| 一、CLINICAL RESEARCHObject ive:Based on the biochemical indexes of patients,the clinical efficacy of Xinyinpian tablets was predicted and the suitable population of Xinyinpian tablets was analyzed and screened.Methods:The study mainly collected patients who had admitted to our department of cardiology in the past two years due to chronic heart failure(HF Chronic heart failure),collected their medical history from patients with qi yin deficiency and blood stasis,and collected physicochemical and other auxiliary examinations during their hospitalization.Retrospective study was carried out,combined with auxiliary examination to summarize the characteristics of patients with qi and yin deficiency and blood stasis,and support vector machine,decision tree analysis,characteristic analysis,and supervised learning model prediction for patients with Xinyinpian tablets.Results:NYHA classification has high sensitivity to diagnose patients with qi-deficiency and blood stasis type heart failure.The area under the ROC curve is AUONYHA=0.73,NYHA sensitivity is 80%,specificity is 62%,LVEF AUCEF=0.64,sensitivity is 55%,specificity is 62%..The central function of patients with Qi-Yin deficiency,blood stasis and water-stasis was grade II,BNP>24.7pg/mL,TC≤5.29 mmol/L,and the effective rate was 5%.Cardiac function grade Ⅲ/Ⅳ,LDL-C≤2.53mmol/L,effective rate 88%.Heart function level Ⅳ,EF>32,effective rate 95%.EF>74,effective 8%.Cardiac functionⅢ/Ⅳ,age≤76 years old,EF≤74,BNP≤1798pg/mL,HCY>15.7umol/L patient effective rate 100%.Female heart function Ⅲ/Ⅳ,age≤76 years old,EF≤74,TG<1.63mmol/L,HCY≤15.7umol/L and medication time less than 30 days,the effective rate of patients is 0.Male cardiac function Ⅳ,age≤76 years old,EF≤71,TG＞0.64mmol/L,HCY≤15.7umol/L patient effective rate 13%.HF≤74,the age of patients>76 years old is 98%effective.Conclusion:Patients with heart function level Ⅱ or higher,accompanied by hyperhomocysteinemia,are more suitable for taking Yinyinpian tablets.二、EXPERIMENTAL STUDY(—)Simultaneous determination of nine index components in Xinvinpian tablets by UPLC-MSObjectiveTo establish a UPLC-MS method for the determination of geniposidic acid,quercetin-3-O-β-D-glucose-7-0-β-D-gentiobioside,leonurine,ginsenoside Rg1,ginsenoside Rb1,astragaloside Ⅳ,ilexsaponin A1,ophiopogonin D and ilexgenin A in Xinyinpian.MethodsThe determination was carried out with a Waters ACQUITY UPLC HSS T3 column(1.8 μm 2.1 mm X 100 mm)and the moble phase comprised of acetonitrile and water containing 0.1%formic acid.The flow rate,column temperature and injeciton volumn were 0.4 mL/min,40℃ and 2 μL,respectively.ResultsThe geniposidic acid,quercetin-3-0-β-D-glucose-7-O-β-D-gentiobiosi de,leonurine,ginsenoside Rg1,ginsenoside Rb1,astragaloside Ⅳ,ilexsap onin A1,ophiopogonin D and ilexgenin A showed a good linearity(r>0.9990)in the ranges of 2.3-2300.0,1.6-400.0,4.7-1800.0,2.9-2300.0,3.3-3400.0,1.7-200.0,2.2-2000.0,3.1-200.0 and 1.9-1800.0 ng/mL.The RSD of percision,reproducibility and stability were all less than 2.00%.The ov erall recoveries were ranged from 97.50-101.90%with the RSD ranging from 0.81-2.81%.ConclusionThis method is simple,speedy and accurate,which can be used for quality control of XinvinPian tablets.(二)MLK3/JNK-autophagy mechanism mediates myocardial fibrosis in mice with chronic heart failureObject iveTo study the molecular mechanism of MLK3/JNK-autophagy in regulating myocardial fibrosis in mice with heart failureMethodsSeventy-five SPF C57 male mice,8 weeks old,except the sham operation group,all constructed TAC models,established a mouse model of chronic heart failure,and randomly divided HF mice into four groups,HF group and MLK3 inhibitor group.