| BackgroundsHypertension, which cause of death is target organ damagement, has become a serious public health problems in the world. Therefore,it has great clincal significance and theoretical value to explore the mechanisms and to find effective measures of prevention and treatment on the hypertensive myocardial fibrosis. The severity of myocardial fibrosis (myocardial fibrosis, MF) has become the main factor to determine the development and prognosis of cardiac target organ damage in hypertensive patients with myocardial fibrosis.Currently,drugs for hypertensive MF mainly include:angiotensin converting enzyme inhibitor(ACEI),angiotensin receptor blockers(ARB),aldosterone receptor inhibitors, beta blockers, statins medicine and natural drug. The negative effects of chemicals, bring people pay more and more attention to the health of human beings and the living environment, such as ACEI and ARB drugs effect of potassium retention, renal insufficiency patients should be used with caution,et al,which made the reliance on natural drugs increase. Natural medicine,such as animal,plant and mineral medicine, which not only has been proved to have certain pharmacological activity by the modern medicine system, but have characteristics of convenient, cheap and no side effect. Therefore, to natural drugs, the research and development rapidly,various artificial prohibition aslo tends to be relaxed in recent years.Grape seed procyanidins extracts(GSPE) is the main effective components of grape seeds contain,has effects of antioxidant, scavenging free radical.Recently, domestic and foreign research also suggests GSPE has effects of anti-atherosclerosis, antioxidant, anti-inflammatory, antitumor, anti-aging. In our previous research also found that,GSPE can retard atherosclerosis in rabbits, has protective effects on db or db mice aorta, peripheral nerve of diabetic rats and target organ damage in ouabain hypertensive rats, also regulate the expression of GSTM in renal tissue of diabetic rats. But, there is no study on the effects and mechanism of hypertensive myocardial fibrosis with GSPE in hypertensive myocardial fibrosis.The main pathological changes of MF are the abnormal degradation of collagen deposition proliferation, caused by proliferation, differentiation and migration in cardiac fibroblast(CFs). Pastly, the research on the function and mechanism of delaying the progress of MF,were doing mainly from oxidative stress, inflammation, and TGF beta-1 orsmad3 signaling pathways. Number of changes or change of the stability in cytoskeleton,occurry in the process of myocardial hypertrophy and failure,have found in recent studies. The cytoskeleton structure can affect cell proliferation,differentiation, migration, cell secretion function etc.cofilin-1,regulatory protein of cytoskeleton protein F-action,which activity regulation mainly through phosphorylation or dephosphoryla-tion. LIM kinase can specific phosphorylate cofilin-1 by uniteing phosphorylation sites of LIM kinase (LIMK1 and LIMK2) on Ser-3 N-terminal. Currently,the ROCK (Rho-activated kinase) and other members of the Rho family can from upstream to activate LIM kinase through chain reaction, then play on the dephosphorylation of cofilin-1. In the field of oncology research has confirmed that Rho or ROCK signal pathway of cytoskeletal protein, in the cardiovascular field has confirmed that RhoA or ROCK signaling pathway plays an important role on the pathophysiological in human hypertension and stroke and chronic heart failure. The present study found that RhoA or ROCK pathway also has correlation with some organs of chronic fibrosis. Our previous study,discusses cytoskeleton profilinl effect on myocardial remodeling and its key role in myocardial remodeling in hypertension, has made meaningful progress. At the same time,we discovered that cofilin-1 change is also remarkable, which belong to the obtained differential expression of actin binding protein by the quantitative marker of comparative proteomics technology (iTRAQ).However, it is not clearthe role of cofilin-1 in cardiac fibrosis and whether GSPE is also affected by the role of cofilin-1, which play a role in anti-myocardial fibrosis.Therefore,we hypothesized:the expression of cofilin-1 will be increased in hypertensive myocardial fibrosis;GSPE has the effect of anti-hypertensive myocardial fibrosis, and its molecular mechanism is down regulated cofilin-1 expression. In this part of the study,we select spontaneously hypertensive racts(SHRs) for