Study On The Clinical Efficacy Of Yiqi Huoxue Lishui Prescription On Chronic Heart Failure And Its Mechanism Of Ventricular Remodelin

Background:Chronic heart failure is the manifestation of a variety of late cardiovascular diseases,patients with severe symptoms,high mortality,poor prognosis,to the patient’s family and society caused huge economic pressure,studies have shown that ventricular remodeling is an important pathophysiological mechanism of heart failure,is an important cause of heart failure exacerbation and death.Myocardial fibrosis is the core mechanism of ventricular remodeling.Based on the regulation effect of Yiqihuoxuelishui Formula on ventricular remodeling and myocardial fibrosis,this study verified its clinical efficacy through clinical experiments and verified its mechanism of inhibiting ventricular remodeling and myocardial fibrosis through animal experiments,providing new research ideas for preventing heart failure with traditional Chinese medicine.Objectives:1.To examine the clinical efficacy of Yiqihuoxuelishui Formula,namely the Chinese Qiinvigorating,blood-activating and diuretic prescriptions,on patients with chronic heart failure(and with the syndrome of Qi deficiency,blood stasis and water retention).2.To clarify that the effects of Yiqihuoxuelishui Formula on inhibiting myocardial fibrosis and delaying ventricular remodeling are related to the regulation of TGF-β1/Smads signaling pathway,and to further explore the targets of action,thus providing evidence for clinical prevention and treatment of heart failure.Methods:1.Clinical ResearchIn this randomized controlled trial(RCT),patients with chronic heart failure(and with the syndrome of Qi deficiency,blood stasis and water retention)receiving treatment in our hospital were randomly assigned to the treatment group(given combined treatment with Yiqihuoxuelishui Formula and western medicine)and the control group(given western medicine treatment)for evaluating the improvement of their cardiac function and clinical efficacy after four weeks of treatment.(1)The primary outcome was left ventricular ejection fraction(LVEF),evaluating the effect of Yiqihuoxuelishui Formula on improving cardiac function.(2)The secondary outcomes including N-terminal prohormone of brain natriuretic peptide(NT-proBNP),New York Heart Association(NYHA)classification,traditional Chinese medicine(TCM)syndrome score,Minnesota Living with Heart Failure Questionnaire(MLHFQ)were used to assess the safety and clinical efficacy of Yiqihuoxuelishui Formula from multiple aspects.(3)Exploratory detection of galectin-3 was performed to evaluate the effect of Yiqihuoxuelishui Formula on myocardial fibrosis,and to identify its prognostic and therapeutic values in patients with chronic heart failure.2.Animal experimental researchIn order to explore whether the protective effect of Yiqihuoxuelishui Formula on myocardial cells in failing heart is realized by regulating TGFβ1/Smads signaling pathway,this study established a model of heart failure post-myocardial infarction in rats and administered different doses of Yiqihuoxuelishui Formula for intervention,compared with the sham-operated group and model group,so as to verify its effect on TGFβ1/Smads signaling pathway from different levels,and to further clarify the targets of action,thus providing experimental basis for TCM treatment for heart failure.(1)At the animal level:verifying the effect of Yiqihuoxuelishui Formula on ventricular remodeling and myocardial fibrosis in rats with heart failure.The color Doppler small animal ultrasound diagnostic instrument was used to measure left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),left ventricular internal diameter end systole(LVIDs),left ventricular internal diameter end diastole(LVIDd).After the Hematoxylin and Eosin(H&E)staining and Masson’s trichrome staining performed on the myocardial tissues of rats,the collagen volume fraction(CVF)was calculated and the ultrastructure of myocardial cells was observed under transmission electron microscope(TEM).The serum galectin-3 level was measured using ELISA.