Relationship between serum exosomes and microglial pyroptosis during brain injury in a young rat model of hepatic ischemia-reperfusion / 中华麻醉学杂志

ABSTRACT

Objective:To evaluate the relationship between serum exosomes and microglial pyroptosis during brain injury in a young rat model of hepatic ischemia-reperfusion (I/R).Methods:Forty-six clean-grade healthy male Sprague-Dawley rats, aged 2-3 weeks, weighing 40-60 g, were allocated into 4 groups using a random number table method: sham operation group (group S, n=10), hepatic I/R group (group I/R, n=13), treatment with serum exosome in sham-operated young rat group (group S-Exosome, n=10), and treatment with serum exosomes in young rats with I/R group (group I/R-Exosome, n=13). The common trunk of the portal vein, left hepatic artery and bile duct was clamped for 60 min resulting in ischemia of 70% of the liver in anesthetized animals.After 6 h of reperfusion, the serum was collected to extract exosomes in S group and I/R group, and the serum exosome suspension 100 μl of S group and I/R group was injected through the tail vein in S-Exosome group and I/R-Exosome group, respectively.The expression of serum exosome marker proteins CD9 and CD81 was determined by Western blot in S group and I/R group.Serum and hippocampi were obtained from each group at 6 h after the corresponding treatment.The expression of NOD-like receptor protein 3 (NLRP3), gasdermin D (GSDMD), pro-caspase-1, cleaved-caspase-1, and apoptosis-associated speck-like protein containing CARD (ASC) in the hippocampus was detected using Western blot, and the expression of GSDMD in hippocampal tissues was determined by immunohistochemistry.The levels of interleukin-1beta (IL-1β), interleukin-18 (IL-18) and tumor necrosis factor-α (TNF-α) in serum and hippocampal tissues and S100β and NSE in serum were determined by enzyme-linked immunosorbent assay.In group I/R-Exosome, 3 rats were selected, and their serum exosomes were extracted, labeled with PKH26 red fluorescence and then injected via the tail vein, and the co-localization between exosomes and microglia was identified by immunofluorescence technique. Results:Compared with group S, the expression of serum CD9 and CD81 was significantly up-regulated in group I/R, the expression of NLRP3, GSDMD, cleaved-caspase-1 and ASC in hippocampal tissues was significantly up-regulated, the levels of IL-18, IL-1β and TNF-α in serum and hippocampal tissues and S100β and NSE in serum were increased in I/R and I/R-Exosome groups ( P<0.05), and no significant change was found in the parameters mentioned above in group S-Exosome ( P>0.05). The positive expression of GSDMD was significantly increased in I/R and I/R-Exosome groups ( P<0.05), no positive expression of GSDMD was found in S and S-Exosome groups ( P>0.05), and the results of immunofluorescence showed the co-localization between exosomes and microglia. Conclusions:The mechanism by which hepatic I/R induces brain injury may be related to serum exosomes-mediated microglial pyroptosis in young rats.