| BackgroundCardiac hypertrophy is characteristic of hypertrophic cardiomyopathy (HCM), myocardial hypertrophy and muscle fibers arranged in disorder are the typical performance. At present, most scholars believe that the pathogenesis of HCM is closely related with genetic and which is an autosomal dominant genetic disease. Embryonic genes such as re-expression of beta myosin heavy chain (β-MHC), brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) gene is considered as molecular markers of cardiac hypertrophy, to detect the expression of these genes can not only positive cardiac hypertrophy exist, can also judge the extent and prognosis of hypertrophy.According to the technology of gene chip, different cardiac hypertrophy models induced gene expression level of the direction, the type and number are different, such as arteriovenous fistula (AVF) and norepinephrine (ISO) production model of myocardial hypertrophy, in 34 of the genes changed, there will be 14 regulated in the opposite direction. The renal abdominal aortic coarctation (AAC), AVF and ISO produced myocardial hypertrophy model, is a classic and widely recognized by domestic and foreign researchers. But it was unclear between these three different cardiac hypertrophy models hypertrophic marker genes ANP, BNP, and β-MHC expression whether there are differences. Therefore, we use AAC, AVF and ISO production model of myocardial hypertrophy in mice, to detect the expression of ANP, BNP and β-MHC. We hope to pick out the production method of obvious expression of cardiac hypertrophy marker gene.The production of animal models requires the selection of suitable strains from different strains of animals, different strains animals, using the same modeling method is also different. Most of these differences are caused by different genetic backgrounds. Different genetic backgrounds, will cause different specific gene expression level, but it is not clear that the impact of human genetic closely related to the HCM hypertrophic molecular markers (ANP, BNP and β-MHC) as the target, HCM model of the different strains mouse will be screened. Therefore, comparison of different strains of ANP, BNP and 3-MHC expression in mouse model of myocardial hypertrophy is necessary.At present, the main methods of treatment of HCM are medication and surgical treatment. Shuangdan oral liquid recorded in China Pharmacopoeia 2015, indications for Yang xin huo xue, hua yu zhi tong. This paper preliminary study results show Shuangdan extracts includes two main effective components (salvianolic acid B and paeonol combination of new compound, SP) has a synergistic effect, and in the model of myocardial ischemia was observed SP has a significant relaxation of vascular function and has a certain role in the prevention and treatment of AVF myocardial hypertrophy model mice, but the effect of SP and the expression of BNP, ANP and (3-MHC relevance, and its mechanism of action has not been clear.According to reports, myocardial hypertrophy and fibrosis are closely related and accompanied by TGF-p1 expression, usually Smad protein is considered to be one of the TGF-β1 signaling pathways downstream medium, the Smad2 and Smad 3 can promote TGF-β1 signal from the cytoplasm to the nucleus conduction, whereas Smad7 is TGF-β1 signal transduction inhibitors, with activated Smad competition binding ligand is activated TGFβ1 receptor, and block the signal transduction, inhibit the target gene transcription and protein expression. Therefore, this study will explore the effects of SP on ANP, BNP and β-MHC expression and TGF-β/Smad signaling pathway in cardiac hypertrophy mice.Objective1. From the three kinds of modeling methods of AAC, AVF and ISO, screening cardiac hypertrophy marker genes ANP, BNP and β-MHC expression model of the most obvious.2. From four different strains BALB/c, C57BL/6, ICR and KM myocardial hypertrophy mice, screening cardiac hypertrophy marker genes ANP, BNP and β-MHC expression strains of the most obvious.3. Based on the selection and preparation of the ideal model of cardiac hypertrophy, explore the effect of SP on ANP, BNP and β-MHC expression and TGF-β/Smad signaling