Sevourane post-conditioning alleviated hypoxic-ischemic brain injury in neonatal rats by inhibiting endoplasmic reticulum stress-mediated autophagy via IRE1 signalings

Post-conditioning with sevourane, a volatile anesthetic, has been proved to be neuroprotective against hypoxic-ischemic brain injury (HIBI). Our previous research showed that autophagy is over-activated in a neonatal HIBI rat model, and inhibition of autophagy confers neuroprotection. There is increasing recognition that autophagy can be stimulated by activating endoplasmic reticulum (ER) stress. Herein, we purposed to explore: i) the association of ER stress with autophagy in the setting of neonatal HIBI; and ii) the possible roles of ER stress-triggered autophagy, as well as IRE1 signaling in the neuroprotection of sevourane post-conditioning against neonatal HIBI. Seven-day-old rats underwent ligation of the left common artery, and a subsequent 2 hour hypoxia (8% O2 / 92% N2). The association of ER stress with autophagy was examined by ER stress inducer (tunicamycin), 4-PBA (ER stress inhibitor), or 3-MA (autophagy inhibitor). Rats in the sevourane post-conditioning groups were treated with 2.4% sevourane for 30 minutes after HIBI stimulation. The roles of ER stress-mediated autophagy, as well as the IRE1-JNK-beclin1 signaling cascade in the neuroprotection afforded by sevourane were explored by ER stress inducer (tunicamycin) and the IRE1 inhibitor (STF-083010). HIBI over ‐ activated ER stress and autophagy in neonatal rats. HIBI-induced autophagy was signicantly aggravated by tunicamycin but blocked by 4-PBA; however, HIBI-induced ER stress was not affected by 3-MA. Sevourane postconditioning signicantly alleviated ER stress, autophagy, cell apoptosis, and cognitive impairments, which were remarkably abolished by tunicamycin. Also, tunicamycin blocked sevourane-induced downregulation of IRE1-JNK-beclin1 signaling pathway. Whereas, IRE1 inhibitor could reverse the effects of


Introduction
Hypoxic-ischemic brain injury (HIBI) is a speci c type of brain injury that result from oxygen deprivation and limited blood ow. HIBI remains among the primary causes of cerebral palsy and other neurodevelopmental disabilities in children, occurring in 1 to 8 per 1000 live births [1]. Although the bene t of therapeutic hypothermia has been shown, over 40% of the cooled infants still die or suffer moderate to severe disabilities [2]. In recent years, the use of medical gases as neuroprotective agents has gained great attention [3]. Sevo urane is an inhaled anesthetic that is widely used in pediatric medicine and sevo urane post-conditioning has been proven to protect against cerebral ischemia damage [3]. Therefore, it is critical to explore the bene ts of sevo urane and the underlying molecular mechanisms.
Autophagy is an intracellular self-degradative process involving the elimination of malformed proteins along with damaged organelles via fusing with the lysosome. Our previous study showed that excessive autophagy is involved in cognitive impairments induced by HIBI in neonatal rats [4]. Growing evidence reveal that ER (endoplasmic reticulum) stress cross talks with autophagy [5]. The ER cellular organelle in involved in protein folding along with secretion, biosynthesisof lipids, and calcium homeostasis. Diverse pathophysiologic and pharmacologic insults could disturb proper functioning of the ER; causing aggregation of misfolded, as well as unfolded proteins in the ER, a condition referred to as ER stress [6]. A previous research documented that the compensatory activation of autophagy removes the aggregated dysfunctional proteins emanating from ER stress [7], suggesting that ER stress could constitute a trigger of autophagy stimulation. Consequently, it is critical to explore the correlation of ER stress with autophagy under neonatal HIBI conditions. ER stress stimulates an adaptive survival mechanism referred to as the UPR (unfolded protein response), responsible for the recovery of ER homeostasis through attenuation of the translation of proteins, promotion of misfolded protein degradation along with induction of ER-resident chaperones. The UPR constitutes three primary signaling systems activating by three prototypical ER localized stress sensors, i.e., IRE1 (inositol-requiring enzyme-1), ATF6 (activating transcription factor-6), as well as PERK (protein kinase RNA-like endoplasmic reticulum kinase) [8]. During the ER stress, it is speculated that three canonical branches of the UPR in distinctive manners activate autophagy. Particularly, in cells inoculated with JNK (c-Jun N-terminal kinase) inhibitor, or IRE1-de cient cells, the ER stress-triggered autophagy was repressed, demonstrating that the IRE1/JNK cascade is necessary for stimulation of autophagy following ER stress [9]. JNK activation leads to Bcl-2 phosphorylation and dissociation with beclin-1, a well-known key regulator of autophagy [10].
Herein, we assessed the association of ER stress with autophagy in neonatal HIBI rats. Besides, we evaluated the neuroprotective effects of sevo urane post-conditioning targeting IRE1 signaling cascades between ER stress and autophagy at the molecular level.

