肝细胞源性神经营养因子MANF经外泌体靶向HSCs的抗肝纤维化作用及机制研究

The mechanisms involved in liver fibrosis are complicated and unclear, especially the communication between the hepatocytes, the hepatic stellate cells (HSCs), and the Kupffer cells. It was recently reported that the hepatocytes excreted exosomes. The nerve growth factors (NGF) derived from hepatocytes induce HSCs apoptosis. We found that the new neurotrophic factor MANF, an ER stress inducible protein, was specifically expressed in hepatocytes and was closely associated with liver fibrosis. We also found that the exosome isolated from hepatocytes under treatment with tunicamycin or TNF-alpha contains MANF protein. Meanwhile, we have reported that MANF inhibited inflammation via blocking NF-kappa B activation. Therefore, we hypothesis that MANF expressed in the hepatocytes and excreted in the exosomes, may regulate HSCs activation during liver fibrosis. To verify this hypothesis, we will investigate the anti-fibrosis of MANF by using of the mice specifically knockouted MANF gene in hepatocytes. We will isolate the exosomes from the primarily culture hepatocytes after treatment with tunicamycin or TNF-alpha and detect MANF level. We also will test whether the exosomes are fused to HSCs. The methods, including cell co-culture, gene knockdown or knockout, gene over-expression will be used to test the effects of MANF derived from hepatocytes on the activation, proliferation and apoptosis of HSCs. We will explore the cellular mechanisms by which MANF regulates HSCs via exosomes delivery.

肝纤维化进程中细胞间信息传递机制不清楚。有报道：肝细胞可分泌外泌体；肝细胞源神经因子能诱导HSCs凋亡。我们发现：肝细胞表达新型神经因子MANF与肝纤维化程度呈正相关，但HSCs中无MANF表达；肝细胞应激时分泌的外泌体含MANF；MANF能抑制炎症。推测：肝纤维化时肝细胞被诱导表达MANF，MANF以外泌体形式调节HSCs活性。为此，将检测病人血清中MANF水平，并分析其与肝纤维化程度相关性；制备肝细胞特异性MANF敲除小鼠模型，并用CCl4诱导慢性肝纤维化，证明MANF具有抗纤维化作用；分离肝细胞外泌体，证明外泌体和MANF能进入HSCs；利用细胞共培养、基因敲低和过表达等技术研究肝细胞源MANF对HSCs功能、增殖及凋亡的影响；进一步探讨SUMO1参与的MANF调节NF-κB机制。上述研究内容均属原始创新，该研究对了解细胞间信息传递方式、阐明肝纤维化机制及发现更有效干预手段奠定基础。

在肝纤维化过程中细胞间信息传递的机制目前尚不十分清楚，尤其是肝细胞、肝星状细胞（HSCs）和Kupffer细胞之间的联系仍需更深一步的研究。我们前期研究发现：MANF作为一种新型神经营养因子，在肝细胞中表达，其表达水平与肝纤维化程度呈正相关。同时，MANF可抑制NF-κB信号通路的激活和炎症反应。因此，我们推测：在肝纤维化进程中，肝细胞来源的MANF可通过外泌体抑制HSCs的激活。我们首先在CCl4诱导的小鼠肝纤维化模型上发现，肝纤维化显著上调肝细胞和巨噬细胞中MANF的水平。但有趣的是MANF不在HSCs细胞中表达。同时发现，肝细胞特异性MANF敲除（HKO）可加重肝纤维化。为了进一步确定MANF的抗肝纤维化作用，我们给予野生型（WT）肝纤维化小鼠重组人MANF，结果发现重组人MANF能够显著缓解小鼠肝纤维化。我们还发现肝细胞MANF缺乏可激活小鼠肝脏中HSCs。此外，我们分离WT和HKO小鼠原代肝细胞，从培养上清中分离外泌体并检测其中MANF的水平。同时利用来源于WT和HKO小鼠原代肝细胞的外泌体处理LX-2细胞，发现肝细胞中MANF敲除后，其分泌的外泌体对LX-2激活的抑制作用降低。本研究证实了MANF的抗肝纤维化作用，揭示了肝细胞中的MANF通过外泌体抑制HSCs激活的新机制。