Studies Of Reverse Effects And Mechanisms Of17-methoxyl-7-hydroxy-benzene-furanchalcone On Pressure Overload-induced Cardiovascular Remodeling Rats

Background：Hypertension is a continuum that starts with a rise in blood pressure,evolves to left ventricular hypertrophy (LVH), proteinuria or endothelialdysfunction, and the major pathophysiological character is pressure overloadand cardiovascular remodeling, including vascular remodeling and cardiacremodeling. Insofar as it is not adequately treated or controlled, finally leads tothe development of complications, and the most relevant of which are stroke andheart failure. Therefore, whether and how to reverse the pressureoverload-induced ventricular remodeling is the key of the recent research. Thedevelopment of new antihypertensive and anticardiovascular remodeling drugsis the focus, which will be expected the decrease in morbidity and mortality ofhypertension. Millettia pulchra (Benth.) Kurz var. Laxior (Dunn) Z.Wei (MKL)is a traditional Chinese medicinal herb that is extensively distributed in theGuangxi Province of China, and17-methoxyl-7-hydroxy-benzene-furanchalcone (MHBFC) is a flavonoid monomer that was originally isolatedfrom a60%ethanol extract from MKL roots. Previous studies havedemonstrated that MHBFC could scavenge hydroxyl radicals and oxyradicals,enhance the cardiocyte survival rate in H2O2and hypoxia/reoxygenation injury,and protect the heart against myocardial ischemia in vitro and in vivo. Based on the above information, we hypothesized that MHBFC might be effective in thetreatment of hypertensive heart disease. Here, for the first time, we were toinvestigate its effect on cardiovascular remodeling induced by pressure overloadas well as the potential mechanisms, which provide the experimental evidenceand theoretical support for further development of MHBFC on reversingcardiovascular remodeling.Objective:To study the reverse effects and mechanisms of MHBFC on pressureoverload-induced cardiovascular remodeling in rats, which provid theexperimental evidence and theoretical support for further development ofMHBFC on reversing cardiovascular remodeling.Methods:Male Sprague–Dawley rats with a body weight of130–160g wereanesthetized under sterile conditions, and the abdominal aorta above the kidneyswas exposed through a abdominal incision and constricted at the suprarenallevel by a4-0silk suture tied around both the aorta and a blunted22-gaugeneedle, which was then pulled out. A similar procedure was performed for shamgroup without the ligature.PartⅠ The protective effects of MHBFC on cardiovascular system inpressure-overload ratsAfter surgery, the rats were divided randomly into5groups (n=6)：(1) shamgroup;(2) model group;(3) Lisinopril15mg/kg group;(4) MHBFC6mg/kggroup;(5) MHBFC12mg/kg group. The drugs were dissolved in distilled waterand orally administered once a day in0.2mL/100g body weight for6weeksfrom the fourth day after surgery. At the end of the experiment, a biologicalsignal quantitative analytical system was used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE andMasson’s stain, the transmission electron microscopes was used to observe themyocardium ultrastructure. RT-PCR method was used to measure the expressionof atrial natriuretic peptide (ANP) mRNA in myocardial tissue.Part Ⅱ Studies of the endothelial mechanisms of MHBFC on pressureoverload-induced cardiovascular remodeling in ratsExperiment1Studies of endothelial mechanisms of MHBFC base on theendothelin system in pressure-overload ratsThe samples obtained from the first part of the experiment were dividedinto4groups:(1) sham group;(2) model group;(3) Lisinopril15mg/kg group;(4) MHBFC12mg/kg group. ELISA was used to determine the plasma ET-1content, RT-PCR method was used to measure the cardiac ET-1and ECE mRNAexpressions, immunochemical method was used to evaluate the ETAand ETBexpressions in myocardial tissue.Experiment2Studies of endothelial mechanisms of MHBFC base on theeNOS-NO signal pathway in pressure-overload ratsAfter surgery, the rats were divided randomly into5groups (n=6):(1) shamgroup;(2) model group;(3) MHBFC12mg/kg group;(4) L-NAME50mg/kggroup (L-NAME50group);(5) L-NAME50mg/kg+MHBFC12mg/kg group(L-NAME50+MHBFC12group). At the end of the experiment, a biologicalsignal quantitative analytical system was used to evaluate the hemodynamicsand heart function, and the histological changes were investigated by HE andMasson’s stain. The