Animals
Experiments were conducted on postnatal day 7 (P7) Sprague-Dawley rats as per the guidelines of the Animal Care Committee of Shengjing Hospital, China Medical University, Shenyang, China. Dams along with their pups were put under a cycle of twelve-hour lighting-darkness conditions. Holding facilities were kept at a constant temperature ranging from 21°C to 23°C and the animals free accessed food and water ad libitum. Signi cant efforts to minimize the number of animals used, as well as any possible suffering were made.

Experimental design
Experiment I: To evaluate the time course expression of ER stress marker GRP78 and autophagy marker LC3B after HIBI, rat pups (n = 32 per group) were grouped randomly into two groups: Sham group and HI group. The left (ipsilateral) hippocampus was collected from the brains of eight pups randomly selected from every group at each time point (3 hours, 6 hours, 12 hours, and 24 hours after HI) and were prepared for western blot.
Experiment II: To elucidate the association of ER stress with autophagy during HIBI, rat pups (n = 12 per group) were assigned randomly into ve groups: Sham, HI, HI + TM (tunicamycin, inducer of ER stress), HI + 4-PBA (sodium 4-phenylbutyrate, inhibitor of ER stress), and HI + 3-MA (3-methyladenine, inhibitor of autophagy). The left (ipsilateral) hippocampus of six pups in all the groups were harvested at 24 h post-HI for western blot. In addition, we sacri ced six rat pups from every group via transcardiac perfusion with PBS and then 4% PFA for preparation of the brain section at 24 hours after HI. Experiment III: To evaluate the neuroprotective effects of sevo urane post-conditioning that target IRE1 signaling cascades between autophagy and ER stress, rat pups (n = 22 per group) were assigned randomly into ve groups: Sham, HI, SPC (sevo urane post-conditioning), SPC + TM, and SPC + TM + IRE1 inhibitor. The left (ipsilateral) hippocampus of six pups in every group were harvested at 24 hours post-HI for western blot. Ten rats in every group were tested in the open eld along with the Morris water maze tests at P35 and P35-40, respectively. The other the six rats from every group were used for Nissl stain.
2.3 Neonatal HIBI model, sevo urane postconditioning, and drug administration Sevo urane was employed to anesthetize seven-day-old rats, and subsequently permanent double ligation of the left common carotid artery performed with a 7-0 surgical silk, within ve minutes. After waking, we transferred the pups back to cages with their mothers for 2 hours. The pups were then placed in a chamber ventilated with 8% O 2 and 92% N 2 at rate of ow of 2 L/min for 2 hours. Rats in the sevo urane groups inhaled 2.4% sevo urane at the rate of ow of 2 L/min for 30 minutes in a chamber under 30% O 2 and 70% N 2 conditions, immediately after HI. Sham control rats were anesthetized and their skin incised and were kept in a chamber under 30% O 2 and 70% N 2 conditions, at the rate of ow of 2 L/min for 2 hours.

