir-H2O2 group,Media-NX group and Etomixir-NX group did not change significantly under basal respiration(P>0.05).Under the condition of maximal respiration,the OCR value of mitochondria in Media-con group and Etomixir-con group had no significant change(P>0.05),the OCR value of mitochondria in Media-H2O2 group and Etomixir-H2O2 group had no significant change(P>0.05),the OCR value of mitochondria in Media-NX group and Etomixir-NX group had no significant change(P>0.05).Meanwhile,compared with Con group,the OCR value of mitochondria in H2O2 group decreased(P<0.05),and the mitochondrial OCR value in NX group increased(P<0.05).8.In H9c2 cells,the OCR values of mitochondria in Media-con group,BPTES-con group,Media-H2O2 group,BPTES-H2O2 group,Media-NX group and BPTES-NX group did not change significantly under basal respiration(P>0.05).Under the condition of maximal respiration,the OCR value of mitochondria in Media-con group and BPTES-con group had no significant change(P>0.05),the OCR value of mitochondria in Media-H2O2 group and BPTES-H2O2 group had no significant change(P>0.05),the OCR value of mitochondria in Media-NX group and BPTES-NX group had no significant change(P>0.05).Meanwhile,compared with Con group,the OCR value of mitochondria in H2O2 group decreased(P<0.05),and the mitochondrial OCR value in NX group increased(P<0.05).9.In H9c2 cells,the OCR values of mitochondria in Media-con group,UK5099-con group,Media-H2O2 group,UK5099-H2O2 group,Media-NX group and UK5099-NX group did not change significantly under basal respiration(P>0.05).Under the condition of maximal respiration,the mitochondrial OCR value of UK5099-con group was lower than that of Media-con group(P<0.05),the mitochondrial OCR value of UK5099-H2O2 group was lower than that of Media-H2O2 group(P<0.05),and the mitochondrial OCR value of UK5099-NX group was lower than that of Media-NX group(P<0.05).At the same time,compared with Con group,the mitochondrial OCR value of H2O2 group was lower(P<0.05),and the mitochondrial OCR value of NX group was higher than that of H2O2 group(P<0.05).10.The results of Western blotting in HFpEF mice and H9c2 cells showed that compared with Sham group and Con group,the p-AMPK/AMPK and PGC-1 ?/GAPDH ratios in TAC and H2O2 group were significantly decreased(P<0.05);compared with TAC group and H202 group,the p-AMPK/AMPK and PGC-1?/GAPDH ratios in NX group were significantly increased(P<0.05).The ratio of p-AMPK/AMPK and PGC-1?/GAPDH in myocardial tissue and cardiomyocytes after Compound C pretreatment were significantly lower than those in NX group(P<0.05).The fourth part is the basic experiment:the study of Nuanxin Capsule on inhibiting mitochondrial apoptosis induced by oxidative stress1.The ratio of Bcl-2/BAX between myocardial tissue and cardiomyocytes in the TAC group was significantly reduced and the ratio of cleaved caspase-3/GAPDH was significantly increased(P<0.05);compared with the TAC group and the H2O2 group,in NX myocardial tissue and myocardial cells,the ratio of Bcl-2/BAX was significantly increased and the ratio of cleaved caspase-3/GAPDH was significantly decreased(P<0.05);after Compound C pretreatment,compared with NX group,the ratio of Bcl-2/BAX decreased significantly and the ratio of cleaved caspase-3/GAPDH increased significantly(P<O.05).2.The ratio of p-AMPK/AMPK between myocardial tissue and cardiomyocytes in the TAC group was significantly reduced and the ratio of p-JNK/JNK was significantly increased(P<0.05);compared with the TAC group and the H2O2 group,in NX myocardial tissue and myocardial cells,the ratio of p-AMPK/AMPK was significantly increased and the ratio of cleaved p-JNK/JNK was significantly decreased(P<0.05);after Compound C pretreatment,compared with NX group,the ratio of p-AMPK/AMPK decreased significantly and the ratio of cleaved p-JNK/JNK increased significantly(P<0.05).3.In H9c2 cells,the results of flow cytometry showed that compared with the Con group,the early(Annexin V-FITC+/PI-quadrant)and late(Annexin V-FITC+/PI+ quadrant)apoptosis rates of the H2O2 group were significantly increased(P<0.05);Compared with the H2O2 group,the early(Annexin V-FITC+/PI-quadrant)and late(Annexin V-FITC+/PI+quadrant)cell apoptosis rate in the NX group were significantly reduced(P<0.05);The early stage(Annexin V-FITC+/PI-quadrant)plus late stage(Annexin V-FITC+/PI+quadrant)cell apoptosis rate after pretreatment of Compound C were significantly higher than that of the NX group(P<0.05).4.In H9c2 cells,the results of flow cytometry showed that compared with the Con group,the high mitochondrial membrane potential of the H2O2 group was significantly lower(P<0.05);compared with the H2O2 group,the high mitochondrial membrane potential was significantly increased(P<0.05);After pretreatment with Compound C,the mitochondrial high membrane potential was significantly lower than that in NX group(P<0.05).5.Western blotting in H9c2 cells showed that the ratio of cytochrome c/GAPDH in the H2O2 group was significantly higher than that in the con group(P<0.05);compared with the H2O2 group,the ratio of cytochrome c/GAPDH in the NX group was significantly lower(P<0.05);The ratio of cytochrome c/GAPDH after Compound C pretreatment was significantly higher than that of NX group(P<0.05).Conclusion1.The first part of the clinical observation studies showed that Nuanxin Capsule reduces left ventricular myocardial energy consumption in patients with HFpEF,improves left ventricular dilatation,reduces NT-proBNP,increases LVEF,and improves the clinical symptoms of heart-yang deficiency type heart failure.2.In the second part of the basic experiment,mitochondrial dysfunction was taken as the breakthrough point.It was found that Nuanxin Capsule directly reduces the production of intracellular ROS and improves cardiac dysfunction,cardiomyocyte damage and mitochondrial dysfunction in HFpEF mice induced by oxidative stress.3.The third part of the basic experiment takes mitochondrial energy metabolism as the entry point.It was found that Nuanxin Capsule increases the mitochondrial glucose oxidative phosphorylation capacity of H9c2 cells by activating AMPK/PGC-1? signaling pathway,thereby increasing ATP production and improving cardiac dysfunction in HFpEF mice under oxidative stress damage,myocardial cell damage and myocardial energy metabolism disorders.4.In the fourth part of the basic experiment,we found that Nuanxin Capsule inhibits mitochondrial apoptosis by activating AMPK/JNK signaling pathway. |
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