醛固酮诱导肝窦内皮细胞外泌体GAS5 lncRNA/miR-21失衡促进肝星状细胞活化的机理研究

Exosomes, which mediate intercellular communication, is gaining more and more attention in recent year. Aldosterone, an important promoter for liver fibrosis, mediates the interaction between liver sinusoidal endothelial cells (LSEC) and hepatic stellate cell (HCS) by inducing oxidative stress, leading to sinusoid capillarisation and liver fibrogenesis. However, how exosomes mediate the intercellular communication between LSEC and HCS has not been reported. Our preliminary studies showed that aldosterone treatment increased mTOR protein level and suppressed the level of autophagy in LSEC, while multivesicular bodys (MVBs) level was increased. Moreover, the level of GAS5 lncRNA was down-regulated while miR-21 level was up-regulated in both LSEC and exosomes. Finally, the aldosterone induced LSEC-derived exosomes could activate HSC, with the detail mechanisms remaining unclear. Consequently, we come up with the idea to explore how exosomes regulate the intercellular communication between LSEC and HSCs. In this study, we intergrate LSEC with HSC by exosomes, and prospect to perform in vivo and in vitro study to confirm the hypothesis: aldosterone induces LSEC autophagy by upregulating mTOR, which promotes exosomes secretion and disturbs the balance between miR-21 and GAS5 lncRNA in exosomes and ultimately regulates the activation and proliferation of HSCs. This project provides a novel pathway to study of liver fibrosis.

基于外泌体的细胞间的通讯方式日益被广泛关注。醛固酮是促肝纤维化的重要物质，其氧化效应可介导肝窦内皮细胞（LSEC）与肝星状细胞（HSC）的相互作用，促进肝窦毛细血管化及肝纤维化形成。然而，外泌体如何介导上述两种细胞间的通讯这一重要科学问题鲜见报道。我们的前期工作发现：醛固酮可引起LSEC mTOR表达升高，自噬减低，多囊泡体增多，LSEC及其外泌体中GAS5 lncRNA水平下降, miR-21表达上调，醛固酮诱导的LSEC外泌体可促进HSC活化，然而机制不明。由此，我们萌发探讨醛固酮如何通过外泌体途径调控LSEC与HSC之间的通讯之设想。本研究以外泌体为纽带将LSEC和HSC连结一体，从体内、外层面验证“醛固酮通过mTOR抑制LSEC自噬，促进外泌体分泌及诱发外泌体中miR-21/GAS5lncRNA失衡，介导肝星状细胞活化增殖”之假说。本研究为拓展醛固酮在肝纤维化的调控机制提供新思路。

本课题从体内、外探讨醛固酮（Aldo）通过激活肝窦内皮细胞（LSEC）自噬及溶酶体的激活影响细胞内多泡小体（MVB），调节其释放的细胞外囊泡质与量的改变，进而对肝星状细胞（HSC）的激活和迁移的影响及其机制。体内研究发现，肝纤维化患者血清Aldo较正常人显著升高，且肝脏自噬水平升高，Aldo受体MR在肝脏中主要位于LSEC内且自噬也主要发生在LSEC内，因此Aldo在肝纤维化的发生中可能与LSEC自噬有关。此外，Aldo持续泵注的大鼠模型发生明显肝纤维化，并伴LSEC自噬发生以及窗孔丢失。体外实验中Aldo诱导LSEC发生自噬及去窗孔化，同时伴随控制外泌体生成类蛋白水平下降；自噬流实验证明在Aldo的诱导下LSEC自噬通量增高，免疫荧光检测发现LSEC的溶酶体增多伴随MVB减少，外泌体释放蛋白RAB27a减少，这些现象可以被Aldo受体抑制剂EPLE所抑制。上述结果表明，Aldo很可能通过促进LSEC自噬发生而影响其分泌的外泌体。文献中以及我们课题组实验都证明正常来源的LSEC外泌体确实对HSC有抑制激活及迁移的作用，但是在加入Aldo后则不再有这种保护作用。本课题组前期根据文献调研猜测的GAS5/mir21轴可能是发挥这个作用的关键分子，然而在实验中我们发现直接对HSC过表达GAS5并不能影响其激活迁移，且在HSC原本mir21确实发挥了很重要的作用，但mir21的基础表达水平很高，仅通过LSEC外泌体传输意义有限。我们猜想外泌体中发挥关键作用的可能有其他的分子及机制，因此我们采用了外泌体全转录组测序技术探究，结果同预期一致，Aldo的刺激明显影响了LSEC分泌的外泌体的质与量，其中外泌体分泌总量下降，通过实验及生信预测，我们发现mir-342-5P很有可能就是外泌体发挥作用关键分子，单个外泌体中该miRNA含量也明显降低，后续实验证明过表达mir-342-5P可以抑制HSC激活及迁移能力，该miRNA很可能是通过抑制TGFβ通路影响HSC的激活及迁移能力。结论：Aldo通过影响LSEC自噬，促使外泌体分泌的总量下降及外泌体中保护性mir-342-5p水平减少，从而介导HSC活化和增殖，最终导致肝纤维化的发生。