Role Of P53-p21 Signaling Pathway In Cardiac Hypertrophy And Effects Of Triptolide

Cardiac hypertrophy is a pathological feature of various cardiac diseases,including hypertension,valvular diseases,coronary heart disease,etc..Althought‘adaptive'hypertrophy can preserve cardiac function at certain extent,prolonged hypertrophy is associated with further development to heart failure.As terminally differentiated cell s,cardiomyocytes stop proliferation soon after birth.However,it can re-enter into cell cycle as stimulated by some pathologic stimulation,then the synthesis of nucleic acid and protein in the cardiomyocytes will increase,which eventually result in hypertrophy.To prevent and reverse cardiac hypertrophy have been proposed as a main target for various cardiovascular diseases.The tumor suppressor p53 is a transcription factor that regulates cell cycle,proliferation and apoptosis of cells.In normal cells,p53 expression is kept at low levels.Accumulation of p53 results in the transactivation of several sets of target genes,such as p21^WAF1/CIP1,which plays critical role in cell cycle as a negative regulator.Triptolide?TP?is an epoxy-2-terpenoid ester compound extracted from the Chinese traditional herb Tripterygium wilfordii,which has been used for the treatment of inflammation and autoimmune diseases,such as rheumatoid arthritis,chronic nephritis and asthma.However,the mechanism of TP on cardiac hypertrophy and its effect on p53-p21 signaling pathway are poorly understood at present.Therefore,the aims of the present study were to explore the mechanism of p53-p21 signal pathway in cardiac hypertrophy and the regulating effect of TP.METHODS1. Neonatal rat ventricular myocytes?NRVMs?were isolated from neonatal SD rats.After being cultured in serum-free DMEM for 24h,the cells were infected with recombinant adenovirus containing p21 or p53 gene at a MOI of 10 for 12h,or transfected with si RNA for 5-7h to block the expression of p21 or p53.After that,cells were subjected to fresh DMEM overnight,then incubated for 24h in a serum-free medium containing 1?mol/L Ang II.Different concentrations of TP?1,3,or 10?g/L?were added meanwhile.The cell size was measured under fluorescent microsopy after staining with rhodamine-labeled phalloidine.The m RNA and protein expression levels of p21,p53 and?-MHC were determined by Real-time PCR and Western blot techniques,respectively.NRVMs were subjected to immunofluorescence with?-actinin and p21 antibody to observe intracellular expression levels of p21 by laser scanning confocal microscopy.2.Thirty mice were randomly divided into 6 groups,i.e.,control,model,TP?0.3,1,3,or 10?g/kg,ip,once daily?groups?n=5 in each group?;and administrated with saline,continuous infusion of isoprenaline?ISO?using osmotic mini-pumps at 40 mg/kg per day,or ISO infusion plus TP?0.3,1,3 or 10?g/kg?treatments for 14 days,respectively.Another 35mice were randomly divided into 7 groups,i.e.,control,model,p53-Ad group,p53 inhibitor,TP,p53-Ad plus TP and p53 inhibitor plus TP group.Mice in control and ISO group were treated as same as above.Those in p53-Ad group were treated with 2×10⁷ TU p53-Ad via femoral vein injection.And those in p53 inhibitor group were treated with pifithrin?2 mg/kg,ip,qd?.3.The general state of all animals was observed during 2 weeks'treatment.At the end of treatments,all animals were weighted and killed for sampling.The heart was weighted to calculate the ratio of heart weight to tibial length?HW/TL?.The cell size was determined by stained with fluorescein isothiocyanate?FITC?-labeled lectin wheat germ agglutinin.Pathological change and fibrosis were analyzed after HE and Masson staining.4.The m RNA and protein expression levels of p21,p53,?-MHC and PCNA in left ventricle were detected by Real-time PCR and Western blot techniques,respectively.RESULTS1. The surface area of the NRVMs in model group elevated significantly compared with control group,and three dosage of TP?1,3 or 10?g/L?treatments reduced myocardial cell size significantly.Moreover,TP decreased m RNA and protein expression levels of p21,p53,?-MHC,which increased dramatically after Ang II stimulation.2. The cell size of cardiomyocyte had a slightly increase after infected with p21-or p53-adenovirus,which aggravated Ang II-induced the expression elevation of p21,p53,and?-MHC.On the contrary,p21 or p53 si RNA could effectively attenuate above hypertrophic responses induced by Ang II.3.Double-immunofluorescence staining showed that the expression of p21 was significantly increased after treated with p53-Ad or Ang II and teansfer from the cytoplasm to the nucleus,but was down-regulated by p53 si RNA infection.4.TP treatment significantly decreased cardiomyocyte size and the m RNA and protein expression levels of p21,p53 and?-MHC.The effects of TP could be reduced by p21 or p53adenovirus,but has a synergistic effect with p21 or p53 si RNA.5.The heart weight index,myocardium injury,inflammatory cell infiltration,cardiomyocyte size,m RNA and protein expressions of p21,p53,?-MHC,PCNA in the ISO group was increased significantily by comparsion to those in control group.TP?0.3,1,3 or10?g/kg?treatment significantly decreased cardiomyocyte size and fibrosis compared with ISO group.The m RNA expression levels of p21,?-MHC and PCNA in high-dose TP group were reduced significantly compared with ISO group,1-10?g/kg TP could significantly decreased protein expression levels of p21,p53,?-MHC and PCNA.6.Heart weight index and cardiomyocyte size were increased by the treatment of p53adenovirus.And Ad-p53 reduced the effects of TP on fibrosis and down-regulated m RNA and protein expressions of p53.CONCLUSION1.p21 and p53 expression level increased in hypertrophied NRVMs and myocardium of mice with cardiac hypertrophy.2. Triptolide could inhibit the hypertrophic response of NRVMs and mice induced by Ang II or ISO,decrease the expression level of p21 and p53.3.Triptolide?0.3-10?g/kg?treatment could dose-dependently ameliorate myocardial tissue damage and myocardial fibrosis.4.Elevated expression of p53 leads to elevation of p21 and downregulation of PCNA,while p21 has a limited positive regulation of p53 expression.5.Inhibit the p53-p21 signaling pathway and cell cycle progression are involved in the effects of TP on cardiac hypertrophy.