immunochemical method was used to evaluate the cardiaceNOS expression, and biochemical method was used to determine plasma NOcontent. Experiment3Studies of endothelial mechanisms of MHBFC base on thePGI2in pressure-overload ratsAfter surgery, the rats were divided randomly into5groups (n=6)：(1) shamgroup;(2) model group;(3) MHBFC12mg/kg group;(4) indomethacin2mg/kggroup (Indo2group);(5) indomethacin2mg/kg+MHBFC12mg/kg group(Indo2+MHBFC12group). At the end of the experiment, a biological signalquantitative analytical system was used to evaluate the hemodynamics and heartfunction, the histological changes were investigated by HE and Masson’s stain.ELISA was used to determine the plasma PGI2content.Results：PartⅠ The protective effects of MHBFC on cardiovascular system inpressure-overload rats1. The change of blood pressure: During the time course, the tail arterialsystolic blood pressure (SBP) in sham group rats maintained at the baseline level,the tail arterial SBP in model group rats increased significantly with atime-dependent manner. After treatment with MHBFC6,12mg/kg, the increaseof tail arterial SBP was inhibited markedly from the fourth week (P＜0.05or P＜0.01vs model group).At sixth week after aortic banding, the carotidASBP,ADBP and AMBP in model group rats increased significantly (P＜0.01), whencompared with the sham group. After treatment with MHBFC6,12mg/kg, theincrease of carotid ASBP, ADBP and AMBP were inhibited significantly (P＜0.05or P＜0.01vs model group).2. The change of heart function: At sixth week after aortic banding, the LVSPand±dp/dtmaxin model group rats increased significantly (P＜0.01vs shamgroup), while LVEDP decreased significantly (P＜0.01vs sham group). Aftertreatment with MHBFC6,12mg/kg, the left ventricular systolic and diastolicfunction were ameliorated significantly (P＜0.05or P＜0.01vs model group). 3. Cardiovascular remodeling: At sixth week after aortic banding, the heartweight/body weight (HW/BW), left ventricular weight/body weight (LVW/BW),right ventricular weight/body weight (RVW/BW), lung weight/body weight(LW/BW), total aorta area (TAA), lumen area (LA), aorta cross-section area(ACSA), ACSA/TAA, aorta diameter (AD), media, lumen and media/lumen inmodel group rats increased significantly (P＜0.01), when compared with shamgroup, indicating a morphological remodeling of heart and aorta. Thecardiomyocyte cross-sectional area, CVF, PVCA and cardiac tissuehydroxyproline in model group rats increased significantly (P＜0.01vs shamgroup). Furthermore, the ANP mRNA expression increased significantly (P＜0.01vs sham group). After treatment with MHBFC6,12mg/kg, the HW/BW,LVW/BW, RVW/BW, LW/BW decreased significantly, cardiomyocytecross-section area, CVF, PVCA, hydroxyproline content in cardiac tissue werereversed, respectively (P＜0.05or P＜0.01vs model group). Myocardial tissueANP mRNA expression was downregulated significantly (P＜0.05or P＜0.01vs model group). Ultrastructural examination revealed that the ultrastructure ofthe cardiomyocytes in model group rats presented mitochondrial changes,characterized by diffuse mild to moderate increase in size (hypertrophy) andnumber (hyperplasia), focally forming several rows of enlarged mitochondriaseparating the myofibrils. Many mitochondria had the appearance of elongatedgiant mitochondria. Longitudinally oriented cristae, either parallel to the longaxis of the mitochondrion or irregular and concentric, were observed in manyareas. After treatment with MHBFC12mg/kg, the cardiomyocytes showedalmost uniform parallel myofibril arrangement. Z-lines dividing the sarcomereswere linear and perpendicular to the myofilaments. Myofibrils alternated withfew rows of ovoid mitochondria. Part Ⅱ Studies of the endothelial mechanisms of MHBFC on pressureoverload-induced cardiovascular remodeling in ratsExperiment1Studies of endothelial mechanisms of MHBFC base on theendothelin system in pressure-overload ratsAt sixth week after aortic banding, the plasma ET-1content was increased,ET-1mRNA, ECE mRNA and ETA, ETBprotein expressions in the myocardialtissue were upregulated in model group rats compared with those in sham grouprats (P＜0.05or P＜0.01). After treatment with MHBFC6,12mg/kg for6weeks, plasma ET-1content was decreased significantly (P＜0.01vs modelgroup), and the upregulation of myocardial tissue ET-1mRNA, ECE mRNA andETA, ETBprotein expressions were inhibited markedly (P＜0.05or P＜0.01vsmodel group).At sixth week after aortic banding, plasma