Ac-YVAD-cmk ameliorated sevoflurane-induced cognitive dysfunction and revised mitophagy impairment

It is common for elderly patients to develop postoperative cognitive dysfunction (POCD), but the pathophysiological mechanisms have not yet been fully explored. NLRP3 inflammasome activation and mitophagy impairment was involved in neurodegenerative disease. This study investigated the interaction of NLRP3 inflammasome and mitophagy in sevoflurane-induced cognitive dysfunction. We found that sevoflurane induced cleaved caspase-1 level, IL-1β and IL-18 maturation, and activated NLRP3 inflammasome in aged mice and the primary hippocampus neuron. The cleaved caspase-1 was demonstrated in microglia of hippocampus. Ac-YVAD-cmk, a selected caspase-1 inhibitor, reduced the expression of cleaved caspase-1, IL-1β, IL-18 and NLRP3 inflammasome activation induced by sevoflurane. Ac-YVAD-cmk ameliorated learning ability impairment in aged mice induced by sevoflurane using Morris water maze. Moreover, Ac-YVAD-cmk reversed the mitophagy flux dysfunction induced by sevoflurane in aged mice by western blotting, immunostaining and mt-Keima reporters. For the first time, we found caspase-1 inhibitor mitigated mitochondria dysfunction and revised mitophagy impairment induced by sevoflurane.


Introduction
The first research reported the relationship between surgery under general anesthesia and postoperative cognitive dysfunction (POCD) in 1955 [1]. There is a significant reduction in cognitive function in POCD patients, which can last for up to six months or even longer [2]. As the most commonly used anesthetic, sevoflurane anesthesia was reported to be associated with cognitive impairment [3]. Cognitive dysfunction induced by sevoflurane may also be caused by neuroinflammation [4], autophagy [5], oxidative stress [6], blood-brain barrier dysfunction [7], and apoptosis [8]. The underlying mechanisms of sevoflurane-induced cognitive impairment remained unknown.
Increasing evidences revealed a causal relationship between sevoflurane-induced cognitive impairment and NLRP3 inflammasomes in hippocampus [9,10]. In response to cellular stress, NLRP3 recruits ASC and pro-caspase-1, which results in cleaved caspase-1 activation and processing of the maturation of IL-1β and IL-18 [11]. Patients with Alzheimer's disease exhibit highly enhanced active caspase-1 expression in their brains, suggesting that the inflammasome plays a role in this neurodegenerative disease [9]. In addition, as an important way for elimination of damaged mitochondrial, mitophagy reduces cellular stress playing a crucial role in neurodegenerative disease and aging [12,13]. Our previous work indicated that mitophagy impairment contributes to sevoflurane-induced cognitive dysfunction in aged rats [14]. However, the interaction between NLRP3 inflammasomes and mitophagy in the sevofluraneinduced cognitive impairment had not been fully clarified. Numerous danger signals activate the NLRP3 inflammasome. A model suggests that NLRP3 is activated by ROS produced in mitochondria. By producing ROS, mitochondria with reduced membrane potential activate NLRP3 inflammasomes [15]. However, induction of mitochondrial outer membrane permeabilization and mitochondrial permeability transition by danger-associated molecular patterns (DAMP), inflammasome activation might leads to block of mitophagy. In present study, we used the caspase-1 inhibitor, Ac-YVAD-cmk to explore the neuroprotection on hippocampus, and to explain the mechanisms underlying the relationship between NLRP3 inflammasomes and mitophagy.

Animals
Zhejiang Science Laboratory Animal Welfare Ethics Review Committee approved this project. (No. ZJU20160074) We conducted all experiments based on its ethical guidelines and in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and in accordance with ARRIVE guidelines. Eighteen-month-old mice were used in this study and obtained from Zhejiang Academy of Medical Sciences. A standard animal care facility provided food and water to these mice for a month, as well as a 12/12 hour light/dark cycle and a constant temperature of 22˚C for the duration of the experiment. Our goal was to minimize pain and discomfort for the animals, and we used the smallest number of animals possible.

