Our present study investigated whether exosome secretion of nucleus pulposus cells (NPCs) is regulated by autophagy. Different autophagic states of NPCs were induced by rapamycin (Rap), bafilomycin A1 (Baf) and other agents, and it was found that exosomes were secreted in an autophagy-dependent manner. Activation or inhibition of autophagy increased or decreased, respectively, the amount of exosomes that were released into the extracellular space. In addition, in order to confirm that Rap-promoted release of exosomes was mediated by autophagy rather than other pathways, we used autophagy associated gene 5 (ATG5) small-interfering RNA (siRNA) to silence the expression of ATG5 gene, which is indispensable for autophagy. The results showed that siRNA against ATG5 (siATG5) induced an accumulation of intraluminal vesicles (ILVs) in NPCs and a concomitant decrease in the amount of exosomes isolated from supernatant. Ras homolog gene (Rho) and Rho-associated coiled-coil forming protein kinase (ROCK) family molecules are capable of cytoskeletal remodeling and affecting vesicle transport. Therefore, we carried out targeted interventions and evaluated the effects of the RhoC/ROCK2 pathway on the secretion of exosomes within autophagic environment. Knockdown of RhoC and ROCK2 with corresponding siRNA significantly inhibited the secretion of exosomes originating from intraluminal vesicles (ILVs) in NPCs, even when NPCs were subsequently treated with Rap. Taken together, our findings suggest that autophagy positively regulates expression levels of RhoC and ROCK2, and that the RhoC/ROCK2 pathway exerts a key function on NPCs-derived exosome secretion.

我们目前的研究调查髓核细胞（NPCs）的外泌体分泌是否受到自噬的调节。雷帕霉素（Rap），巴非霉素A1（Baf）和其他药物诱导NPC的不同自噬状态，发现外泌体以自噬依赖性方式分泌。自噬的激活或抑制分别增加或减少了释放到细胞外空间的外泌体的数量。此外，为了确认Rap促进的外泌体释放是通过自噬而非其他途径介导的，我们使用了自噬相关基因5（ATG5）小干扰RNA（siRNA）来沉默ATG5基因的表达，这是必不可少的自噬。结果表明，针对ATG5的siRNA（siATG5）诱导了NPC中腔内小泡（ILV）的积累，并同时减少了从上清液中分离的外泌体的数量。Ras同源基因（Rho）和Rho相关的卷曲螺旋形成蛋白激酶（ROCK）家族分子能够进行细胞骨架重塑并影响囊泡转运。因此，我们进行了有针对性的干预，并评估了RhoC / ROCK2途径对自噬环境中外泌体分泌的影响。剔除RhoC和ROCK2与相应的siRNA，即使随后用Rap治疗NPC，也会显着抑制源自NPC中腔内囊泡（ILV）的外泌体的分泌。两者合计，我们的发现表明自噬正调控RhoC和ROCK2的表达水平，