肝缺血再灌注大鼠外泌体对小胶质细胞焦亡的影响

Effect of hepatic ischemia-reperfusion rats-derived exosomes on microglial pyroptosis

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=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5×10^(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=6):对照组(C组)、107个/ml肝缺血再灌注外泌体组(107组)、10^(8)个/ml肝缺血再灌注外泌体组(10^(8)组)和10^(9)个/ml肝缺血再灌注外泌体组(10^(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=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10^(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。结果肝缺血再灌注外泌体与小胶质细胞存在共定位。10^(8)个/ml和10^(9)个/ml肝缺血再灌注外泌体上调NLRP3、ASC、GSDMD和cleaved-caspase-1的表达(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(P<0.01)。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=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),107 cells/ml I/R-exosomes treated group(group 107),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-1βand 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-1βand TNF-αin the supernatant were elevated in group I/R-exosome(P<0.01).Conclusions Hepatic I/R rats-derived exosomes can promote microglial pyroptosis.