The Effect Of Qiliqiangxin Capsule On Microvessel Injury, Ventricular Remodeling And Metabolic Remodeling In Heart Failure Rats

Objective: Under the guidance of the Vessel-Collateral Theory, theThoracic Aorta Constriction (TAC) was applied to develop the chronic heartfailure model of rat. The major influence of microvessel injury and effects ofQiliqingxin capsule (QLQX) were disclosed through observing the damage ofmicrovessel structure and function and apoptosis and fibrosis of myocardialtissue and energy metabolism dysfunction of myocardial mitochondria in theanimal model; The Langendorff was adopted to determine the effects ofQLQX on the energy metabolism and mitochondria related protein and signalpathway p-AMPK/PPARαin heart failure. From above experiments, the roleof microvessel and ventricular remodeling and metabolism remodeling toheart failure was explored. These data would give scientific supports to theYing-nutrient and Wei-defense “unblocking collaterals with subsequentconvergence and substance-qi transformation” in the Vessel-CollateralTheory.Methods:The study includes four sections:1The effects and mechanism of QLQX on myocardial microangiopathy inheart failure ratsTAC was used to develop heart failure rats induced by overload pressure.Four weeks later, the survival rats were randomly divided into seven groups(n=15): Control group, Captopril group and high, medium, low-dose QLQXgroups (QLQX-H, QLQX-M, QLQX-L), Model group, Sham operation group(Sham). The rats were administered with QLQX by gavage at the dose of10ml/kg once a day for6weeks, then the same volume of solvent to Sham,Control and Model group. After the last administration and fasted12h, every rat was detected the changes of hemodynamic through carotid arteryintubation. The serum of rats was collected to detect the content of pro-Nterminal brain natriuretic peptide (N-proBNP), angiotensinII (AngII), andaldosterone (ALD) by radioimmunoassay. The ELISA was adopted to detectnorepinephrine (NE), ET-1and vWF. The ultrastructure of microvesselendothelial cells in myocardial tissue was observed by transmission electronmicroscopy. The immunofluorescence technique was used to observe theeffects of QLQX on microvessel density of hypertrophic myocardial tissue inpressure-overload rats. The mRNA expression of ICAM-1VCAM-1andeNOS were detected by Real-time PCR. The content of serum was dected toreflect the endothelial function. The histological staining was adopted toobserve the changes of myocardial cells, cardiac fibroblasts, microvessel andthe intervention effect of QLQX on pathogenesis of heart failure.2The pathological mechanism of ventricular remodeling in heart failure ratsand the intervention of QLQXIt was the same with part1that experimental animals modeling, grouping,administration and derived method. The HE staining and transmission electronmicroscope were used to observe the ultra-structural changes in myocardium.The changes of myocardial collagen fiber hyperplasia were determinedwith Masson trachoma staining. The contents of myocardial hydroxyprolinewere detected by the method of alkaline hydrolysis. Western blot was used todetect the protein expression of COLⅠand COLⅢ in myocardial tissue.The myocardial cell apoptosis rate was detected by flow cytometry andTUNEL. Real-time quantitative PCR was used to analyze the mRNAexpression of nutrition-1(CT-1), cytochrome C (CytC) in myocardial tissue.3The effects and mechanism of QLQX on metabolic remodeling in heartfailure rats.It was the same with part1that experimental animals modeling, grouping,administration and derived method. The serum was adopted to detect thecontent of lactic acid (LA), lactate dehydrogenase (LDH), free fatty acid (FFA)and uric acid (UA). The content of ATP, ADP and AMP was determined with high performance liquid chromatography to calculate the total adenosine pooland energy charge in myocardial tissue. Myocardial cell mitochondriamembrane potential (MMP) was detected by flow cytometry. Dissolvedoxygen electrode was used to detect mitochondrial oxidative activity (RCR).Real-time PCR was used to analyze the expression of CPT-Ⅰ, GLUT4andPGC-1α mRNA.Western blot was used to detect p-AMPK, AMPK, PPARαand PGC-1α protein expression level.4The effect and mechanism of QLQX on energy metabolism in the perfusionfailure heart in vitroThe Langendorff perfusion apparatus was adopted to observe the functionof isolated hearts. Low calcium K-H solution induced isolated-heart failure.QLQX was set five concentration (0.025,0.05,0.1,0.2,0.4g/L),andLanatoside as positive control, to observe