Effects Of Triptolide On Expression Of Cyclin-dependent Kinase Inhibitor 1a And Cardiac Hypertrophy In Vitro And In Vivo

Although the differentiation of mammalian cardiomyocytes is extremely active during embryonic period, it will stop proliferation and withdraw from the cell cycle soon after birth. Cardiomyocytes can re-enter into cell cycle as stimulated by some pathologic factors. And then the synthesis of protein and nucleic acid in the cardiomyocytes will increase, which in turn result in enlargement of cell size, cardiac hypertrophy and eventually heart failure. Cardiac hypertrophy is a compensatory response to chronic overload under certain disease conditions such as hypertension and coronary artery disease. And it is an independent risk factor for the progress of heart failure. It’s possible to reduce the morbidity and mortality of heart failure by reversing cardiac hypertrophy. To prevent and reverse cardiac hypertrophy have been a main target of various cardiovascular diseases such as hypertension and chronic heart failure.Cyclin-dependent kinase inhibitor 1a(CDKN1a) plays critical role in cell cycle as a negative regulator. CDKN1 a is highly expressed in adult heart, and induce cardiac hypertrophy via inhibiting the activation of cyclin and cyclin-dependent kinase(CDK) complexes which make the most cells stay in G0/G1 stage. It has been reported that the expression and activation of CDKN1 a can be regulated by triptolide(TP), an active ingredient from the traditional Chinese herb Tripterygium wilfordii HooK F. However, its effects and mechanisms on cardiac hypertrophy are still unclear. Therefore, the present study was designed to investigate the regulatory mechanism of CDKN1 a in cardiac hypertrophy and the effects of triptolide on the expression of CDKN1 a and cardiac hypertrophy in vitro and in vivo.METHODS1. H9c2 cell line was passaged and cultured in vitro. The tolerated concentration of TP on H9c2 was detected using MTT assay.2. The hypertrophic response of H9c2 cells was induced by angiotensin II(1 μmol/L) incubating for 24 h. Four concentrations of TP(0.3, 1, 3, 10 μg/L) were used to treat cells. The cell size was measured under fluorescent microsopy using ImageJ software after staining with rhodamine-labeled phalloidine. The m RNA and protein expression of α-MHC, β-MHC, ANP, CDKN1 a mRNA were determined by Realtime-PCR and Western blot techniques, respectively.3. Thirty male mice were randomly divided into six groups, i.e, control, model, TP(10, 30, 100 μg·kg-1·d-1) and TP control goup, n=5 in each group; and administrated with saline, continuous infusion of isoprenaline(ISO) at 40 mg·kg-1·d-1 for 14 days, ISO infusion plus TP(10, 30, 100 μg·kg-1·d-1, ip, qd, 14d) treatments, or TP treatment solely(30 μg·kg-1·d-1, ip, qd), respectively. And other twenty mice were randomly divided into four groups i.e. control, model, CDKN1 a inhibitor and CDKN1 a adenovirus group. The mice in control and ISO group were treated as same as above. Those in CDKN1 a inhibitor group were treateded with ISO infusion and sterigmatocystin(3 mg/kg, ip, qd). And mice in CDKN1 a adenovirus group were treated with 2×107 TU Ad-CDKN1 a adenovirus via femoral vein injection.4. The general state of all animals was observed during whole experiment period. After 2 weeks’ treatment, mice were killed for sampling. The heart weight index was measured with weighting method. The pathological change and fibrosis were analyzed after HE and masson staining and cell size was determined after lectin staining.5. The mRNA and protein expression levels of α-MHC, β-MHC, ANP, CDKN1 a in left ventricle were detected by Realtime-PCR and Western blot techniques, respectively.RESULTS1. The proliferation of H9c2 cells had no significant change after incubated with 0.1-10 μg/L TP for 24 hours. Stimultaneously, incubation with 0.1-30 μg/L TP for 48 hours inhibited cell proliferation markedly.2. The surface area of the H9c2 cells in Ang II group elevated significantly compared with control group, and those in 0.1-10 μg/L TP groups decreased significantly by comparsion of Ang II treated group. Moreover, TP(1, 3, 10 μg/L) significantly downregulated the m RNA and protein expression levels of β-MHC, ANP, CDKN1 a, and increased those of α-MHC, respectively.3. ISO infusion induced servere myocardium injury and inflammatory cell infiltration and increased heart weight index significantily by comparsion of those in control group. TP(30, 100 μg/kg) and sterigmatocystin treatment attenuated tissue injury and decreased heart weight significantly compared with ISO group, respectively.4. TP(10, 30, 100 μg/kg) or sterigmatocystin treatment could reduce myocardial fibrosis and cardiomyocyte size extraordinarily compared with those in ISO group.5. Compared with controls, the m RNA and protein expression levels of myocardial α-MHC decreased and those of β-MHC, ANP, CDKN1 a increased significantly in ISO group(p<0.01). After treated with TP(30,100 μg/kg) or sterigmatocystin expression of α-MHC increased and that of β-MHC, ANP, CDKN1 a decreased markedly, respectively. Besides that mentioned above, the expression level of α-MHC m RNA and ANP protein in 10 μg/kg TP group were not significantly changed compared with ISO group.CONCLUSION1. CDKN1 a expression level increased in hypertrophed H9c2 cells and mice with cardiac hypertrophy.2. Triptolide treatment could inhibit the hypertrophic response of H9c2 cells induced by angiotensin II incubation.3. Triptolide could effectively attenuate cardiac hypertrophy and myocardial fibrosis induced by isoprenaline infusion in mice.4. CDKN1 a is involved in the pathological process of cardiac hypertrophy, and regulating the expression of CDKN1 a and subsequently correcting the imbalance of α-MHC and β-MHC contribute to the anti-cardiac hypertrophy mechanism of triptolide.