关键词: 腹膜间皮细胞, 上皮-间充质转化(EMT), NLRP3, 间充质干细胞外泌体

Abstract: Objective To investigate the effects of bone marrow mesenchymal stem cell-derived exosomes (BMSCs-Exo) on the regulation of epithelia-mesenchymal transition (EMT) in human peritoneal mesothelial cells (HPMCs) treated with glucose-based peritoneal dialysis fluid (PDF). Methods BMSCs-Exo were verified by transmission electron microscopy(TEM), nanoparticle tracking analyzer(NTA) and Western blot. Cultured HPMCs(HMrSV5) were divided into 5 groups: control group, high glucose-based PDF (1.5%, 2.5%, and 4.25%) group, siNLRP3 group, siNC group and BMSCs-Exo treated group. Expression of E-cadherin, vimentin, α-smooth muscle actin (α-SMA) and NLRP3 inflammasome-related proteins were detected by Western blot. Real time RT-PCR was used to detected the expression of α-SMA, E-cadherin and TGF-β1 mRNAs in HMrSV5 cells. The concentration of TGF-β1, IL-1β and IL-18 in the culture supernatant was determined by ELISA. Results The exosomes isolated were spherical and double-membrane vesicles with 40-150 nm in diameter, which expressed CD9, CD81, TSG101 and Alix protein. Our results showed that the level of α-SMA and vimentin were significantly up-regulated and E-cadherin (epithelial marker) was significantly decreased in HMrSV5 cells treated with high glucose PDF compared with the normal HMrSV5 cells. The expression of NLRP3,pro-caspase-1 and pro-IL-1β were also significantly up-regulated in HMrSV5 cells treated with high glucose PDF compared with the normal HMrSV5 cells. The level of TGF-β1, IL-1 β and IL-18 in high glucose PDF treated HMrSV5 cells culture supernatant was up-regulated in a dose dependent manner. The protein level of α-SMA was decreased and E-cadherin level was increased by an NLRP3 siRNA to inhibit the activation of NLRP3. Compared with 4.25% PDF treated cells, E-cadherin expression was up-regulated, while the expression of α-SMA and vimentin were down-regulated in BMSCs-Exo treatment cells (P＜0.05). Furthermore, the protein expression of NLRP3, pro-caspase-1 and pro-IL-1β in 4.25% PDF-treated HMrSV5 cells were significantly reduced by BMSCs-Exo. BMSCs-Exo also reduced the level of TGF-β1, IL-1β and IL-8 in the 4.25% PDF-treated HMrSV5 cells culture supernatants (P＜0.05). Conclusions High glucose PDF-induced EMT in HPMCs might be mediated by NLRP3 inflammatory signaling pathway, which can be inhibited by BMSCs-Exo.

Key words: peritoneal mesothelial cells, epithelial-mesenchymal transition, NLRP3, bone marrow mesenchymal stem cell-derived exosomes