experiments in vivo, echocardiography, myocardial tissue staining method and electronic X-ray electron microscope were used to observe myocardial structure, function, fibrosis tissue pathology,changes in the cytoskeleton structure for definiting the effects of GSPE on hypertensive myocardial fibrosis; applied tissue immunofluorescence staining clear out the expression and localization of cofilin-1 in the myocardium; use enzyme linked immunosorbent assay (ELISA), western blot, real time quantitative RT-PCR methods from molecular level to clear the effect of GSPE anti-myocardial fiber fibrosis effect and molecular regulation mechanism.Objectives1. To study the effect of GSPE on anti-myocardial fibrosis in SHR.2. To study the molecular mechanism of GSPE on anti SHR myocardial fibrosis medi-ated by cofilin-1.Methods1. Experimental animals and groups:SHR(n=24)and WKY(n=8),male,20 weeks old) were fed with normal diet and were kept under observation for 1 week prior to the start of experiments,then therapy 22 weeks,which use about 10g GSPE. SHR were separated to 3 groups,(1)SHR group with high dose of GSPE (SHRH):250mg or kg or d,intragastric administration, n=8;(2) SHR group with low dose of GSPE (SHRL):100mg or kg.d,intragastric administration, n=8;(3)control SHR(SHRC):intragastric administration with normal saline,n=8.WKY(n= 8) were choosen as the empty control,to observe the development of hypertension.The animals were killed at the end of 22 weeks.2. General informations measurement:Blood pressure and heart rate detection:every 2 weeks and before SHRs were killed,we detect rat tail artery systolic pressure, diastolic pressure, mean arterial pressure and heart rate by the BP-98A a smart mouse tail noninvasive blood pressure meter.Indicators of blood biochemical and heart failure detevtion:check the serum levels of TC,TG,LDL-C,NT-proBNP before the experiment and after the death of rats.3. Determination of cardiac structure and function:Observe the left ventricular systolic and diastolic function in rats by two-dimensional, M and tissue Doppler echocardiography,before the start of the experiment and GSPE treatment 22 weekend.Weigh the rats weight (weight body, WB) before the rats were sacrificed, weigh the rats heart weight (weight HW, BW) after be executed, and calculate HW/BW (mg/g).4. Determination of histopathological changes in myocardial fibrosis:The degree of myocardial fibrosis measurement:observe pathological changes of myocardial tissue with HE staining,observe area of collagen fibers with massion staining,distribution of collagen fibers was observed by Sirius red staining;immun-ohistochemical method for determination of OC-SMA and collagen-I expression in myocardial tissue of rats.Observation of myocardial ultrastructure:myofilaments, he sarcomere, mitochondria and nucleus of subcellular structure with transmission electron microscope.5. Detection of myocardial fibrosis in molecular level:Western blot and RT-PCR methods for determination of α-SMA,collagen I, MMP-9, TIMP-1 expression; immunofluorescence staining,Western blot and RT-PCR methods for determination of cofilin-1 expression; Western blot and RT-PCR methods for determination of the expression of R0CK1 in myocardial tissue of rats; Western blot and RT-PCR methods for determination of TGF beta I,smad3, p-smad3 expression.6. Detection of oxidative stress index and molecular level in myocardial tissue:ELISA method for the determination of H2O2, NO, MDA, SOD protein expression in myocardial tissue of rats;western blot method for measuring the expression of Nrf2 in rat cardiac muscle.Results1. The effects of GSPE on the general situation of SHRsBefore the treatment of GSPE, there was no significant difference about body weight among the WKY and SHR groups (P>0.05), heart rate, systolic pressure, diastolic pressure and mean arterial pressure in WKY group were significantly lower than that in SHR group (P< 0.01),but SHR group had no difference (P> 0.05);The 22 weeks of GSPE treatment, there was still no significant difference about body weight among the WKY and SHR groups (P>0.05), and heart rate, systolic pressure, diastolic pressure, mean arterial blood pressure is significantly lower in WKYC groups than that in SHR group (P< 0.01),but compared with the control group, heart rate, systolic pressure, diastolic pressure, mean arterial pressure were all decrase in therapy SHR group (P< 0.05), but the high and low dose group had no significant difference (P> 0.05).Before and after GSPE treatment, there