| objective:To investigate the inhibitory effect of estradiol on the proliferation of cardiac fibroblasts induced by angiotensin II in mice,that has a relationship with the inhibition of the expression of intermediate-conductance Ca2+-activated K+channel.Method:1.The culture and identification of neonatal mouse cardiac fibroblasts: After neonatal mouse heart ventricles were obtained by operationcardiac fibroblasts were cultured by the way of enzyme digestion differential adherent and identified by immunofluorescence with specific antibody.2.Establishment of Cardiac Fibroblast Proliferation Model : The cultured CFS was incubated at the following concentrations of Ang II: 10-4,10-5,10-6,10-7 and 10-8mol/L for 12 h,24h,48 h and 72 h,No Ang II as control group.The proliferation of CFS was detected by MTT assay.3.To observe the effect of estradiol and TRAM-34 on the proliferation activity of cardiac fibroblasts in neonatal mice induced:(1)Control group(Ang II);(2)Ang II+E2 group:After pretreatment with Ang II at a concentration of 10-6mol/L for 30 min,add 10-4,10-5,10-6,10-7,10-8 mol/Lconcentration of E2;(3)Ang II+TRAM-34 grou : After pretreatment with Ang II at a concentration of 10-6mol/L for 30 min,add10-4,10-5,10-6,10-7,10-8mol/L concentration of TRAM-34.4.The expression of KCa3.1m RNA was detected by PCR and the phosphorylation of p38-MAPK was detected by ELISA:CFS was previously treated with Ang II(10-6mol/L)for 30 min,The experiment grouped as follows:(1)contol group;(2)Ang II group;(3)Ang II+E2×10-5mol/L group;(4)Ang II+TRAM-34×10-4mol/L group incubate for 48 h.MTT assay cell proliferation activity,The expression of KCa3.1 m RNA was detected by PCR,The phosphorylation of p38-MAPK was detected by ELISA.5.Results:1.Ang II can induce mice CFS proliferation,and has a time and concentration dependency,compared with blank control group,the proliferation of 10-6,10-5,10-4mol/L was significant(P <0.01).compared with10-6mol/L,the 10-5,10-4mol/L concentration no significant difference(P>0.05).The concentration of 10-6mol/L was used as the model of proliferation.E2 and TRAM-34 inhibited the proliferation of mouse CFS significantly,and had a concentration and time-dependent.There was no significant difference in the proliferation of CFS between 10-5mol/LE2 and 10-4mol/L TRAM-34 for 48h(P> 0.05).Effects of E2 and TRAM-34 on KCa3.1 m RNA Expression:Compared with contol group and Ang ? group,the expression of KCa3.1m RNA was significantly increased(P <0.01).Compared with contol group,Ang?+ E2 ×10-5 mol/L group and Ang?+ TRAM-34×10-4mol/L group could significantly inhibit the expression of KCa3.1m RNA and statistically significant(P<0.05).Compared with Ang?group,Ang?+ E2×10-5mol/L group and Ang?+ TRAM-34 × 10-4mol/L group could significantly inhibit the expression of KCa3.1m RNA and statistically significant(P <0.01).There was no significant difference between Ang?+ E2×10-5mol/L group and Ang?+ TRAM-34×10-4mol/L group(P> 0.05).Effects of E2 and TRAM-34 on phosphorylation ofP38-MAPK:Compared with contrl group,Ang? could significantly increase the phosphorylation level of p38-MAPK(P <0.01).Compared with contrl group,Ang? could significantly increase the phosphorylation level of p38-MAPK(P <0.01);Compared with Ang?group,Ang II + E2×10-5mol/L group significantly reduced the phosphorylation level of p38-MAPK,which was statistically significant(P <0.01).Compared with Ang?group,Ang II +TRAM-34×10-4mol/L had no significant effect on p38-MAPK phosphorylation(P> 0.05).Ang II + E2×10-5mol/L group and Ang II + TRAM-34×10-4mol/L group were significantly different,with statistical significance(P<0.01).conclusion:Ang? could induce the proliferation of CFS in mice.E2 and TRAM-34 could inhibit the proliferation of CFS.There was no significant difference in the proliferation of CFS between 10-5mol/LE2 and 10-4mol/L TRAM-34.Estrogen inhibited the proliferation of cardiac fibroblasts The mechanism is related to the decrease of p38-MAPK phosphorylation and the inhibition of KCa3.1 m RNA expression. |
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