Experimental Study On Effect Of Tong Xin Luo On Myocardial Remodeling In Spontaneously Hypertensive Rat

BackgroundLeft ventricular hypertrophy is one of the most common changes in primary hypertension.The two aspects of pathological changes were as follows:myocardial cell's hypertrophy,necrosis and apoptosis;extracellular matrix remodeling of myocardium.Myocardial remodeling induced not only stiff cardiac ventricle and decreased cardiac function but also myocardial ischemia and cardiogenic sudden death.How to treat hypertension is very important.At present,one of the most significant gist is to prevent and reverse myocardial remodeling of hypertension patients.For recent decades,a number of basical and clinical studies on myocardial remodeling of hypertension have been founded.The phasic achievements were obtained,but the mechanism,early diagnosis and therapeutic measures of myocardial remodeling were still unexplored.It is important for treatment of myocardial fibrosis to explore deeply the mechanism and the methods to block the pathway of myocardial remodeling development.Myocardial remodeling means that excess collagen fibers accumulated and the contents of the collagen increased notably or the compositions of the collagen altered. The extracellular matrix(ECM)in most tissues is composed of a complex arrangement of fibrillar collagen,elastin,microfibrillar proteins,proteoglycans and adhesive proteins such as laminin and fibronectin.Among the ECM proteins, collagens constitute up to 85%,among which typeâ… and typeâ…¢constitute two-thirds.The degradation of the ECM are mainly modulated by matrix metalloproteinases(MMPs),and the activities of MMPs can be specifically inhibited by a family of protease inhibitors,termed tissue inhibitors of matrix metalloproteinase (TIMPs),which are endogenous physiological inhibitors of MMPs.That is,the delicate balance between MMPs and TIMPs is very important to maintain the normal metabolism of the collagen.In briefly,the development of myocardial remodeling in hypertension is a process that numerous active factors participated and co-regulated, and a result that collagenation exceeds its degradation.Accordingly,inhibiting proliferation of cardiac fibroblasts and deposition of collagen are the key of the precaution and reversion of myocardial remolding and of improving heart function. Nuclear factor-κB(NF-κB)is a well-characterized transcription factor that elicits a hypertrophic response in cardiac myocytes.Tong-xin-luo capsule(TXL)is a medicine consisting of traditional Chinese herbs and insects used for cardiovascular diseases. Some researches indicated the important effect of TXL on myocardial remodeling. But many problems is unclear,such as effect of TXL on myocardial remodeling and the main intracellular signal transduce pathway of TXL regulating ECM remodeling.The deep study of those questions not only can conduce to interpret the molecular-biological mechanism of ECM remodeling,but also can bring new application area for Tong xin luo.The purpose is to establish new target point and method for early diagnosis and treatment to prevent and reverse myocardial remodeling of hypertension.Objectives1.To explore the effect of Tong xin luo on myocardial remodeling in spontaneous hypertensive rats(SHR).2.To explore molecular biological mechanism of Tong xin luo regulating ECM remodeling.Methods16 male 8-week-old SHR were randomly divided into two groups:SHR-controlled group(SHR group)(n=8)and Tong xin luo group(n=8).The same week-old Wistar-kyoto rats group(WKY group)(n=8)was also randomly selected.The Tong xin luo group animals were dosed orally with Tong xin luo(1.5 g.kg^-1.d^-1)and the other group animals were given with 0.9%saline every day for 12 weeks.The detection contents were listed below:(1)The animals were weighed every week and the blood pressure was measured every two weeks.(2)The common echocardiographic indexes were detected before and at the end of the experiment.(3) Hemodynamics indexes were measured by cardiac catheterization at the end of the experiment.(4)After the rats were killed,we get left ventricle mass(LVM)and calculate the left ventricular relative mass(LVM/BW).