| Objetctive To investigate the effects of simvastatin on angiotensinⅡinduced myocardial fibroblasts(CFBs) proliferation and collagen secretion in newborn rats and explore the potential antifibrotic mechanism.Methods1.CFBs of neonatal Sprague - Dawley(SD) rats were isolated and subcultured,all with the method of trypsin digestion and differential anchoring velocity.2.Control group:CFBs were subcultured without any stimulus.3.AngⅡstimulating group:the CFBs were stimulated with 10-6mol/L AngⅡfor 48 hours.4.Simvastatin treating group:use 10-6mol/L AngⅡas stimulated stimulus,and then divided them into three groups in which simvastatin with concentration of 10-7mol/L, 10-6mol/L and 10-5mol/L respectively were added to incubate CFBs for 48 hours.5.Simvastatin and MVA treating group:divide CFBs into two groups after adding 10-6mol/L AngⅡand 2×10-4mol/L MVA as stimulus,and then incubated CFBs in these two groups respectively,one group with 10-5mol/L Simvastatin and the other with 10-4mol/L Simvastatin6.Collect CFBs form each group,and then measure the contents of hydroxyproline by colorimetric assay,Cell cycle distribution by flow cytometry,proliferation of CFBs by MTT colorimetric assay,the expression of TGF-β1mRNA by RT-PCR analysis.Result1.When CFBs were stimulated with 10-6mol/L AngⅡfor 48 hours,absorbance value was 0.368±0.017,significantly higher than control 0.231±0.014(P＜0.05);The percentage of CFBs in G0-G1 stage was 53.03%±3.44%,significantly lower than control 74.22%±4.15%(P＜0.05);The percentage of CFBs in S stage was 25.32%±1.67%significantly higher than control 14.37%±2.97%(P＜0.05);The percentage of CFBs in G2-M stage was 21.98%±2.12%,significantly higher than control 11.40%±1.59%(P＜0.05); Hydroxyproline content was(2.52±0.14),significantly higher than control 1.23±0.09 (P＜0.05);Relative expression of TGF-β1 mRNA was 0.91±0.06,significantly higher than control 0.53±0.04(P＜0.05).2.When CFBs were incubated with 10-6mol/L AngⅡand simvastatin at different concentration of 10-7mol/L,10-6mol/L,10-5mol/L for 48 hours,absorbance values were 0.238±0.013,0.209±0.022,0.143±0.020,all significantly lower than the value in AngⅡstimulating group(P＜0.05),and decreased in dose-dependent manner(P＜0.05);The percentages of CFBs in G0-G1 stage were 64.07%±2.84%,68.09%±1.82%,75.64%±2.83 %,all significantly higher than the percentage in AngⅡstimulating group(P＜0.05),the percentages of CFBs in S stage were 19.75%±1.50%,16.80%±0.93%,13.16%±1.20%, all significantly lower than the percentage in AngⅡstimulating group(P＜0.05),the percentages of CFBs in G2-M stage were 16.34%±1.67%,15.10%±0.96%,11.20%±1.92 %,all significantly lower than the percentage in AngⅡstimulating group(P＜0.05). Hydroxyproline contents were 1.46±0.12,1.39±0.07,1.25±0.08,all significantly lower than the content in AngⅡstimulating group(P＜0.05).There was no significant difference among different concentration simvastatin(P＞0.05).Relative expression of TGF-β1 mRNA were 0.78±0.04,0.69±0.05,0.58±0.06,all significantly lower than the index in AngⅡstimulating group(P＜0.05),there was no significant difference among different concentration simvastatin(P＞0.05).3.When CFBs were incubated with 10-6mol/L AngⅡ+10-5mol/L simvastatin + 2×10-4mol/L MVA or 10-mol/L AngⅡ+ 10-4mol/L simvastatin+2×10-4mol/L MVA, absorbance values were 0.351±0.024,0.343±0.018,the value was not different between AngⅡstimulating group and Simvastatin + MVA treating group(P＞0.05).There was no significant difference between different concentration simvastatin(P＞0.05);The percentages of CFBs in G0-G1 stage were 47.86%±7.35%,48.21%±7.47%,the percentages of CFBs in S stage were 27.49%±3.76%,28.22%±5.01%,the percentages of CFBs in G2-M stage were 25.10%±3.54%,24.23%±2.70%,The percentage in all cell cycle of CFBs were not different between AngⅡstimulating group and Simvastatin + MVA treating group(P＞0.05),there was no significant difference between different concentration simvastatin(P＞0.05);Hydroxyproline contents were 2.38±0.11,2.36±0.09, the content was not different between AngⅡstimulating group and Simvastatin + MVA treating group(P＞0.05),there was no significant difference between different concentration simvastatin(P＞0.05).Relative expression of TGF-β1 mRNA were 0.87±0.04,0.85±0.05,the index was not different between AngⅡstimulating group and Simvastatin + MVA treating group(P＞0.05),there was no significant difference between different concentration simvastatin(P＞0.05).Conclusion1.AngⅡcan stimulate the proliferation of CFBs and collagen synthesis,upregulate the expression of TGF-β1 mRNA.2.Simvastatin can suppress AngⅡinduced proliferation of CFBs and collagen synthesis,downregulate the expression of TGF-β1 mRNA.3.MVA can reverse the effect of simvastatinit which suggests that simvastatin works perhaps by suppressing MVA synthesis and downregulating the expression of TGF-β1 mRNA.
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