was no significant difference in blood lipids and heart failure (P>0.05).2. The effects of GSPE on the structration and function of SHRs(1) Cardiac ultrasound parameters:Compared with before GSPE treatment, GSPE treatment for 22 weeken, WKYC group diastolic function index E’ or A’ significantly decreased (P<0.01), IVSd significantly increased (P<0.01), LVPWd was significantly thickened (P<0.01).Compared with before GSPE treatment, GSPE treatment for 22 weeken, SHR groups diastolic function index E’or A’significantly decreased (P<0.01), LVEF increased (P<0.01 or P<0.05), LVIDd reduction (P<0.01 or P<0.05), IVSDd significantly thickened (P<0.01), LVPWd was significantly thickened (P<0.01).Before GSPE treatment, compared with the WKYC group:SHR groups E’or A’significantly decreased (P< 0.01). IVSd were significantly increased (P< 0.01).GSPE for 22 weeks, compared with WKYC group;SHR group E’or A’ decreased significantly (P< 0.01); SHRC group LVEF significantly increased (P< 0.05); SHR groups IVSd thickening (P< 0.01 or p<0.05), LVPWd significant thickening (P< 0.01).GSPE treatment for 22 weeks, compared with SHRC:E’ or A’ in GSPE treatment groups of SHR significantly increased (P<0.01), LVEF significantly decreased (P<0.01), LVIDd increased (P<0.01 or P<0.05), IVSd thinning (P<0.05), LVPWd thinning (P<0.01).GSPE treatment for 22 weeks, SHRH group compared with SHRL group:E’ or A’ increased(P<0.05), LVEF decreased (P<0.05), IVSd thinning more significantly (P<0.05), lvpwd thinning more significant (P<0.01).(2) the heart of gross specimen showed that, compared with WKYC group,SHR groups heart size increased. Quantitative analysis of heart weight/body weight ratio (HW/BWmg/g). Results showed that:compared with group WKYC,SHR groups were increased (P<0.05 or P<0.01); compared with SHRC, treatment of SHR group had decreased, but no significant statistical difference (P> 0.05).3. GSPE effects on histopathology of myocardial fibrosis in SHR HE staining of myocardium:Myocardial cell morphology, stripes, intercalated disc and nucleus were normal;muscle fiberswere dyed clear in WKYC rats.Observation of longitudinal myocardium:in SHR group,myocardial cells had been swelling, contents were granular;most cells were sparse, the gap was not clear;most of the myocardial fiber had been fracture and fusion as wavy;some stripes, intercalated disc were still clear;nuclear were enlargement or shrinkage, interstitial fibrosis could be seen in partial view of myocardial;the SHR therapy group compared to the control group, myocardial cell swelling and myocardial fiber fracture were all decreased,most striated and intercalated disc were still clear, the vision of myocardial interstitial fibrosis was reduced.Observation of cross-sectional myocardium:cell diameter were broadening, single myocardial cell area increased significantly;the SHR therapy group compared to the control group, cell diameter broaden light, single myocardial cell area had been narrow.Massion staining of myocardium:Longitudinal observation:compared with WKYC group, myocardial interstitial and collagen fibers in subendocardium all increased significantly, while with the arrangement and walking disorder.Cross-sectional observation:collagen fibers arounded vascular lumen increased significantly. Compared with SHRC group, myocardial interstitial and collagen fibers in subendocardium and around the vascular lumen were all reduced significantly in SHR treatment group,whose changes improved obviously in SHRH group.The ratio of The collagen area (CVF) and the ratio of peritubular collagen area or lumen area (PVCA or LA) results:compared with WKYC rats, the collagen expression were significantly increased (CVF,P<0.01; PVCA or LA,.P<0.01) in SHRgroups;compared with SHR control group, CVF and PVCA or LA were significantly reduced (P<0.01)in SHR treatment group,which decreased significantly (P<0.05)in SHRH group.Sirius red staining of myocardial:Collagen fiber distribution less in WKYC groups,there were less collagen fiber in peripheral vascular.SHR groups showed that collagen fibers were increased, arranged in disorder, fracture, what see more in peripheral vascular, and based on type I collagen fibers; compared SHR treated group with the control group, distribution of collagen fiber reduced, arrangement reduced mussily, less faults, perivascular distribution reduced significantly,which improve significantly in he high dose group.Observation of changes of cytoskeleton structure:In WKYC group, the content of myofibrillar