the effects of QLQX on isolatedcardiac left ventricle pressure, left ventricular load changes and coronary flowand detect the total adenylate pool and energy charge.The mRNA expressionof α-MHC,β-MHC, Mfn2, Drp1was detected by Real-time PCR. Western blotwas used to analyze the protein expression of AMPK, PPARα, p-Akt andp-PI-3K in myocardial tissue.Results:1The effects and mechanism of QLQX on myocardial microangiopathy inheart failure ratsResults of hemodynamics in rats: Compared with the sham group, SBP,LVSP, LVEDP in Model group rats were increased and±dp/dtmax decreasedsignificantly (P<0.01). Compared with the Model group, QLQX decreasedSBP, LVSP, LVEDP levels, and increased±dp/dtmax level, estatisticallysignificant changes in QLQX-H and Captopril group (P<0.01). Comparedwith the sham group, no significant difference was detectd in AngⅡ, ALD,NE, NT-proBNP. Compared with the Sham group, these indexes weresignificantly increased in the Model group (P<0.01).Compared with the Modelgroup, the level of Ang II, ALD in QLQX-M, QLQX-H and Captopril groupdecreased significantly (P<0.01). Compared with the Model group, the treatment group decreased NE and NT-proBNP levels, especially QLQX-M(P<0.05), QLQX-H and Captopril group (P<0.01).The ultrastructure of myocardial micro-vascular: In Model group therewas obvious edema around the capillary, a little in basement membrane, andlumen was irregular, no obvious mitochondrial structure, closely connecttionwas fuzzy, pinocytosis vesicles decreased. Capillary endothelial structure wasbetter in treatment groups, for example, mitochondrial fusion andvacuolization decreased, pinocytosis vesicles relatively increased and tightjunction recoverd obviously.The microvessel density of cardiac muscle tissue: Compared with shamgroup, the CD34positive count and microvascular relatively density decreasedsignificantly in Model group (P<0.01). The statistical difference of DAPI wasnot showed. Compared with the positive count in Model group, the CD34positive count increased in QLQX-L group (P<0.05), QLQX-M and QLQX-H(P<0.01); compared with the Model group, the relative microvascular densityof cardiac muscle tissue increased in QLQX group, the QLQX-M andQLQX-H group showed statistically difference (P<0.01).Serum ET-1, vWF levels: Compared with sham group, the ET-1and vWFcontents in Model group increased obviously (P<0.05). Compared with Modelgroup, the ET-1contents in QLQX group decreased significantly (P<0.01).Compared with Model group, the vWF contents decreased significantly inQLQX-M, QLQX-H, Captopril group (P<0.01) and QLQX-L group (P<0.05).The expression of inflammatory cytokines ICAM-1, VCAM-1and eNOSMrna: Compared with sham group, the ICAM-1and VCAM-1expression ofcardiac tissue of rats in Model group increased significantly (P<0.01).Compared with sham group, the eNOS mRNA expression in Model groupdecreased (P<0.01); compared with Model group, the QLQX group couldpromote the expression of eNOS mRNA at different extents (P<0.05, P<0.01),the QLQX-H group showed remarkable effects (P<0.01).2Effects of QLQX on ventricular remodeling in pressure-overload heartfailure rats 2.1Myocardial tissue morphological morphology: Compared with the shamgroup, there showed myocardial fiber hypertrophy, partly fracture visible,atrophy of muscle fibers, and diameter in Model group. QLQX inhibitedcardiac hypertrophy, reduced muscle fiber fracture, atrophy and interstitialfibrosis on different degree. Especially QLQX-H group, there was onlypartially myocardial fiber atrophy and myocardial fiber occasionallydegeneration.Compared with the Sham group, the ultrastructure of cardiac fibersdisplaied disorderly-arranged in Model group. Other changes including Z linesvisible fracture or disappear, myocardial interstitial and obvious edema aroundnuclear, and glycogen decreased. Mitochondrial morphology varied.Membrane structure is not clear, partly membrane fused, and the cristadisordered, fused or disappeared. Treatment groups significantly relieved themyocardial cell stomata edema and mitochondrial injuried, increased theglycogen amount; glycogen particles relatively increased and Z lines werearranged in order.2.2The effects of QLQX on Collagen fiber hyperplasia of myocardial tissue:Compared with the control and sham group, HW/BW index of Model ratsincreased obviously (P<0.01).Compared with Model group, the treatmentgroups may lower HW/BW index (P<0.05, P<0.01), especially the index ofQLQX-H rats decreased significantly.The Masson trichromatic staining ofmyocardial tissue showed that myocardial fibers arranged closely, and