Effect of hepatic ischemia-reperfusion rats-derived exosomes on microglial pyroptosis
10.3760/cma.j.cn131073.20211012.00112
- VernacularTitle:肝缺血再灌注大鼠外泌体对小胶质细胞焦亡的影响
- Author:
Yidan WANG
1
;
Lili JIA
;
Min WEI
;
Jingshu LYU
;
Ying SUN
;
Yiqi WENG
;
Wenli YU
Author Information
1. 天津医科大学一中心临床学院，天津 300192
- Keywords:
Liver;
Reperfusion injury;
Exosomes;
Microglia;
Pyroptosis
- From:
Chinese Journal of Anesthesiology
2022;42(1):60-64
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To evaluate the effect of hepatic ischemia-reperfusion (I/R) rats-derived exosomes on microglial pyroptosis.Methods:Twenty clean-grade healthy male Sprague-Dawley rats, aged 2-3 weeks, weighing 20-50 g, were divided into 2 groups ( n=10 each) using a random number table method: sham operation group (group S) and hepatic I/R group (group I/R). The serum of rats in S group and I/R group was collected, and exosomes were isolated from the sera using differential centrifugations.Microglial cells were co-cultured with PKH26-labeled exosomes for 6 h. The intake of exosomes in microglial cells was determined using immunofluorescence staining.Primary microglial cells were seeded onto 6-well culture plates at a density of 5×10 5 cells/ml and were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), 10 7 cells/ml I/R-exosomes treated group (group 10 7), 10 8 cells/ml I/R-exosomes treated group (group 10 8), and 10 9 cells/ml I/R-exosomes treated group (group 10 9). Microglia in each group were co-cultured with the corresponding concentration of I/R-exosomes for 6 h. The expression of NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cleaved-caspase-1 and gasdermin-D (GSDMD) was detected using Western blot.Primary microglial cells were divided into 3 groups ( n=24 each) by a random number table method: control group (group C), sham operation-exosomes treated group (group S-exosome) and I/R-exosomes treated group (group I/R-exosome). In S-exosome group and I/R-exosome group, exosomes 10 8 cells/ml in S group and I/R group were given, respectively, to incubate cells for 6 h. The expression of NLRP3, ASC and GSDMD mRNA was determined by real-time polymerase chain reaction, and the levels of interleukin-18 (IL-18), IL-1β and tumor necrosis factor-alpha (TNF-α) in the cell culture supernatant were measured by enzyme-linked immunosorbent assay. Results:The results from immunofluorescence staining showed that I/R-exosomes colocalized with microglia.The 10 8 cells/ml I/R-exosomes and 10 9 cells/ml I/R-exosomes up-regulated the expression of NLRP3, ASC, GSDMD and cleaved-caspase-1 in microglial cells ( P<0.01). Compared with group C and group S-exosome, the expression of NLRP3, ASC and GSDMD mRNA in microglial cells was up-regulated, and the levels of IL-18, IL-1β and TNF-α in the supernatant were elevated in group I/R-exosome ( P<0.01). Conclusions:Hepatic I/R rats-derived exosomes can promote microglial pyroptosis.
