| Part One:Clinic TrialObjection:To evaluate the clinical efficacy and safety of Qiangxin Decoction in patients with diastolic heart failure(heart and kidney yang deficiency syndrome).Methods:Patients in this study are from the inpatient department of the Department of Cardiology in Shanghai Municipal Hospital of T.C.M.from 1/7/2017 to 1/11/2018,which is in accordance with the heart and kidney yang deficiency syndrome,and the diagnosis as diastolic heart failure(NYHA Ⅱ-Ⅲ).They were randomly assigned to the control or treatment group.The control group received western medicine treatment with perindopril 4mg qd.po.+metoprolol qd.po.,if the patient has edema,then add furosemide 20mg qd.po.+spironolactone 20mg qd.po..In the other hand,treatment group would take QiangXin Decoction based on the treatment of the control group.The QiangXin Decoction consists of aconite 6g,cinnamon 6g,cassia twig 12g,psoralen 30g,salvia 30g,peach kernel 15g,safflower 6g,white peony 12g,polygonatum 18g,alisma 30g,big belly skin 30g,400ml bid.po..The course of treatment is 4 weeks.At the 0th,2nd,and 4th week,the data on the efficacy of patients in two groups were recorded,including NYHA grading,LEE’s score,TCM score,NT-proBNP,echocardiography,heart rate blood pressure,heart rate variability,etc.,and daily safety events.The data was analyzed using SPSS 21.0.Results:A total of 142 patients were enrolled in the study.8 patients in the control group,4 patients in the treatment,12 patients in total are shed,130 patients finished the study in the end.There was comparability between the two groups for baseline.In terms of NYHA grading,after 4 weeks’ treatment,22 patients(35.9%)was NYHA grading Ⅰ,28 patients(44.4%)was NYHA grading Ⅱ,13 patients(20.6%)was NYHA grading Ⅲ,and none was NYHA grading Ⅳ,while in the treatment group 32 patients(47.8%)was NYHA grading Ⅰ,26 patients(38.8%)was NYHA grading Ⅱ,9 patients(13.4%)was NYHA grading Ⅲ,and none was NYHA grading Ⅳ.There was no significant difference in the distribution of NYHA grading between two groups after 4 weeks of treatment(P>0.05).In terms of the efficacy based on NYHA grading,there were 35(55.6%)effective cases,28(44.4%)ineffective cases and none aggravated cases in the control group,while 36(53.7%)effective cases,31(46.3%)ineffective cases and none aggravated cases in the treatment group.There was no significant difference in the efficacy based on NYHA grading between two groups after 4 weeks of treatment(P>0.05).In terms of the LEE’s score,at 4 weeks of treatment,the treatment group was lower than the control group(P<0.05).In the comparison of time points in each group,LEE’s score in the control group was lower than its in the first place(P<0.05),while the treatment group decreased significantly after 2 weeks treatment(P<0.001).In terms of the efficacy based on LEE’s score,there were 6(9.5%)markedly effective cases,26(41.3%)effective cases,31(49.2%)ineffective cases and none aggravation in the control group,while there were 6(9.0%)markedly effective cases,45(67.2%)effective cases,16(23.9%)ineffective cases and none aggravated in the treatment group.There was a statistically significant difference in the distribution of the efficacy based on LEE’s score after 4 weeks’treatment(P<0.05).In terms of the TCM score,after 4 weeks’ treatment,there was a statistically significant difference between two groups in the TCM scores(P<0.001).The mean value of difference between the treatment group and the control group was 6.190(95%CI:3.602-8.779).In the comparison of the time points in each group,the total TCM score in the treatment group was lower than that in the control group(P<0.001).Among the symptoms and signs,the scores of ’palpitations’,’abdominal distension’,’qi shortness’,’chilling’,’chest distress’ in the treatment group were lower than those in the control group(P<0.05).In terms of the efficacy based on the TCM scores,there was 1(1.6%)markedly effective case,37(58.7%)effective cases,17(27.0%)ineffective cases,7(11.1%)aggravated cases in the control