醛固酮对介导大鼠肝星状细胞收缩的非钙离子依赖信号通路的影响

Aldosterone stimulates hepatic stellate cells contraction via Ca^2＋independent pathways

目的探讨醛固酮（Aldo）对介导大鼠肝星状细胞（HSC）收缩的非Ca^2＋依赖性信号传导通路的影响。方法对HSCT6细胞给予Aldo10μmol／L处理，用聚硅酮膜法检测HSCT6细胞的收缩性；在激光共聚焦显微镜下动态观察HSC-T6细胞胞内游离钙离子浓度的变化。RTPCR检测Aldo受体阻断剂安体舒通、蛋白激酶C特异性抑制剂Stauro、Rho激酶特异性抑制剂Y27632、肌球蛋白轻链激酶特异性抑制剂ML-7对RhoRock通路中Rock2、RhoAGTP、RhoGEFmRNA表达水平的影响。对各组间数据行单因素方差分析，两两比较采用LSD法。结果Aldo可诱导HSC-T6细胞收缩；Aldo对HSC-T6细胞胞内游离钙离子浓度变化无明显影响。Aldo可诱导Rock2、RhoAGTP、RhoGEFmRNA的表达增强（0．770±0．049、0．960±0．096、0．180±0．006，P值均〈0．01），而其阻断剂安体舒通可明显抑制这三种元件mRNA的表达（0．440±0．166、0．370±0．180、0．050±0．001，尸值均〈0．01）。Aldo＋Y27632组上述三种元件mRNA的表达较Aldo组减弱。Aldo＋ML-7＋Stauro组三种元件的mRNA表达水平（0．940±0．066、1．330±0．192、0．160±0．007）较对照组（0．140±0．023、0．540±0．111、0．110±0．012）增强（P〈0．05），Aldo＋Y27632＋ML-7＋Stauro组RhoGEF（0．290±0．004，P〈0．01）的表达较ML-7＋Stauro两者联合抑制组（0．160±0．007）增强。结论Aldo可诱导HSC的收缩，这与Rho激酶介导的非Ca^2＋依赖性信号通路相关。

Objective To investigate the mechanisms of Aldosterone stimulating hepatic stellate cells（HSCs） contraction via Ca^2＋independent pathways. Methods HSC-T6 cell line was pre-disposed with Aldo 10 μmol/L. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The concentration variation of intracellular free calcium in rat HSC was observed by laser confocal microscopy. Besides, HSC-T6 cell line was under pre-disposal treatment with the blocking agents of Aldo receptor - antisterone, protein kinase C （PKC） special blocking agent-Stauro, Rho kinase blocking agent-Y27632 and MLCK special blocking agent-ML-7 respectively prior to stimulation with aldosterone. RT-PCR was used to detect the expression of Rock2, RhoAGTP and RhoGEF in Ca^2＋ independent pathways mediated by Rho- kinase. Results Aldo could induce HSCs contraction. The concentration of intracellular free calcium in rat HSCs had no change after pre-disposal treatment with Aldo. The mRNA expressions of Rock2, RhoAGTP and RhoGEF increased significantly after treatment with Aldo （0.770± 0.049, 0.960 ±0.096, 0.180 ±0.006, P 〈 0.01）.When inhibited with antisterone, the mRNA expressions of the three elements were （0.440 ±0.166, 0.370± 0.180 and 0.050± 0.001, P 〈 0.01）, lower than that of Aldo group, but higher in ML-7＋Stauro ＋ Aldo groups （0.940 ±0.066, 1.330 ±0.192 and 0.160 ± 0.007, P 〈 0.05） as compared to the control group （0.140 ± 0.023, 0.540± 0.111 and 0.110 ±0.012）. In the Y27632 ＋ ML-7 ＋ Stauro＋Aldo group, the mRNA expression of RhoGEF （0.290 ±0.004, P 〈 0.01）was higher than that of the ML-7 ＋ Stauro ＋ Aldo group （0.160 ± 0.007）. Conclusion Aldo could induce HSCs contraction via Ca^2＋ independent pathways and Rho-Rock pathway involved in the process.