rare, ordered, Z line and shading with clear; intercalated disc structure; mitochondria along the myofibril arranged parallel to the long axis, matrix particles homogeneous and complete dense mitochondrial cristae; nuclei were round or oval, nuclear staining uniform quality, clear nucleolus, nuclear membrane is smooth and complete.In SHR groups,myofibrillar content were richly,arranged in disorder, destroy the structure of sarcomere, Z line breaks into a wave shape, most of the regional myofilament dissolved disappear and hyperplasia of mitochondria were replaced; abnormal mitochondrial morphology, number and arrangement disorder, part of mitochondrial swelling, particles appeared in the electron lucent zone due to lack of matrix, mitochondrial cristae unclear; nuclear hypertrophy, deformity, irregular nuclear membrane depression, chromatin aggregation of small clumps in the nucleus, especially in the inner membrane, obviouslly in nucleolus;rough endoplasmic reticulum and Golgi flourishing, which prompt to the strong function of synthesis and secretion.Compared SHR treatment group with the control group, the Z line fracture change degree of ease, myofilament dissolved disappear and the range be replaced by proliferation of mitochondria were reduced; rough endoplasmic reticulum and Golgi body is slightly flourish,which suggested that the synthesis and secretion function was slightly reduced.Immunohistochemistry of myocardium:The proteins of α-SMA and collagen-I were expressed mainly in the cytoplasm and the myocardium.In WKYC group, they were expressed smally, assumesed as funicular, parallel or wavy expression and arranged parallel to the long axis of muscle fiber,and around small blood vessels showed a discontinuous distribution. In SHR groups,the expression increased significantly, arranged in disorder, mainly distributed at perivascular.The expression in SHR treatment group than that in SHR control group d ecreased.Semi quantitative statistical analysis showed that:compared with WK YC group, α-SMA and collagen I expression increased obviously (p<0.01 or p<0.05)in SHR groups; compared with SHRC group, α-SMA and collagen I expression decreased (p<0.05), and depended on dose (P<0.05).4. Effect of GSPE on the molecular level of myocardial fibrosis in SH R4.1 Effect of GSPE on the protein expression of α-SMA,collagenI,MMP-9 and TIMP-1 in SHR myocardium fibrosis tissueWestern blot results:compared with WKYC group, α-SMA,collagen I, TIMP-1 expression increased significantly (P<0.01 or P<0.05)in SHR groups, but these were significantly reduced in SHR treated groups than that in SHR control group (P<0.01 or P<0.05), and showed a dose dependent manner (P<0.01 or P<0.05).Compared with WKYC group,MMP-9 expression decreased significantly (P<0.01 or P<0.05)in SHR group, but these were significantly resaised in SHR treated group than that in SHR control group (P<0.01 or P<0.05), and showed a dose dependent manner (P<0.01 or P<0.05).RT-PCR results:compared with WKYC group, TIMP-1mRNA expression increased significantly (P<0.01 or P<0.05)in SHR groups, but these were significantly reduced in SHR treated groups than that in SHR control group (P<0.01 or P<0.05), and showed a dose dependent manner (P<0.01 or P<0.05). Compared with WKYC group,MMP-9mRNA expression decreased significantly (P<0.01 or P<0.05)in SHR groups, but these were significantly resaised in SHR treated groups than that in SHR control group (P<0.01 or P<0.05), and showed a dose dependent manner (P<0.01 or P<0.05).4.2 Effect of GSPE on the expression of cofilin-1 protein in SHR myocardial fibrosisThe expression and localization of cofilin-1:immunofluorescence staining,the expression of cofilin-protein was mainly expressed in the myocardium in each group. Western blot results:compared with WKYC group, cofilin-1 expression increased significantly (P<0.01)in SHR groups, but these were significantly reduced in SHR treated groups than that in SHR control group (P<0.01 or P<0.05); compared with group SHRL group, the expression had a decreasing trend in SHRH, but no significant difference (P>0.05).RT-PCR results:compared with WKYC group, cofilin-1mRNA expression increased significantly (P<0.05)in SHR groups, but these were significantly reduced in SHR treated groups than that in SHR control group (P<0.01 or P<0.05); compared with group SHRL group, the expression had a decreasing trend in SHRH, but no significant difference (P>0.05).4.3 Effect of GSPE on the expression of ROCK1 protein in SHR myocardial