Animal treatment
A sevoflurane vaporizer and an anesthetizing chamber with two connections was used to induce general anesthesia in aged mice The aged mice of sevoflurane group were exposed to 2% sevoflurane delivered by a humidified 30% O 2 carrier gas for 5 h. According to Ac-YVADcmk administration, the aged mice were randomly assigned to 4 groups: control group (Ctrl), sevoflurane group (SEV), Ac-YVAD-cmk group (AC) and sevoflurane plus Ac-YVAD-cmk group (SEV+AC). The aged mice of SEV and SEV+AC groups were exposed with 2% sevoflurane for 5h. The aged mice of Ctrl and AC groups were exposed to the carrier gas without sevoflurane in the same period time. According to previous studies [16], the mice in the AC and SEV+AC groups were administrated with Ac-YVAD-cmk (12.5 μmol/kg, i.p.) 1 h before sevoflurane treatment. According to MCC950 administration, the aged mice were randomly assigned to 4 groups: control group (Ctrl), sevoflurane group (SEV), MCC950 group and sevoflurane plus MCC950 group (SEV+MCC950). The aged mice of SEV and SEV+MCC950 groups were exposed with 2% sevoflurane for 5h. The aged mice of Ctrl and MCC950 groups were exposed to the carrier gas without sevoflurane in the same period time. Aged mice were injected intraperitoneally (i.p.) with 50 mg/kg MCC950 or vehicle control (DMSO/PBS) 1 h before sevoflurane treatment according to previous study [17]. In order to ensure sufficient ventilation, a single sample of arterial blood was obtained at the end of sevoflurane anesthesia or sham exposure via cardiac puncture from 5 mice of each group. These mice were not used for any other part of the study. Using a blood gas analyzer, arterial carbon dioxide partial pressure (PaCO2), arterial oxygen pressure (PaO2), blood oxygen saturation (SaO2), and power of hydrogen (PH) were measured. (Kent Scientific Corp., Torrington, CT, USA). The levels of pH, PaCO2, PaO2, Glucose, and SaO2 are not significantly different among the groups. All the mice were sacrificed after behavior tests. The mice were anesthetized with ketamine (60mg/kg) and xylazine (5mg/kg) intraperitoneally, then perfused with 0.01 mol/L phosphate buffered saline (PBS, pH 7.2-7.4) followed by 4% paraformaldehyde in 0.1 mol/L PB through the ascending aorta in immunostaining assay. In the immunoblot assay, ATP assay and the lipid peroxidation malondialdehyde (MDA) assay, the mice were anesthetized with ketamine (60mg/kg) and xylazine (5mg/kg) intraperitoneally, then perfused with 0.01 mol/L phosphate buffered saline (PBS, pH 7.2-7.4), the hippocampus were removed into lysis buffer.

Morris water maze test
The spatial memory abilities were measured by using the Morris Water Maze (MWM) 24 h after the sevoflurane exposure and Ac-YVAD-cmk administration. The protocol was completed in according to previous reports [18]. A circular black pool (diameter: 120 cm; depth: 21 cm) was filled with opaque water using white non-toxic ink to reach 1.0 cm above the platform surface (diameter, 10 cm), and the water temperature was kept at 22˚C. In the training phase, all animals received four training trials per day for a total of four days. The mice were placed into the pool at a special starting position and allowed to discover the hidden platform for 60 s. Mice were guided to the platform if they could not locate the platform within 1 min. The latency time (the time to reach the hidden platform) was recorded for assessing the spatial learning. After each trial, the mice were wiped dry and a heat lamp was used to faster temperature recovering before returning to home cages. In the testing phase, the platform was removed, and each mouse was allowed to swim freely in the pool for 2 min. The platform crossing times and the quadrant time were recorded for measuring memory function.

Novel object recognition test
The novel object recognition test (NOR) was performed as described previously [19]. Briefly, mice were allowed to explore the chamber freely for 15 min on the first day. Then the mice were exposed to two identical objects (A) for 5 min during the training phase. After 24h, the mice were exposed to the familiar object (A) and a novel object (B) for 5 min during the test phase. The total distance traveled for the first 15 min on the first day was recorded and the recognition index was calculated: A recognition index was calculated for each animal and reported as the ratio TB/(TA + TB), where TA = time spent exploring the familiar object A and TB = time spent exploring the novel object B. Exploration was defined as sniffing or touching the object with the nose or forepaws.