(2)At the molecular level:exploring the effect of Yiqihuoxuelishui Formula on TGFβ1/Smads signaling pathway and identifying the mechanisms of its inhibiting myocardial fibrosis and improving ventricular remodeling.The protein expressions of TGFβ1,Smad2,Smad3,p-Smad2,p-Smad3 and other proteins were detected using western blot;and the mRNA expressions of TGFβ1,Smad2 and Smad3 were detected using RT-qPCR to identify the targets of action of Yiqihuoxuelishui Formula for improving ventricular remodeling and myocardial fibrosis.Results:1.Clinical Research(1)Ultrasonic cardiogramThe ultrasonic cardiogram was used to evaluate the influence of Yiqihuoxuelishui Formula on cardiac structure and function in patients with heart failure,and the quantified comparisons of LVEF,LVFS,LVIDs and LVIDd were performed in each group.After the combined treatment with western medicine and Yiqihuoxuelishui Formula,LVIDd decreased compared with the control group,indicating that Yiqihuoxuelishui Formula improved the ventricular structure of patients with heart failure.There were no significant differences in LVEF,LVFS and LVIDs between the treatment group and control group(P>0.05).However,within the treatment group,LVEF,LVIDs and LVIDd were improved after the combined treatment,while the significant differences before and after treatment were not observed within the control group,in which patients were treated with only the basic western medicine(P>0.05).The results showed that western medicine combined with Yiqihuoxuelishui Formula could improve ventricular structure and cardiac function in patients with heart failure.(2)NT-proBNPThe NT-proBNP level increased after the incidence of heart failure,while decreased after treatment with Yiqihuoxuelishui Formula,showing significant differences before and after treatment(P<0.01).After administrating Yiqihuoxuelishui Formula,the reduction of NTproBNP level in the treatment group was significantly more than that in the control group(P<0.01).(3)NYHA classificationAfter treatment,the distributions of NYHA classification in both groups were significantly improved(P<0.01),and the improvement in the treatment group was significantly better than that in the control group(P<0.01).The therapeutic effective rates of the treatment group and control group were 84.44%and 44.19%,respectively.The efficacy in the treatment group was significantly better than that in the control group,and the difference was statistically significant(P<0.01).(4)TCM syndrome score and quality of lifeThe TCM syndrome scores in both groups were significantly improved after treatment(P<0.01),and the improvement after treatment with Yiqihuoxuelishui Formula was significantly better than that in the control group(P<0.01).The MLHFQ was used to investigate patients’ quality of life.As for the within-group comparison,the quality of life was significantly improved in both groups after treatment(P<0.01).As for the between-group comparison,after the combined treatment,the improvement of life quality in the treatment group was significantly better than that in the control group(P<0.01).(5)Galectin-3Among patients with heart failure,the Galectin-3 level increased in different degrees.In the Yiqihuoxuelishui Formula group,the Galectin-3 level decreased significantly after treatment(P<0.05).There was no such significant difference between the treatment group and control group(P>0.05).2.Animal experimental research(1)24h urine volumesThe 24h urine volumes of rats in each group after four weeks of intervention were collected using metabolism cages.Compared with the sham-operated group,the urine volume in the model group was significantly decreased(P<0.05);compared with the model group,the urine volumes in the TCM-L,TCM-H and ACEI groups were significantly increased(P<0.05),and the rise was particularly marked in the ACEI group.(2)Lung indexAfter sampling,the lung weights of rats in each group were recorded and the lung index was calculated.Compared with the sham-operated group,the lung index of the model group was significantly increased(P<0.05);compared with the model group,those of the TCM-L,TCM-H and ACEI groups were significantly decreased(P<0.05),and the reduction in the TCM-H group was the most noteworthy.