(5)Myocardial ultrastructral and histopathological changes were observed.(6)The content of collagen were quantified by Masson-staining.(7)The protein levels of P65,P50 were detected by immunohistochemistry.(8)The proteins of phospho-IκBα,P₆₅,P₅₀were detected by Western-blot.(9)The mRNA expression of MMP-9,TIMP-1,P₆₅, P₅₀,Collagenâ… ,Collagenâ…¢mRNA were detected by quantification real-time RT-PCR.(10)The complex composed of IκBαand heterodimer of P₆₅/P₅₀was detected by Co-immunoprecipitate.Results1.Blood pressure measurementDuring the whole experiment,compared with the WKY group,the blood pressure in SHR group and Tong xin luo group was significantly increased(P＜0.05); the blood pressure in Tong xin luo group was insignificantly slower than in SHR group(P＜0.05).2.Body weight and left ventricular relative mass(LVM/BW)measurementDuring the whole experiment,compared with the W-KY group,the body weight in SHR group and Tong xin luo group were significantly decreased(P＜0.05).The body weight in fenofibrate group were insignificantly different from SHR-controlled group (P＞0.05).At the end of the experiment,compared with WKY group,the left ventricular relative mass(LVM/BW)in SHR group was significantly increased(P＜0.01); compared with SHR group,the LVM/BW in Tong xin luo group was significantly decreased(P＜0.05).3.Echocardiographic detectionThe common echocardiographic indices:Before the experiment,compared with WKY group,LVEDD were significantly decreased(P＜0.05)and the LVPWD and IVSD were significantly increased than in SHR group and Tong xin luo group(P＜0.05);there were no significant difference between SHR group and Tong xin luo group(P＞0.05);there were no significant difference among WKY group,SHR group and Tong xin luo group on FS,EF,E wave,E/A,Ea wave,Ea/Aa and DT of E wave (P＞0.05).At the end of the experiment,compared with WKY group,the LVPWD and IVSD in SHR group were significantly increased(P＜0.01),but LVEDD,E wave,Ea wave,E/A ratio and Ea/Aa ratio were significantly decreased(P＜0.01),IVRT and DT of E wave were extended(P＜0.01);Compared with SHR group,the LVPWD and IVSD in Tong xin luo group were significantly decreased(P＜0.05),but LVEDD,E wave,Ea wave,E/A ratio and Ea/Aa ratio were significantly increased(P＜0.05), IVRT and DT of E wave were shortened(P＜0.05);there were no significant difference among WKY group,SHR group and Tong xin luo group on FS and EF (P＞0.05).Integrated backscatter detection:Before the experiment,there was no significant difference of average image intensity(Aâ…¡),peak to peak intensity(PPI)and corrected acoustic intensity(CAI)in left ventricular posterior wall(LVPW)and interventricular septa(IVS)among the three groups(P＞0.05).At the end of the experiment, compared with WKY group,Aâ…¡and CAI were obviously increased(P＜0.05)and PPI of the SHR group was obviously decreased(P＜0.05)in LVPW and IVS. Compared with SHR group,Aâ…¡and CAI of Tong xin luo group were obviously decreased(P＜0.05)and PPI of Tong xin luo group was obviously increased(P＜0.05) in IVS and LVPW.4.Hemodynamic indices determinationAt the end of the experiment,compared with WKY group,left ventricular end-diastolic pressure(LVEDP)in SHR group increased significantly(P＜0.01),and the peak left ventricular pressure descending rate(-dp/dtmax)and the peak left ventricular pressure ascending rate(+dp/dtmax)in SHR group decreased significantly (P＜0.01).Compared with SHR group,LVEDP in Tong xin luo group decreased significantly(P＜0.05),and±dp/dtmax increased significantly(P＜0.05).5.Ultrastructural change observation by transmission electron microscopyThe left ventricular myocytes from WKY 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 SHR group arranged irregularly.The pericellular membrane was interrupted and unclear.The local myofibril was disintegrated.The myofilarnent 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 SHR group,the ultrastructural change of Tong xin luo group were obviously improved.The myocytes from Tong xin luo group arrange more regularly than SHR group.The phenomenon that the local myofibril was disintegrated was decreased.The T tubule was moderately dilated.The mitochondria were more