fibrosisWestern blot results:compared with WKYC group, ROCK1 expression increased significantly (P<0.01)in SHR groups, but these were significantly reduced in SHR treated groups than that in SHR control group (P<0.01 or P<0.05); compared with group SHRL group, the expression had a decreasing trend in SHRH, but no significant difference (P>0.05).RT-PCR results:compared with WKYC group, ROCKlmRNA expression increased significantly (P<0.01)in SHR groups, but these were significantly reduced in SHR treated groups than that in SHR control group (P<0.05);compared with group SHRL group, the expression had a decreasing trend in SHRH, but no significant difference (P>0.05).4.4 Effect of GSPE on the expression of TGFβ-1 orsmad3 signaling pathway protein in SHR myocardial fibrosisWestern blot results:compared with WKYC group, TGFβ-1,Smad3 and p-smad3 expression increased significantly (P<0.01)in SHR groups, but these were significantly reduced in SHR treated groups than that in SHR control group (P<0.01 or P<0.05), and showed a dose dependent manner (P<0.01).RT-PCR results:compared with WKYC group, TGFP-1mRNA expression increased significantly (P<0.01)in SHR groups, but these were significantly reduced in SHR treated groups than that in SHR control group (P<0.01), and showed a dose dependent manner (P<0.05).5. Effect of GSPE on myocardial oxidative stress in SHRDetermination of oxidative stress by ELISA:compared with WKYC group, H2O2 and NO content were significantly increased (P<0.01 or P<0.05)in SHR groups, there was no significant difference between the SHR groups (P>0.05). Compared with WKYC group, MDA and SOD content were significantly decreased (P<0.01)in SHR groups,there was no significantly difference with MDA between tne SHR groups(P>0.05),but SOD was significantly raised in SHR treated groups than that in SHR control group (P<0.01), and showed a dose dependent manner (P<0.05).Nrf2 expression results by western blot:Compared with WKYC group, Nrf2 expression decreased significantly (P<0.01)in SHR groups, but these were significantly increased in SHR treated groups than that in SHR control group (P<0.01 or P<0.05), and showed a dose dependent manner (P<0.01).Conclusion1. GSPE can reduce myocardial fibrosis in spontaneously hypertensive rats (SHR) and protect on myocardial structure and function injury.2. GSPE has an anti-hypertensive myocardial fibrosis effect through inhibit cofilin-1 and ROCK1 protein expression.3. Cofilin-1 expression in myocardium was increased in SHR, and main distributed in the myocardial interstitium,which play a important role in midiating MF.BackgroundMyocardial fibrosis (MF) is a common pathological changes of various heart disease, such as hypertension heart disease,myocardial infarction, cardiomyopathy, diabetic cardiomyopathy.Cardiac fibroblasts (CFs) are the main effective cells, which are present in the interstitial space, through proliferation, differentiation and migration, increasing collagen synthesis and abnormal deposition resulting inf MF.Therefore, it has great significance to clarify the mechanism of CFs differentiation, to find effective treatment and prevention measures, to further clarify the mechanism of myocardial fibrosis and to find effective therapeutic drugs.Cofilin-1 is a kind of low molecular weight (21 actin) binding protein of actin, and its basic function is to enhance the conversion of actin filaments. Cofilin-1 can increase the rate of F-actin disassembly,increase the circulation rate of the G-actin, that can meet the physiological movement of the cell, the cleavage activity plays a very important physiological role in promoting actin transformation of architecture (lattice to bundles) and in actin remodeling.Previous studies have found that, in vitro,cofilin-1 mainly affects cell migration and cell migration; in vivo, cofilin-1 research mainly in the field of cancer, such as, cofilin-1mRNA overexpression were detected in breast tumor cell invasion subgroups, cofilin-1 expressed highly in oral squamous cell carcinoma, renal cell carcinoma and ovarian cancer dads.In recent years, the study found that cofilin-1 lost live can lead to proteinuria, increase the damage of kidney by being involved in the anti-inflammatoryin in renal hypertension and increase the damage of arterial structure and function by mediatingin anti-oxidative stress in large artery. In cardiovascular field,It is reported that cofilin-1 expression in aortic dissection tissues compared with normal tissues was significantly reduced, and smooth