(3)Changes in the cardiac structure and functionThe change of cardiac ultrasound indexes in each group of rats was measured by the echocardiography of small animals.The results showed that compared with the shamoperated group,the model group presented decreased LVEF and LVFS(P<0.05),enlarged ventricles(increased LVIDd and LVIDs;P<0.05),thinner ventricular wall(decreased LVAWd,LVAWs,LVPWd and LVPWs;P<0.05),higher left ventricular index(P<0.05),and increased left ventricular end-diastolic volume(LV Vol;d).After four weeks of treatment,the cardiac function and structure of rats in the ACEI,TCM-L and TCM-H groups were improved.Specifically,there were significant differences in improving the cardiac function(LVEF and LVFS increased)and increasing the thickness of the left ventricular wall in infarction areas(LVPWd increased;P<0.05),while there were only improving trends in other indicators.(4)Morphological changes in myocardial tissuesIn the sham-operated group,the myocardial cells of rats were closely arranged with complete morphology and clear nuclei.Compared with the sham-operated group,the number of myocardial cells in the model group was reduced,and the cells were swollen,infiltrated by a large number of inflammatory cells.The pathological morphology and structure of the myocardial cells in the TCM-L and TCM-H groups were improved,and the same trends also existed in positive medicine.(5)Degree of myocardial fibrosisThe Masson’s trichrome staining was used to observe the myocardial fibrosis of rats in each group,and the results showed that the myocardial cells of rats in the sham-operated group were intact and the myocardial fibers were arranged neatly and tightly.Compared with the sham-operated group,the myocardial cells of rats in the model group were disordered and the mesenchyme was filled with a large number of blue-dyed collagen fibers(P<0.05).Compared with the model group,the myocardial cells in the ACEI,TCM-L and TCM-H groups were arranged neatly,and there were less blue-dyed collagen fibers(P<0.05).(6)Morphological changes of myocardial fibers and mitochondriaThe TEM was used to observe the mitochondrial ultrastructure of myocardial cells in each group.The myocardial fibers of rats in the sham-operated group were arranged tightly and orderly,intercalated discs were complete,and mitochondria were arranged neatly between cardiac fibers,mostly were round,oval or rod.Compared with the sham-operated group,the myocardial fibers in the model group decreased in number,with disordered arrangement,blurred intercalated discs as well as adherent,swollen and deformed mitochondria.Compared with the model group,the myocardial mitochondrial structure of the ACEI,TCM-L and TCM-H groups were improved,and the improvement in the ACEI group was the best.(7)Galectin-3 expression levelELISA was used to detect the serum Galectin-3 expression level of rats,which in the model group significantly increased compared with that in the sham-operated group(P<0.05).After intervention,the serum Galectin-3 levels in the ACEI,TCM-L and TCM-H groups significantly decreased(P<0.05),with the ACEI group showing the most significant effect.(8)Levels of TGF-β1/Smads mRNA expressionCompared with the sham-operated group,the TGF-β1,Smad2 and Smad3 mRNA expression in myocardial tissues of rats in the model group significantly increased(P<0.05).Compared with the model group,the levels of TGF-β1,Smad2 and Smad3 mRNA expression in the ACEI,TCM-L and TCM-H groups significantly decreased(P<0.05).(9)Levels of TGF-β1/Smads protein expressionWith the western blot examining levels of TGF-β1/Smads protein expression,the results showed significant increase in the TGF-β1,p-Smad2 and p-Smad3 protein expression in myocardial tissues of rats in the model group when compared with the sham-operated group(P<0.05).Compared with the model group,the TGF-β1,p-Smad2 and p-Smad3 protein expression in the ACEI,TCM-L and TCM-H groups significantly decreased(P<0.05).Conclusions:1.The combined treatment with Yiqihuoxuelishui Formula and basic western medicine may improve the clinical symptoms of patients with chronic heart failure(and with the syndrome of Qi deficiency,blood stasis and water retention),ameliorate their cardiac function,reduce the level of NT-proBNP and Galectin-3 as well as the TCM syndrome score,and enhance their quality of life.2.The Yiqihuoxuelishui Formula may improve the body function in those with heart failure,reduce the damage of myocardial cells in rats with chronic heart failure,ameliorate myocardial fibrosis,inhibit ventricular remodeling after heart failure,and promote cardiac function,which may be realized through the regulation of TGF1/Smads signaling pathway.