(URMC099),autophagy inhibitor group(3MA),rapamycin(RAPA)autophagv inducer group.One week after successful model,the corresponding volume and drug were injected intraperitoneally for 30 days.After 30 days,echocardiography was performed,and the samples were taken through paraffin section of heart,masson staining,WGA staining,immunohistochemistry,immunofluorescence,and the changes of myocardial cell morphology and myocardial fibrosis.The inhibitors URMC099,3MA were used to detect the expression of MLK3,P-MLK3,JNK,P-JNK,LC3,Beclin-1,Lampl autophagy,Collagen Ⅰ,Collagen Ⅲ,α-SMA fibrotic protein and gene.Results:1.Inhibition of MLK3 effectively inhibits autophagy and improves cardiac function.Compared with the control group,LVEF and FS were significantly decreased in the RAPA group and the HF group,and LVDd and LVDs were significantly increased(P<0.05).Compared with HF group,LVMC group,3MA group,LVEF,FS increased significantly,LVDd,LVDs decreased relatively(P<0.05)2.Inhibitor URMC099,3MA can significantly inhibit the expression of BNP and ANP genes.Compared with control,the levels of BNP and ANP in HF group were significantly increased(P<0.01).The levels of BNP and ANP in MLK3 inhibitor group and 3MA inhibitor group were significantly lower than those in HF group(P<0.05).3.MLK3,an autophagy inhibitor,effectively prevents cardiac hypertrophy and reduces myocardial fibrosis.Compared with the control group,the heart weight/body weight ratio of the HF group was significantly higher(P<0.05),and the ratio of the HF group was lower than that of the MLK3 and 3MA inhibitor groups(P<0.05).Compared with the control group,the HF group had myocardial fibrosis.The degree of myocardial fibrosis was significantly reduced in MLK3 and 3MA groups compared with HF(P<0.01).Compared with control group,the surface area of myocardial cells in HF group was large(P<0.05);The cell surface area of the group was decreased(P<0.05)compared with the MLK3 and 3MA inhibitor groups.4.MLK3,autophagy inhibitor,can inhibit MLK3,P-MLK3,P-JNK protein,autophagy protein LC3II/I,Beclinl,Lampl,myocardial fibrosis related proteins Coll-Ⅰ,Coll-Ⅲ,α-SMA expression.Compared with the control group,MLK3,P-MLK3,and P-JNK were also up-regulated in the myocardial tissue of the HF group(P<0.05),while the expression of LC3Ⅱ/Ⅰ,Beclinl,and Lampl associated with autophagy activity increased significantly.(P<0.05),the expression of Coll-Ⅰ,Coll-Ⅲ and α-SMA collagen related to myocardial fibrosis expression was up-regulated simultaneously(P<0.05).Compared with HF group,MURMC group,3MA group MLK3,P-MLK3,P-JNK protein,autophagy marker proteins LC3Ⅱ/Ⅰ,Beclinl,Lampl expression were significantly down-regulated,myocardial fibrosis-related proteins Coll-Ⅰ,Coll-Ⅲ The expression ofα-SMA collagen was down-regulated(P<0.05).5.MLK3 and autophagy inhibitor can effectively inhibit the expression of Coll-Ⅰ,Coll-Ⅲ and α-SMA genes in mouse myocardial tissue.Compared with the control group,the expressions of Coll-Ⅰ,Coll-Ⅲ and α-SMA in the HF group were up-regulated(P<0.05).Compared with the URMC group,the expressions of Coll-Ⅰ,Coll-Ⅲ and α-SMA in the 3MA group were down-regulated.(P<0.05).ConclusionMLK3/JNK may ameliorate myocardial fibrosis by reducing heart function in mice with chronic heart failure by down-regulating autophagy.