Basic Study And Drug Intervention On Cardiovascular Remodeling In Rats With Metabolic Syndrome

BackgroundThe constellation of metabolic abnormalities including centrally distributed obesity, decreased high-density lipoprotein cholesterol(HDL-C),elevated blood pressure(BP), and hyperglycaemia is known as the metabolic syndrome(MS).The MS is very common in the population and a precursor state for cardiovascular disease.The overall risk of MS is more than the sum of the risk of its sole components,and they reinforce each other.Left ventricular remodeling is one of the independent risk factors of cardiovascular diseases and mainly represented by eccentric left ventricular hypertrophy,occurring at the background of combination of abdominal obesity, hypertension,and diabetes.The more the factors of metabolic abnormalities are,the more obviously left ventricular hypertrophy appeared.Previous studies have shown that single factor,such as diabetes and hypertension,is contributed to the changes of left ventricular(LV) structure and function.But at present,cardiac remodeling and the relationship between the molecules and cardiac remodeling in MS was still not reported systematically.In the current study,we created a rat model of MS and these rats thus represent a good model of human MS.Base on this model,the aim of this study is to investigate the changes of left ventricular(LV) structure and function in MS,try to elucidate the underlying mechanisms,and find the the drug which reverse or retards myocardial impairment.Insulin resistance(IR) is a common phenomenon and plays an important role in the pathophysiology of MS.Moreover,anti-inflammatory and proinflammatory molecules purportedly play an important role in developing IR.Effects of inflammatory cytokines on IR,such as TNF-α,IL-1 and IL-6 are very popular studied. A key mechanism by which inflammatory cytokines were thought to induce IR involved serine phosphorylation of insulin receptor substrate(IRS)-1I.Some studies have demonstrated IRS-1 was phosphorylated at serine site by several pathways, including IKK/NF-κB(IκB kinase/nuclear factor-κB),JNK(c-jun N-terminal kinase), SOCS(suppressors of cytokine signaling).They were activated by inflammatory cytokines and inhibited normal tyrosine phosphorylation of IRS-1.Therefore,the strength of insulin signaling was reduced via the IRS-1/phosphatidylinositol(PI) 3-kinase pathway,resulting in diminished metabolism of glucose and fat in insulin target tissues.Some studies demonstrated PI3Kinase/Akt activation is essential and collectively controls programmed cell death,cellular metabolism and organ protection.Huang-Lian-Jie-Du-Tang(HLJDT),is an important multiherb remedy in traditional Chinese medicine,and consists of four medicinal compositions.HLJDT has been used for improvement of inflammation,glucose,lipids and insulin resistance. Although anti-inflammatory effect of HLJDT is definite,very few studies have been done to demonstrate the working mechanisms that HLJDT improves inflammation-mediated insulin resistance in MS using recently developed biochemical methodologies.The aims of the present study were to observe the changes of cardiac structure and function,investigate the effect of inflammation on myocardial remodeling,and elucidate HLJDT prevents myocardial remodeling and the molecular mechanisms in rats with MS.It is anticipated to establish new target point and method for early treatment of myocardial damage with MS,preventing the development of cardiovascular complications.Objective1.To establish a rat model of MS.2.To observe the changes of left ventricular structure and function in MS rats.3.To evaluate the effect of HLJDT on left ventricular structure and function in MS rats.4.To prove the activation of IKK and JNK induced by inflammation,and evaluate the effects of IKK and JNK activation on normal insulin signal.5.To study the effect and the possible mechanism of HLJDT on left ventricular remodeling in rat model of MS.MethodsFourty-five male Wistar rats were randomly divided into control group(n=10) and model group(n=35).Control rats were fed with standard chow and tap water.Rats in model group were fed with free access to a high-fat,high-glucose and high-salt diet with sucrose solution(HF-diet).After HF-diet feeding for 16 weeks,twenty-one MS rats were further divided into two groups,MS group(n=10),continuing of HF-diet feeding;HLJDT group(n=11),continuing of HF-diet feeding plus treatment with HLJDT 1.04g/100g by garage.Rats of control and MS