3.1 Expression levels of GRP78 and LC3 increased in time-dependent manner post HI
Firstly, ER stress and autophagy activity in the hippocampus were measured by western blot at different time points (3h, 6 h, 12 h, and 24 h) after HI. Glucose-regulated protein 78 (GRP78), also referred to as BiP, is a resident protein of the ER and upon ER stress, it first dissociates from ER membrane to help destroyed proteins refold and degrade [11]. Induction of GRP78 is considered as a marker for ER stress [12] and activation of autophagy was examined by immunoblotting of LC3B. As shown in Figure 1, GRP78 expression level was upregulated from 3 h to 24 h post-HI (Figure 1A, B, 3 h, p < 0.05; 6 h, p < 0.01; 12 h, p < 0.05; 24 h, p < 0.05) and the peak GRP78 level was observed at 6 h post-HI. Increase of LC3BII expression started at 6 h and reached a peak at 24 h (Figure 1A, C, 6 h: p < 0.05; 12 h: p < 0.05; 24 h: p < 0.01) after HI treatment. Our results indicated that both ER stress and autophagy are involved in the pathological process of neonatal HIBI. In addition, we chose HI-24 h model for the subsequent experiments.
3.2 ER stress triggered autophagy in the neonatal HIBI rats
Next, to determine the relationship between ER stress and autophagy in the context of neonatal HIBI, the rats were administrated with tunicamycin (ER stress inducer), 4-PBA (ER stress inhibitor), or 3-MA (autophagy inhibitor). p62 is a ubiquitin binding protein involved in selective autophagy and reduction of p62 has been regarded as a marker for increase of autophagic flux [13]. Results showed that HI insult over-activated ER stress and autophagy as indicated by upregulation of GRP78 (Figure 2A, B, p < 0.05 vs Sham group) and LC3BII (Figure 2A, C, p < 0.01 vs Sham group), and downregulation of p62 (Figure 2A, D, p < 0.05 vs Sham group). HI-induced ER stress and autophagy reinforced by combination with TM (Figure 2, GRP78: p < 0.05 vs HI group; LC3BII: p < 0.05 vs HI group; p62, p < 0.05 vs HI group), but counteracted by 4-PBA administration (Figure 2, GRP78: p < 0.05 vs HI group; LC3BII: p < 0.05 vs HI group; p62, p < 0.05 vs HI group). However, 3-MA was able to restrain autophagy activity (Figure 2, LC3BII: p < 0.05 vs HI group; p62: p < 0.05 vs HI group) without significant effects on ER stress (Figure 2, no significant difference in GRP78 expression). The above data indicated that ER stress was an upstream event of autophagy during neonatal HIBI.
3.3 ER stress-autophagy are involved in the cell apoptosis induced by HIBI
The cell apoptosis in the hippocampus was assessed by TUNEL assay. As illustrated in Figure 3, positive apoptosis cells were observed in HI group (CA1: p < 0.01 vs Sham group; CA3: p < 0.01 vs Sham group), and treatment with TM enhanced HI-induced apoptosis in the hippocampal CA1and CA3 subregions (CA1: p < 0.05 vs HI group; CA3: p < 0.01 vs HI group). Interfering ER stress by administration with 4-PBA restrained HI-induced cell apoptosis (CA1: p < 0.05 vs HI group; CA3: p < 0.05 vs HI group). Similarly, the autophagic inhibitor 3-MA suppressed HI-induced apoptosis via inhibition of autophagy (CA1: p < 0.05 vs HI group; CA3: p < 0.05 vs HI group).
3.4 Sevoflurane post-conditioning alleviated ER stress-mediated autophagy via regulation of the IRE1 pathway
Our previous researches indicated that sevoflurane-conferred neuroprotection against HIBI is related to inhibited excessive autophagy [14,15]. TM was applied in order to elucidate whether inhibition of autophagy by sevoflurane was mediated through suppressing ER stress. Sevoflurane post-conditioning attenuated HI-induced upregulation in GRP78 and LC3BII, and reduction in p62 (Figure 4A, B, HI vs. Sham: p < 0.05, p < 0.01, p < 0.01; SPC vs. HI: p < 0.05, p < 0.05, p < 0.05; for GRP78, LC3BII, and p62, respectively), but these effects were blocked by TM (Figure 4A, B, SPC + TM vs. SPC: p < 0.05, p < 0.05, p < 0.05 for GRP78, LC3BII, and p62, respectively).