noobvious hyperplasia of collagen fiber in Control group and the shamgroup.There were mass collagen hyperplasia and spread to the myocardialfiber bundles in Model group. QLQX could inhibit the collagen fiberproliferation and reduce collagen fibrosis size in myocardial tissue and bloodvessels.Compared with the sham group, the content of hydroxyproline increasedsignificantly in Model group(P<0.01).Compared with the Model group, thehydroxyproline content in treatment group decreased at different degree, inwhich the QLQX-L group, captopril group (P<0.05), QLQX-M and QLQX-H group decreased significantly (P<0.01).Compared with sham group, the content of COL I, COL III in Modelgroup increased significantly(P<0.01).Compared with Model group, captopriland QLQX-H decreased the expression of COLⅠ(P<0.01,P<0.05). Comparedwith Model group, there was a significant statistical difference between thecaptopril group, QLQX-M and QLQX-H groups in COL Ⅲ protein,significantly in QLQX-H group.2.3Myocardial cell apoptosis:The results with TUNNEL analysis showed that the amout of apoptoticcells of myocardial tissue in Model group significantly increased andapoptosis cells decreased after treatment. The results with Flow cytometryanalysis showed that the apoptotic myocardial cells significantly increased(P<0.01) in Model group.Compared with sham group, QLQX reducedapoptotic myocardial cells in pressure-overload heart failure rats statisticallysignificantly (P<0.01).Compared with sham group, the mRNA expression of CT-1and CytCsignificantly increased in Model group (P<0.01). Compared with the Modelgroup, QLQX reduced CT-1, CytC expression (P<0.01), Captopril groupdecreased CT-1, CytC mRNA expression (P<0.05, P<0.01).3The effects of QLQX on the metabolic remodeling of rats suffered fromcardiac failureCompared with Model group, the QLQX-L and QLQX-H group coulddecrease the blood serum LA content of pressure overload rats significantly(P<0.05). The QLQX decreased LDH and UA level greatly (P<0.01).Compared with Model group, FFA content in QLQX groups decreased atdifferent concentrations with statistical difference. QLQX-H group showedobvious effect (P<0.01), which was better than captopril group (P<0.05).Compared with sham group, the total adenosine and energy charge valuedecreased significantly (P<0.01). Compared with Model group, treatmentgroup increased the adenosine pool and energy value greatly, in which theQLQX-L increased the total adenosine pool content and energy value (P<0.05, P<0.01).Compared with sham group, the membrane potential of mitochondria ofcardiac muscle decreased in the Model group (P<0.01). Compared with Modelgroup, the MMP of mitochondria of cardiac muscle increased in treatmentgroup, and QLQX-M and QLQX-H group showed significant increase(P<0.01). Treatment groups could improve the respiratory function ofmitochondria and enhance the RCR (P<0.05, P<0.01).Compared with sham group, the mRNA expression of CPT-Ⅰand GLUT4and PGC-1α in Model group decreased significantly (P<0.01). Comparedwith Model group, the mRNA expression of above index in QLQX-H groupshowed significant difference (P<0.05, P<0.01).Compared with sham group, the protein expression of p-AMPK in Modelgroup showed no statisticly difference (P>0.05). Compared with Model group,the expression level of p-AMPK in treatment group increased significantly, inwhich the QLQX-M and QLQX-H group increased obviously (P<0.01). Thetotal amount of AMPK in each group didn’t show significant difference(P>0.05). Compared with sham group, the protein expression level of PPARαand PGC-1α in Model group decreased obviously (P<0.01). Compared withModel group, the expression of the two protein in QLQX-M and QLQX-Hgroup increased significantly (P<0.01).4The effects and mechanism of QLQX on energy metabolism in the perfusionfailure heart in vitroOnly being perfused with K-H liquid, the left ventricular pressure in eachgroup didn’t show significant difference (P>0.05). When mimicing the cardiacfailure with low calcium K-H liquid perfusion, the left ventricular pressure ineach group decreased obviously and the cardiac loading increased at meanwhile. Compared with Model group, the left ventricular pressure in all QLQXgroups at different concentration increased accompanying withconcentration increasing, in which the left ventricular pressure of QLQX-0.2,QLQX-0.4group increased significantly (P<0.01), and this showed certaindose-effect relationship. After being adding QLQX, the left ventricular loading in each concentration group showed decrease in different degree. After beingadding