Role of immune-related GTPase M1 in cortical neurons autophagy of mice with sepsis-induced brain injury

Abstract

To investigate the role of immune-related GTPase M1 (IRGM1) in cortical neurons autophagy in mice with sepsis induced brain injury (SIBI). Methods: Sixty wild-type C57BL/6 mice and sixty IRGM1 gene knockout C57BL/6 mice were randomly divided into 4 groups: a sham-operated wild-type (SWT) group, a cecal ligation and puncture (CLP) model wild-type (MWT) group, a sham-operated knockout (SKO) group, and a CLP model knockout (MKO) group. Models of mice with sepsis were established by CLP. Six hours of after CLP, the neurobehavioral scores for mice were recorded. The mice were diagnosed with SIBI and enrolled for the studies in next step if the neurobehavioral score was less than 6 in the MWT and MKO groups. The sham operation group only opened the abdominal cavity without CLP. Pathological changes in mouse cerebral cortex were observed by HE staining. Electron microscope was used to observe the ultrastructure of autophagy in cortical neurons. The expression of IRGM1 and INF-γ mRNA in the cerebral cortex of mice were detected by Real time quantitative PCR. The protein expression of microtubule-associated protein 1 light chain 3 (LC3)-II, LC3-I, sequestosome-1 (SQSTM1) and IRGM1 were measured by Western blot. Immunofluorescence staining was used to examine the expression of IRGM1 in mouse cortical neurons. Results: In the MWT group, the cortical neurons showed dilated endoplasmic reticulum, swelling mitochondria, and increased number of autophagosomes after 6 or 24 h of CLP in contrast to the SWT group. At 6 h after CLP, the expression of LC3-II in the cerebral cortex began to up-regulate, and the up-regulation was maintained till 96 h after CLP; on the contrary, SQSTM1 began to decline after 6 h of CLP. Compared with SWT group, IRGM1 was strongly up-regulated in the cerebral cortex of mice at both mRNA and protein levels in the MWT group after 12 h of CLP, and the mRNA expression of IFN-γ was also increased significantly (P<0.05). At 24 h after CLP, the IRGM1 expression of cortical neurons in the MWT group was significantly higher than that in the SWT group. The baseline of autophagy activity was quite low in the cerebral cortex cells in the SWT and the SKO groups. There was almost no detected expression of LC3-II; conversely, the expression of SQSTM1 was very high after 12 h of CLP. However, the expression of LC3-II was significantly up-regulated and the expression of SQSTM1 was down-regulated in the MWT group (P<0.05). On the other hand, there was almost no detected LC3-II expression in cerebral cortex in the MKO group, and the expression of SQSTM1 was up-regulated. At 6 h after CLP, the incidence of SIBI was 90% (27/30) in the MWT group, and 96.67% (29/30) in the MKO group. At 12 h of CLP, the neurobehavioral scores in the MKO group was significantly lower than that in the MWT group (4.97±0.71 vs 5.43±0.86; t=2.284, P=0.026). HE staining showed that mice in the MKO group suffered severe cerebral cortex injury, and the number of nerve cells was significantly reduced compared with that in the MWT group. Conclusion: The IRGM1 exerts a protective effect on the brain of the mice with SIBI, and its mechanism might be related to the regulation of autophagy in mouse cortical neurons.