Cell culture and treatment
The primary hippocampal neuronal culture was performed as described [20]. Briefly, the dissected hippocampus from E17 fetal mice was used. The primary hippocampal neurons were cultured for 20 days before treatment and harvested for subsequent detection.

Immunostaining
The mice were anesthetized and perfused with 0.01 mol/L phosphate buffered saline (PBS, pH 7.2-7.4) followed by 4% paraformaldehyde in 0.1 mol/L PB through the ascending aorta. The brains were then removed, and post-fixed in the same solution for 2 h before cryoprotection in PB containing 15% sucrose at 4˚C for 2 days, and the brains were removed into PB containing 30% sucrose at 4˚C for another 2 days for dehydration. The brains were wrapped and embedded in aluminum foil with O.C.T and stored at -70˚C until sectioning is performed. Every 25 μm thickness at hippocampus region, serially cut through the brain in cryostat, were collected.

Measurement of mitochondrial activities
A fluorometric assay kit (S0027) from Beyotime were utilized to quantify the ATP concentration and the assay was completed according to manufacturer's instruction. The hippocampus was homogenized in lysis buffer. The supernatant was added to the substrate solution following centrifugation. Compared with BCA assay for protein quantitation, the calculation for concentration of ATP was according to the unit protein content (μmol/μg).

Measurement of lipid peroxidation
The hippocampus was homogenized in IP lysis buffer. The supernatant was collected by centrifugation at 12 000×g for 10 min, and the protein concentration was tested by a bicinchoninic acid protein assay kit (Beyotime, P0012S). The Lipid Peroxidation malondialdehyde (MDA) assay kit was used to quantify the MDA concentration by (Beyotime, S0131). The calculation for concentration of MDA was according to the unit protein content (μm/mg).

Detection of ROS production in situ
Dihydroethidium could be oxidized to red fluorescent molecule ethidium by superoxide. We used the DHE (Sigma-Aldrich) to detect the in situ ROS production in hippocampus. The brain sections were incubated with DHE and captured by fluorescent microscope all the parameters used were kept consistent during capturing. And the DHE fluorescence intensity was quantified according to ImageJ software.

Mitochondrial reactive oxygen species
According to previous work [14], we measured regional mitochondrial ROS accumulation using the Mito-SOX reagent (M36008, Thermo Fisher, USA) and the intracellular ROS levels using the fluorescent probe dihydroethidium (DHE). Briefly, H4 cells and the neurons were treated with Ac-YVAD-cmk (40 μM) for 30 min in advance of 4.1% sevoflurane exposure for 6 h. Then 5 μM Mito-SOX working solution was applied for mitochondrial ROS accumulation. After incubating for 10 min at 37˚C without light exposure treatment, excitation wavelengths were measured at 510 nm and emission at 580 nm by a fluorescence microplate reader (Spec-traMax M5/M5e). After treatment with sevoflurane and Ac-YVAD-cmk, H4 cells and the neurons were incubated with 1 μM DHE (Bestbio, China) for 60 min at 37˚C. Excitation wavelengths were measured at 518 nm and emission at 610 nm by a fluorescence microplate reader (SpectraMax M5/M5e).

Mt-keima reporter assay
After seeding H4 cells for 16 h, mt-Keima reporters were transfected into H4 cells by Lipofectamine 3000 (Thermo Fisher, USA, L300015) and incubated for 8 h as described previously [14]. All experiments were performance after mt-Keima reporters transfection. H4 cells were treated with Ac-YVAD-cmk (40 μM) 30 min before 4.1% sevoflurane exposure. After sevoflurane treatment for 6 h, the cells were visualized under an epifluorescence microscope using 488 nm and 561 nm lasers immediately. All the parameters used were kept consistent during capturing. The 561-channel fluorescence signal indicated mitophagy flux.

Transmission electron microscope
As described previously [14], the tissues from the hippocampus were fixed with 2.5% glutaraldehyde overnight at 4˚C. The tissues were post-fixed with 1% osmium tetraoxide for 2 h, after rinsing with PBS. The tissues were then rinsed with distilled water, followed by a graded ethanol dehydration series ending with propylene oxide. After infiltration in a mixture of one-half propylene oxide and one-half resin, the tissues were embedded in resin. Sections (120 nm) were cut and stained with 4% uranylacetate for 20 min and with 0.5% lead citrate for 5 min.
Hippocampal neurons were observed on a transmission electron microscope (TEM) (Phliphs Tecnai 10, Holland) in the Center of Cryo-Electron Microscopy at Zhejiang University.