muscle cells and endothelial cells of the arrangement, the proliferation, migration is closely related to, and is involved in the signaling pathway involved in Rho/ROCK. Pho M etc,also reported that cofilin-1 is an important marker for the differentiation of cardiac valve disease. In the first part, cofilin-1 expression increased in SHR myocardial interstitium;expression of a-SMA,which on behalf of CFs cell differentiation increased markedly; moreover,grape seeds procyanidine extracts(GSPE)can also down regulated expression of cofilin-1 and a-SMA in myocardial fibrosis tissue. These results suggest that, GSPE play a role in anti-myocardial fibrosis through regulate the expression of cofilin-1 in CFs, reduce the differentiation of CFs to fibroblasts myocardial(MFs) and the synthesis of collagen.Transforming growth factor beta-1 (TGFβ-1) is the most important pro fibrosis growth factor, which is the common pathway of MF caused by many factors.Previous studies have found thatrin vivo, gene transfection of TGFβ-1 can induce MF; in vitro, inhibit cell growth, promote cell differentiation, regulate extracellular matrix production and degradation in a variety of roles; and series at the turn with a variety of signaling pathways as Rho/ROCK etc. Collagen,as the main body of extracellular matrix components, including Ⅰ, Ⅱ, Ⅲ, Ⅳand V type,whose roles of synthesis increased and degradation decreased and abnormal deposition are the pathological basis of myocardial fibrotic responses. Matrix metalloproteinase(MMPs) is an endogenous family proteases, which can degrade extracellular matrix proteins, such ascollagen I,MMP-9 is the most widely studied.the main role of tissue inhibitor of metalloproteinase(TIMPs) is to inhibit the excessive degradation of MMPs in extracellular matrix protein, TIMP-1 and 3 distribute in the heart.Therefore, MMP-9 and TIMP-1 are widely used in the detection of collagen degradation.In addition, CFs differentiate into MFs, with a stronger ability to synthesize and secrete collagen, and to highly express α-SMA, so,as a marker for detection of CFs differentiation,a-SMA has been used in many researchs.The first part of this study also found that,cofilin-1,TGF (3-1,α-SMA, collagen I, TIMP-1 expression were all significantly increased and MMP-9 decreased significantly in hypertensive myocardial fibrosis; at the same time,we found that, after treated with GSPE, cofilin-1,TGF β-1, α-SMA, collagen I and TIMP-1 expression significantly decreased and MMP-9 was increased significantly.Therefore, in vitro, we adopted the TGF β-1 stimulates CFs differentiation, gave cofilin-1 interference with slow virus and GSPE intervention, used western blot detect the cell differentiation and collagen synthesis, degradation to further explore the molecular mechanism of GSPE anti-myocardial fibrosis by mediating cofilin-1, which can further elucidate the molecular regulation mechanism of cofilin-1 in myocardial fibrosis and GSPE targets for intervention.Objectives1. To study the molecular regulation mechanism of cofilin-1 in CFs differentiation and collagen synthesis.2. To study the molecular mechanism of GSPE inhibition of CFs differentiation and collagen synthesis mediated by cofilin-1.Methods1. Culture and differentiation of CFs:(1)In the time gradient experiment, human CFs (HCFs) were respectively stimulated by TGFP-1 lOng/ml with 0,24,48 and 72 hours;in the experiment of concentration gradient, we applied 0,5,10 and 50ng/ml TGFβ-1 to stimulate HCFs for 48 hours. Then,collected the cells, extracted protein, and detected the expression of α-SMA and cofilin-1.(2)According to the different transfection virus, the cells were divided into 4 groups:1)control group of PBS:no intervention,only with PBS;2)group of TGF(3-1 stimulation;3)group of TGFβ-1 stimulation and LV-CON-RNAi:TGFβ-1 stimulation and transfection with slow virus carrying diorder siRNA;4)group of TGFβ-1 stimulation and LV-CFL1-RNAi:TGFβ-1 stimulation and transfection with slow virus carrying CFL1-RNAi (28562-1);(3)According to the different intervention of GSPE,the cells were divided into 4 groups:l)control group of TGFβ-1 stimulation:cells with TGFβ-1 stimulation;2)group of TGFβ-1 stimulation and LV-CFL1-RNAi;3)group of TGFβ-1 stimulation and LV-CFL1-RNAi and GSPE;4)group of GSPE and TGFβ-1 stimulation:2. Inverted fluorescence microscope:detection of the virus transfection effect which carrying report gene GFP.3. Western blot:collecting cells, extracting protein, detecting the expression levels of α-SMA, cofilin-1,collagen I, MMP-9and TIMP-1 protein on CFs.Results1. Effects of CFs differentiation on the expression of cofilin-1 proteinThe expression of cofilin-1 protein increased with the degree of CFs differentiation, that was to say,the expression of cofilin-1 protein enhanced with α-SMA expression enhanced, when the expression of α-SMA protein was the strongest, cofilin-1 protein expression was also the strongest.