| Background:Diabetes is one of the most important non-communicable diseases which threats to human health. Diabetic cardiomyopathy (Diabetic Cardiomyopathy, DCM) is a chronic complication of diabetes. DCM can induce heart failure, arrhythmias, cardiogenic shock, and sudden death, and aggravate prognosis suffering from other diseases. DCM is is still the major death cause in diabetic patients. DCM pathogenesis is very complex, mainly associated with the Renin-angiotensin-aldosterone system, oxidative stress, inflammatory cytokines, growth factors, extracellular matrix, and myocardial fibrosis etc..However, myocardial fibrosis is a characteristic pathology of diabetic cardiomyopathy.Cardiomyocytes and cardiac fibroblasts are the major cellular components of the heart. The number of myocardial cells is 90% of the total, the number of fibroblasts is 60%-70% of the total. Under physiological conditions, cardiac fibroblast cells are called fibroblasts, and its function is stationary. In myocardial fibrosis, various signaling factors such as transforming growth factor-? (TGF-?), tumor necrosis factor (TNF-a), nuclear transcription factor (NF-? B), and nuclear factor of activated T (NFAT) other stimulate fiber cells into fibroblasts and secrete large amounts of fiber (type I, type III collagen) and matrix, further forms scar tissue to prevents cardiac rupture. At the same time, due to excessive secretion of fibers and matrix, cause deposition of extracellular matrix, resulting in myocardial fibrosis.Recent studies have found that TGF-? is closely related with the formation of myocardial fibrosis of diabetes. It is most closely related cytokines with tissue fibrosis, involved in inflammation and regulation of extracellular matrix deposition. It can play a biological role through Smad signaling pathway, protein kinase C pathway, mitogen-activated protein kinase (MAPK) pathway, which TGF-?1/Smad signaling pathway is considered to be the most significant role in the most classic way.Currently, the treatment of myocardial fibrosis lacks of effective means. By studying regulatory mechanisms of medicine for diabetes myocardial fibrosis, explore the feasibility of Chinese medicine to prevent diabetes myocardial fibrosis.Purpose:To explore the effect on Tongxinluo regulating fibroblasts which using high sugar treatment fibroblasts. Further explore the effects about Tongxinluo treat myocardial fibrosis and cardiac function by using high-fat diet and low-dose streptozotocin (Streptozotocin, STZ) intraperitoneal injection induced diabetic cardiomyopathy (DCM) model. On this basis, to detect the expression levels of TGF-?1/Smads pathway in myocardial tissue and to find new targets for prevention and treatment of myocardial fibrosis using traditional Chinese medicine.Methods:1. In vitro:Cultured rat cardiac fibroblasts were divided into control group, Tongxinluo low, medium and high dose group, valsartan group. Then using MTT method detect the cell Proliferation and vitality. On this basis, protein expression levels of collagen type ?,?, TGF-?1 were detected by the application of immune enzyme-linked immunosorbent assay (ELISA). Using ICC method detects the protein levels of TGF-?1. Using ICC and Western blot method detects the protein levels of phospho-Smad2, Smad3 and Smad7.2. In vivo:After high fat diet fed rats 4W, Using insulin sensitivity index(ISI) determine insulin resistance, then STZ solution of a small dose was intravenous injection to prepare a model of diabetes. The successful model rats is divided into diabetic cardiomyopathy (DCM), low-dose group Tongxinluo (TXL-L), TXL dose group (TXL-M), TXL high dose group (TXL-H), positive drug group (positive), another 10 healthy rats as a control group (CON)(n=10).Detection indicators are as follows: ? observe and record the general condition(including weight, hair color, diet, defecation, etc.) and blood glucose concentration. ? 12 weeks after modeling, using cardiac catheterization to detect left ventricular systolic pressure (left ventricular systolic pressure, LVSP), left ventricular end-diastolic pressure (left ventricular end-diastolic pressure, LVEDP), left ventricular pressure maximal rate of rise and fall (± dp/dtmax).? Calculated rat heart weight index and left ventricular mass index. ? Using HE staining observed change of myocardial tissue. ? Using masson staining measured myocardial interstitial collagen volume integral. ? Using immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) detected protein expression of TGF-?1. ?Using immunohistochemical staining and Western blot to detect protein levels of phospho-Smad2, phospho-Smad:3 and Smad7.Results:1. In vitro experiments:(1)MTT results:? High glucose (25mmol/L) duration of action filter Results: With the extension of high-sugar processing time, OD value gradually increased and reached the peak at 24h, and after 48h significantly reduced. The results suggest that fibroblasts viability is strongest at 24h after high sugar treatment... ? TXL effect screening:Compared with the control group, with the extension of time, OD value of the model group was significantly increased. After TXL of different dose intervention, OD values decreased significantly, suggesting that TXL are significantly inhibited proliferation and viability of fibroblasts.(2) ELISA results:The expression level of collagen type ?, ? in high sugar treatment group (Model) were significantly higher than the control group (CON), while the expression level of TXL treatment group is significantly lowed than Model group, in which high dose group is the most obvious.(3) TGF-?1 protein level detection:ICC and ELISA test results showed that TGF-?1 levels in high glucose treatment groups (Model) was significantly higher than the CON group, while the expression level of TXL treatment group is significantly lowed than Model group, in which high dose group is the most obvious. And TXL high dose treatment group was significantly lower than positive drug group.