Effects Of Xuezhikang On The Proliferation And Collagen Production Of Cardiac Fibroblasts Induced By AngiotensinⅡ And Its Possible Molecular Machenism

Myocardiac fibrosis can be caused by myocardial ischemia, overload, hypoxia, inflammation, which occurs in different cardiovascular diseases. Myocardial fibrosis can reduce the compliance of the ventricle and increase the stiffness of the myocardium, which lead to diastolic dysfunction, the increased myocardial electric excitation dispersion caused by myocardial fibrosis can induce ventricular arrythmia. In addition, myocardial fibrosis may affect the uptake of oxygen and nutrients and energen production of myocardium. All of above are the pathology foundation of the difficult heart failure, which made the prevention and improvement of myocardial fibrosis become a hot spot. Myocardial fibrosis is mainly characterized by cardiac fibroblast (CFs) activation, proliferation, collagen producing and deposition; CFs is the main effect cell in myocardial fibrosis, and plays a critical role in the ECM formation and myocardial fibrosis developing, therefore the proliferating activity and functional changes of CFs are the determining factors in the development of myocardial fibrosis.Statins are inhibitors of HMG-CoA reductase, which are used as lipid-lowing medicine to treat disorders of plasma lipids. In addition, statins have been proved to have other effects. For example, statins may reduce the left ventricular mass; Lovastatin inhibit angiotensin II induced hypertrophy of cultured newborn rat myocardial cells in vitro; and Atorvastatin inhibit CFs proliferation and the collagen synthesis. Xuezhikang is a traditional Chinese medicine that is also used to treat the disorders of plasma lipids .As an extract of Cholestin, Xuezhikang was made from product fermented by Chinese red yeast. The chemical component of this drug is complex, containing a family of natural materials. Besides its effect on decreasing lipid level of plasma, it has been reported that Xuezhikang may ameliorate ventricle diastolic function, when compared with the placebo group, the left ventricle hypertrophy develop more slowly in Xuezhikang treated group, which suggests Xuezhikang may delay left ventricle remodeling caused by pressure load increasing. These results provide a new point of view for its clinical applying.In this research, we investigated the effects of Xuezhikang on myocardial fibrosis in a vitro model; exploited the effects of Xuezhikang on collagen production and proliferation of CFs and the signal pathway invoved to provide experimental and theoretical basis for using this medicine to prevent and treat myocardial fibrosis.1.Establishment of in vitro model for myocardial fibrosisObjective:CFs are the main effective cells in myocardial fibrosis, they play a critical role in extracellular matrix formation. The variations of their proliferating properties and functions are determining factors in myocardial remodeling. Angiotensin II (Agn II), which has the activities of growth factor, can up regulate the expression of cytokines, promote cell proliferation and regulate the metabolism of extracellular matrix. Based on this, we established a in vitro primary cell culture model by stimulating the newborn rat CFs with Agn II , which can be used to observe the the effects of medicines on CFS.Methods:CFs were isolated with trypsin digestion method from neonatal SD rats, 2nd and 3ird passages of the cells were used in experiments. The cells were identified under inverted microscope and by immunocytochemistry stain. The livability of the cultured cells were estimated by trypanblue. MTT method was used to obeserve the effect of AngII on the proliferation of CFs.Results: Under microscrope, the primary cardiac fibroblasts isolated appeared irregular triangular in morphology, the cytoplasm was transparent and thin, with 2~3 nucleus which were bigger than normal. No spontaneous beat could be seen. Trypanblue staining showed that viable cells were >97%. When stained by immunohistochemistry, the cells showed positive for vimentin and fibronectin, and negative for alpha smooth muscle actin(α-SMA). That suggested the cells isolated were cardiac fibroblasts and purity of the cells was about 98%. The effect of Ang II on the proliferation of CFs was performed by MTT method, and the result showed that Ang II at the concentrations of 10-8mol/L, 10-7mol/L,10-6mol/L and 10-5mol/L promoted the proliferation of CFs, and this effect was dosage dependent, with most significant effect at concentration of 10-6mol/L. The effect of Ang II at the concentrations of 10-6mol/L on the proliferation of CFs was performed for 24 h,48 h,72h, and the result showed the effect was time dependant.Conclusion: The results above suggested that trypsin digestion combined with differential attachment method could be used successfully to culture neonatal rats cardiac fibroblasts; Ang II at the concentration of 10-6mol for 48h could enhance the proliferation of CFs and this system could be used as a in vitro model for the research of drugs on the myocardial fibrosis.2. Effects of Xuezhikang on the proliferation of CFs induced by angiotensinⅡObjective:Statins have been proved to be a effective inhibitor of myocardial hypertrophy induced by Ang II .Xuezhikang contains Monacolins. Whether or not this medicine has the similar effect on inhibiting the myocardial fibrosis have not been reported. Therefore, in this study, we observed the effect of Xuezhikang on the proliferation and TGF-β1, in order to exploit whether Xuezhikang could or not prevent or regress myocardial fibrosis in cellular level and its possible mechaniam.Methods:The model used in this study is the same as above. The 2nd passage of CFs were adjusted to the