Statistical analysis
GraphPad Prism 8.0 software was used to process these data. All data were represented as mean ± standard deviation, and which were analyzed via one-way analysis of variance (ANOVA) and Tukey's post hoc test. P<0.05 was considered as statistical significance.

Sevoflurane enhanced the microglial inflammasome activation in aged mice
The 18-month-old mice were treatment with 2% sevoflurane for 5 h. To explore the impact of NLRP3 inflammasome activation on aged mice after sevoflurane treatment, cleaved caspase-1, IL-1β and IL-18 maturation were measured by western blotting (Fig 1A). According to the semiquantitative analysis, sevoflurane enhanced the expression level of NLRP3, cleaved caspase-1, IL-1β and IL-18 ( Fig 1C). The primary hippocampal neuron was exposed by 4.1% sevoflurane for 6 h. The protein levels of cleaved caspase-1 and IL-1β were detected in primary hippocampal neuron after sevoflurane treatment by western blotting (Fig 1B). The semi-quantitative analysis suggested that sevoflurane increased the expression level of cleaved caspase-1 and IL-1β in primary hippocampal neuron (Fig 1D). Further, to explore the special localization of inflammasomes in nerve cells after sevoflurane treatment in aged mice, the immunostaining was measured in hippocampus of aged mice (Fig 1E). The results indicated that sevoflurane increased the level of cleaved caspase-1 and IL-1β in CA3 of aged mice and the NLRP3 inflammasomes activator proteins immune-colocalized with microglia activator protein (CD11b) (S1 Fig). We measured the colocalization of NLRP3 and Cleaved caspase-1 in hippocampus of aged mice after sevoflurane treatment by immunostaining assay. Sevoflurane increased the expression level of NLRP3 and Cleaved caspase-1 in hippocampus of aged mice. (Fig 1F and 1G) Moreover, we used Pearson's correlation coefficient (PCC) to quantify colocalization of NLRP3 and Cleaved caspase-1. The PCC is a well-established measure of correlation has range of +1 (perfect correlation) to −1 (perfect but negative correlation) with 0 denoting the absence of a relationship [25]. In our work, the mean PCC in control group is 0.0873. Sevoflurane increased the mean PPC to 0.2393 significantly (p<0.001). (Fig 1H) The above data suggested in hippocampus of aged mice after sevoflurane treatment, cleaved caspase-1 was activated by the high level of NLRP3.

Ac-YVAD-cmk reduced the inflammasome activation induced by sevoflurane
In order to determine the inflammasome activation in aged sevoflurane mice after sevoflurane treatment, the mice were treatment with the caspse-1 inhibitor Ac-YVAD-cmk before sevoflurane treatment. The inflammasome activation in hippocampus of aged mice were detected by western blotting. The semi-quantitative analysis indicated Ac-YVAD-cmk reduced the high expression level of NLRP3, cleaved caspase-1, IL-1β and IL-18 in aged mice after sevoflurane treatment. (Fig 2A and 2B) In immunostaining assay, Ac-YVAD-cmk decreased the upregulated level of cleaved caspase-1 induced by sevoflurane in CA3. (Fig 2C and 2D) Ac-YVADcmk decreased the upregulated level of NLRP3 and cleaved caspase-1 induced by sevoflurane in primary hippocampus neurons. (S3A and S3B Fig). The above results suggested Ac-YVADcmk reduced the inflammasome activation induced by sevoflurane, and inflammasome activation may contribute to sevoflurane-induced neurotoxicity.

Ac-YVAD-cmk rescued learning ability impairment induced by sevoflurane in aged mice
Several researches reported that the neurotoxicity of sevoflurane induced learning impairment [14,26]. To explore the effect of Ac-YVAD-cmk in learning ability, the spatial learning ability of aged mice were measured by Morris water maze 24 h after the sevoflurane with or without Ac-YVAD-cmk treatment. The escape latency (Fig 3A) was longer in SEV group than that in control group. The platform crossings (Fig 3B) and quadrant time (Fig 3C) were decreased in sevoflurane mice compared with the control mice. Ac-YVAD-cmk decreased the escape latency and increased the platform crossings in the aged mice after sevoflurane treatment. (Fig  3A and 3B).

