| Objective:Copy Myocardial fibrosis model rats by intraperitoneal injection of isoproterenol hydrochloride,with cellular molecular biology techniques and methods,to observe the expression of TGF-?1/LIMK1 signaling pathway during myocardial fibrosis,and To observe the effect of Jiedu Tongluo Recipe on the regulation of TGF-?1/LIMK1 signal pathway and the mechanism of MF intervention.Methods:Experiment 1:50 SD rats were randomly divided into 5 groups.Normal control group was given injection of saline,the other group was given intraperitoneal injection of isoproterenol 5mg.kg-1,at 3 days,7 days,14 days,and 28 days,recorded the rat weight and taken the SD male rat hearts quickly under ether anesthesia.After measuring heart weight,remove the ventricle,placed in 4%paraformaldehyde fixative,for histological HE staining,Immunohistochemical method to measure?-actin??-SMA?and vimentin?Vim?positive expression area,take out the apex of the heart and quickly store it in liquid nitrogen,determination of hydroxyproline content by spectrophotometry.Experiment 2:according to the results of Experiment 1,60 SD rats were divided into control group?model group?captopril group?Chinese medicine low dose group?middle dose group?high dose group,Normal control group was given injection of saline,the other group was given intraperitoneal injection of isoproterenol 5mg.kg-1.The traditional Chinese medicine group was administered 10 g,20 g,30 g/kg of Jiedu Tongluo Fang granule suspension daily;captopril group was given a captopril tablet suspension at a dose of5mg.kg-1 daily;the normal control group was given daily gavage with equal volume of tap water,hearts of SD male rats were taken out under ether anesthesia quickly after recording rat weight,measure heart weight and calculate heart weight index;Take out the heart and quickly store it in liquid nitrogen,spectrophotometric determination of hydroxyproline.Remove the ventricle and put in 4%paraformaldehyde fixative,for histology HE and Masson staining,for histology HE and Masson staining,Immunohistochemical method to detect the positive expression area of type I collagen?Col I?,type III collagen egg?Col III?,?-SMA,Vim,and monoserine protein kinase?LIMK1?;eyeball collect blood,collect serum and place in-20°C refrigerator,the collected sera were grouped and subjected to cell culture.Experiment 3:The animal serum prepared in advance was divided into control group?model group?captopril group?traditional Chinese medicine low dose group?traditional Chinese medicine medium dose group?traditional Chinese medicine high dose group which used to culture cardiac fibroblasts,Western-blot method was used to detect the relative expression of?-SMA,Vim,ROCK1,and LIMK1proteins in each constituent fibroblast;Real-time fluorescent quantitative PCR to detect the mRNA content of TGF-?1,RhoA,ROCK1,and LIMK1 in fibroblasts;MTT method to detect TGF-induced fibroblast proliferation phase,after stimulating fibroblasts with TGF,real-time fluorescent quantitative PCR was used to detect the mRNA content of RhoA,ROCK1,and LIMK1 in fibroblasts.Results:Experiment 1:The Construction of rat myocardial fibrosis model:1.HE staining results:Myocardial cells in the control group were clear,cells were consistent in size and space with clear cytoplasmic staining,after isoprenaline injection 3days,7 days,14 days,28 days,disorders of cell arrangement in rat myocardial tissue cells,the nuclear condensation in necrotic areas,the cytoplasm decreased or missing and connective tissue hyperplasia.This change was most severe in the 3d group,which was relatively reduced in the 7d and 14d groups,relatively improved in the 28d group.2.Heart weight index:Compared with the control group,there was no significant difference in heart weight index in the 3d group,P>0.05.The heart weight index in the 7d group,the 14d group,and the 28d group decreased,P<0.05.3.Determination of hydroxyproline content:Compared with the control group,the content of hydroxyproline in the 3d group,the 7d group,the 14d group,and the 28d group increased significantly,P<0.05,and showed an increasing trend with time.4.The Immunohistochemistry results:Compared with the control group,in the myocardial tissues of the 3d and 7d groups,significant positive expressions of SMA and VIM proteins were seen,and P<0.05.There was no significant difference between the 14th and 28d groups compared with the control group P>0.05.Experiment 2:Observation of pharmacodynamics of Jiedu Tongluo Fang in Animals vivo:1.Heart weight index:Application of isoproterenol to the rat myocardial fibrosis model,modeling