pathway.Methods1. The C57BL/6 mice were randomly divided into control group and model group, model group were made model of myocardial hypertrophy by AVF, AAC and ISO. Then KM, ICR, C57BL/6 and BALB/c in four strains of mice were randomly divided into control group and model group, which will adopt the previous results to make the model of myocardial hypertrophy. After 4 weeks, the mice body weight (BW) was weighed, sacrificed and the heart weight (HW) and left ventricular weight (LVW) were weighed, at Last, calculated cardiac index (HW/BW) and left ventricular mass index (LVW/BW). A section of heart tissue samples after separation, fast washing, filling, and frozen in liquid nitrogen, for later detected by qRT-PCR. Another part of the heart tissue were rinsed with physiological saline, fixed in 10% formaldehyde, after 24h, washing, dehydration, hardening, transparent, infiltration and paraffin embedding, sectioning, hematoxylin and eosin (HE) staining, photograph, tissue chip production, and detect the protein expression of ANP, BNP and P-MHC by immunohistochemical method.2. The C57BL/6 mice were randomly divided into control group and model group, the latter by AAC method to make the model of myocardial hypertrophy, After 2 weeks the models rats were randomly divided into:model, positive drug and SP large, medium and small dose group, and intragastric administration to fourth weekends. Positive drug group every day according to 35mg/kg give Carter Plymouth tablets, SP large, medium and small dose group respectively every day according to 60,30 and 15 mg/kg give SP solution, the control group and model group were given the same volume of solvent. After the last administration, the mice BW was weighed, sacrificed and HW and LVW were weighed, at Last, calculated HW/BW and LVW/BW. Some cardiac tissues packaging frozen and used for qRT-PCR and Western blot; another part of the heart tissue HE staining, photograph and tissue chip making, detect the protein expression of ANP, BNP and (3-MHC by immunohistochemical method.Result1. Comparison of ANP, BNP and β-MHC expression changes of cardiac hypertrophy model induced by different methodsCardiac hypertrophy index HW/BW and LVW/BW were significantly increased by AAC, AVF and ISO method were used to make the model of myocardial hypertrophy mice. At high magnification, visible myocardial hypertrophy, sparse and interstitial fibrosis, suggested cardiac hypertrophy mice model is successful by AAC, AVF and ISO. compared with AAC group, ISO group HW, LVW/BW and AVF group HW/BW significantly reduce, AVF and ISO groups mice ventricular wall thickening, ventricular cavity stenosis lighter, prompted the hypertrophy degree will superior to AVF and ISO method by AAC method making cardiac hypertrophy model mice.Myocardial hypertrophy mice heart left ventricular ANP, BNP and β-MHC mRNA transcription and protein expression levels were significantly increased by the AAC and AVF methods, Similarly, ANP and BNP mRNA transcription and protein expression levels were significantly increased by ISO method, but β-MHC mRNA transcription and protein expression level was only increasing trend. Comparison of three methods AAC, AVF and ISO established cardiac hypertrophy model, showed that AAC mice left ventricular ANP, BNP and MHC mRNA transcription and protein expression level significantly increased, suggesting that the AAC upregulation of myocardial hypertrophy model mouse left ventricular ANP, BNP and β-MHC mRNA and protein levels which is superior to the AVF and ISO method.2. Differential expression of myocardial hypertrophy marker genes ANP, BNP and β-MHC in four mice strainsC57BL/6, BALB/c, KM and ICR were used to make the model of myocardial hypertrophy, among them, C57BL/6, BALB/c and KM mice cardiac hypertrophy index LVW/BW and HW/BW increased significantly, but HW/BW and LVW/BW was only increasing trend in ICR mice. HE staining showed that four strains mouse cardiac muscle cell hypertrophy, partial muscle fiber breakage, the cell gap increased, suggesting that the use of C57BL/6, KM, BALB/c mouse model of myocardial hypertrophy is successful, ICR mice is not ideal. Comparison