| Cardiac hypertrophy is considered to be a compensatory adaptation in response to various sorts of intrinsic and extrinsic stimuli,which is characterized by reactivation of fetal genes,increase of sarcomeres,and enlargement of cell size.Although initially occurs as a compensatory response,the continued presence of hypertrophy eventually lead to decompensation,which is absolutely increase for the morbidity and mortality of a variety of cardiovascular diseases.Therefore,to reverse the pathological myocardial hypertrophy has been become an important strategy for the prevention and treatment of cardiovascular diseases.During early embryonic stages of mammalian,the cardiac myocytes proliferate rapidly with high mitotic index.However,the cell cycle promoting factors cyclins and cyclin-dependent kinases?CDK?are downregulated soon after birth,while cell cycle inhibitors?cyclin-dependent kinase inhibitors,CDKI?are induced,and it has been thought that mammalian cardiomyocytes exit the cell cycle and become terminally differentiated.Recent studies indicated that postnatal mammalian cardiomyocytes are still capable of cell cycle reentry.The cell cycle regulators can reactivate and express with stimulating of pathologic factors.The cardiomyocytes therefore obtain the ability to undergo DNA and proteins synthesis,but they can not successfully complete mitosis,which results in increase in cell size and cardiac hypertrophy.The cell cycle is controlled by a series of key components including various cyclin–CDK complexes and CDKI regulatory molecules that exert negative effect.Hence,the activity of cell cycle regulators is not only required for cell division,but also involved in hypertrophic growth.Accordingly,it is important to study the expressions of cell cycle regulators in the process of myocardial hypertrophy and explore the possible mechanisms involved.There regulators and their temporal expressions might be novel therapeutic target for prevention and treatment of cardiac hypertrophy.Triptolide?TP?,the major active component of the Chinese medicinal herb Tripterygium wilfordii Hook F,can regulate the expressions of various cell cycle regulatory factors in tumor cells.However,the effects of TP on cell cycle regulators in myocardial hypertrophy and its mechanism are at present poorly understood.Therefore,the aims of the present study were to explore the fluctuated expressions of cell cycle regulators in cardiac hypertrophy and effects of triptolide in vitro and in vivo.METHODS1.H9c2 myocytes were stimulated with 1.0 ?mol·L-1 angiotensin II?Ang ??for scheduled times.Cells were stained by rhodamine labeled phalloidin and the cells area was measured by ImageJ software.The mRNA expression levels of cyclin B1,D1,E1,CDK1,2,4,6,and CDK inhibitor p21 were detected by real-time PCR at different time points?0,5,10,30 min,and 1,2,3,6,12,24,48 h,respectively?.2.Neonatal rat ventricular myocytes?NRVM?from 1 to 3 days old Sprague-Dawley rats were isolated and cultured.After being cultured in serum-free DMEM for 24 h,NRVM were incubated for scheduled times?0,5,10,30 min,and 1,2,3,6,12,24 h,respectively?in a non-serum medium containing 1.0 ?mol·L-1 Ang ?.Cells were analyzed with flow cytometry to detect the cell cycle.3.NRVMs were coincubated with 1.0 ?mol·L-1 Ang ? and 1.0 ?g·L-1 triptolide?TP?for scheduled times.Cells were stained by rhodamine labeled phalloidin and the cells area was measured by ImageJ software.mRNA expression levels of ?-myosin heavy chain??-MHC?and cyclin A1,B1,D1,E1,CDK1,2,4,6,and p21,p27 were detected by real-time PCR at different time points.The protein expression levels of selected molecules which showed dramatic change in mRNA expression level were determined with Western blotting method.4.Seventy-two male mice were randomly divided into isoprenaline?ISO?and TP treated group,and administrated with ISO(10 mg·kg-1,sc,bid)or ISO plus TP(10 ?g·kg-1·d-1,ip,qd)for 0,1,3,7,14,21 days,respectively?n=6 in each group?.5.The general state of all animals was observed during whole experiment period.After different time of treatment,the heart weight?HW?and tibial length?TL?were measured to calculate the ratio of HW to TL?HW/TL?.The pathological change and myocardial fibrosis were analyzed after HE and masson staining,and cell size was determined after lectin staining.6.The mRNA expression levels of ?-MHC and cyclin A2,B1,D1,E1,CDK1,2,4,6,p21,p27 in left ventricle were determined by real-time PCR.And the protein expression levels of ?