(4) Protein expression levels of phopho-Smad2, Smad3 and Smad7 detection:ICC and Western blot analysis showed, protein expression of phospho-Smad2, Smad3 in the Model group was significantly higher than the CON group, while the level of expression Tongxinluo treatment group is significantly lowed than Model group, in which high dose group is the most obvious. Theres is no significant difference betweent TXL dose treatment group and positive drug group, but the high-dose treatment group was significantly lower than positive drug group. Expression of Smad? localized in the cytoplasm. There is a small number positive cells in CON group. Model group showed obvious Smad7 positive cells, and TXL treatment group was seen a lot of positive cells expression, in which high dose group is the most obvious.2. In vivo experiments:(1) Body weight, insulin sensitivity index and blood glucose detection and general condition observation:There is no significant difference in initial body weight of rats. In the first 4 weeks of the experiment, compared with the normal control group (normal diet), the weight was significant increase in other rats (high fat diet), indicating that weight will increase significantly after fed high-fat diets, further promoting form of insulin resistance. Blood glucose and insulin sensitivity index(ISI) test results showed that high fat diet for 4 weeks, fasting insulin levels of DCM group, TXL each treatment group, positive drug were significantly increased, and insulin sensitivity index was significantly reduce. From the model to the end of the experiment, the fasting blood glucose values of DCM group, Tongxinluo treatment group and positive drug group were significantly higher than those of normal control group.(2) General state observation:Generally condition was good, rats eating, drinking urine and no significant change in CON group. Rats had "a little" symptoms DCM group. Compared with the DCM group, general condition was improved in each TXL treatment group rat, particularly most obvious improvement.(3) Hemodynamic changes:the end of the experiment, left ventricular systolic pressure (LVSP), maximal rate of left ventricular pressure (+dp/dtmax), left ventricular pressure maximal rate of decrease (-dp/dtmax) in DCM group was significantly lower than CON group, while Tongxinluo treatment group is significantly higher than DCM group, in which high dose group is the most obvious. Theres is no significant difference betweent TXL middle, high dose treatment group and positive drug group. Left ventricular end diastolic pressure (LVEDP) in DCM group was significantly higher than the CON group, while Tongxinluo treatment group is significantly lower than DCM group, in which high dose group is the most obvious. Theres is no significant difference betweent TXL middle, high dose treatment group and positive drug group.(4) Rat heart weight index, left ventricular mass index:Cardiac index and left ventricular mass index in DCM group was significantly higher than the CON group. Cardiac index and left ventricular mass index in TXL group was significantly higher than the DCM group, in which high dose group is the most obvious, and better than the positive drug group.(5) HE staining:Cardiac muscle was normal structure in CON group. And cardiac muscle in DCM group arranged in disorder, cell gap increases, the stromal cells presenting fibrosis, cell size is irregular. Compared with the DCM group, the situation of Tongxinluo group was significantly improved, in which high dose group is the most obvious.(6) Masson staining and collagen volume fraction analysis:There is a small amount of interstitial in CON group. Cardiac muscle of rats DCM group arranged in disorder, significantly increased the number of collagen fibers, collagen volume increase, showed significant interstitial fibrosis. Compared with the DCM group, the situation of Tongxinluo group was significantly improved, in which high dose group is the most obvious, the collagen volume in TXL middle, high dose group was significantly lower than positive drug group.(7) Protein expression levels of TGF-?1 detection:immunohitochemistry and Western blot analysis showed, protein expression of TGF-?1 in the DCM group was significantly higher than the CON group, while the level of expression in Tongxinluo treatment group is significantly lowed than DCM group, in which high dose group is the most obvious. Theres is no significant difference betweent TXL middle,high dose treatment group and positive drug group.(8) Protein expression levels of phopho-Smad2, Smad3 and Smad7 detection: immunohitochemistry and Western blot analysis showed, protein expression of phospho-Smad2, Smad3 in the DCM group was significantly higher than the CON group, while the level of expression in Tongxinluo treatment group is significantly lowed than DCM group, in which high dose group is the most obvious. Theres is no significant difference betweent TXL middle, high dose treatment group and positive drug group.Expression of Smady in DCM group is higher than CON group, while the level of expression in Tongxinluo treatment group is significantly higher than DCM group, in which high dose group is the most obvious. Theres is no significant difference betweent TXL middle, low dose treatment group and positive drug group, but the high-dose treatment group was significantly higher than positive drug group.Conclusion:1. Tongxinluo can inhibit the proliferation and viability of fibroblasts, thereby inhibiting the activity of collagen type I and III, and reducing the secretion of type ?and ? collagen.2. The above-mentioned effect of Tongxinluo could be achieved through the activation of TGF-?1/Smads pathway.3. Tongxinluo reduce myocardial interstitial fibrosis and improve the heart function of diabetic rats through regulating TGF-?1/Smads signal pathway, indicating that Tongxinluo have a protective effect of cardiac function of diabetic cardiomyopathy rats. |
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