Western blot analysis
The ipsilateral hippocampus was isolated and kept at -80℃ immediately until use. The RIPA lysis buffer was employed to homogenize the and further span at 14,000 rpm at 4℃ for 30 minutes. We collected the supernatant and then employed the BCA Protein Assay Kit to quantify the proteins. The proteins were then fractionated on a 10% SDS-PAGE gel. After that, the fractionated proteins were blotted onto PVDF membranes. Then, 5% BSA was employed to block the membranes. After that, the membranes were inoculated with the primary antibodies consisting of; anti-GRP78 (1:500, Abcam, UK), anti-LC3B (1:500, Cell Signaling Technology, USA), p62 (1:500, Cell Signaling Technology, USA), IRE-1 (1:500, Abcam, UK), p-IRE-1 (1:500, Abcam, UK), JNK (1:500, Abcam, UK), p-JNK (1:500, Cell Signaling Technology, USA), beclin1 (1:500, Cell Signaling Technology, USA) overnight at 4°C. Thereafter, the membranes were inoculated with speci ed secondary anti-bodies for 2 hours at room temperature. Immunolabeling was done with the SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scienti c, USA). The GE Amersham Imager 600 was employed to image the immunoblots and the Image J software utilized to analyze the protein bands. β-actin served as the normalization standard.

TUNEL assay
The in situ Apoptosis Detection Kit (Roche, Switzerland) was employed to perform the TUNEL assay as described by the manufacturer at 24 hours after HI. Brie y, the sections were dewaxed and heated in citrate buffer, followed by inoculation in the blocking buffer (PBS enriched with 10% FBS) for 30 minutes. Next, we inoculated the sections with TdT along with dUTP at 37℃ for 1 hour. DAPI staining of the nuclei was performed. The uorescence was captured using a Nikon Eclipse NI microscope. The image J software was employed to count the number of TUNEL-positive cells. The data were shown as apoptosis index, quanti ed as the ratio of (TUNEL-positive cells) / (total cells) × 100%.

Behavioral tests
The open eld test was carried out to assess locomotor and exploratory activities. Each rat was placed at the center of a 100 cm × 100 cm × 50 cm black plastic chamber for minutes. The EthoVision XT software (Noldus, Wageningen, The Netherlands) was employed to record and analyze the tests. After that, 75% alcohol was used to clean the arena after every test to eliminate olfactory cues.
The Morris water maze (MWM) tasks were conducted to explore long-term learning, as well as memory ability. The MWM was composed of a round black pool, with a diameter of 160 cm and depth of 60 cm that was lled with water to a height of 30 cm at room temperature (20 ± 1°C). We placed a cylindrical platform with a diameter of 12-cm into the pool, immersed 1.5 cm deep into the water and 30 cm from the wall. Extra-maze distal cues which had different colors and dimensions were suspended on the black curtains of the pool and were kept constant during the whole experiment. Training sessions took ve days and were taken four times a day with an interval of 30 minutes. We placed 10 rats per group into the water to face the pool wall in one of four quadrants. Every rat was allowed 60 seconds to trace the hidden platform. The daily order of entry into these quadrants was randomized. When the escape latency (time used to nd the platform) was within 90 seconds, the rats were allowed to remain on the platform for 20 seconds. When the rats could not trace the platform within 90 seconds, we guided them to the platform on which they stayed for 20 seconds and then recorded the escape latency as 90 seconds. In spatial probe task, rats were put into the water and left to swim freely for 90 seconds after removing the platform. An automatic video camera system (Shanghai Mobiledatum Ltd, China) was placed above the pool to record every task.

Nissl staining
Coronal sections about 3.5 mm from caudal to bregma were harvested for Nissl staining. A digital microscope camera was employed to image the pyramidal cell layer of the CA1 region along with the CA3 region. Microphotographs of cells from three Nissl-stained sections were determined in every rat. Lastly, the image J software was employed to determine the number of healthy, Nissl-positive, neuronal cells.

Statistical analyses
Statistical analyses were implemented in the GraphPad Prism V.7.0 software. Data are given as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and subsequent Student-Newman-Keuls post-hoc test were implemented to compare between and within multiple groups. Data for escape latency were compared using two-way repeated measures ANOVA and subsequent Dunnett's multiple comparisons tests. Data for spatial probe test were compared using Dunn's multiple comparison test. P<0.05 signi ed statistical signi cance.