groups were given the same volume of saline by gavage.After treated 12 weeks,all the rats were killed and the hearts were kept at -80℃.The following parameters were measured during the study:(1) Body weight(BW) was measured every two weeks in the morning,together with systolic blood pressure (SBP) by the tail-cuff method using a Rat Tail Manometer;(2) Before and after HF-diet feeding and at the end of experiment,samples of venous blood were collected to detect triglycerides(TG),cholesterol(TC),HDL-C,LDL-C,fasting blood glucose (FBG) and insulin(FINS) levels,and calculate insulin resistance index(IRI);(3) At the end of experiment,samples of venous blood were collected to detect the levels of IL-6 and TNF-α;(4) The common echocardiographic indexes were detected before and after HF-diet feeding and at the end of experiment.At the end of experiment,all the animals were sacrificed,and the hearts were kept for the following study:(1) electron microscopic examination of myocardial ultrastructral changes and pathological study of myocardium;(2) masson and sirius red staining of collagen;(3) TUNEL technique of cardiocyte apoptotic activity;(4) immunohistochemistry of MCP-1,ICAM-1 and NF-κB P65/P50;(5) real-time RT-PCR measurement of collagenⅠ/Ⅲ,TGF-β,ICAM-1,IL-6,TNF-α,IKKβand NF-κB mRNA;(6) Western Blot for IκB,NF-κB,JNK,IRS-1 and Akt.Results1.Metabolic parameters detectionBefore the experiment,mean body weight(BW),tail blood pressure(SBP),fasting insulin(FINS),glucose(FBG),insulin reisistance index(IRI) and lipid profiles(TG, TC,HDL-C,LDL-C) were similar between the control and model groups(P＞0.05).After 16 weeks,the HF-fed rats were found to develop insulin resistance, hypercholesterolemia,low HDL-C,hyperglycemia,hypertension and obesity compared to the NC group(P＜0.01).This means the rat model of MS was achieved by fed with HF-diet for 16 weeks.At the end of the experiment,compared with NC group,BW,SBP,TC,FINS,FBG and IRI in MS group were significantly increased(P＜0.01).Compared with MS group, BW,SBP,FINS,FBG and IRI in HLJDT group were significantly decreased (P＜0.01～0.05) and HDL-C was decreased(P＜0.05).2.Serum IL-6 and TNF-αlevel measurement by ELISAAt the end of the experiment,compared with NC group,the IL-6 and TNF-αlevel in MS group were significantly increased(P＜0.01).The elevated IL-6 and TNF-αlevel were decreased in HLJDT group(P＜0.01).3.Echocardiographic detectionThe common echocardiographic indices:Before the experiment,there were no significances between the normal-fed and HF-fed rats(P＞0.05).After the HF-diet feeding for 16 weeks,compared with NC group,the interventricular septal wall thickness(IVS),posterior wall thickness at end diastole(LVPW),relative wall thickness(RWT) and left ventricular relative weight(LVRW) in model group were significantly increased(P＜0.01).E/A ratio was obviously decreased(P＜0.01).At the end of the experiment,compared with NC group,the IVS,LVPW,RWT and LVRW in MS group were significantly increased(P＜0.01).E/A ratio was obviously decreased (P＜0.01) and DT of E wave was longer(P＜0.05).Compared with MS group,IVS, LVPW,RWT and LVRW in HLJDT group were significantly decreased(P＜0.01).E/A ratio was obviously increased(P＜0.05).Integrated backscatter detection:Before the experiment,there was no significant difference of the calibrated integrated backscatter percent(myocardial IBS-pericardial IBS,IBS%) and cyclic variation of IBS(CVIB) in LVPW and IVS between NC and model groups(P＞0.05).After the HF-diet feeding for 16 weeks, compared with NC group,IBS%of model group was obviously increased(P＜0.01) and CVIB was obviously decreased(P＜0.01) in LVPW and IVS.At the end of the experiment,compared with NC group,IBS%of MS group was obviously increased (P＜0.01) and CVIB was obviously decreased(P＜0.01) in LVPW and IVS.Compared with MS group,IBS%of the HLJDT group was obviously decreased(P＜0.05) and CVIB was obviously increased(P＜0.05) in IVS and LVPW.4.Ultrastructural change observation by transmission electron microscopyThe left ventricular myocytes from NC group arranged regularly.The pericellular membrane was uninterrupted and intact.The thick and thin myofilament arranged regularly.The sarcomere and light dark band were clear.The uniformly sized mitochondria were abundant and showed round or oval shape.The nuclear membrance was smooth and intact.A typical distribution of heterochromation in the form of clusters at the nuclear membrane was present in all cardiomyocyte nuclei. Intercalated disk was clear,intact and normally