Apoptotic Effect Of Matrine On Proliferative Cardiac Fibroblasts And Its Mechanism

AIM:The aim of this study is to investigate apoptotic effect of matrine on proliferative mouse cardiac fibroblasts and it's mechanism.METHODS:Cardiac fibroblasts were isolated from 3-day-old Kunming mice by collagenase disruption and cultured. Firstly, normal cardiac fibroblasts and angiotensin II -intreated cardiac fibroblasts were respectively treated for 24 hours by 1.0~4.0mmol/L matrine and their apoptosis ratios were measured by flow cytometry.Secondly, according to the results of above experiments, the conscentrations frome 2.0mmol/L to 4.0mmol/L were selected. Then the cardiac fibroblasts were respectively treated for 24 hours with 10-7mmol/LangiotensinII, 2.0mmol/Lmatrine+angiotensin II, 3.0mmol/L matrine+angiotensin II and 4.0mmol/L matrine+angiotensin II . The cardie fibroblasts maintained in Dulbecco's modified Eagle's medium(DEME) containing 1% new born calf serum were as control. Morphology of cell were observed by inverted phase contrast microscope and cell numbers were measured by use of a Coulter Counter. Morphology of apoptotic cardiac fibroblasts were observed by fluorescence microscope with PI/Hoechst33342 double staining. Cell cycle phases, apoptotic ratio of cardiac fibroblasts and expression of the apoptosis-related gene Bcl-2, Bax were measured by flow cytometry. Activity of Caspase-3 and expression of activated fragment of Caspase-3 from the total cell extracts were detected by Western blotting analysis.Finally, the cardiac fibroblasts were respectively treated for 12 hours, 24hours, 48hours and 72hours by 3.0mmol/L matrine+angiotensin II. The cardie fibroblasts maintained in Dulbecco's modified Eagle's medium(DEME) containing 1% new born calf serum were as control. All the detections and their methods were as the same as above.RESULTS:1.Treatment on normal cardiac fibrolblasts and proliferative cardiac fibroblasts with 1~4 mmol/Lmatrine for 24 hours revealed that when the concentratin is 1.0mmol/L, neither normal cardiac fibrolblasts nor proliferative cardiac fibroblasts did appear apoptosis; when 2.0mmol/L, proliferative cardiac fibroblasts started apoptosis; when3.0mmol/L, both normal cardiac fibrolblasts and proliferative cardiac fibroblasts appeared apoptosis and the apoptotic ratio of proliferative cardiac fibroblasts was markedly higher than that of normal cardiac fibrolblasts; when 4.0mmol/L, both normal cardiac fibrolblasts and proliferative cardiac fibroblasts also appeared apoptosis, but the apoptotic ratios of these two groups were no significant difference.2. Treatment on proliferative cardiac fibroblasts with 2~4 mmol/Lmatrine for 24 hours revealed that cell numbers were decreased and the apoptotic ratios were increased gradually with the concentration increased(P<0.05); PI/Hoechst33342 double staining could distinguish apoptotic cells and normal cells, typical apoptosis features of nucleolus were observed under fluorescence microscope in 3.0 mmol/Lmatrine+ angiotensin II and the features were more prominent with the concentration increased. Matrine also affected cell cycle obviously, compared with control and angiotensin II. Matrine mainly causing the number of cell in Gl phase was increased (P<0.05) and that of in S phase was decreased (P<0.05). Expression of the apoptosis-suppression gene Bcl-2 was reduced (P<0.05), while expression of the apoptosis-induction gene Bax was enhanced and the effect was dose dependented (P<0.05). At the same time activity of Caspase 3 was induced and expression of activated fragments of Caspase-3 were detected in 3.0mmol/L matrine+angiotensin II and 4.0mmol/L matrine+angiotensin II.3.Treatment on proliferative cardiac fibroblasts with 3.0 mmol/Lmatrine for 12h,24h,48h and 72h revealed that the apoptosis ratio was increased gradually from 24 hours to 48 hours (P0.05); PI/Hoechst33342 double staining could distinguish apoptosis cells and normal cells, typical apoptosis features of nucleolus were observed under fluorescence microscope when proliferative cardiac fibroblasts were treated by 3.0 mmol/Lmatrine for 24hours and the feat...