two blockers, the pressure decreased significantly (P<0.01), and thecardiac load increased at some extent, and the cardiac load after addingblocker MK-886showed significant increase (P<0.05).Compared with normal K-H perfusion, the coronary flow in Model groupdecreased obviously (P<0.01). Compared with Model group, theQLQX-0.05,QLQX-0.1,QLQX-0.2and QLQX-0.4group increased thecoronary flow obviously (P<0.05, P<0.01).Compared with QLQX-0.2, thecoronary flow with two blockers decreased, but there was no statisticlydifference between these two blocker groups.Compared with control group, the energy charge value of cardiac muscletissue in Model group decreased (P<0.01).Compared with Model group, thepositive medication, QLQX-0.1, QLQX-0.2and QLQX-0.4showed (P<0.01)statistically significance.Compared with Model group, the mRNA expression level of Drp1inQLQX-0.4group decreased (P<0.05). Compared with QLQX-0.2group, theexpression with blockers increased, and but showed no significant difference.Compared with Model group, the positive control medication, QLQX-0.2and0.4group increased the expression of Mfn2obviously (P<0.01), andQLQX-0.1showed statistically difference (P<0.05). The positive controlmedication, QLQX-0.1, QLQX-0.2and QLQX-0.4group increased theα-MHC mRNA (P<0.01). Compared with QLQX-0.2group, two groups withblocker decreased the expression of α-MHC mRNA. Compared with Modelgroup, the positive control medication, QLQX-0.2and QLQX-0.4groupdecreased the expression of β-MHC greatly (P<0.01). Compared withQLQX-0.2group, two groups with blocker increased the mRNA expression ofβ-MHC.Compared with normal K-H perfusion group, the expression level ofp-PI-3K and p-Akt in Model group decreased (P<0.01).Compared with Modelgroup, the treatment increased the expression level of p-PI-3K and p-Aktprotein (P<0.05, P<0.01). Compared with QLQX-0.2, the two blockers up-regulated the protein expression of p-Akt, but this didn’t show statisticdifference (P>0.05).Compared with QLQX-0.2group, the expression ofp-PI-3K with two blockers didn’t show statistic difference (P>0.05).Compared with positive medication, the QLQX-0.4increased the proteinexpression of p-Akt (P<0.05).Conclusion:1It is the first time that Ying and Wei theory in Vessels-CollateralTheory was used as guidance to discuss the important role of microvasculardamage in heart failure. Heart failure is multi-dimensional spatio-temporalcomplex pathological processes including ventricular remodeling and energymetabolism disorder, caused by abnormal hemodynamic and neurohumoraltrigger. Through experiment, microvascular injury, abnormal energymetabolism, pathological changes of ventricular remodeling in case of heartfailure and the internal relation between them are discussed. The theory ofscientific value of the Ying-nutrient and Wei-defense “unblocking collateralswith subsequent convergence and substance-qi transformation” of theVessel-Collateral Theory was verified, and it offers new thinking for theprevention and treatment of cardiac failure.2The effects and mechanism of QLQX on microvascular provention,ventricular remodeling, and energy metabolism were elaborated through theanimal experiment in vivo. TAC was applied to develop the chronic heartfailure model of rat and demonstrate that QLQX could improving the functionof failing heart by adjusting the vasoactive substances, inhibittinginflammatory injury, promoting myocardial angiogenesis and structure,improving hemodynamics, and inhibitting neurohumoral excessivelyactivation. QLQX inhibited cardiac fibroblast proliferation. At the same time,QLQX capsule reduced myocardial apoptosis, the mechanism may be relatedto the mitochondrial apoptotic pathway mediated by CT-1and CytC. QLQXcapsule regulates metabolic pathways of glycolipid through p-AMPK/PPARαpath, improves energy metabolism of rats with chronic heart failure, increasesthe expression of PGC-1α, promotes mitochondrial biosynthesis, protects mitochondrial function and oxidative respiration activity of myocardial tissueof rats with heart failure, reduces the concentration of LA, FFA and UA incirculating blood and reduces endothelial injury.3It was demonstrated the protective effects of QLQX on the structure andfunction of myocardial mitochondrial through the up-regulation of Mfn2mRNA expression and down-regulation expression of Drp1mRNA, andQLQX inhibit myocardial mitochondrial apoptosis pathway mediated by CytCthrough down-regulating Drp1mRNA expression. This illustrates thecorrelation between mitochondria of energy metabolism and structuralremodeling of the failing heart.