of four strains mice C57BL/6, BALB/c, KM and ICR myocardial hypertrophy model, showed that C57BL/6 mice cardiac hypertrophy index HW/BW and LVW/BW increased the most obvious, followed by KM mice, suggesting that the cardiac hypertrophy index produced by C57BL/6 mice was larger than that of BALB/c, KM and ICR.C57BL/6, BALB/c, KM and ICR were used to make the model of myocardial hypertrophy, among them, KM and C57BL/6 mouse left ventricular ANP, BNP and P-MHC mRNA transcription and protein expression levels were significantly increased, but BALB/c mice BNP and ICR mice ANP, BNP mRNA transcription and protein levels only increased trend. Comparison of four strains mice C57BL/6, BALB/c, KM and ICR myocardial hypertrophy model, Show C57BL/6 mice left ventricular ANP, BNP and β-MHC mRNA transcription and protein expression level significantly increased, suggesting that the C57BL/6 mice ANP, BNP and β-MHC mRNA transcription and protein expression level were superior to BALB/c, KM and ICR in myocardial hypertrophy model.3. Effect of SP on expression of ANP, BNP, β-MHC and TGF-β/Smad signaling pathway in cardiac hypertrophy miceC57BL/6 mice cardiac hypertrophy model was prepared by AAC, show model mice index HW, LVW/BW increased significantly, increases the size of the heart, myocardial hypertrophy, part of the muscle fiber fracture, the intercellular space increased, suggesting that this experimental model is successful. The large and medium doses of SP and CAP can significantly reduce the hypertrophy index HW/BW and LVW/BW of the model mice, decrease the cardiac volume, reduce the degree of muscle fiber breakage, and the gap between the cells, there was no significant difference between CAP and SP, Suggest that SP can significantly reduce C57BL/6 myocardial hypertrophy mice heart hypertrophy index HW/BW, LVW/BW and myocardial pathological injury, the SP strength and captopril tablets are equivalent.C57BL/6 mice cardiac hypertrophy model was prepared by AAC, the expression levels of β-MHC, BNP and ANP mRNA transcription and protein were signifiqantly increased in the left ventricle of cardiac hypertrophy model mice; The large and medium doses of SP and CAP can significantly reduce the levels of β-MHC, BNP and ANP mRNA transcription and protein expression in the left ventricle of the model mice, and there was no significant difference between SP and CAP, Suggest that SP can significantly reduce ANP, BNP and β-MHC mRNA transcription and protein expression levels in the left ventricular of myocardial hypertrophy model C57BL/6 mice, the SP strength and captopril tablets are equivalent.C57BL/6 mice cardiac hypertrophy model was prepared by AAC. The TGF-P1, Smad2 and Smad3 mRNA transcription levels were significantly increased in the heart of the model mice, compared with the control group, there was no significant difference in the level of Smad7 mRNA transcription in the model group. The large and medium doses of SP and CAP can significantly down regulate the transcription of TGF-β1, Smad2, Smad3 mRNA and Smad3 protein expression, obviously increase the Smad7 mRNA transcription and protein expression levels in the left ventricle of model mice, Show SP on the mechanism of prevention and treatment of cardiac hypertrophy may be related to the regulation of TGF-β/Smad signaling pathway.Conclusion1. Using AVF, AAC and ISO three ways to make cardiac hypertrophy models, ANP, BNP and β-MHC expression for the screening conditions in the left ventricular, the results showed that the model of myocardial hypertrophy produced by AAC method was the best.2. ANP, BNP and P-MHC expression for the screening conditions in the cardiac hypertrophy mice left ventricular, the model of C57BL/6, BALB/c, KM and ICR mouse cardiac hypertrophy was made by AAC method, the C57BL/6 strain mouse model was the best.3. SP significantly inhibited C57BL/6 mice cardiac hypertrophy and expression of ANP, BNP and β-MHC in cardiac hypertrophy mice was made by AAC method.4. The mechanism of SP inhibiting the cardiac hypertrophy in mice may be related to the regulation of TGF-β/Smad signaling pathway. |
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