structured.A little fibroblast and collagenous fibers distributed in extracellular matrix.The microvessel lumen was normal and the structure of endothelial cell was normal.The left ventricular myocytes from MS group arranged irregularly.The pericellular membrane was interrupted and unclear.The local myofibril was disintegrated.The myofilament was distorted and interrupted.The sarcomere was in a bad apposition. The swelling mitochondria increased and accumulated.The figure print mitochondria could be found.The nuclear shape was abnormal with deep notch.There was accumulation euchromatin.The unclear intercalated disk was distorted and interrupted. A lot of collagenous fibers distributed in extracellular matrix.The microvessel lumen was narrow.The endothelial cell was swelling obviously and protruded to the lumen.Compared with MS group,the ultrastructural changes of HLJDT group were obviously improved.The myocytes from HLJDT group arrange more regularly than MS group.The phenomenon that the local myofibril was disintegrated was decreased. The T tubule was moderately dilated.The mitochondria were more regular than MS group and the finger print mitochondrion was not found.The unclear shape was more regular than MS group.Intercalated disk was improved.The collagenous fibers in extracellular matrix decreased obviously and the swelling endothelial cell of microvessel was alleviated.5.Pathological detectionThe myocytes from NC group were arranged regularly in HE-staining slice.The size of the nuclear was uniform.The staining cytoplasm was homogeneous.The myocytes from MS group arranged irregularly.The unclear was irregular and the interrupted myofibril arranged irregularly.The HLJDT group was obviously improved.The myocytes from HLJDT group arranged more regularly than MS group. The interrupted myofibril was not common.The nuclear shape was more regular than MS group.6.The content of collagen detection by Masson-stainingThe myocyte was red or yellow and the collagen was green or blue.The collagen tissue was appropriate arranged among cardiomyocytes in the NC group.However, collagen tissue increased markedly,and disrupted in some area in MS group. Compared with MS group,the collagen tissue decreased and arranged regularly in the HLJDT group.Quantitative analysis results:The content of collagen in MS group was higher significantly than that of NC group(P＜0.01).Compared with MS group,the content of collagen of HLJDT group were obviously decreased(P＜0.05).7.Localization of apoptosis by TUNEL assayTUNEL positive cells were expressed as a percentage of normal nuclei.Compared with the NC group,the number of positive cells was significantly increased in the MS group(P＜0.01).Compared with MS group,the number of positive cells decreased in the HLJDT group(P＜0.01).8.Imrnunohistochemistry detectionCompared with NC group,NF-κB P65/PS0,MCP-1 and ICAM-1 expression showed significant increase in MS group.Compared with MS group,rats in HLJDT group showed significant decrease of NF-κB P65/P50,MCP-1 and ICAM-1 expression.9.The mRNA expression of several genes Compared with NC group,the levels of CollagenⅠ/Ⅲ,TGF-β,ICAM-1,IL-6, TNF-α,IKKβand NF-κB mRNA expression in MS group showed significant increase (P<0.01).Compared with MS group,rats in HLJDT group showed significant decrease of CollagenⅠ/Ⅲ,TGF-β,ICAM-1,IL-6,TNF-α,IKKβand NF-κB mRNA expression after 12 weeks treatment(P<0.01～0.05).10.The protein expression of several factorsCompared with NC group,protein expression level of IκB in MS group showed significant decrease(P<0.01).Compared with MS group,rats in HLJDT group showed significant increase of IκB protein expression after 12 weeks treatment (P<0.05).Compared with NC group,total and nuclear protein expression levels of NF-κB in MS group showed significant increase(P<0.05).Compared with MS group,rats in HLJDT group showed significant decrease of NF-κB protein expression after 12 weeks treatment(P<0.05).Compared with NC group,the levels of phospho-JNK and phospho-IRS-1(Ser307 site) protein expression in MS group showed significant increase(P<0.01),and Akt protein expression showed significant decrease(P<0.01).Compared with MS group, rats in HLJDT group showed significant decrease of phospho-IRS-1 and increase of Akt protein expression after 12 weeks treatment(P<0.01～0.05).The level of phospho-JNK was not statistical significance between MS and HLJDT groups (P>0.05).Compared with NC and HLJDT groups,the levels of JNK and IRS-1 protein expression in MS group showed