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We also used the novel objective recognition test 24 h after the sevoflurane with or without Ac-YVAD-cmk treatment. The overall distance of traveling was not significant difference ( Fig  3D), suggesting sevoflurane did not cause motor function impairment. The recognition index was decreased in the SEV group compared to the Ctrl group, and Ac-YVAD-cmk rescued the decreased recognition index after sevoflurane treatment. (Fig 3E) The above data indicated that Ac-YVAD-cmk rescued learning ability impairment induced by sevoflurane in aged mice.

Ac-YVAD-cmk mitigated mitochondria dysfunction induced by sevoflurane
Our previous work reported that mitophagy dysfunction was one of the mechanisms underlying sevoflurane-induced learning impairment [14]. In order to explore the relationship between NLRP3 inflammasomes and mitochondria function underlying learning ability impairment induced by sevoflurane, the mitochondria function was measured. The generation of reactive oxygen species (ROS) was detected qualitatively in fresh frozen hippocampal sections by DHE staining. Sevoflurane increased the ROS fluorescence level in hippocampal sections of aged mice. And the hippocampus from the sevoflurane and Ac-YVAD-cmk group showed weaker DHE staining compared with the sevoflurane mice. (Fig 4A and 4B) In MAD assay, Ac-YVAD-cmk revised the high level of lipid peroxidation induced by sevoflurane in aged mice ( Fig 4C). Moreover, we found the aged mice after sevoflurane treatment exhibited lower baseline ATP concentrations in the hippocampus as compared with the control aged mice. Ac-YVAD-cmk increased ATP concentrations in hippocampal mice of aged mice as compared with sevoflurane mice (Fig 4D).
In order to test the effect of Ac-YVAD-cmk on mitochondrial ROS, we measured regional mitochondrial ROS accumulation using the Mito-SOX reagent and the intracellular ROS levels using the fluorescent probe dihydroethidium in H4 cells. H4 cells were treated with Ac-YVAD-cmk (40 μM) 30 min before 4.1% sevoflurane exposure. The data indicated that sevoflurane increased the intracellular ROS level by 3.3-fold and the mitochondrial ROS level by 11.8-fold. And Ac-YVAD-cmk relieved the intracellular ROS level and mitochondrial ROS accumulation induced by sevoflurane (Fig 4E). Ac-YVAD-cmk also decreased the mitochondrial ROS level significantly when the primary hippocampus neurons were exposure to sevoflurane. (S3C Fig) The above results suggested Ac-YVAD-cmk mitigated the sevofluraneinduced mitochondria dysfunction.

Ac-YVAD-cmk reversed the mitophagy impairment induced by sevoflurane
Mitophagy is the removal of damaged mitochondria through autophagy to regulate mitochondrial quality. In order to effect of Ac-YVAD-cmk on mitophagy flux in hippocampus of aged mice after sevoflurane, western blotting was used. Sevoflurane enhanced the LC3B II/I and accumulated the level of P62, and increased the expression level of Tomm20. (Fig 5A and 5C) The results suggested that sevoflurane inhibited mitophagy in hippocampus. And in the sevoflurane mice, Ac-YVAD-cmk blocked the high level of LC3B II/I, P62 and Tomm20, reversed the mitophagy flux to normal level.
In order to detect the mitophagy flux, the H4 cells were transfected with mt-Keima reporters. Administration of the mitophagy inducer CCCP significantly increased red fluorescence in H4 cells, suggesting that mitophagy was induced (S4C Fig). H4 cells were treated with sevoflurane and Ac-YVAD-cmk. H4 cells treated with sevoflurane had lower red fluorescence compared with control cells, indicating sevoflurane caused mitophagy dysfunction. Ac-YVAD- cmk rescued the mitophagy flux by increasing the red fluorescence. These data suggested that Ac-YVAD-cmk alleviated the mitophagy impairment induced by sevoflurane.
We measured the colocalization of a mitochondrial matrix protein, HSP60 and lysosomal membrane protein, LAMP2 in hippocampus of aged mice after sevoflurane and Ac-YVADcmk treatment by immunostaining assay. The expression level of HSP60 were higher in the sevoflurane mice than that of control mice, Ac-YVAD-cmk decreased the high level of HSP60 after sevoflurane treatment. Sevoflurane reduced the expression level of LAMP2 in hippocampus. In the SEV+AC group, the expression level of LAMP2 were higher than that of in the SEV group. (Fig 5E and 5F) Moreover, we used Pearson's correlation coefficient (PCC) to quantify colocalization of HSP60 and LAMP2. Ac-YVAD-cmk increased the PPC which was reduced by sevoflurane. (Fig 5G) The above results indicated sevoflurane promoted mitochondria accumulation and lysosome dysfunction resulting mitophagic flux inhibition and further verified the positive role of Ac-YVAD-cmk on the mitophagy flux dysfunction induced by sevoflurane.