NO content was decreased inmodel group compared with that in sham group (P＜0.01). Treatment withMHBFC6,12mg/kg increased the plasma NO content significantly, and thesignificant negative linear correlations were found between RVW/BW, cardiactissue hydroxyproline, SBP and plasma NO.Experiment2Studies of endothelial mechanisms of MHBFC base on theeNOS-NO signal pathway in pressure-overload rats1. The change of blood pressure: The tail arterial SBP in L-NAME group ratsincreased significantly with a time-dependent manner compared with that inmodel group rats, respectively (P<0.05or P<0.0l). Combined with MHBFC12mg/kg, the tail arterial SBP in the2nd,4th,6th weeks decreased significantlycompared with that in L-NAME group rats, respectively (P<0.05or P<0.0l).2. The change of heart function: After6weeks of NO synthase inhibition, anenhanced contractility but a reduced diastolic compliance were taken place in L-NAME group rats (P＜0.01vs sham group), MHBFC could significantameliorate the LV function, and the ASBP and LVSP decreased significantly(P＜0.01vs L-NAME group).3. Cardiac remodeling: At sixth week after aortic banding, the LVW/BW,RVW/BW and cardiomyocytes cross-section increased significantly in L-NAMEgroup rats (P＜0.05or P＜0.01vs sham group). The combination of MHBFC12mg/kg with L-NAME ameliorated cardiac hypertrophy significantly (P＜0.01vs L-NAME group). Histological examination demonstrated that both CVF,PVCA and myocardial tissue hydroxyproline increased significantly inL-NAME group compared with that in sham group rats (P＜0.01), and thecombination of MHBFC12mg/kg with L-NAME decreased cardiac tissuehydroxyproline significantly (P＜0.01vs L-NAME group).4. The change of eNOS-NO: At sixth week after aortic banding, the eNOSprotein expression level was decreased significantly in mode group rats (P＜0.01vs sham group). Treatment with MHBFC12mg/kg, the eNOS proteinexpression level was increased significantly (P＜0.01vs model group). TheeNOS protein expression level was decreased significantly in L-NAME grouprats, but combination with MHBFC12mg/kg, the eNOS protein expressionlevel was increased significantly (P＜0.01vs L-NAME group). The plasma NOwas decreased significantly in L-NAME group rats, but combination withMHBFC12mg/kg, the plasma NO was increased significantly (P＜0.01vsL-NAME group).Experiment3Studies of endothelial mechanisms of MHBFC base on thePGI2in pressure-overload rats1. The change of blood pressure: At sixth week after aortic banding, the tailartery SBP and ASBP in Indo2group rats increased significantly with a time-dependent manner. Combination with MHBFC12mg/kg amelioratedindomethacin induced severe hemodynamics significantly (P＜0.05or P＜0.01vs Indo2group).2. The change of heart function: At sixth week after aortic banding, the cardiaccontractility in Indo2group rats increased significantly, but a reduced cardiacdiastolic compliance significantly. Combined with MHBFC12mg/kg inhibitedthe increase of cardiac contractility, ameliorated cardiac compliance.3. Cardiac remodeling: At sixth week after aortic banding, HW/BW, LVW/BW,RVW/BW and cardiomyocytes cross-section increased significantly in Indo2group (P＜0.01vs sham group). Combination with MHBFC12mg/kgameliorated cardiomyocytes hypertrophy significantly (P＜0.01vs Indo2group). Compared with sham group, the CVF, PVCA and hydroxyprolineincreased significantly in Indo2group (P＜0.01), and combination withMHBFC12mg/kg inhibited the deterioration of CVF and PVCA (P＜0.05or P＜0.01vs Indo2group), inhibited the increase of cardiac hydroxyproline (P＜0.01vs Indo2group).4. The change of plasma PGI2content: At sixth week after aortic banding, theplasma PGI2content decreased significantly in model group rats (P＜0.01vssham group). Long-term treatment with MHBFC12mg/kg increased plasmaPGI2significantly (P＜0.01vs model group). Chronic administration of indodecreased plasma PGI2content significantly, and combination with MHBFC12mg/kg inhibited the decrease of plasma PGI2content significantly (P＜0.01vsIndo2group).Conclusions:1. MHBFC6,12mg/kg can significantly reverse pressure-overloadcardiovascular remodeling induced by abdominal aorta binding in rats with a certain extent of dose-dependent manner;2. The protective mechanism of MHBFC on cardiovascular system inpressure-overload rats may be related to its regulation of endothelial function,augmenting NO and PGI2secretion, and inhibiting ET-1biosynthesis andsecretion, and ET-1system activation.