was performed 3 days after injection.Compared with the control group,the heart weight index of the model group did not increase significantly,and P>0.05,compared with the model group,the cardiac index of the captopril group and the low,medium and high dose groups of Chinese medicine did not change significantly,and P>0.05;2.Results of determination of hydroxyproline:Compared with the control group,the hydroxyproline content in the model group increased significantly,P<0.05;compared with the model group,the hydroxyproline content in the captopril group and the low-dose,medium-dose,and high-dose groups of Chinese medicine significantly decreased,and P<0.05.3.HE staining:In the control group,the cells were neatly arranged,the intercellular spaces were uniform in size,and the cytoplasmic staining was uniform,The model group,captopril group,and the low-dose,medium-dose,and high-dose groups of Chinese medicine showed different degrees of myocardial fibrosis.It is mainly composed of nucleus shrinkage,myocardial fiber lysis,and breakage.The cells are arranged irregularly,the cell structure is destroyed,and the cytoplasm is stained unevenly,the degree of myocardial fibrosis was lighter in the captopril group and the low-dose,medium-dose,and high-dose groups-dose of Chinese medicine.4.Masson staining:Compared with the control group,the model group,the captopril group,and the different doses of the traditional Chinese medicine group had different degrees of myocardial tissue damage.The specific manifestations were pyknosis of the nucleus,disordered cell arrangement,and irregular intercellular spaces.And a large amount of blue-stained fibrous collagen appeared and damaged myocardial cells,and a small amount of inflammation infiltrated the necrotic part.Compared with the model group,the degree of myocardial fibrosis was lighter in the captopril group and the Chinese medicine groups at various doses,Compared with the control group,the collagen fiber volume ratio?CVF?in the rat myocardial tissue increased significantly in the model group,and P<0.05.Compared with the model group,the captopril group and the Chinese medicine low,medium and high dose groups were significantly reduced.And P<0.05.5.Immunohistochemical method to determine the expression area of ColI,ColIII,?-SMA,Vim,LIMK1protein:?1?Type I collagen and type III collagen protein appear brown in immunohistochemical staining,Compared with the control group,the positive expression area of type I collagen and type III collagen protein in the model group was significantly higher than that in the control group,P<0.05.Compared with the model group,the positive expression area of type I collagen and type III collagen protein in the captopril group and the low,medium and high dose groups of Chinese medicine was significantly lower than the model group,P<0.05.?2?The positive expression of?-SMA and Vim proteins is brown,Compared with the control group,the expression areas of?-SMA and Vim protein in the model group were significantly higher than those in the control group,and P<0.05;compared with the model group,the?-SMA and Vim protein expression area in the captopril group and the low,medium,and high dose groups of Chinese medicine decreased significantly,P<0.05;?3?Compared with the control group,the expression of LIMK1 protein in the model group was significantly hi gher than that in the control group,P<0.05.Compared with the model group,the captopril group and the low,medium,and high dose groups of Chinese medicine were significantly lower than the model group,P<0.05.Experiment 3:The Effect of Jiedu Tongluo Fang on TGF-?1/LIMK1 signaling pathway.1.Western-blot method to detect the content of?-SMA,Vim,ROCK1,and LIMK1protein in each constituent fibroblast:?1?Compared with the control group,the expression levels of?-SMA and Vim proteins in the model group increased,with statistical differences,P<0.05.Compared with the model group,the expression levels of?-SMA and Vim proteins in the captopril group were significantly lower than those in the model group,with a significant difference,P<0.05.Compared with the model group,the expression levels of?