环孢霉素A抑制血管升压素诱导的心脏成纤维细胞增殖和胶原合成(英文)

Inhibitory effect of cyclosporin A on growth and collagen synthesis of rat cardiac fibroblasts induced by arginine vasopressin

目的探讨环孢霉素A(CsA)对血管升压素(AVP)诱导心脏成纤维细胞(CFs)增殖和胶原合成作用的影响。方法采用胰酶消化法培养新生Sprague-Daw ley大鼠CFs,无血清培养基培养24 h以上的细胞在AVP刺激的基础上给予不同浓度的CsA。采用MTT法测定细胞数目,应用流式细胞仪分析细胞周期,羟基脯氨酸比色法测定细胞培养上清液羟基脯氨酸含量,钙调神经磷酸酶(CaN)活性通过分光光度计测定。结果环孢霉素A可剂量依赖性地抑制AVP诱导的心脏成纤维细胞数目的增加,0.05,0.5及5μmol.L-1CsA对CFs的MTT吸收度值的抑制率分别为12%,24%和29%(均P<0.05);细胞周期分析显示,0.5μmol.L-1CsA可降低AVP诱导的S期百分数和增殖指数(P<0.05);0.5及5μmol.L-1CsA可降低细胞培养上清液羟基脯氨酸含量,抑制率分别为29%和33%,有统计学差异(均P<0.05);CsA可完全阻断AVP诱导CFs细胞内CaN活性,而CsA对基础状态下的CaN活性和细胞存活率无明显影响。结论CsA通过阻断CaN信号通路抑制AVP诱导的CFs增殖和胶原合成,可抑制心肌纤维化进程,为心肌纤维化的防治提供了新的治疗靶点。

Aim To investigate the effects of cyclosporin A （CsA） on growth and collagen synthesis of cardiac fibroblasts （CFs） induced by arginine vasopressin （AVP）. Methods CFs of neonatal Sprague-Dawley rats were isolated by trypsinization and cultured; growth-arrested CFs were stimulated with 1 ×10^-7 mol·L^-1 AVP in the presence or absence of CsA （0.05, 0.5 and 5 μmol · L^-1）. MTT and flow cytometry techniques were adopted to measure cell number and analyze cell cycle respectively. Collagen synthesis was determined by measurement of hydroxyproline content in culture supernatant with colorimetry. Calcineurin activity was estimated by chemiluminescence. Trypan blue staining to test the viability of CFs. Results O. 05, O. 5 and 5 μmol · L^-1 CsA inhibited the increase of CFs number induced by 1 × 10^-7 mol · L^-1AVP in a dose-dependent manner, with the inhibitory rates by 12% , 24% and 29%, respectively （P 〈0.05）. Furthermore, cell cycle analysis showed 0. 5 μmol · L^-1 CsA decreased the S stage percentage and proliferation index of CFs stimulated by AVP （ P 〈 0. 05 ）. In culture medium, the hydroxyproline content induced by AVP decreased by O. 5 and 5 μmol · L^-1 CsA （ P 〈 0. 05 ） , with the inhibitory rates of 29% and 33% , respectively. CsA completely inhibited the increment of calcineurin activity induced by AVP （P 〈 0. 01 ） , but CsA itself had no effect on the baseline of calcineurin activity and CFs viability. Conclusion CsA inhibits proliferation and collagen synthesis of CFs by virtue of blocking calcineurin signaling pathway and might provide a novel target for prevention and treatment to cardiac fibrosis.