Reports indicate that ER stress can trigger autophagy through IRE1 pathway [16]. The specific IRE1 inhibitor STF-083010 was administrated to further confirm that whether the IRE1/JNK/beclin1 pathway is involved in the neuroprotection of sevoflurane. Proteins in the IRE1 pathway, including p-IRE1/IRE1, p-JNK/JNK, and beclin1, were also assessed by Western blot. p-IRE1/IRE1, p-JNK/JNK ratio are used to stand for activation levels of IRE1 and JNK. IRE1 inhibitor reversed the effects of TM on the expression of GRP78, LC3BII, and p62 (Figure 4A, B, SPC+TM vs. SPC, p < 0.05, p < 0.05, p < 0.05; SPC+TM+IRE1 inhibitor vs. SPC+TM: p < 0.05, p < 0.05, p < 0.05; for GRP78, LC3BII, and p62, respectively), indicating that sevoflurane restrained ER stress-triggered autophagy through IRE1 pathway during HIBI. As presented in Figure 4, obvious increases in rations of p-IRE1/IRE1, p-JNK/JNK, and expression level of beclin1 were observed in ipsilateral hippocampus of rats compared with sham group (Figure 4C, D, HI vs. Sham: p < 0.05, p < 0.05, p < 0.05 for p-IRE1/IRE1, p-JNK/JNK, and beclin1, respectively). However, treatment with sevoflurane remarkably restrained the above changes (Figure 4C, D, SPC vs. HI: p < 0.05, p < 0.05, p < 0.05 for p-IRE1/IRE1, p-JNK/JNK, and beclin1, respectively), which were reversed by combination with TM (Figure 5, SPC+TM vs. SPC: p < 0.05, p < 0.05, p < 0.05 for p-IRE1/IRE1, p-JNK/JNK, and beclin1, respectively). IRE1 inhibitor counteracted the effects of TM (Figure 5, SPC+TM+IRE inhibitor vs. SPC+TM: p < 0.05, p < 0.05, p < 0.05 for p-IRE1/IRE1, p-JNK/JNK, and beclin1, respectively). The above results suggested that sevoflurane post-conditioning suppressed ER stress-autophagy via IRE1/JNK/beclin1 signaling pathway.
3.5 Sevoflurane post-conditioning improved cognitive performance and prevented neuronal loss in rats following HI
To evaluate locomotor activity and anxiety-like behavior in a novel environment, rats underwent the open field test at P35. As shown in Figure 5, no differences were observed among groups in total distance traveled, time spent in the center region, or the number of feces. These results suggested that treatment factors in this study did not influence locomotor activity or anxiety-like behavior in rats.
From day P35, Morris water maze tests were performed to evaluate spatial learning and memory. As shown in Figure 5, rats in all groups showed a significant downward trend in escape latency. Compared with the sham group, rats in the HI group exhibited longer escape latency to reach the platform (Figure 5D, day 29 post-HI, p < 0.05; day 30 post-HI, p < 0.001; day 31 post-HI, p < 0.001; day 32 post-HI, p < 0.001; day 33 post-HI, p < 0.001) and fewer platform crossings (Figure 5E, p < 0.001), indicating that rats displayed impaired learning and memory function following HIBI model. Sevoflurane post-conditioning successfully shortened escape latency (Figure 5D, day 30 post-HI, p < 0.001; day 31 post-HI, p < 0.001; day 32 post-HI, p < 0.001; day 33 post-HI, p < 0.001) and increased platform-crossing times (Figure 5E, p < 0.05), but these effects were reversed by TM (Figure 5 D, E, escape latency, day 30 post-HI, p < 0.01; day 31 post-HI, p < 0.01; day 32 post-HI, p < 0.01; day 33 post-HI, p < 0.01; platform crossing times, p < 0.05). However, treatment with IRE1 inhibitor blocked the negative effects of TM (Figure 5 D, E, escape latency, day 31 post-HI, p < 0.01; day 32 post-HI, p < 0.01; day 33 post-HI, p < 0.01; platform crossing times, p < 0.05).
Nissl staining procedures were conducted to determine neuron status among groups after behavioral tests. Neurons with increased intracellular space were scattered in an irregular arrangement in the hippocampus of the rats in the HI and SPC +TM groups, whereas neurons in the SPC and SPC + IRE1 inhibitor groups preserved a better functional status. Sevoflurane significantly attenuated HI-induced reduction of neuronal density in the CA1 and CA3 hippocampal areas (HI vs. Sham: p < 0.001, p < 0.001; SPC vs. HI: p < 0.001, p < 0.001; for CA1 and CA3, respectively). Treatment with TM increased neuronal density in the hippocampus (SPC+TM vs. SPC: p < 0.001, p < 0.001 for CA1 and CA3, respectively), while IRE1 inhibitor blocked the effects of TM (SPC+TM+IRE1 inhibitor vs. SPC+TM: p < 0.001, p < 0.01 for CA1 and CA3, respectively).
These results indicated that sevoflurane post-conditioning alleviated HI-induced cognitive impairments, possibly by regulating ER stress-mediated autophagy via IRE1 signaling pathway.