-MHC and cyclin D1,CDK4,CDK6,P21 were detected by Western blotting method.RESULTS1.Cell size of H9c2 increased soon after stimulation of Ang ?.The mRNA expressions of cyclin E1,CDK4 and CDK2 all reached the peak at 5min after stimulation of Ang ?.mRNA expression of cyclin D1 was increased dramatically at 10 min,followed by a decrease trend.However,the mRNA expression of cyclin B1 and CDK6 both showed two peaks.p21 mRNA level was up to the peak at 30 min,decreased to be lowest at 3h.Although its expression increased gradually after 3h,p21 mRNA remained low level.2.Compared with H9c2 cell,NRVM size increased more obviouly after stimulation of Ang ?.The cell size increased dramatically after Ang ? stimulation for 6h compared to the control.However,TP treatments decreased cell size significantly by comparison of that in the Ang ? group.Cell cycle analysis indicated that the myocytes number in phase of S+G2 increased and that in G1 phase decreased significantly compared with control group.3.The mRNA expressions of ?-MHC,cyclin A1,p21 and p27 in NRVM increased soon after stimulated for 5min.p21 mRNA expression was up to the maxima at 30 min.After 30 min,mRNA expressions of all cell cycle factors showed a decrease trend,?-MHC mRNA level was lowest at 6h,followed by an increase trend and reached the maximum at 48 h,which was consistent with the results of cell size.The mRNA expressions of CDK1,2,4,6,and cyclin B1,D1,p21 were lowest at 2h.And mRNA expressions of cyclin A1 and p27 mRNA reached lowest at 1h.And that of cyclin A1 was up to the peak at 3h.Only the expressions of CDK1,p21,p27 mRNA increased significantly after stimulation of Ang ? for 2448h?P<0.01 or 0.05?.TP treatment not only significantly decreased NRVM size,but also effectively prevented the abnormal expressions of cell cycle regulators compared to vehicle?Ang ??in NRVM.4.Western blot analysis indicated the expression levels of ?-MHC,CDK4,CDK6 in NRVM gradually reduced after stimulation of Ang ?.P21 express increased significantly at 30 min compared to control,followed by a decrease trend.The protein expression of CDK4 and P21 reached lowest at 2h,and that of ?-MHC,CDK6 reached lowest at 30 min.Then protein expression levels were increased markedly and all reached the mximum after stimulation for 1224h compared with those in the control group.Interestingly,it has been shown that the effects of Ang ? on the protein expression was more obvious compared to mRNA.TP could down-regulate the expression levels of cell cycle regulators and attenuates cardiac hypertrophy significantly.5.All of the mice in the ISO groups showed waxing fleeciness,lackluster fur,breathlessness and less activity,and reactions became more and more serious with the stimulation time lasting.Mice in TP-treated groups were more active by comparison of those in the ISO group.6.After treated with ISO for 3 days,heart index?HW/TL?and myocardial cross sectional area increased significantly by comparsion of those in control group.Morphological analysis showed myocardium injury,inflammatory cell infiltration and collagen deposition in ISO-treated animals.TP could significantly decrease the heart index,improve tissue injury and attenuate myocardial fibrosis compared with ISO groups.7.ISO treatment decreased the myocardial mRNA expressions of ?-MHC,cyclin D1,E1,CDK2,4,6 gradually in mice.mRNA expressions of ?-MHC,cyclin D1,CDK6 reached valley at day 1,and that of cyclin E1,CDK2 and CDK4 reached valley at day 3.Followed by an increase trend,?-MHC mRNA level was up to the peak at day 14.The mRNA expressions of cyclin A2,B1,CDK1 were increased dramatically at day 1,and cyclin B1 mRNA was up to the peak at day 3.The expression levels of other factors all reached the peak at day 7,followed by a decrease trend.After 21 days of ISO treatment,the mRNA expressions of cyclin B1,E1 were decreased markedly compared with those in control group,respectively.Compared with control,the expression of p21 was up-regulated and p27 was decreased after stimulation of ISO.p27 mRNA expression reached the lowest point at day 3,and increased to peak a day 14 with the expression of p21 mRNA.TP treatment could correct abnormal expressions of ?-MHC and cell cycle regulators cyclin,CDK,CDKI effectively.8.The protein expression levels of ?-MHC and cell cycle regulators were determined using Western blotting method.The result demonstrated that the expression of ?-MHC was increased gradually in the ISO groups,and up to the peak at day 14.The expression levels of cyclin D1,CDK6 increased dramatically and all reached the peak at day 3,followed by a decrease trend.The expressions of CDK4,P21 were lowest at day 3,followed by increased.However,TP treatment could correct expression of ?-MHC and the unbalance of cell cycle regulators.CONCLUSION1.The cell cycle regulators in H9c2 cells,NRVM myocytes and myocardium of mice showed unbalanced expressions in the process of hypertrophic response.2.Triptolide treatment could inhibit the hypertrophic response of H9c2 cells and NRVM myocytes induced by angiotensin II incubation,and effectively attenuate cardiac hypertrophy and myocardial fibrosis induced by ISO in mice.3.TP can correct the abnormal expressions of various cell cycle regulators,which is involved in its mechanisms of attenuating cardiac hypertrophy. |
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