increase,but there was not statistical significance (P>0.05).11.Correlation analysisThere were significantly positive correlation of serum IL-6 and TNF-αlevels with BW,SBP,FBG,FINS,IRI and collagen(P<0.01).In contrast,there was a significantly negative correlation of serum IL-6 and TNF-αlevels with HDL-C (P<0.05).Conclusion 1.A rat model of MS was established by a high-fat,high-glucose and high-salt diet with sucrose solution.The animal model was valuable for the study of the mechanism in cardiac remodeling of MS,2.MS rats showed cardiac remodeling,such as considerable apoptosis of cardiocytes, collagen deposition and myocardial hypertrophy.The early change was the diastolic dysfunction.3.HLJDT can prevent cardiac from damage.4.The the activation of IKK and JNK induced by inflammation was proved in MS, and this showed obviously correlation with the cardiac remodeling.5.HLJDT can prevent cardiac from damage throughout inhibiting inflammation-induced IKK activation,thereby improve insulin signal transduction in MS. BackgroundMetabolic syndrome(MS) is a group of metabolic conditions that occur together, promotes the development of cardiovascular disease(CVD) and type 2 diabetes mellitus(T2DM),and leads to increased mortality due to T2DM and CVD. Prevalence of MS is rapidly increasing in developing countries,and previous reports showed the prevalence of MS is about 14%to 18%in the adult population,and increases with increasing living standard and unhealthy life style.Each component of MS may be the independent risk factor of cardiovascular diseases.Beyond the importance of each separate risk factor,the aggregation of abdominal obesity,impaired glucose metabolism,dyslipidemia and hypertension, summarized as the MS,characterizes individuals with a significant increase in the risk for CVD.The risk of coronary heart disease and stroke in the MS patients are 3 folds than in health people,and cardiovascular mortality was also markedly increased in those subjects.This means the changes of macro-vascular structure and function in MS patients maybe contribute to the development of CVD.Previous studies showed in the MS patients,artery intima-media thickness(IMT) and stiffness and dysfunction of vessel endothelium were increased,and these predict the early pathological changes of vascular.Thus,on account of high morbidity and mortality of CVD in MS, it is very important to pay attention to popular tendency,study the mechanisms of vascular remodeling,multifactorial intervention to delay the progression of CVD,and effectively decrease the cardiovascular end-point events in patients with MS.MS is defined as a cluster of cardiometabolic risk factors which are related to insulin resistance(IR).Research has centred on identifying important inflammatory markers,and tumor necrosis factor-alpha has been highlighted as a key mediator of insulin resistance,as well as interleukin-6(IL-6).Compared with the controls, patients with MS showed increased C-reactive protein,interleukin-6,tumor necrosis factor-alpha and intercellular adhesion molecule.A set of experiments mimic the "typical metabolic insulin resistance" showed that inhibition of the metabolic branch of insulin signaling leads to an enhanced mitogenic action(MAPK activation),regulating genetic transcription and promoting cell proliferation.In 1993,p38MAPK was found,which a new pathway in MAPKs, and always studied as a signal transmission of inflammation.In different cells,p38MAPK could be activated by different stimulus,including high glucose,free fatty acids, cholesterol,and proinflammatory molecules.The activation of p38MAPK can regulate multiple genic transcription and expression,such as NF-κB and ATF-2.Now a deal of studies found the activation of p38MAPK was correlated with vascular remodeling.Huang-Lian-Jie-Du-Tang(HLJDT) consists of four medicinal compositions, which is Coptis chinensis,Scutellaria baicalensis,Phellodendron amurense and Gardenia jasminoides.HLJDT has been used for improvement of inflammation, glucose,lipids and insulin resistance.Further studies revealed that HLJDT decreases cardiovascular events is related to its anti-inflammation effects.There are few studies on vascular remodeling in MS.The aims of the present study were to observe the changes of vascular structure and function,investigate the effect of inflammation on vascular remodeling,and elucidate HLJDT prevents vascular remodeling and the molecular mechanisms in rats with MS.It is anticipated to establish new target point and method