group,while there were 15(22.4%)markedly effective cases,31(46.3%)effective cases,16(23.9%)ineffective cases and 6(9.0%)aggravated cases in the treatment group.There was a statistically significant difference in the distribution of the efficacy based on the TCM scores between the two groups after 4 weeks’ treatment(P<0.05).In terms of the NT-proBNP,there was no significant difference between two groups(P>0.05),but the NT-proBNP decreased gradually in the treatment group(P<0.05).In terms of echocardiography,there was no significant difference between two groups in LVEDD,LVESD,LAD,LVEF,E/A before and after the treatment(P>0.05),whereas LVEDD in two groups decreased significantly(P<0.001),and E/A in both groups gradually decreased after treatment(P<0.05).In terms of Tissue Doppler Imaging,there was a statistically significant difference(P<0.001)between the two groups at 4 weeks’treatment.The treatment group was higher than the control group and mean value of e’in the treatment group gradually decreased significantly with the progress(P<0.001).The mean value of E/e’ in the treatment group was lower than the control group(P<0.05).In terms of autonomic nervous function,about heart rate,there was a statistically significant difference in heart rate between two groups at 2 weeks’ treatment and 4 weeks’ treatment(P<0.001).There was no significant difference in systolic blood pressure and diastolic blood pressure between two groups(P>0.05).The blood pressure in the group decreased significantly with the process of treatment(P<0.001).In terms of heart rate variability,after 2 weeks’ and 4 weeks’ treatment,there was statistically significant difference of SDNN between two groups(P<0.05),moreover,SDNN in both groups were significantly increased with the process of treatment(P<0.001).After 2 weeks’ and 4 weeks’ treatment,there was a statistically significant difference between two groups(P<0.05).SDANN increased in both groups after 4 weeks’treatment(P<0.05).There was a statistically significant difference in rMSSD between the two groups(P<0.001).Mean value of pNN50 in the control group was 8.95±2.07,11.84±2.58,12.83±2.47(%)at 0th,2th,4th weeks,respectively,while the treatment group was 8.71±2.95,11.24±2.92,14.75±2.91(%),respectively.After 4 weeks’treatment,there was a statistically significant difference between the two groups(P<0.001),which the treatment group was higher than the control group and pNN50 in two groups gradually increased with the process of treatment(P<0.001).There was no significant difference in TP between two groups(P>0.05).In terms of safety evaluation,5 cases had adverse reactions in the control group,the adverse reaction rate was 7.0%.Among them,there were 2 cases of edema,1 case of chest distress and 2 cases of low blood pressure.There were 3 cases of adverse reactions in the treatment group,the adverse reaction rate was 4.2%.Among them,3 cases were diarrhea,and the adverse reactions were mild in both groups.In addition,the changes in blood routine examination,liver and kidney function examination and other indicators before and after treatment were within the normal range,and no obvious abnormal changes were observed.Conclusion:1.QiangXin Decoction can improve the positive signs of patients with diastolic heart failure.2.QiangXin Decoction can improve the clinical symptoms and signs in T.C.M.of patients with diastolic heart failure.3.QiangXin Decoction can improve diastolic dysfunction of left ventricular in patients with diastolic heart failure.4.QiangXin Decoction can increase the parasympathetic function of patients with diastolic heart failure.5.QiangXin Decoction may cause mild diarrhea in few patients with diastolic heart failure,but it is still safe to use.Part Two:Experiment StudyObjection:Evaluation of the efficacy of Acetyl-Higenamine(AH)on cardiac fibrosis and hypertrophy through in vitro and in vivo experiments and then explore its related mechanisms.Methods:Experiment 1:In vitro study of the efficacy of AH on cardiac fibrosis and