(三)Study on the prevention and treatment of myocardial fibrosis in mice with chronic heart failure by Xinyinpian TabletsObject iveObservation on the preventive and therapeutic effects of Xinyin Tablet on myocardial fibrosis in mice with chronic heart failure.Methods90 male Sprague-C57 C57 male mice,8 weeks old,except the sham operation group,all constructed TAC models,established a mouse model of chronic heart failure,and randomly divided HF mice into five groups,HF group and Xinvinpian Tablet,medium and low dose groups,perindopril PDPL.One week after the model was successfully administered,the corresponding volume and medicine were given.After 60 days of continuous administration,cardiac ultrasound was performed.The samples were taken through paraffin section of the heart and stained with masson to observe changes in myocardial fibrosis.The expression of MLK3,P-MLK3,JXK,P-JNK,LC3,Beclin-1,Lampl autophagy,Collagen Ⅰ,CollagenⅢ,α-SMA fibrosis protein and gene in mouse myocardial tissue.Results1.The high and medium doses of Xinyinpian Tablet can effectively improve the heart function of mice after heart failure.Compared with the control group,the LVEF and FS of the HF group were significantly decreased,and the LVDd and LVDs were significantly increased(P<0.05).Compared with the HF group,the LVEF and FS were significantly increased in the perindopril group,the high-dose and middle-dose groups,and the LVDd and LVD were relatively decreased(P<0.05).Compared with the HF group,the improvement of LVEF,FS,LVDd and LVDs in the low-dose group(xyp0.3g/Kg)was not significant(P>0.05).2.Xinvinpian tablets high and medium dose group,perindopril group can improve myocardial fibrosis in mice after heart failure.Compared with the control group,the degree of myocardial fibrosis was significantly aggravated in the HF group compared with the control group(P<0.01).Compared with the HF group,the degree of myocardial fibrosis in the high-and medium-dose group and the perindopril group was significantly reduced(P<0.01).Compared with the HF group,the improvement of myocardial fibrosis in the low-dose group was not significant(P>0.05).3.Xinvinpian Tablet can effectively inhibit myocardial fibrosis by inhibiting MLK3/JNK and inhibiting autophagy.Compared with the control group,MLK3/JNK,P-MLK3,and P-JNK proteins were up-regulated in the HF group(P<0.05).Compared with the HF group,the expression of MLK3/JNK,P-MLK3 and P-JNK protein in the perindopril group,Xinyin high and middle dose groups was down-regulated(P<0.05).Compared with the control group,the expression of LC3,Beclin-1 and LAMP1 in the HF group was up-regulated(P<0.05).Compared with the HF group,the expression of LC3,Beclin-1 and LAMP 1 protein in the perindopril group,the high-dose and middle-dose groups was down-regulated(P<0.05).Compared with the control group,the expression of α-SMA,collagenⅠ and Collagen Ⅲ collagen in the HF group was significantly increased(P<0.01).Compared with the HF group,the perindopril group,the heart-negative high-and medium-dose group α-SMA The expression of collagenI and collagenⅢ protein was down-regulated(P<0.05).4.Xinyinpian Tablet can inhibit the expression of BNP and ANP genes.Compared with the control group,the expression of BNP and ANP in the HF group was up-regulated(P<0.01).Compared with the HF group,the expressions of BNP and ANP were down-regulated in the perindopril group,the high-grade and low-dose groups(P<0.05).5.Xinyinpian Tablet can inhibit the expression of Coll-Ⅰ,Coll-Ⅲ and a-SMA genes.Compared with the control group,the expression of Coll-Ⅰ,Coll-Ⅲ and a-SMA genes in the HF group was up-regulated(P<0.01).Compared with HF group,the expressions of Coll-Ⅰ,Coll-Ⅲ and a-SMA were down-regulated in the high,middle and low dose groups of Perindopril and Xinyin tablets(P<0.05).ConclusionThe high and middle dose groups of Xinyinpian Tablet can improve heart function in heart failure mice.Xinyinpian Tablet improves myocardial fibrosis in heart failure mice by regulating MLK3/JNK-autophagy. |
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