The Investigation Of Hypertension Complicated With LVH Constitution And The Mechanism Of Berberine's Anti-cardiac Hypertrophy Based On Autophagy

Part Ⅰ:Study on the antagonistic effect of berberine on myocardial hypertrophy based on the regulation of autophagy by Lnc RNA-MIAT/m TOR signaling pathwayObjectiveTo explore how berberine can antagonize myocardial hypertrophy by mediating autophagy through lnc RNA/m TOR signaling pathway.MethodsAnimal experiment:using the model of myocardial hypertrophy induced by pressure overload caused by coarctation of abdominal aorta,24 SD rats weighing about 200g were randomly divided into sham operation group（CON,n=8）and abdominal aortic coarctation group（CAA,n=8）,berberine+abdominal aortic coarctation group（BBR+CAA group,n=8）.Berberine（30mg/kg.d）was given intragastrically once a day after coarctation of abdominal aorta.Four weeks after operation,left ventricular ejection fraction（LVEF）,Intraventricular septum in diastole（IVSd）and Left ventricular posterior wall in diastole（LVPWd）were measured by echocardiography,and the pathological changes of myocardium were observed by HE staining.The expressions ofβ-MHC and ANF,Beclin1,ATG5,p62,Lnc RNA MIAT,HOTAIR and CHRF were detected by q PCR.cell experiment:Experiment one:The H9C2 cardiomyocytes were treated with angiotensin II with concentration gradient(0、10^-8、10^-7、10^-6mol/L)for 48 hours and divided into H9C2control group（CON group）,10^-8mol/L Ang II group,10^-7mol/L Ang II group and 10^-6mol/L Ang II group.The morphology of cardiomyocytes and the number of cardiomyocytes per unit area were observed by inverted microscope.The average surface area（μm²）of cardiomyocytes was calculated by image analysis software.The expressions of Lnc RNA MIAT,HOTAIR and CHRF in cardiomyocytes were detected by q PCR.Experiment two:According to the results of experiment one,The H9C2cardiomyocytes was treated with 10^-6mol/L AngⅡand different concentrations of berberine（0μmol/L,0.5μmol/L,5μmol/L,50μmol/L）meanwhile.The experiment was divided into five groups:H9C2 control group（CON group）,10^-6mol/L Ang II group,10^-6mol/L Ang II+0.5μmol/L BBR group,10^-6mol/L Ang II+5μmol/L BBR group,10^-6mol/L Ang II+50μmol/L BBR group.After 48 hours of treatment,the cardiomyocyte area were detected by fluorescence microscopeunder immunofluorescence staining and the expressions of Lnc RNA in cardiomyocytes were detected by q PCR.Experiment three:The experiment was divided into three groups:normal H9C2cardiomyocyte control group（NC group）,plasmid unloaded cardiomyocyte hypertrophy model group(10^-6mol/L AngⅡ+si NC group)and si RNA-MIAT transfected cardiomyocyte hypertrophy model group(10^-6mol/L AngⅡ+si MIAT group).Cell interference technique was used to interfere with the expression of lnc RNA MIAT.The cardiomyocyte area were detected by fluorescence microscope under immunofluorescence staining and the expression of autophagy related signal pathway proteins AMPK,m TOR and LC3Ⅱwas detected by Western bloting.ResultsAnimal experiment:Abdominal aortic constriction operation induced a marked increase in interventricular septum,left ventricular posterior wall thickness and left ventricular weight（LVW）and the ratio of LVW/body weight,as well as the expression ofβ-MHC and ANF,all of which were significantly suppressed by berberine.The results showed that autophagy was inhibited during myocardial hypertrophy,and berberine could antagonize myocardial hypertrophy by enhance autophagy.At the same time,we found that berberine could down-regulate the expression of Lnc RNA MIAT,which can regulate autophagy.cell experiment:The results showed that the expression of Lnc RNA MIAT was significantly increased in H9C2 myocardial hypertrophy cell induced by AngⅡ,which was positively correlated with the concentration of AngⅡ,especially with the concentration of 10^-6mol/L.Berberine could antagonize changes of myocardial hypertrophyand down-regulate the expression of lnc RNA MIAT significantly,which was negatively correlated with the concentration of berberine.The expression of Lnc RNA MIAT was significantly decreased After si MIAT interference,the autophagy of cardiomyocytes was enhanced,and its effect was achieved by enhancing the activity of AMPK and inhibiting the activity of m TOR.ConclusionBerberine can antagonize myocardial hypertrophy by increasing autophagy,which may be mediated by Lnc RNA MIAT/AMPK/m TOR signaling pathway.Part Ⅱ:Study on TCM constitution of patients with Hypertension complicated with left Ventricular hypertrophyObjectiveThe purpose of this study was to study the distribution of TCM constitution types in patients with hypertension complicated with left ventricular hypertrophy,and to summarize the distribution characteristics of TCM constitution types,so as to provide clinical objective basis for further prevention and treatment of traditional Chinese medicine（TCM）.MethodsThe outpatients and inpatients with hypertension complicated with left ventricular hypertrophy in the Department of Cardiovascular Medicine,Baoan TCM Hospital affiliated to Guangzhou University of traditional Chinese Medicine from January 2017 to December 2018 were collected.Through the investigation and filling in the questionnaire of TCM physique,the distribution characteristics of TCM constitution types were analyzed.Results209 patients with hypertension complicated with left ventricular hypertrophy were included,including 129 males and 80 females,with a minimum age of 30 years and a maximum age of 91 years（mean 61.4 ±14.05 years）.The shortest course of disease was 7 months and the longest was 384 months.The average course of disease was 92.61 ±86.88 months.The constitution types of traditional Chinese medicine were phlegm-dampness,damp-heat and yin deficiency,which were 71 cases,52 cases and 50 cases,respectively,accounting for 33.97%,24.88% and 23.93%,respectively.The physique types with less frequency were qi depression,yang deficiency,peace and blood stasis,which were 3 cases,4 cases,7 cases and 8cases,respectively,accounting for 1.44%,1.91%,3.35% and 3.83%,respectively.The order of TCM constitution types is phlegm-dampness > dampness-heat > yin deficiency > qi deficiency > blood stasis > peace > yang deficiency > qi depression.There were significant differences in left ventricular mass index among different constitution types.ConclusionPhlegm-dampness,dampness-heat and yin deficiency are the main TCM constitution types in patients with hypertension complicated with left ventricular hypertrophy.The degree of myocardial hypertrophy of Pinghe quality and qi depression was mild,phlegm-dampness,dampness-heat,blood stasis and yin deficiency were more serious,qi deficiency and yang deficiency were the heaviest.