关键词: 高糖腹膜透析液, 人腹膜间皮细胞, NLRP3, IL-1&beta

Abstract: Objective： To explore the effect of high glucose-based peritoneal dialysis fluids on NLRP3-IL-1β in human peritoneal mesothelial cells. Methods: HMrSV5 cells (SV40 immortalized human peritoneal mesothelial cell line) were grown in type Ⅰ collagen-coated dishes in DMEM/F12 containing 10% fetal calf serum (FCS). All experiments on HMrSV5 cells were performed between passages 5 and 10-The cells were divided into 7 groups: control, 1-5% dextrose, 2-5% dextrose, 425% dextrose, rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A-Immunoblotting was used to evaluate the expression of IL-1β-Small interfering RNA (siRNA) targeting NLRP3 was used to downregulate the expression of NLRP3 and Western blot was used to evaluate the expression of IL-1β in human peritoneal mesothelial cells exposed to 4-25% dextrose- In the meanwhile, resveratrol （RSV） was used to induce autophagy, 3-methyladenine (3-MA) and siRNA against Beclin 1 or ATG5 were used to block auto-phagy, flow cytometric was used to analyze the respiring (mitotracker deep red), total (mitotracker green) and reactive oxygen species (ROS)generating mitochondria (mitoSOX); Western blot was used to evaluate the expression of IL-1β- Results: The IL-1β relative expressions were 0, 0-175±0-082, 0-418±0-163, 2-357±0-288, 2-642±0-358, 3-271±0-462, and 0-123±0-091, indicating that the cells exposed to high glucose-based peritoneal dialysis fluids and cells treated with mitochondria respiratory chain key enzyme complex Ⅰ, and complex Ⅲ inhibitors increased the IL-1β expression- And we found that NLRP3 knock-down significantly blocked the upregulation of IL-1β- In addition, the fluorescence intensity of total mitochondria and ROS-generating mitochondria in the following groups: control, negative control, RSV, 3-MA, ATG5 siRNA, Beclin1 siRNA were 1-76±0-42, 1-83±0-55, 1-85±0-62, 7-36±0-92, 5-35±0-77, 5-06±0-62 and 821-68±95-12, 868-15±102-82, 723-39±92-56, 1 660-08±113-65, 1 433-01±107-24, 1 562-36±112-88 respectively- The increased concentrations of mitochondrial ROS and IL-1β upregulation were confirmed in the inhibition but not the induction of auto-phagy-We also found that downregulation of ATG5 and Beclin1 sensitized cells for the release of IL-1β induced by MSU (monosodium urate) or nigericin which was the NLRP3 inflammasome activator- RSV treatment attentuated this effect- Conclusion: Long-term application of high glucose-based peritoneal dialysis fluids can trigger the consistent activation of NLRP3-IL-1β in peritoneal mesothelial cells-Timely initiation of autophagy may block the NLRP3-IL-1β activation and provide a basis for the further development of a potential therapeutic strategy for delay of chronic inflammation and peritoneal fibrosis associated with peritoneal dialysis.

Key words: High glucose-based peritoneal dialysis fluids, Human peritoneal mesothelial cells, NLRP3, IL-1β