Discussion
The underlying pathogenesis of POCD is still unknown. Our previous work revealed the role of mitophagy on the cognitive impairment in aged mice after treatment with sevoflurane [14]. In present work, we provided evidence that NLRP3 inflammasomes was involved in postoperative cognitive impairment. Utilization of caspase-1 inhibitor, Ac-YVAD-cmk mitigated sevoflurane-induced learning ability impairment. Moreover, we found that for the first time Ac-YVAD-cmk mitigated mitochondria function and revised mitophagy flux induced by sevoflurane. The current work disclosed Ac-YVAD-cmk treatments for POCD are beneficial for the improvement outcome, and provide a proof involving the interaction between inflammasomes and mitophagy.
Dysregulated activation of inflammasomes, mitophagy dysfunction are relevant to pathogenesis of neurodegenerative diseases [27,28]. Disturbance of the mitochondria following mitochondria ROS generation triggered inflammasome activation [29]. Meanwhile, mitophagy is required for clearance the damaged mitochondria [30]. Mitophagy dysfunction accumulated the production of mitochondrial ROS and increased the translocation of mitochondria DNA into the cytosol. The above risks intensified the inflammasome activation [31,32]. A series of reports revealed ameliorated cognitive dysfunction through the activated of mitophagy and subsequently inhibition of the NLRP3 inflammasome [10,27,33]. These reports suggested that mitophagy dysfunction regulated inflammasome activation. In addition, inflammasome activation leads to caspase-1 dependent mitochondrial damage regulating mitophagy flux. The recent research suggested inflammasomes mediated Parkin cleavage inhibiting mitophagy [34]. The danger-associated molecular patterns (DAMP) triggers mitochondrial outer membrane permeabilization and mitochondrial permeability. And inflammasome activation might leads to block of mitophagy. In present study, in the aged mice