Contents of GRP78 and LC3 increased time dependently post HI
Firstly, ER stress along with autophagy activity in the hippocampus were measured by western blotting at different time points (3hours, 6 hours, 12 hours, and 24 hours) post HI. GRP78 (glucose-regulated protein 78), also referred to as BiP, is a ER resident protein and when under ER stress, it dissociates rst from the ER membrane to help destroyed proteins refold and degrade [11]. Stimulation of GRP78 is considered as a biomarker of ER stress [12] and stimulation of autophagy was examined by immunoblotting of LC3B. As indicated in Figure 1, GRP78 expression level was upregulated from 3 h to 24 h post-HI ( Figure 1A, B, 3 hours, p < 0.05; 6 hours, p < 0.01; 12 hours, p < 0.05; 24 hours, p < 0.05) and the peak GRP78 level was reported at 6 hours after HI. Increase of LC3BII expression started at 6 hours and attained the peak at 24 hours ( Figure 1A, C, 6 hours: p < 0.05; 12 hours: p < 0.05; 24 hours: p < 0.01) after HI treatment. Our data illustrated that ER stress along with autophagy participate in the pathological process of neonatal HIBI. Besides, we selected HI-24 hours model for the successive experiments.

ER stress triggered autophagy in the neonatal HIBI rats
Next, to assess the association of ER stress with autophagy in the context of neonatal HIBI, the rats were inoculated with tunicamycin (ER stress inducer), 4-PBA (ER stress inhibitor), or 3-MA (autophagy inhibitor). The p62 ubiquitin binding protein participates in the selective autophagy and reduction of p62 is a biomarker for increase of autophagic ux [13]. Results showed that HI insult over-activated ER stress and autophagy as indicated by upregulation of GRP78 as illustrated in Figure 2A, B (p < 0.05 in contrast with the Sham group) and LC3BII (Figure 2A, C, p < 0.01 in contrast with the Sham group), and downregulation of p62 (Figure 2A, D, p < 0.05 in contrast with the Sham group). HI-triggered ER stress and autophagy reinforced by combination with TM ( Figure 2, GRP78: p < 0.05 in contrast with HI group; LC3BII: p < 0.05 in contrast with HI group; p62, p < 0.05 in contrast with HI group), but counteracted by 4-PBA administration (Figure 2, GRP78: p < 0.05 in contrast with HI group; LC3BII: p < 0.05 in contrast with HI group; p62, p < 0.05 in contrast with HI group). However, 3-MA was able to restrain autophagy activity ( Figure 2, LC3BII: p < 0.05 in contrast with HI group; p62: p < 0.05 in contrast with HI group) without dramatic in uences on ER stress (Figure 2, no remarkable difference in GRP78 expression). Altogether, these data illustrated that ER stress occurred upstream of autophagy during neonatal HIBI.