regular than SHR group and the finger print mitochondrion was not found.The unclear shape was more regular than SHR group.Intercalated disk was improved.The collagenous fibers in extracellular matrix decreased obviously and the swelling endothelial cell ofmicrovessel was alleviated.6.Pathological detectionThe cardiocytes in WKY group arranged regularly in the slides stained with HE. The size of the nuclear was uniform.The stained cytoplasm was homogeneous.The cardiocytes in SHR group arranged irregularly.The nuclear was irregular and the interrupted myofibril arranged irregularly.The Tong xin luo groups were obviously improved.The cardiocytes in Tong xin luo group arranged more regularly than SHR group.The interrupted myofibril was not common,and the nuclear shape was more regular than SHR group.7.The content of collagen detected by Masson-stainingThe cardiocytes were stained red and collagen was stained green or blue.The collagen tissue was appropriately arranged among cardiomyocytes in WKY group. However,collagen tissue increased markedly,and disrupted in some area in the SHR group.Compared with SHR group,the collagen tissue decreased and arranged regularly in Tong xin luo group.Quantitative analysis results:The collagen volume fraction(CVF)in SHR group was higher significantly than that of WKY group(P＜0.01).The CVF in Tong xin luo group was lower significantly than that of SHR group(P＜0.05).8.Immunohistochemistry detectionThe positive reaction of P₆₅and P₅₀protein were stained brown and mainly absent in myocardial cellular nucleus.There was uniform distribution of weak brown staining in WKY group,but P₆₅and P₅₀staining in SHR group was strong and located at fargoing cardiac muscle tissues,and the expression of P₆₅and P₅₀protein were higher significantly than that of WKY group(P＜0.01);The staining was weak and located within limits in Tong xin luo group,and the expression of P₆₅and P₅₀protein were lower significantly than that of SHR group(P＜0.05).9.The results of Western-blotThe level of cytoplasmic phospho-IκBa protein in the SHR group was significantly higher than that in the Tong xin luo group(P＜0.05),while the level of cytoplasmic phospho-IκBa protein in the Tong xin luo group were significantly higher than in the WKY group(P＜0.01).10.The results of RT-PCRThe expression of P65 mRNA in SHR group was higher significantly than that of WKY group(P＜0.01).The expression of P65 mRNA in Tong xin luo group was lower than that of SHR group(P＜0.05).The expression of MMP-9 mRNA in SHR group was higer significantly than that of WKY group(P＜0.01);The expression ofMMP-9 mR.NA in Tong xin luo group was higher than that of SHR group(P＜0.05).The expression of TIMP-1 mRNA in SHR group was higher significantly than that of WKY group(P＜0.01);The expression of TIMP-1 mRNA in Tong xin luo group was lower significantly than that of SHR group(P＜0.05);the ratio of MMP-9/TIMP-1 in SHR group was significantly higer than in WKY group(P＜0.01),and the ratio of MMP-9/TIMP-1 in SHR group was significantly lower than in Tong xin luo group(P＜0.05).The expression of collagenâ… and collagenâ…¢mRNA in SHR group was higher significantly than that of WKY group(P＜0.01);the expression of collagenâ… and collagenâ…¢mRNA in Tong xin luo group was lower significantly than that of SHR group(P＜0.05).11.The results of Co-immunoprecipitationCompared with the SHR and the TXL group,the level of the complex in WKY groups were significantly lower,while the level of cytoplasmic complex composed of IκBa and P₆₅/P₅₀heterodimer in the SHR group was significantly lower than that in the Tong xin luo group.Conclusions1.There is myocardial remodeling in 20 weeks spontaneously hypertensive rats, especially extracellular matrix remodeling,and there is abnormal diastolic function.2.Tong xin luo has an inhibitory effect on myocardial remodeling in the SHR and it can also improve cardiac diastolic dysfunction.3.Tong xin luo can inhibit the expression of P65/P50 protein and the degradation of cytoplasmic complex composed of IκBa and P₆₅/P₅₀heterodimer,which result in inhibiting the transcription of its downstream gene such as collagenâ… ,collagenâ…¢, MMP-9 and TIMP-1.