hypertrophy.First,use neonatal rat cardiac fibroblasts,and use TGF-β(10ng/ml)to induce the fibrosis reaction.The efficacy of AH was evaluated by detecting α-SMA protein or related gene.AH is divided into some different concentration,including 0.1,0.3,1,3uM to explore its working concentration.Furthermore,by detecting other fibrosis-related protein or gene and wound healing test to confirm the effect of AH on cardiac fibrosis.Then,neonatal rat cardiomyocytes were used to evaluate the effect of AH on cardiac hypertrophy induced by phenylephrine(100uM),and that was observed by immunostaining.Experiment 2:In vivo study of the effects of different doses of AH on iso-induced cardiac fibrosis and pathological hypertrophy in mice.60 SPF 8-week-old female c57BL/6 mice were randomly divided into blank group,iso group,iso+AH low dose group,iso+AH medium dose group,iso+AH high dose group and iso+Higenamine(Hige)group(positive control).Except the blank group,each group received iso subcutaneous injection 30mg/kg/d,and iso+AH groups from low to high dose received extra AH intraperitoneal injection from 1,3,10mg/kg/d,respectively,iso+Hige group received intraperitoneal injection of 10mg/kg/d.This experiment last for 21 consecutive days.On the 22th day,mice would have echo test and then the hearts were detected by pathological staining and cardiac hypertrophy,fibrosis-related protein or genes were detected.Experiment 3:In vivo study of the effects of oral AH on iso-induced cardiac fibrosis and pathological hypertrophy in mice.40 SPF 8-week-old female c57BL/6 mice were randomly divided into blank group,AH group,iso group and iso+AH group.Except the blank group,each group received iso subcutaneous injection 30mg/kg/d,AH groups were given AH 20mg/kg/d orally.This experiment last for 35 consecutive days.The hearts were taken for pathological staining and hypertrophy,fibrosis-related protein or gene detection.Experiment 4:In vitro study of the mechanism of effects of AH on TGF-β-induced neonatal rat cardiac fibroblasts.Fibrosis was activated in neonatal rat cardiac fibroblasts by TGF-β(10ng/ml),then the phosphorylated or non-phosphorylated protein or gene associated with Smad signaling pathway would be detected.Moreover,usingβ-adrenoreceptor inhibitors to explore whether the upstream target is β-adrenoreceptors.Results:Experiment 1:When the concentration of the AH group was greater than luM,the expression of α-SMA protein and its gene in the AH groups were lower than that in the TGF-β group(P<0.05).The half maximal inhibitory concentration(IC50)of AH is about 2uM.In other fibrosis-related genes,AH also inhibits the expression of collagen I,collagen III,fibronectin,transgelatin,osteopontin,myosin,troponin in TGF-β-induced fibrosis of primary culture of neonatal rat cardiac fibroblasts(P<0.05).In wound healing test,AH can slow the cell migration induced by TGF-β in primary culture of neonatal rat cardiac fibroblasts(P<0.05).In addition,AH also inhibited PE-induced primary culture of neonatal rat cardiomyocyte hypertrophy(P<0.05).Experiment 2:Echocardiography showed the LVEDV in iso+AH high-dose group and iso+Hige group were lower than the iso group(P<0.05),mean value of which were 0.13±0.02,0.16±0.02(ml),respectively,and the mean difference was 0.072±0.016(ml)(95%CI:0.040-0.103)and 0.045±0.013(ml)(95%CI:0.018-0.072),respectively.The LVESV in the iso+AH high-dose group was lower than the iso group(P<0.05),with an average of 0.07±0.01(ml),and the mean difference was 0.027±0.012(ml)(95%CI:0.002-0.052).The LVIDd in the iso+AH high-dose group was significantly lower than the iso group(P<0.001),with an average of 3.76±0.22(mm)and the mean difference was 0.635±0.141(mm)(95%CI:0.344-0.926),moreover,iso+Hige group was also lower than iso group(P<0.05),with mean value of 4.03±0.31(mm),and mean difference of 0.373±0.121(mm)(95%CI:0.125-0.620).In echocardiogram,the contraction and diastolic function of the hearts gradually increased with the increase of the drug dose in the AH-loaded group.Compared with the