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following sevoflurane treatment, we found Ac-YVAD-cmk decreased the expression of cleaved caspase-1, IL-1β and IL-18 inhibiting the activation of NLRP3 (Fig 2). As a distinguished inhibitor of caspase-1, Ac-YVAD-cmk mitigated the sevoflurane-induced mitochondria dysfunction through reducing the lipid peroxidation and mitochondrial ROS accumulation ( Fig  3). Moreover, our work suggested, for the first time, that Ac-YVAD-cmk blocked the high level of LC3B II/I, P62 and Tomm20 induced by sevoflurane in aged mice and revising the mitophagy flux by using mt-Keima reporters in H4 cells. Ac-YVAD-cmk increased the colocalization with HSP60 and LAMP2 in hippocampus which was reduced by sevoflurane. We found for the first time Ac-YVAD-cmk ameliorated sevoflurane-induced learning ability impairment through NLRP3 inflammasomes activation subsequent revising mitophagy flux.
A couple of reports suggested that NLRP3 inflammasome regulated autophagy. NLRP3 was reported to interact with Beclin 1, a protein promoted in autophagy initiation, through NACHT domain [35]. Our previous work indicated sevoflurane induced mitophagy dysfunction through inactivation of Parkin expression. Overexpression of PARK2, but not mutant PARK2 lacking enzyme activity, in neuron cell lines declined ROS and Tomm20 accumulation and reversed mitophagy dysfunction following sevoflurane treatment [14]. In bone marrowderived macrophages, inflammasome activation caspase-1-mediated cleavage of Parkin is required to caspase-1-dependent inhibition of mitophagy [34]. In the present work, Ac-YVAD-cmk might mediate cleavage of Parkin through the caspase-1 activation resulting revising mitophagy impairment.
Ac-YVAD-cmk, a well-known selective inhibitor of caspase-1, has been reported to exert a protective effect on the brain, liver, and lung in mouse models of septic shock in previous studies [4,29,36]. Ac-YVAD-cmk decreased the release of mature IL-1β/IL-18 and microglia activation in perihematomal tissue of ICH rat improving the behavioral performance [23]. Ac-YVAD-cmk pretreatment attenuated isoflurane-induced NLRP-caspase-1 pathway and learning ability impairment [4]. In the current study, we found Ac-YVAD-cmk ameliorated NLRP3 inflammasome activation induced by sevoflurane, revised mitophagy flux, improved cognitive performance. Pretreatment of Ac-YVAD-cmk is regarded as a novel therapeutic strategy for neuroinflammation-induced learning ability impairment.
The E17 mice hippocampal neurons were widely used in neurobiological research. Some research teams utilized primary hippocampal neurons to explore the neurotoxicity of sevoflurane [22,37,38]. Primary neurons receive and transmit chemical or electric signals and directly take part in the signal transduction. In neurons, synaptic plasticity is one of the important neurochemical foundations of learning and memory [39]. However, it's not easy to transfect primary neurons with chemical reagents [40]. The low transfection efficiency limits the application of primary neurons. In our current study, the results of western blots in primary hippocampus neurons suggested that NLRP3 inflammasomes activation may be involved in cognition dysfunction induced by sevoflurane, and Ac-YVAD-cmk reduced the inflammasome activation induced by sevoflurane. We also measured regional mitochondrial ROS accumulation and the intracellular ROS levels in primary hippocampal neuron. Ac-YVAD-cmk relieved the mitochondrial ROS accumulation induced by sevoflurane. These results suggested Ac-YVAD-cmk alleviated the sevoflurane-induced cognition dysfunction, partly due to mitigation of mitochondria damage. The glial cells perform a role in regulating homeostasis of the nervous system which provides adequate protection and support in the functioning of the nervous system [41]. H4 human neuroglioma cells have been utilized as an alternative in several previous studies [14,21,42], despite their immortal nature. High transfection efficiency in H4 cells help us to explore many complex physiological and pathological mechanisms in nervous system. In order to detect the mitophagy flux, mt-Keima reporters were transfected into H4 cells by Lipofectamine 3000. Ac-YVAD-cmk rescued the mitophagy flux by increasing the red fluorescence. We utilized H4 cells to further verify the positive role of Ac-YVAD-cmk on the mitophagy flux dysfunction induced by sevoflurane, resulting relieved cognitive impairment. According to the characteristic of primary hippocampus neuron and H4 human neuroglioma cells, we fully utilized these two types of nerve cells to explore the mechanisms of cognitive impairment in aged mice induced by sevoflurane. We verified that Ac-YVAD-cmk ameliorated sevoflurane-induced cognitive dysfunction and revised mitophagy impairment.
As a form of cell death, pyroptosis is characterized by the activation of NLRP3 inflammasomes, the formation of cell membrane pore and mediated by gasdermin (GSDM) family protein (GSDMD and GSDME) [43,44]. Proinflammatory Caspases (Casps) induce cleaving the protein GSDMD, which targets cellular membranes and leads to plasma membrane permeabilization [45]. In our study, we found sevoflurane induced the expression of Cleaved GSDMD in hippocampus of aged mice (S5A and S5B Fig). Moreover, the outer layer of the nuclear membrane was compromised by sevoflurane, as the ultrastructural damages of cells in hippocampus shown by TEM. The rupture of the plasma membrane in extremis led to the extravasation of cellular contents, as shown by the reduced abundance of intracellular dense particles (S5C Fig). The above results suggested that pyroptosis was involved in the cognitive impairment induced by sevoflurane. Some specific inhibitor of pyroptosis, like necrosulfonamide should be used to clarify the role of pyroptosis in cognitive impairment further.
In the current study, we identified NLRP3 inflammasomes activation as a cause of sevoflurane-induced cognitive impairment. We promote that, for the first time, Ac-YVAD-cmk ameliorated NLRP3 inflammasome activation induced by sevoflurane and revised mitophagy flux.