-SMA and Vim protein in theand the low,medium,and high dose groups of Chinese medicine decreased,with a significant difference,P<0.05.Compared with the model group,the low-dose and medium-dose groups of Chinese medicine decreased,with significant differences,P<0.05;?2?Compared with the control group,the expression level of ROCK1 protein in model fibroblasts increased significantly,P<0.05.Compared with the model group,the expression level of ROCK1 protein in the captopril group,the traditional Chinese medicine low,medium-dose group and the high-dose traditional Chinese medicine fibroblasts decreased significantly,and Significant statistical difference,P<0.05.?3?Compared with the control group,the expression of LIMK1 protein in the model group increased,with statistical differences,P<0.05,Compared with the model group,the expression of captopril decreased,with a significant difference,P<0.05.Compared with the model group,the low,medium,and high dose groups of Chinese medicine decreased,and there were statistical differences,P<0.05.2.Real-time PCR Detection of TGF-?1,RhoA,ROCK1,LIMK1 mRNA content in fibroblasts:?1?Quantitative real-time PCR to detect the mRNA expression of TGF-?1 in each constituent fibroblast,compared with the control group,the expression level of TGF-?1 mRNA in the model group increased significantly,and P<0.05.Compared with the model group,the expression of TGF-?1 mRNA in the captopril group and the low,medium and high dose groups of Chinese medicine decreased significantly,P<0.05.?2?Real-time PCR detection the mRNA expression of RhoA and ROCK1 in each component of fibroblasts:Compared with the control group,the mRNA expression levels of RhoA and ROCK1 in the model group increased significantly,P<0.05.Compared with the model group,the expression levels of RhoA and ROCK1 mRNA in the captopril group and the low,medium and high dose groups of Chinese medicine decreased significantly,P<0.05.?3?Real-time PCR detecting the expression of LIMK1 mRNA in each constituent fibroblast,the results show that,compared with the control group,the expression of LIMK1 mRNA in the model group increased significantly,P<0.05.Compared with the model group,the expression of LIMK1 mRNA in the captopril group,the low,medium,and high dose groups of Chinese medicine decreased significantly,P<0.05.3.MTT method to detect TGF-?1 induced fibroblast proliferation phase:After stimulating fibroblasts with different concentrations of TGF-?1,calculate cell proliferation rate based on absorbance,the results showed that when different concentrations of TGF-?1 stimulated fibroblasts,the cells showed proliferation,TGF-?1 in low concentration group,medium concentration group and high concentration group showed cell proliferation after 24 hours,and peaked in 48 hours and declined after 72hours.The low concentration group showed the strongest proliferation rate at different time points.The medium concentration group had the lowes t proliferation rate at each time point.So in the study of fibroblast proliferation,the low concentration(0.025 ng.ml-1)of TGF-?1 was selected as the drug dose,and the fibroblasts were intervened at a time point of 48 hours.4.After stimulating fibroblasts with TGF,real-time PCR:detect the mRNA content of RhoA,ROCK1,LIMK1 in fibroblasts:The results show that,?1?Compared with the control group,the RhoAmRNA content in the model group increased significantly.And P<0.05.Compared with the model group,the content of RhoAmRNA in thecaptopril group,the low dose,medium and high dose groups of Chinese medicine was significantly reduced.P<0.05.?2?Compared with the control group,the mRNA content of ROCK1 and LIMK1 in the model group increased significantly,P<0.05.Compared with the model group,the mRNA content of ROCK1,LIMK1 in the captopril group and the low,medium and high dose groups of Chinese medicine was significantly reduced,P<0.05.Conclusion:1.Intraperitoneal injection of rats with is oprenaline hydrochloride injection 5mg.kg-1,The pathological changes of myocardial tissue in rats after 3 days,and the hydroxyproline content in myocardial tissue increased,the expression of?-SMA and Vim proteins in myocardial tissue increased,this s hows that the rat myocardial fibrosis model was successfully prepared after the injection of isoproterenol 3 days.2.When application of Jiedu Tongluo Fang in rat myocardial fibrosis model,myocardial fibrosis was significantly improved.By inhibiting the secretion and deposition of myocardial collagen to inhibition of myocardial fibrosis.3.When in vitro application of Jiedu Tongluo Fang on rat fibroblasts,By down-regulating the RhoA?ROCK1,and LIMK1 factors in the TGF-?1/LIMK1 signaling pathway,Inhibition of?-SMA and Vim protein expression in fibroblasts,thereby inhibiting myocardial fibrosis.4.When TGF-?1 induces rat fibroblast proliferation,Jiedu Tongluo Fang can inhibit fibroblast proliferation,And achieved by down-regulating the RhoA,ROCK1,and LIMK1 factors in the TGF-?1/LIMK1 signaling pathway... |
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