ER stress-autophagy participates in the cell apoptosis triggered by HIBI
The cell apoptosis in the hippocampus was explored by TUNEL assay. As demonstrated in Figure 3, positive apoptosis cells were seen in HI group (CA1: p < 0.01 in contrast with Sham group; CA3: p < 0.01 in contrast with Sham group), and treatment with TM enhanced HI-induced apoptosis in the hippocampal CA1and CA3 subregions (CA1: p < 0.05 in contrast with HI group; CA3: p < 0.01 in contrast with HI group). Interfering ER stress by administration with 4-PBA restrained HI-induced cell apoptosis (CA1: p < 0.05 in contrast with HI group; CA3: p < 0.05 in contrast with HI group). Similarly, the autophagic inhibitor 3-MA suppressed HI-induced apoptosis via inhibition of autophagy (CA1: p < 0.05 in contrast with HI group; CA3: p < 0.05 in contrast with HI group).
Reports document that ER stress can stimulate autophagy through IRE1cascade [16]. The speci c IRE1 inhibitor STF-083010 was administrated to further con rm that whether the IRE1-JNK-beclin1 cascade is involved in the neuroprotection of sevo urane. Proteins in the IRE1 cascade consisting of p-IRE1/IRE1, beclin1, and p-JNK/JNK were also assessed via Western blot. p-IRE1/IRE1, p-JNK/JNK ratio are employed to stand for activation levels of IRE1 and JNK. IRE1 inhibitor reverted the imapcts of TM on the expression of GRP78, LC3BII, and p62 ( Figure 4A, B, SPC+TM in contrast with SPC, all p < 0.05; SPC+TM+IRE1 inhibitor in contrast with SPC+TM: all p < 0.05; for GRP78, LC3BII, and p62, respectively), indicating that sevo urane restrained ER stress-induced autophagy via the IRE1 cascade 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 in contrast with sham group (Figure 4C, D, HI in contrast with Sham: all p < 0.05 for p-IRE1/IRE1, p-JNK/JNK, and beclin1, respectively). Nevertheless, inoculation with sevo urane dramatically restrained the above changes ( Figure 4C, D, SPC in contrast with HI: all p<0.05 for p-IRE1/IRE1, p-JNK/JNK, and beclin1, respectively), which were reversed by combination with TM ( Figure 5, SPC+TM in contrast with SPC: all 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 in contrast with SPC+TM: all p<0.05 for p-IRE1/IRE1, p-JNK/JNK, and beclin1, respectively). The above results suggested that sevo urane post-conditioning suppressed ER stress-autophagy via IRE1-JNK-beclin1 signaling cascade.
3.5 Sevo urane post-conditioning improved cognitive performance and prevented neuronal loss in rats following HI To explore the anxiety-like behavior along with the locomotor activity in a novel environment, the rats underwent an open eld task at P35. As indicated in Figure 5, no differences were reported among groups in overall travelled distance, time taken in the center region, or the number of feces. These results suggested that treatment factors in this study did not influence anxiety-like behavior or locomotor activity in rats.
From day P35, MWM tasks were carried out to explore spatial learning along with the memory ability. As shown in Figure 5, rats in all groups exhibited a signi cant downward trend in escape latency. In contrast with the sham group rats, the HI rats 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. Sevo urane 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 the number of times of crossing the platform ( 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. There was an irregular scattered organization of the neurons that had increased intracellular space 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. Sevo urane signi cantly attenuated HI-induced reduction of neuronal density in the CA1 and CA3 hippocampal areas (HI in contrast with Sham: p < 0.001, p < 0.001; SPC in contrast with HI: p < 0.001, p < 0.001; for CA1 and CA3, respectively). Treatment with TM increased neuronal density in the hippocampus (SPC+TM in contrast with SPC: p < 0.001, p < 0.001 for CA1 and CA3, respectively), while IRE1 inhibitor blocked the effects of TM (SPC+TM+IRE1 inhibitor in contrast with SPC+TM: p < 0.001, p < 0.01 for CA1 and CA3, respectively).
These results indicated that sevo urane post-conditioning alleviated HI-induced cognitive impairments, possibly by regulating ER stress-mediated autophagy via IRE1 signaling cascade.