iso+Hige group,the waveform separation distance of iso+AH high-dose group changed more significantly.In the images of hearts,the left ventricular papillary muscle in iso group was not accessible becaouse of the cardiac hypertrophy,while that in the iso+AH high-dose group and the iso+Hige group could be explored.In terms of survival rate,there were two mice died in iso group at the 7th and 9th day of the experiment,two in iso+AH low-dose group at the 9th and 13th day,and one in iso+AH medium-dose group at 14th day,but there was no statistical difference between the survival time of each group(P>0.05).In terms of the hearts in each group,in iso+AH high-dose group(10mg/kg),HW/BW showed a statistically decrease(P<0.05),while HW/TL showed a statistically significant decrease(P<0.001),which was the same as the iso+Hige group,and the HW/TL in iso+AH mid-dose group was also lower than that in the iso group(P<0.05).In terms of fatal gene expression,the Nppa in iso+AH medium group,iso+AH high-dose group,iso+Hige group were lower than those in iso group(P<0.05).There was only a decrease in AH high-dose group of Nppb compared with iso group(P<0.05).The β-MHC in iso+AH high-dose group and iso+Hige group were lower than those of the iso group(P<0.05).In masson’s staining,the fibration staining areas of iso+AH medium-dose and high-dose group were significantly lower than those of the iso group(P<0.001),and iso+Hige group was lower than iso group(P<0.05).In Sirius red staining,the fibration staining areas of iso+AH medium-dose and high-dose group and iso+Hige group were lower than that of the iso group(P<0.05).In the expression of fibrosis-related gene expression,the expression of col-1 in the AH medium-dose group was lower than that in the iso group(P<0.05),and there was a significant decrease compared with the iso group in AH high-dose group of the expression level of col-1 and col-3(P<0.001).Experiment 3:In the survival of each group of mice,during the 35 days of experiment,the mice in the iso group died two on the 12th and 13th day of the experiment,and no death occurred in the other groups,but there was no statistical difference between each group(P>0.05).In terms of the hearts,HW/BW and HW/TL in the iso+AH group had a statistically significant decrease compared with the iso group(P<0.05).In HE staining,the iso+AH group can alleviate the disorder of myocardial cell nucleus induced by iso.In the detection of fatal gene expression,the expression of ANP,BNP,β-MHC in iso+AH group was lower than that in iso group(P<0.05).In terms of fibration staining,the fibration area of masson’s and Sirius red in the iso+AH group showed a statistically significant decrease compared with the iso group(P<0.05).Experiment 4:TGF-β activates phosphorylation of Smad2/3 signaling at approximately 30 min in primary culture of neonatal rat cardiac fibroblasts.However,AH could decrease the expression level of p-Smad2/3 which was activated in TGF-β group(P<0.05),and AH could inhibit the expression of Smad4 protein and its gene(P<0.05),while up-regulated Smad7 protein and its gene(P<0.05).When using β-adrenoreceptor inhibitors,the levels of α-SMA in the TGFβ+AH group,TGFβ+AH+CGP-20712A,TGFβ+AH+ICI-118551 groups were lower than those in the TGFβ group(P<0.05),indicating that there was no difference between the groups(P<0.05).Conclusion:1.In vitro study,AH can improve TGFβ-induced fibrosis in neonatal rat fibroblast and PE-induced hypertrophy in neonatal rat cardiomyocytes.The half maximal inhibitory concentration(IC50)is about 2uM.2.In vivo study,intraperitoneal injection of AH(10mg/kg)and oral administration of AH(20mg/kg)can both improve iso-induced cardiac hypertrophy and fibrosis in c57BL/6 mice.3.Structural modification of acetylation enhances the intervention of H on myocardial fibrosis induced by iso in mice.4.AH can inhibit the TGF-β/Smad signaling pathway in neonatal rat fibroblasts in vitro,but its upstream is independent of β-adrenoreceptor. |
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