GW4869 inhibits the release of exosomes from bone marrow mesenchymal stem cells

Sun Shu-li; Xiao Pei-xin; Ding Hui; Wang Jing; Liu Zi-quan; Liu Jin-yang; Wang Xue; Shi Sha; Lv Qi; Fan Hao-jun

doi:10.3969/j.issn.2095-4344.0407

Abstract：BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) and BMSCs-derived exosomes have similar functions, but the regulatory mechanism underlying the release of exosomes is still unclear. OBJECTIVE: To investigate the role of GW4869, an inhibition of neutral sphingomyelinase 2, in the release of exosomes in BMSCs and the influence of GW4869 on BMSCs proliferation. METHODS: Rat BMSCs were divided into three groups: normal control group, 24-hour GW4869 treatment group and withdrawal of GW4869 for 24 hours group (24-hour GW4869 treatment followed by 24-hour successive culture with drug withdrawal). Cultured cells were collected to extract exosomes by ultracentrifugation. Western blot was used to detect exosome-associated proteins CD63 and tumor susceptibility gene 101 (TSG101). The concentration and size distribution of exosomes were measured using nanoparticle tracking analysis. BCA was used to test the level of total proteins in exosomes. Live cell imaging system was used to observe the influence of GW4869 on BMSCs proliferation. RESULTS AND CONCLUSION: (1) Western blot results showed that exosomes expressed marker proteins such as CD63, TSG101. (2) Findings from the nanoparticle tracking analysis confirmed that the size of released exosomes was about 114 nm. (3) Significantly reduced release of exosomes was found in the two treatment groups compared with the normal control group (P < 0.01), but there was no significant difference between 24-hour GW4869 treatment group and withdrawal of GW4869 for 24 hours group (P > 0.05). (4) No significant difference in the proliferation of BMSCs was found among the three groups (P > 0.05). To conclude, 24-hour W4869 can inhibit the release of exosomes by BMSCs and this inhibitory effect is still sustained within 24 hours after drug withdrawal. However, GW4869 has no influence on the proliferation of BMSCs.