for early treatment of cardiovascular damage with MS,preventing the development of cardiovascular complications.Objective1.To observe whether there are vascular remodeling and vascular inflammation in rats with MS.2.To study the effect and the possible mechanism of HLJDT on vascular remodeling in rat model of MS.Methods Fourty-five male Wistar rats were randomly divided into control group(n=10) and Model group(n=35).Control rats were fed with standard chow and tap water.MS rats were fed with flee access to a high-fat,high-glucose and high-salt diet with sucrose solution(HF-diet).After HF-diet feeding for 16 weeks,twenty-one MS rats were further divided into two groups,MS group(n=10),continuing of HF-diet feeding; HLJDT group(n=11),continuing of HF-diet feeding plus treatment with HLJDT 1.04g/100g by gavage.Rats of control and MS groups were given the same volume of saline by gavage.After trated 12 weeks,all the rats were killed and the hearts were kept at -80℃.The following parameters were measured during the study:(1) Body weight(BW) was measured every two weeks in the morning,together with systolic blood pressure (SBP) by the tail-cuff method using a Rat Tail Manometer;(2) Before and after HF-diet feeding and at the end of experiment,samples of venous blood were collected to detect triglycerides(TG),cholesterol(TC),HDL-C,LDL-C,fasting blood glucose (FBG),insulin(FINS) levels,insulin resistance index(IRI),IL-6 and TNF-αafter 12 hours of fasting.At the end of experiment,all the animals were sacrificed,the aortas were kept for the following study:(1) pathological stusy of aorta;(2) immunohistochemistry of ICAM-1 and VCAM-1;(3) real-time RT-PCR lneasurement of MMP-2,ICAM-1, VCAM-1,IL-6 and TNF-αmRNA;(4) Western Blot for p38MAPK,ATF-2 and MMP-2.Results1.The experiment animalsA total of 31 rat finished the study,10 rats in control group,10 rats in MS group and 11 rats in HLJDT group.2.Comparison of metabolic indexed2.1 Comparison of metabolic indexed between control and model group after HF-diet feedingBefore HF-diet feeding,mean body weight,tail blood pressure,fasting insulin (FINS),glucose(FBG),and lipid profiles(triglyceride,cholesterol,HDL-C,LDL-C) were similar between the control and MS rats(P＞0.05).After 16 weeks,the HF-fed rats were found to develop insulin resistance,hypercholesterolemia,low HDL-C, hyperglycemia,hypertension and obesity compared to the NC group(P＜0.01).This means the rat model of MS was achieved by feed with HF-diet for 16 weeks.2.2 Comparison of metabolic indexed among three groups after HLJDT treatment After the 12-week treatment with HLJDT,BW,SBP,FINS,FBG and IRI were markedly reduced(P＜0.01～0.05),and HDL-C was increased in HLJDT group compared with MS group(P＜0.05).3.Pathological detectionIn NC and HLJDT groups,HE stain demonstrates that the intima of rats were smooth,the endothelial cells were flat and attached to the internal elastic lamina; while in MS group,the intima was coarse and thickening partly,there are breakages of endothelial cells.4.Immunohistochemistry detectionCompared with control group,the levels of ICAM-1 and VCAM-1 expression in MS group showed significant increase(P＜0.01).Compared with MS group,rats in HLJDT group showed significant decrease of ICAM-1 and VCAM-1 expression after 12 weeks treatment(P＜0.01).5.The mRNA expression of several genesCompared with control group,the levels of MMP-2,ICAM-1,VCAM-1,IL-6 and TNF-αmRNA expression in MS group showed significant increase(P＜0.01). Compared with MS group,rats in HLJDT group showed significant decrease of MMP-2,ICAM-1,VCAM-1,IL-6 and TNF-αmRNA expression after 12 weeks treatment(P＜0.01 ).6.The protein expression of p38MAPK,ATF-2 and MMP-2Compared with NC group,the levels of phopho-p38MAPK,phopho-ATF-2 and MMP-2 protein expression in MS group showed significant increase(P＜0.01). Compared with MS group,rats in HLJDT group showed significant decrease of phopho-p38MAPK,phopho-ATF-2 and MMP-2 protein expression after 12 weeks treatment(P＜0.01～0.05). Conclusion1.A rat model of MS showed hypertrophical remodeling of aorta and the vascular inflammation was enhanced.All these abnormalities maybe contribute to the high prevalence of cardiovascular complications in MS.2.Inflammation could activate p38MAPK and up-regulate the expression of MMP-2, thereby aggratate vascular remodeling in MS.3.HLJDT can prevent vascular remodeling through reducing inflammatory cytokines expression,thereby inhibit p38MAPK activation and reduce the expressions of ATF-2/MMP-2 in MS.