α-SMA and cofilin-1 protein expression showed a time-dependent increase in CFs,which stimulated by TGFβ-1(P<0.01);α-SMA and cofilin-1 protein expression were significantly increased at 24 hours (P<0.01),48 hours up to the highest, and there was no significant difference between 48 hours and 72 hours (P>0.05).α-SMA and cofilin-1 protein expression showed a oncentration-dependent increase in CFs,which stimulated by TGFβ-1(P<0.01); α-SMA and cofilin-1 protein expression were significantly increased at 5ng/ml group (P<0.01),10ng/ml group up to the highest, and there was no significant difference between 1 Ong/ml group and 50ng/t group(P>0.05).2. The efficiency and effect of cofilin-1 interference with slow virus on HCFsCON-RNAi, CFL1-RNAi(28560-1),CFL1-RNAi(28561-1)and CFL1-RNAi(28562-1) respectively transfected HCFs,72 hours after transfection,observed lentivirus carrying the GFP gene expression, found that, the efficiency of infection was greater than 70%, and then collected the cells, extracted proteins were detected. Results showed that, CFL1-RNAi(28560-1),CFL1-RNAi(28561-1)and CFLl-RNAi(28562-1) transfection reduced the cofilin-1 expression with 52%,88% and 92%, and screen out the most effective interference sites CFL1-RNAi(28562-1) for subsequent cell experiments in the group of LV-CFL1-RNAi.3. Enhancement on CFs differentiation and collagen synthesis and the inhibitoron effect of cofilin-1 on collagen degradationCompared with the control group of PBS, the expression of α-SMA, Collagen I and TIMP-1 proteins were significantly increased (P<0.01), and the expression of MMP-9 protein was significantly lower (P<0.01) in HCFs stimulated by TGFβ-1.Compared with the control group of TGFβ-1 stimulation,in LV-CFL1-RNAi group, α-SMA, Collagen I and TIMP-1 proteins expression were significantly decreased (P< 0.01),MMP-9 protein expression was significantly increased (P<0.01);in LV-CON-RNAi group,the proteins expression had no significant change (P> 0.05).4. Inhibitory effect of GSPE on the expression of cofilin-1 in CFsCompared with the control group of TGFβ-1 stimulation,the expression of cofilin-1 were significantly decreased in the groups of GSPE+TGFβ-1 stimulation (P< 0.01) and LV-CFL1-RNAi+TGFβ-1 stimulation (P< 0.01) and GSPE+LV-CFLl-RNAi+TGFβ-stimulation (P< 0.01).Compared with the group of LV-CFL1-RNAi+TGFβ-1 stimulation,there was no significant change on the expression of cofilin-1 in the group of GSPE+LV-CFL1-RNAi+TGFβ-1 stimulation(P> 0.05);the expression of cofilin-1 were significantly increased in the groups of GSPE+TGFβ-1 stimulation(P< 0.01).5. Inhibition on CFs differentiation and collagen synthesis and the enhancement effect of GSPE on collagen degradationCompared with the control group of TGFβ-1 stimulation,in the group of GSPE+TGFβ-1 stimulation, α-SMA, Collagen I and TIMP-1 proteins expression were significantly decreased (P< 0.01),MMP-9 protein expression was significantly increased (P<0.01);Compared with the control group of TGFβ-1 stimulation,in the group of LV-CFL1-RNAi+TGFβ-1 stimulation, α-SMA, Collagen I and TIMP-1 proteins expression were significantly decreased (P< 0.01),MMP-9 protein expression was significantly increased (P<0.01);Compared with the control group of GSPE+TGFβ-1 stimulation,in the group of LV-CFL1-RNAi+TGFβ-1 stimulation, α-SMA, Collagen I and TIMP-1 proteins expression were significantly decreased (P< 0.01),MMP-9 protein expression was significantly increased (P<0.01);Compared with the group of LV-CFL1-RNAi+TGFβ-1 stimulation,there was no significant change on the expression of cofilin-1 in the group of GSPE+LV-CFL1-RNAi+TGFp-1 stimulation(P> 0.05).Conclusion1. Cofilin-1 protein can promote the differentiation of CFs, invrease the collagen synthesis, decrease the collagen degradation.2. GSPE can reduce the differentiation of CFs, reduce the collagen synthesis, increase collagen degradation, then play the role of anti myocardial fibrosis by inhibiting the expression of cofilin-1 in CFs;meanwhile,cofilin-1 may be the target protein of GSPE intervention. |
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