concentration of 5×104/ml and seeded into 24 well plate. When the cells growth to 80% confluent, the medium was discarded and the cells were washed with PBS for two times and then the different stimulate factors were added according to the grouping: (1). Control group: DMEM with 10% FCS; (2). Ang II group: Ang II (10-6 mol/L)+DMEM containing 10% FCS; (3). Ang II (10-6 mol/L)+DMEM containing 10% FCS+30μg/L Xuezhikang; (4). Ang II (10-6 mol/L)+DMEM containing 10% FCS+150μg/L Xuezhikang; (5). Ang II (10-6 mol/L)+DMEM containing 10% FCS+750μg/L Xuezhikang; 48 hours later, the cells were trypsinized with 0.25% trypsin and collected. The alive cell number were counted on hemometer and the OD were measured using MTT method to investigate the proliferation of CFs in different groups. Flow cytometric analysis were performed after the cells were stained with PI and ELISA was used to detect the expression of TGF-β1.Results:The results gotten by cell counting showed that Xuezhikang could dose-dependently inhibit the proliferation of CFs induced by Ang II, and the same results could be gotten by MTT method. Flow cytometry analysis indicated that: the percentage of cells in G0/G1 decreased but that of cells in S, G2/M phase increased in Ang II group, and Xuezhikang groups had increased percentage of cells in G0/G1 phase and reduced percentage of cells in S, G2 /M phase. ELISA demonstrated that after 48 hour of treatment with AngⅡand Xuezhikang, AngⅡcould significantly increase TGF-β1 protein .Xuezhikang could significantly suppress this increase.Conclusion: These results proved that Xuezhikang could suppress the proliferation of CFs stimulated by AngⅡand this effect might be mediated by decreasing the expression of TGF-β1.3. Effects of Xuezhikang on collagen production of CFS induced by AngⅡand its possible molecular machenismObjective:the process of myocardial fibrosis involve the phenotype changes of CFs, which bring about the synthesis increasing and arrangement disturbance of collagen. It has been reported that, in addition to promoting the proliferation, Ang II can also enhance the synthesis of collagen of the CFs. In the second part of this study, it has been observed that Xuezhikang could inhibit the proliferation of CFs, but the influence of Xuezhikang on the mRNA of type I and III collagen , TGF-β1and the expression of c-fos, an early reactive gene, have not been involved. In this part of the research, we tested the variations of these issues.Methods:The preparation and identification of primary CFs cell culture was in the same way as in the first part of this study. The cell cultures were divided into groups the same way as in the second part experiment. The method of Sirius staining was used to detect the production of collagens; RNA was extracted with TRIZOL and the mRNA expression of TGF-β1, c-fos, and collagens was detected by RT-PCR, with GAPDH as inner reference. The products amplified were electrophoresed on agarose gel and the density of the bands were measured and analysesed using a geography analysis software.Results:The results showed that: Ang II at the concentration of 10-6 mol enhanced the production of collagens of CFs; Xuezhikang at different concentrations inhibit the collagen production of the cells stimulated by Ang II and the effect was dose dependent. Compared with control group, the mRNA expression of TGF-β1, c-fos and collagens elevated in Ang II group and Xuezhikang at the concentrations of 150μg/L or more could significantly decrease the expression.Conclusion: These results suggested that Xuezhikang could inhibit the collagen production and TGF-β1 expression of CFs induced by Ang II at triscription and translation levels; in gene level, Xuezhikang decreased the expression of c-fos, which might be the mechanism for the Xuezhikang to suppress the proliferation, collagen sythesis of CFs stimulated by Ang II. 4. Effects of Xuezhikang on left ventricular diastolic function and heart rate variability of patients with hypertentionObjective:Hypertension is a common disease with a high morbidity in our country that can caused cardiac hypertrophy and fibrosis. The cardiac pathological changes caused by hypertension mainly include left ventricle remodeling and myocardial fibrosis which are characterized by increased collagen volume ratio and cardiomyocyte hypertrophy. Myocardial fibrosis increases the stiffness of ventricle and affects the diastolic function.The above parts of this study had shown that Xuezhikang could improve myocardial fibrosis. In this part of investigation, we observed the effect of Xuezhikang on the degree of remodeling by observating of left ventricular diastolic function and heart rate variability(HRV). Methods:51 patients with hypertention were enrolled. They were divided randomly into two groups:⑴25 patients received the conventional treatment for hypertention.⑵26 patients administered Xuezhikang in addition to the conventional treatment. 20 of healthy individuals matched in age and sex were recruited as control group. With doppler tissue imaging(DTI) and pulsed-wave doppler blood flow, Em,Am,E,A and the time domain indices of HRV were measured .Results:⑴Compared with control group ,the parameters E/Em, Em/Am of left ventricular diastolic function and the parameter SDNN,RMSSD,PNN50 of HRV varied significantly with patients of hypertention, P < 0.05.⑵Compared with conventional treatment, Em/Am, SDNN,RMSSD,PNN50 increase significantly and E/Em were significantly lower with patients treated with Xuezhikang, P<0.05.⑶. Linear correlation analysis demonstrated that SDNN and Em/Am were positively correlated, P<0.01. Conclusion: Therapy with Xuezhikang could improve left ventricular diastolic function and heart rate variability of patients with hypertention.