Discussion
HIBI is among the primary causes of neonatal mortality and could cause neurodevelopmental disabilities, with limited therapy options. Excessive autophagy caused by HIBI may cause neuronal death [4]. Our previous studies revealed that the volatile anesthetic sevo urane could exert neuroprotective effects against HIBI via autophagy inhibition [14,15]. Yet, the underlying mechanisms remain unclear. Herein, we provided evidence that ER stress contributed to autophagy in HIBI neonatal rats. In addition, sevo urane post-conditioning alleviated ER stress-dependent autophagy through modulating IRE1-JNK-beclin1 cascades.
Autophagy is among the pathogenetic mechanisms of hypoxic-ischemic cerebral injury [4,17]. Autophagy occurs constitutively at a basal level to sustain cellular homeostasis and function, but can also be triggered by both physiological and pathological stimuli. Previously, we documented that in neonatal rat HIBI model, induction of highly activated autophagy by HI insult it is detrimental and contributes to neuronal apoptosis and cognitive dysfunction [4]. It is emerging increasingly clear that ER stress is one of the signaling cascades involved in regulation of autophagy [18,5]. GRP78 is a central modulator of the ER stress signaling cascade with established function as an ER chaperone protein. During ER stress, it dissociates rst from the ER membrane to aid the destroyed proteins to refold and degrade [12]. Thus, activation of GRP78 is considered as a biomarker of ER stress. Herein, both ER stress along with autophagy are activated in the pathological process of neonatal HIBI, as evidenced by increased GRP78 and LC3BII expression, as well as decreased p62 expression. Our results supported results from previous studies that ER stress along with autophagy are involved in ischemic stroke [19,20]. Of note, to elucidate the association of ER stress with autophagy in the setting of neonatal HIBI, the rats were inocualted with TM (ER stress inducer), 4-PBA (ER stress inhibitor), or 3-MA (autophagy inhibitor). Results showed that HIBI-induced autophagy was signi cantly enhanced by TM but counteracted by 4-PBA; however, HIBIinduced ER stress was not affected by 3-MA. The above data suggested that ER stress is also a prospective inducer of autophagy in neonatal HIBI rats. Similarly, ER stress mediated-autophagy has been implicated in the pathogenesis of traumatic brain injury [21].
Numerous studies have documented that sevo urane post-conditioning exhibits neuroprotective effects against HIBI, and the protective mechanisms may be attributed to its suppressing autophagy properties. Since ER stress is the upstream cascade inducing autophagy in HIBI, we hypothesized that sevo urane restrained autophagy through inhibiting ER stress. To con rm this hypothesis, ER stress inductor TM was administrated. Herein, there were remarkable decreases in GRP78 and LC3BII protein levels, whereas increase in p62 protein level following sevo urane post-conditioning, but these effects were blocked by TM. Alteration of the protein expressions mentioned above revealed that sevo urane restrained autophagy through inhibiting ER stress.
Our results also suggested that the IRE1-JNK-beclin1 cascade was involved in the mechanisms of neuroprotection of sevo urane during the inhibition of ER stress-induced autophagy. The UPR is an adaptive cascade that aims to reestablish cellular homeostasis after ER stress. When the UPR occurs, three ER-resident transmembrane proteins (IRE1, ATF6, and PERK) sense it through a luminal domain and transmits to the cytoplasm and nucleus in order to facilitate folding of proteins, attenuation of translation and ER-linked misfolded protein degradation [22]. Interestingly, in a study of IRE1-PERK-ATF6-de ciency in ER stress, autophagy was suppressed in the IRE1 signaling cascade, but not in the PERK and ATF6 cascades [9]. In embryonic broblasts, IRE1defect reduced aggregation of LC3-positive vesicles [23]. Under physiological conditions, the ER chaperone GRP78 docks to the regulatory luminal domain of IRE1 and suppresses its activity, whereas in ER stress, GRP78 dissociates from IRE1, allowing it to oligomerize, trans-autophosphorylate, and subsequently activate its endoribonuclease domain [24]. As expected, p-IRE1/IRE1 ratio was increased with HIBI-induced ER stress but decreased by sevo urane. It has been reported that ER stress-mediated autophagy is modulated via IRE1 cross talk with TRAF2 to induce JNK activation/phosphorylation [9]. Previous investigations have also further illustrated that elevated JNK signaling is linked to autophagy stimulation [25]. One of the key checkpoints in the modulation of autophagy is the interaction between the autophagic effector beclin-1 and the members of the antiapoptotic Bcl-2 family [26]. Activation of JNK triggered the phosphorylation of Bcl-2 and disrupts the generation of Bcl-2/beclin-1 autophagy repressor complexes [27,28]. Western blot analysis showed that sevo urane signi cantly inhibited HI-induced the upregulation of p-IRE1/IRE1, p-JNK/JNK, and beclin-1. TM reversed the effects of sevo urane on IRE1/JNK/beclin-1 signaling, as well as the autophagy biomarkers LC3B-II and beclin1, and IRE1 inhibitor counteracted the effects of TM. Therefore, the mechanisms by which sevo urane affects the ER stress-mediated autophagy are attributed to the inhibited of IRE1/JNK/beclin-1 (Figure 7).
In summary, our data have shed some light on the mechanisms of sevo urane in treatment for HIBI. We established that the ER stress-autophagy signaling is a pivotal mechanistic event that participates in HIBI, and sevo urane post-conditioning signi cantly protects against HIBI in neonatal rats by repressing ER stress-mediated autophagy through IRE1-JNK-beclin1 signaling cascade.    The density of bands analyzed by Image J, with β-actin serving as the normalization control. Data are given as mean ± SD. n = 6 for each group. *p < 0.05 and **p < 0.01 in contrast with the indicated group.  Figure 5D, *p < 0.05 and ***p < 0.001 in contrast with Sham group. ###p < 0.001 in contrast with HI group. ^^p < 0.01 in contrast with SPC group. &&p < 0.01 in contrast with SPC+TM group. In Figure 5E, *p < 0.05 and ***p < 0.001 in contrast with the indicated group.  Mechanisms responsible for protective in uences of sevo urane post-conditioning against neonatal hypoxic-ischemic brain injury.