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目的:评价肝缺血再灌注大鼠外泌体对小胶质细胞焦亡的影响。方法:清洁级健康雄性SD大鼠20只，2~3周龄，体重20~50 g，采用随机数字表法分为2组(n n＝10)：假手术组(S组)和肝缺血再灌注组(I/R组)。取S组及I/R组大鼠血清，使用超速离心法提取外泌体。将PKH26标记的外泌体与小胶质细胞共孵育6 h，采用免疫荧光法检测外泌体的摄取。将原代小胶质细胞以5×10n 5个/ml的密度接种于6孔板，采用随机数字表法分为4组(n n＝6)：对照组(C组)、10n 7个/ml肝缺血再灌注外泌体组(10n 7组)、10n 8个/ml肝缺血再灌注外泌体组(10n 8组)和10n 9个/ml肝缺血再灌注外泌体组(10n 9组)。依次给予相应浓度肝缺血再灌注外泌体共孵育6 h。采用Western blot法检测NOD样受体蛋白3(NLRP3)、凋亡相关微粒蛋白(ASC)、裂解含半胱氨酸的天冬氨酸蛋白水解酶1(cleaved-caspase-1)和消皮素D(GSDMD)的表达。采用随机数字表法将原代小胶质细胞分为3组(n n＝24)：对照组(C组)、假手术外泌体组(S-exosome组)和肝缺血再灌注外泌体组(I/R-exosome组)。S-exosome组和I/R-exosome组分别给予10n 8个/ml的S组、I/R组外泌体孵育6 h。采用RT-PCR法检测NLRP3、ASC和GSDMD mRNA的表达，采用ELISA法检测上清液IL-18、IL-1β和TNF-α的浓度。n 结果:肝缺血再灌注外泌体与小胶质细胞存在共定位。10n 8个/ml和10n 9个/ml肝缺血再灌注外泌体上调NLRP3、ASC、GSDMD和cleaved-caspase-1的表达(n P<0.01)。与C组和S-exosome组比较，I/R-exosome组NLRP3、ASC和GSDMD mRNA的表达上调，上清液IL-18、IL-1β和TNF-α的浓度升高(n P<0.01)。n 结论:肝缺血再灌注大鼠外泌体可促进小胶质细胞焦亡。“,”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 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 5×10n 5 cells/ml and were divided into 4 groups (n n=6 each) using a random number table method: control group (group C), 10n 7 cells/ml I/R-exosomes treated group (group 10n 7), 10n 8 cells/ml I/R-exosomes treated group (group 10n 8), and 10n 9 cells/ml I/R-exosomes treated group (group 10n 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 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 10n 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.n Results:The results from immunofluorescence staining showed that I/R-exosomes colocalized with microglia.The 10n 8 cells/ml I/R-exosomes and 10n 9 cells/ml I/R-exosomes up-regulated the expression of NLRP3, ASC, GSDMD and cleaved-caspase-1 in microglial cells (n 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 (n P<0.01).n Conclusions:Hepatic I/R rats-derived exosomes can promote microglial pyroptosis.