Piezo1离子通道介导的流体剪切应力抑制MLO-Y4骨细胞凋亡

Piezo1 ion channel-mediated fluid shear stress inhibits the apoptosis of MLO-Y4 osteocytes

目的:研究Piezo1机械敏感性离子通道对骨细胞的调控作用。方法:应用课题组研发的流体剪切力(FSS)加载装置,使用免疫荧光探究FSS对Piezo1通道在MLO-Y4中影响的最佳强度;对Piezo1使用FSS、激动剂Yoda1或阻滞剂GsMTx4进行干预。使用Western Blot探究Cleaved Caspase-3、Bcl-2、Bax凋亡相关蛋白的表达量;使用Hoechst-33258以及流式细胞凋亡检测MLO-Y4骨细胞的凋亡比例。结果:免疫荧光染色结果表明FSS对MLO-Y4骨细胞中Piezo1的表达呈现先增加后降低的单峰趋势,其中以9 dyne/cm2加载30 min为最佳刺激条件。使用Yoda1抑制MLO-Y4细胞凋亡(P<0.05),使用GsMTx4促进MLO-Y4细胞凋亡(P<0.05)。Piezo1介导的FSS抑制MLO-Y4细胞凋亡(P<0.05)。结论:Piezo1机械敏感性离子通道介导的FSS在9 dyne/cm2条件下抑制MLO-Y4骨细胞凋亡,且FSS在该条件下可以有效逆转Piezo1阻滞剂GsMTx4在4μmol/L作用0.5 h下促进MLO-Y4细胞凋亡作用。

Objective To investigate the regulation of mechanosensitive ion channel Piezo1 on osteocytes. Methods Immunofluorescence staining was used to investigate the best condition for Piezo1 channel-mediated FSS loaded by the selfdeveloped fluid shear stress(FSS) loading device on MLO-Y4 osteocytes. Piezo1 was intervened with FSS, Piezo1 activator Yoda1,or Piezo1 blocker Gs MTx4. The expression levels of apoptosis-related proteins(Cleaved Caspase-3, Bcl-2, and Bax) were analyzed using Western Blot analysis, and the apoptosis rates of MLO-Y4 osteocytes was detected by Hoechst-33258 and flow cytometry.Results The result of immunofluorescence staining indicated that in MLO-Y4 osteocytes exposed to FSS, the expression of Piezo1had a unimodal tendency of increasing firstly and then decreasing, and that the optimal stimulation condition was 9 dyne/cm2loading for 30 minutes. Yoda1 inhibited the apoptosis of MLO-Y4 osteocytes(P<0.05), and GsMTx4 promoted the apoptosis of MLO-Y4osteocytes(P<0.05). Piezo1-mediated FSS inhibited the apoptosis of MLO-Y4 osteocytes(P<0.05). Conclusion When loading FSS at 9 dyne/cm2, the FSS mediated by mechanosensitive ion channel Piezo1 can inhibit the apoptosis of MLO-Y4 osteocytes, and FSS can effectively reverse the apoptosis of MLO-Y4 osteocytes promoted with Piezo1 blocker GsMTx4 at 4 μmol/L for 0.5 h.