Tanshinone ⅡA On Cardiac Fibrosis In Rats And Its Mechanisms

BackgroundsMyocardial fibrosis is a common pathological changes of heart function,it could be caused by a variety of diseases,such as hypertension, myocardial infarction,pulmonary hypertension, metabolic syndrome, valvulopathy, arrhythmia. In return,Maycardial fibrosis increases morbidity of cardiovascular disease, such as arrhythmia, heart failure, stroke.It has a long history on the study of myocardial fibrosis, most professors consider it is caused by expressive deposition of extracellular matrix(especially type I and type III collagens) currently.Numerous studies have demonstrated that Activated Renin- angiotensin aldosterone System play a critical function in myocardial fibrosis. As the main active substance in the RAAS angiotensin Ⅱ plays an important role in myocardial fibrosis. With widespread using of angiotensin Converting Enzyme Inhibitors, angiotensin Receptor blockers, aldosterone Receptor antagonist, beta blockers in clinic in recent years, great progress has been made in the prevention and treatment of myocardial fibrosis, but the study of myocardial fibrosis is still in progress.In recent years, with the research of TGF-β1/Smads signal pathway, professors discovered that TGF- β 1/Smads signal pathway is closely related to myocardial fibrosis. TGF- beta 1 / Smads signal pathway system is made up of TGF- beta and Smads family. TGF- beta 1 can induce cardiac fibroblasts proliferation,differentiation to myofibroblasts, witch has a dual function of smooth muscle cells and CFs, and can synthesize ECM, increased myocardial fibrosis. TGF- beta 1can increase expression of collagen, fibrinolytic enzyme activators inhibitor 1,connective tissue growth factor, tissue inhibitor of metallproteinases, increase synthesis of ECM and decrease degradation of ECM, resulting in excessive extracellular matrix deposition, promoting the occurrence and development of myocardial fibrosis. Target TGF- beta and Smads protein, block TGF- beta 1 /Smads signaling pathways to resist myocardial fibrosis has become a hotspot, and achieved certain results, but as a result of TGF- beta 1 / Smads signal pathways and cross multiple signaling pathways, making the clinical efficacy of the drug which block TGF- beta 1 / Smads signal is not sure, indeed serious side effects will appear.Tanshinone Ⅱ A is fat-soluble active monomers extracted from salvia miltiorrhiza, due to its effect of antioxidant, protecting myocardial cell, platelet aggregation resistance, stable artery atheromatous plaque, expansion of coronary artery, it is widely used in clinical treatment of cardiovascular disease, such as coronary heart disease, arrhythmia. In recent years, professors found that, TSNⅡA achieve the effect of anti fibrosis by inhibiting myocardial fibroblasts proliferation,reducing the synthesis of ECM. It still needs further research weather TSN Ⅱ A presence of anti fibrosis effect, and whether its anti fibrosis effect achieved by inhibiting the TGF- beta 1 / Smads signal pathway.ObjectiveIn this experiment, rats model were established by planted micropump subcutaneously which can slow-release Ang Ⅱ, different doses of TSN ⅡA were given, observe the myocardial fibrosis in rats, expression of TGF- beta 1, p-Smad2/3 protein, mRNA and protein level of its downstream factor, CTGF, PAI- 1.Research the anti fibrosis effect of TSN ⅡA, discuss weather the anti fibrosis effect of TSN Ⅱ A is related to down-regulating the activity of beta 1 / Smads signal pathway, and provide experimental basis for the clinical application of TSN ⅡA in anti fibrosis.MethodsCardial fibrisis rat models were established by planted micropump subcutaneously which can slow-release Ang Ⅱ. 32 healthy S-D rats, weight 150 ~150 g, select 24 rats to build model randomly. All rats alive postoperative, model rats were divided into tree groups randomly: model group, given 70 mg/(kg, d) saline by gavage; low-dose of TSN ⅡA group, given 35 mg/(kg·d) TSN ⅡA by gavage;high-dose of TSN ⅡA group, given 70 mg/(kg·d) TSN ⅡA by gavage; 8 in each group, given 70 mg/(kg · d) saline to control group by gavage. Gavage for six weeks,measure the weight, heart rate, tail-artety blood pressure of rats. Kill the rats by cervical dislocation, weigh left ventricular weight, calculate the left ventricular mass index; Take part of the left ventricular tissue for Masson staining to observe cardiac fibrosis; the rest preservated in liquid nitrogen for the following experiments: detect hydroxyproline of myocardial tissues by chemical colorimetric determination; detect Mrna expression of CTGF and PAI 1 by Real- time PCR;detect CTGF, PAI- 1, TGF- beta 1, p- Smad2/3 protein expression by Western-blot.Results(1)Planted micropump subcutaneously slow-release Ang Ⅱ for two weeks,gavage for six weeks, After statistical testing, the change of body weight, heart rate without statistical significance, blood pressure, LVW, LVMI increased in model group, the change of blood pressure without statistical significance after the intervention of TSN Ⅱ A, but LVW, LVMI were reduced compared with model group, and the LVW, LVMI of high dose of TSN ⅡA group were lower than low dose of TSNⅡA group.(2)The HYP content of rats myocardial tissues in model group is higher than the control group, reduced after the intervention of TSN ⅡA, and it is lower in high dose of TSN ⅡA group than low dose of TSN ⅡA group; Masson staining for rats myocardial tissues show that, the rat myocardial fibrosis was obviously in model group, relieved after the intervention of TSN ⅡA,and the fibrosis is more obviously in low dose of TSN ⅡA group than hige dose of TSN ⅡA group.(3)According to the results of RT-PCR and Western blot test, the m RNA and protein expression of PAI- 1 and CTGF,the protein expression of TGF- beta 1 and p- Smad2/3 are higher in model group than control group, reduced after the intervention of TSN ⅡA,and they are lower in high dose of TSN ⅡA group than low dose of TSN ⅡA group.Conclusions(1)Planting micropump subcutaneously which can slow-release Ang Ⅱ is a effective method to establish the cardial fibrisis rat models, and accompanied by the rising of blood pressure.(2) In the model group which can slow-released Ang Ⅱ,the PAI-1、CTGF is overexpression,accompanied by the activation of TGF- beta 1 / Smad2/3 signaling pathway.(3)TSN Ⅱ A can inhibit the TGF- beta 1 / Smad2/3 signaling pathways,decrease the expression of PAI- 1, CTGF, improving myocardial fibrosis induced by Ang Ⅱ, and with dose dependent.